research progress on stem cell therapies for articular cartilage regeneration

Articular cartilage regeneration by activated skeletal stem cells

Affiliations.

• 1 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
• 2 Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA.
• 3 Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
• 4 Department of Bioengineering, Stanford University, Palo Alto, CA, USA.
• 5 Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
• 6 Department of Stanford Nano Shared Facilities, Stanford University, Palo Alto, CA, USA.
• 7 Department of Surgery, University of Michigan, Ann Arbor, MI, USA.
• 8 Department of Orthopaedic Surgery, Stanford University, Palo Alto, CA, USA.
• 9 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. [email protected].
• 10 Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA. [email protected].
• 11 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. [email protected].
• 12 Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA. [email protected].
• PMID: 32807933
• PMCID: PMC7704061
• DOI: 10.1038/s41591-020-1013-2

Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage 1 . The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation 2-4 . Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis 5-7 . We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

Publication types

• Research Support, N.I.H., Extramural
• Research Support, Non-U.S. Gov't
• Research Support, U.S. Gov't, Non-P.H.S.
• Cartilage, Articular / cytology
• Cartilage, Articular / physiology*
• Cell Differentiation
• Cells, Cultured
• Chondrocytes / cytology
• Chondrocytes / physiology
• Chondrogenesis / physiology
• Fetal Tissue Transplantation
• Fetus / cytology
• Heterografts
• Mesenchymal Stem Cells / physiology
• Mice, Inbred C57BL
• Mice, Transgenic
• Regeneration / physiology*
• Stem Cells / cytology
• Stem Cells / physiology*
• Tissue Engineering / methods

Grants and funding

• S10 OD021514/OD/NIH HHS/United States
• K99 AG049958/AG/NIA NIH HHS/United States
• R01 DE026730/DE/NIDCR NIH HHS/United States
• HHMI/Howard Hughes Medical Institute/United States
• R01 AR071379/AR/NIAMS NIH HHS/United States
• U01 HL099776/HL/NHLBI NIH HHS/United States
• R21 DE024230/DE/NIDCR NIH HHS/United States
• R00 AG049958/AG/NIA NIH HHS/United States
• R01 DE027323/DE/NIDCR NIH HHS/United States
• T32 GM119995/GM/NIGMS NIH HHS/United States
• U24 DE026914/DE/NIDCR NIH HHS/United States
• R21 DE019274/DE/NIDCR NIH HHS/United States
• R01 GM123069/GM/NIGMS NIH HHS/United States
• R56 DE025597/DE/NIDCR NIH HHS/United States
• R01 DE021683/DE/NIDCR NIH HHS/United States

Cartilage: From Biology to Biofabrication pp 453–490 Cite as

Stem Cells Therapy for Cartilage Regeneration in Clinic: Challenges and Opportunities

• Mina Shahnazari 3 ,
• Sara Malih 4 , 5 ,
• Reza Naeimi 3 ,
• Marzieh Savari 3 ,
• Niloofar Shokrollah 3 ,
• Parisa Samadi 6 &
• Mohsen Sheykhhasan 3 , 7  
• First Online: 12 July 2023

246 Accesses

For both academics and clinicians, the repair and regeneration of articular cartilage have offered a challenging array of issues. Injuries to articular cartilage have a poor chance of healing since it is an avascular tissue. Small defects may eventually heal on their own without treatment, but the repair tissue is inferior to the body’s own hyaline cartilage because it is made of fibrocartilage. Due to its regenerative capabilities, the idea of stem cell therapy has sparked intense research into its potential application for treating cartilage lesions, including OA. The purpose of this chapter is to present a perspective on stem cell-based therapy for cartilage repair and to highlight recent developments in advanced cell therapy, in particular, the use of embryonic stem cells, mesenchymal stem cells, and induce pluripotent stem cells for treating diseases associated with cartilage defects, particularly OA.

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Researchers find method to regrow cartilage in the joints

In laboratory studies, Stanford School of Medicine researchers have found a way to regenerate the cartilage that eases movement between bones.

August 17, 2020 - By Christopher Vaughan

Michael Longaker

Researchers at the Stanford University School of Medicine have discovered a way to regenerate, in mice and human tissue, the cushion of cartilage found in joints.

Loss of this slippery and shock-absorbing tissue layer, called articular cartilage, is responsible for many cases of joint pain and arthritis, which afflicts more than 55 million Americans. Nearly 1 in 4 adult Americans suffer from arthritis, and far more are burdened by joint pain and inflammation generally.

The Stanford researchers figured out how to regrow articular cartilage by first causing slight injury to the joint tissue, then using chemical signals to steer the growth of skeletal stem cells as the injuries heal. The work was published Aug. 17 in the journal Nature Medicine .

“Cartilage has practically zero regenerative potential in adulthood, so once it’s injured or gone, what we can do for patients has been very limited,” said assistant professor of surgery Charles K.F. Chan , PhD. “It’s extremely gratifying to find a way to help the body regrow this important tissue.”

The work builds on previous research at Stanford that resulted in isolation of the skeletal stem cell, a self-renewing cell that is also responsible for the production of bone, cartilage and a special type of cell that helps blood cells develop in bone marrow. The new research, like previous discoveries of mouse and human skeletal stem cells, were mostly carried out in the laboratories of Chan and professor of surgery Michael Longaker , MD.

Articular cartilage is a complex and specialized tissue that provides a slick and bouncy cushion between bones at the joints. When this cartilage is damaged by trauma, disease or simply thins with age, bones can rub directly against each other, causing pain and inflammation, which can eventually result in arthritis.

Charles K.F. Chan

Damaged cartilage can be treated through a technique called microfracture, in which tiny holes are drilled in the surface of a joint. The microfracture technique prompts the body to create new tissue in the joint, but the new tissue is not much like cartilage.

 “Microfracture results in what is called fibrocartilage, which is really more like scar tissue than natural cartilage,” said Chan. “It covers the bone and is better than nothing, but it doesn’t have the bounce and elasticity of natural cartilage, and it tends to degrade relatively quickly.”  

The most recent research arose, in part, through the work of surgeon Matthew Murphy, PhD, a visiting researcher at Stanford who is now at the University of Manchester. “I never felt anyone really understood how microfracture really worked,” Murphy said. “I realized the only way to understand the process was to look at what stem cells are doing after microfracture.” Murphy is the lead author on the paper. Chan and Longaker are co-senior authors.

For a long time, Chan said, people assumed that adult cartilage did not regenerate after injury because the tissue did not have many skeletal stem cells that could be activated. Working in a mouse model, the team documented that microfracture did activate skeletal stem cells. Left to their own devices, however, those activated skeletal stem cells regenerated fibrocartilage in the joint.

But what if the healing process after microfracture could be steered toward development of cartilage and away from fibrocartilage? The researchers knew that as bone develops, cells must first go through a cartilage stage before turning into bone. They had the idea that they might encourage the skeletal stem cells in the joint to start along a path toward becoming bone, but stop the process at the cartilage stage.

The researchers used a powerful molecule called bone morphogenetic protein 2 (BMP2) to initiate bone formation after microfracture, but then stopped the process midway with a molecule that blocked another signaling molecule important in bone formation, called vascular endothelial growth factor (VEGF). 

“What we ended up with was cartilage that is made of the same sort of cells as natural cartilage with comparable mechanical properties, unlike the fibrocartilage that we usually get,” Chan said. “It also restored mobility to osteoarthritic mice and significantly reduced their pain.”

As a proof of principle that this might also work in humans, the researchers transferred human tissue into mice that were bred to not reject the tissue, and were able to show that human skeletal stem cells could be steered toward bone development but stopped at the cartilage stage.

The next stage of research is to conduct similar experiments in larger animals before starting human clinical trials. Murphy points out that because of the difficulty in working with very small mouse joints, there might be some improvements to the system they could make as they move into relatively larger joints.

The first human clinical trials might be for people who have arthritis in their fingers and toes. “We might start with small joints, and if that works we would move up to larger joints like knees,” Murphy says. “Right now, one of the most common surgeries for arthritis in the fingers is to have the bone at the base of the thumb taken out. In such cases we might try this to save the joint, and if it doesn’t work we just take out the bone as we would have anyway. There’s a big potential for improvement, and the downside is that we would be back to where we were before.”

Longaker points out that one advantage of their discovery is that the main components of a potential therapy are approved as safe and effective by the FDA. “BMP2 has already been approved for helping bone heal, and VEGF inhibitors are already used as anti-cancer therapies,” Longaker said. “This would help speed the approval of any therapy we develop.”

Joint replacement surgery has revolutionized how doctors treat arthritis and is very common: By age 80, 1 in 10 people will have a hip replacement and 1 in 20 will have a knee replaced. But such joint replacement is extremely invasive, has a limited lifespan and is performed only after arthritis hits and patients endure lasting pain. The researchers say they can envision a time when people are able to avoid getting arthritis in the first place by rejuvenating their cartilage in their joints before it is badly degraded.

 “One idea is to follow a ‘Jiffy Lube’ model of cartilage replenishment,” Longaker said. “You don’t wait for damage to accumulate — you go in periodically and use this technique to boost your articular cartilage before you have a problem.”

Longaker is the Deane P. and Louise Mitchell Professor in the School of Medicine and co-director of the Institute for Stem Cell Biology and Regenerative Medicine. Chan is a member of the Institute for Stem Cell Biology and Regenerative Medicine and Stanford Immunology.

Other Stanford scientist taking part in the research were professor of pathology Irving Weissman, MD, the Virginia and D. K. Ludwig Professor in Clinical Investigation in Cancer Research; professor of surgery Stuart B. Goodman, MD, the Robert L. and Mary Ellenburg Professor in Surgery; associate professor of orthopaedic surgery Fan Yang, PhD; professor of surgery Derrick C. Wan, MD; instructor in orthopaedic surgery Xinming Tong, PhD; postdoctoral research fellow Thomas H. Ambrosi, PhD; visiting postdoctoral scholar Liming Zhao, MD; life science research professionals Lauren S. Koepke and Holly Steininger; MD/PhD student Gunsagar S. Gulati, PhD; graduate student Malachia Y. Hoover; former student Owen Marecic; former medical student Yuting Wang, MD; and scanning probe microscopy laboratory manager Marcin P. Walkiewicz, PhD.

The research was supported by the National Institutes of Health (grants R00AG049958, R01 DE027323, R56 DE025597, R01 DE026730, R01 DE021683, R21 DE024230, U01HL099776, U24DE026914, R21 DE019274, NIGMS K08GM109105, NIH R01GM123069 and NIH1R01AR071379), the California Institute for Regenerative Medicine, the Oak Foundation, the Pitch Johnson Fund, the Gunn/Olivier Research Fund, the Stinehart/Reed Foundation, The Siebel Foundation, the Howard Hughes Medical Institute, the German Research Foundation, the PSRF National Endowment, National Center for Research Resources, the Prostate Cancer Research Foundation, the American Federation of Aging Research and the Arthritis National Research Foundation.

About Stanford Medicine

Stanford Medicine is an integrated academic health system comprising the Stanford School of Medicine and adult and pediatric health care delivery systems. Together, they harness the full potential of biomedicine through collaborative research, education and clinical care for patients. For more information, please visit med.stanford.edu .

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Stem cells in articular cartilage regeneration

• Giuseppe Filardo 1 ,
• Francesco Perdisa 1 ,
• Alice Roffi 2 ,
• Maurilio Marcacci 1 , 2 &
• Elizaveta Kon 1 , 2  

Journal of Orthopaedic Surgery and Research volume  11 , Article number:  42 ( 2016 ) Cite this article

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Mesenchymal stem cells (MSCs) have emerged as a promising option to treat articular chondral defects and early OA stages. However, their potential and limitations for clinical use remain controversial. Thus, the aim of this systematic review was to examine MSCs treatment strategies in order to summarize the current clinical evidence for the treatment of cartilage lesions and OA.

A systematic review of the literature was performed on the PubMed database using the following string: “cartilage treatment” AND “mesenchymal stem cells”. The filters included publications on the clinical use of MSCs for cartilage defects and OA in English language up to 2015.

Our search identified 1639 papers: 60 were included in the analysis, with an increasing number of studies published on this topic over time. Seven were randomized, 13 comparative, 31 case series, and 9 case reports; 26 studies reported the results after injective administration, whereas 33 used surgical implantation. One study compared the 2 different modalities. With regard to the cell source, 20 studies concerned BMSCs, 17 ADSCs, 16 BMC, 5 PBSCs, 1 SDSCs, and 1 compared BMC vs PBSCs.

Conclusions

The available studies allow to draw some indications. First, no major adverse events related to the treatment or to the cell harvest have been reported. Second, a clinical benefit of using MSCs therapies has been reported in most of the studies, regardless of cell-source, indication or administration method. Third, young age, lower BMI, smaller lesion size for focal lesions and earlier stages of OA joints, have been shown to correlate with better outcomes, even though the available data strength doesn’t allow to define clear cutoff values.

Articular cartilage lesions are a debilitating disease, often resulting in fibrillation and subsequent degradation of the surrounding articular surface, possibly involving the subchondral bone as well, thus favoring the development of osteoarthritis (OA). OA affects up to 15 % of the adult population and represents the second greatest cause of disability worldwide [ 1 ], with a massive impact on society both in terms of quality of life for the individuals and high costs for the healthcare system [ 2 ]. Several approaches have been proposed for the management of cartilage degeneration, ranging from pharmacological to surgical options, aimed at reducing symptoms and restoring a satisfactory knee function [ 3 , 4 ]. However, none of them has clearly shown the potential of restoring chondral surface and physiological joint homeostasis in order to prevent OA, which in the final stage often requires prosthetic replacement.

Among the solutions proposed to delay the need for metal resurfacing of the damaged articular surface, mesenchymal stem cells (MSCs) have recently emerged as a promising option to treat articular defects and early OA stages [ 5 ]. MSCs are multipotent progenitor cells that can differentiate into selected lineages including chondrocytes, with capability of self-renewal, high plasticity, and immunosuppressive and anti-inflammatory action [ 6 , 7 ]. Moreover, Caplan and colleagues [ 8 ] recently underlined that these cells, derived from perivascular cells called “pericytes”, have a key role in the response to tissue injuries not just by differentiating themselves, but also by inducing repair/regeneration processes at the injury site through the secretion of several bioactive molecules [ 9 ]. In light of these properties, MSCs represent an excellent candidate for cell therapies and their healing potential has been explored also in terms of cartilage tissue regeneration and OA processes modulation [ 6 ]. The first investigations involved MSCs derived from bone marrow, which have been applied either as a cell suspension after being expanded by culture (BMSCs), or used as a simple bone marrow concentrate (BMC), thanks to their relative abundance [ 6 ]. Despite an extensive preclinical research and promising clinical results, some drawbacks related to the cell harvest and culture led to the development of different alternative options, with stem cells derived from adipose tissue (ADSCs), synovial tissue (SDSCs), and peripheral blood (PBSCs) [ 10 , 11 ]. Besides these sources already explored and reported in the clinical use, cells derived from fetal tissues are being currently investigated at preclinical level [ 12 ]. Although numerous advancements have been made, the understanding of MSCs mechanism of action as well as their potential and limitations for the clinical use remain controversial. Many questions are still open on the identification of patients who might benefit more from this kind of treatment, as well as the most suitable protocol of administration (no. of cells, concentrated or culture-expanded, best harvest source, etc.).

Based on these premises, the aim of this systematic review was to examine the literature on MSCs treatment strategies in the clinical setting, in order to summarize the current evidence on their potential for the treatment of cartilage lesions and OA.

Materials and methods

A systematic review of the literature was performed on the PubMed database by two independent reviewers using the following string: “cartilage treatment” AND “mesenchymal stem cells”. The filters included publications on the use of MSCs for cartilage defects and OA in the clinical field and in English language, published from 2000 to the end of 2015. Articles were first screened by title and abstract. Subsequently, the full texts of the resulting articles were screened and those not reporting clinical results of MSCs for cartilage and OA treatment were excluded. The reference lists of the selected articles were also screened to obtain further studies for this review.

Our search identified 1639 papers after the screening process, 60 were included in the analysis (Fig.  1 ), which showed an increasing number of studies published on this topic over time (Fig.  2 ). Among the 60 selected studies, 7 were randomized, 13 comparative, 31 case series, and 9 case reports; 26 studies reported the results after injective administration, whereas 33 used surgical implantation. One study compared the two different modalities. With regard to the cell source, 20 studies concerned BMSCs, 17 ADSCs, 16 BMC, 5 PBSCs, 1 SDSCs, and 1 compared BMC versus PBSCs. While all the included studies are summarized in detail in Table  1 according to cell source and treatment strategy, the most relevant findings will be discussed in the following paragraphs.

Scheme of research methodology

The systematic research showed an increasing number of clinical studies published over time

An increasing number of papers have been focused on this cell source in the past few years, both as BMSCs and BMC. Cultured BMSCs and BMC differ for composition, since adult bone marrow contains heterogeneous blood cells at various differentiation stages [ 13 ]. Thus, the harvest includes plasma, red blood cells, platelets, and nucleated cells, a small fraction of which contains adult MSCs that can be isolated through culture expansion [ 14 ]. However, even if not expanded, the heterogeneity of cell progenitor types in BMC might positively influence tissue regeneration [ 15 ]. Moreover, cell culture not only offers a higher number of cells but also presents high costs and some regulatory problems, since these products might be considered as pharmacological treatments by regulatory agencies. Thus, one-step techniques using BMC for the delivery of autologous cells in a single time are gaining increasing interest in the clinical setting. Besides these considerations, positive findings are leading the research towards the use of both cell-based strategies.

Cultured BMSCs: injective treatment

In 2008, Centeno and colleagues [ 16 ] first reported the promising clinical and MRI improvements at early follow-up after single intra-articular (i.a.) injection of autologous cultured BMSCs in a patient with knee degenerative cartilage disease, and similar findings at short term were later shown also by the groups of Davatchi [ 17 ], Emadedin [ 18 ], and Sol Rich [ 19 ]. Orozco et al. confirmed a rapid and progressive clinical improvement of knee OA in the first 12 months [ 20 ], which was maintained at 24-month follow-up, together with improved cartilage quality at MRI [ 21 ]. Finally, Davatchi et al. [ 22 ] updated their report, showing gradual mid-term deterioration of the outcomes in advanced OA.

Among comparative studies, Lee et al. [ 23 ] tested two administration strategies for focal knee cartilage defects and found no differences either by using BMSCs implantation under periosteum flap or microfractures (MFX) plus BMSCs i.a. injection, thus endorsing the less invasive approach.

Three randomized controlled trials (RCTs) have also been published. Wong et al. [ 24 ] treated knee unicompartmental OA with varus malalignment by combined high tibial osteotomy (HTO) and MFX. Patients randomly received post-operative i.a. injection of BMSCs-hyaluronic acid (HA) or HA alone as control. Both groups improved their scores, but BMSCs produced better clinical and MRI outcomes. Vangsness et al. [ 25 ] administered a single i.a. injection in patients after medial partial meniscectomy. Patients were randomized in two treatment groups (low- or high-dose allogeneic cultured BMSCs with HA) and a control group (HA-only). Both treatment groups showed improved clinical scores versus control, and MRI showed signs of meniscal volume increase at 24 months. Finally, Vega et al. [ 26 ] randomized two treatment groups for knee OA: a significantly greater improvement was shown after a single allogeneic BMSCs injection compared to control HA.

Cultured BMSCs: surgical delivery

Adachi et al. [ 27 ] observed cartilage and bone regeneration in a biopsy after cultured BMSCs implantation on hydroxyapatite-ceramic scaffold for osteochondral knee lesion (OLK). Haalem et al. [ 28 ] implanted BMSCs on a platelet fibrin glue (FG) scaffold, showing significant improvement and complete MRI filling of the cartilage defect. Kasemkijwattana et al. [ 29 ] seeded cells on a collagen scaffold with positive results in two traumatic knee lesions. Similarly, Kuroda et al. [ 30 ] had good results implanting BMSCs on collagen membrane with periosteum coverage in a judo-player knee, with hyaline-like tissue at a 12-month histology evaluation. Wakitani et al. used the same technique with positive findings also for patellofemoral lesions [ 31 ], stable at mid-term follow-up [ 32 ]. They also performed a comparative evaluation of this technique for focal defects in OA knees: two groups were treated with HTO, with or without BMSCs augmentation [ 33 ]. BMSCs-group showed better histology, but clinical scores comparable to the cell-free group. Nejadnik et al. compared BMSCs implantation with first-generation ACI in two groups of patients and observed comparable benefits [ 34 ].

Finally, Richter et al. [ 35 ] investigated the outcomes offered by BMSCs onto a collagen matrix for chondral ankle lesions, confirming no complications and a promising clinical improvement at 24 months of follow-up.

BMC: injective treatment

A single study by Varma et al. [ 36 ] reported promising results with BMC injection after arthroscopic debridement for knee OA, with increased benefits compared to debridement alone.

BMC: surgical delivery

The group of Giannini published several studies of scaffold-associated BMC implantation in knee and ankle joint defects. In their first study [ 37 ], they showed clinical and MRI improvements at 24 months after BMC implantation into collagen powder or HA matrix for osteochondral lesions of the talus (OLTs). Later [ 38 ], they reported a significant worsening between 24 and 48 months of follow-up, but the final result was still satisfactory compared to the basal level. Patients with longer symptoms before surgery had worse clinical outcomes. They also observed no degeneration progression at 24 months in five hemophilic ankle lesions [ 39 ], and similar results were confirmed in a larger group of patients treated for OLTs or ankle OA defects [ 40 ]. Also, this study showed a worsening trend after 24 months with a higher failure rate, which underlined the influence of OA degree and patient BMI. Moreover, a further study by Buda et al. [ 41 ] confirmed a similar trend of gradual worsening up to 72 months after scaffold-assisted BMC implantation.

Giannini et al. [ 42 ] also performed comparative evaluations: positive and similar clinical outcomes were found in three groups of patients treated with one-step BMC-HA matrix implantation versus open ACI or arthroscopic MACT for OLTs at 36 months of follow-up. These results were later confirmed at 48 months after collagen scaffold implantation, seeded either with BMC or cultured chondrocytes, with better tissue quality at MRI for the BMC group [ 43 ]. Moreover, a RCT by Cadossi et al. [ 44 ] highlighted that biophysical stimulation with pulsed electromagnetic fields (PEMFs) might improve the results at 12 months after collagen matrix-BMC implantation for OLTs.

Matrix-assisted BMC implantation was also investigated for the treatment of OLKs. The promising results using BMC on HA matrix were first reported by Buda et al. at short-term follow-up, with positive MRI and histology findings [ 45 , 46 ], and then confirmed by Gobbi et al. [ 47 ], who observed superior outcomes using BMC instead of chondrocytes for the treatment of large patellofemoral defects. Similar results were obtained also by seeding BMC on collagen scaffolds: Gigante et al. [ 48 ] used BMC-enhanced AMIC technique with positive short-term clinical results, but limited tissue quality at histology [ 49 ], and Gobbi et al. [ 50 ] observed hyaline appearance and better short-term improvement in patients younger than 45 years and with single and smaller lesion. Finally, Skowronski et al. [ 51 ] documented stable mid-term outcomes after the treatment of large chondral lesions.

The possibility of using autologous PBSCs obtained by culture expansion from a venous sample was first introduced by Saw et al. [ 52 ], who treated chondral knee lesions with subchondral drilling and five postoperative i.a. injections of PBSCs and HA, reporting no adverse reactions and positive histological findings. Turajane and colleagues [ 53 ] showed short-term clinical improvement using the same technique in early knee OA patients. Later, the group of Saw [ 54 ] also performed a RCT, documenting comparable clinical outcomes at 24 months, but better MRI and histological evaluations versus HA control.

With regard to surgical application, Fu et al. [ 55 ] reported optimal results at 7.5 years in a lateral trochlea lesion treated with patellar realignment plus periosteum-covered PBSCs implantation in a kick boxer, and Skowronski et al. [ 56 ] implanted PBSCs with a collagen membrane in a group of patients, reporting a stable improvement up to 72-month follow-up.

ADSCs present a lower chondrogenic potential when compared with BMSCs [ 57 ]. Nonetheless, they can be obtained from liposuction, a simple and cheap procedure, and their clinical use is rapidly increasing, thanks to their easy availability and abundance [ 10 ]. Whereas the use of cultured cells has rarely been reported, the preferred technique involves cell harvest, collagenase digestion, and isolation of the stromal vascular fraction (SVF), a heterogeneous cell population that, among pre-adipocytes and immune cells, also includes ADSCs [ 58 ].

Injective treatment

Jo et al. [ 59 ] published the only available study on cultured ADSCs, applied at different doses: their preliminary clinical data showed no adverse events, and a clinical-MRI improvement at 6 months after injecting the highest dose.

Most of the literature focused instead on SVF. Regarding knee OA, Pak et al. [ 60 ] first obtained a promising clinical improvement 3 months after i.a. injection of subcutaneous SVF with HA, dexamethasone, and PRP in a patient. Later, they [ 61 ] confirmed safety and effectiveness of SVF injections in a larger cohort of patients treated into different joints. Bui et al. [ 62 ] also reported short-term clinical and MRI improvement after injection of SVF and PRP. However, the group of Koh was the main investigator of SVF use, starting from the infrapatellar fat pad source, in a case–control study [ 63 ]: all patients underwent debridement and the treatment group received an additional SVF-PRP injection. No major adverse events and a tendency for better outcomes were observed in the SVF group. The improvement was confirmed at 24 months in a further study [ 64 ]. The number of injected cells correlated with both clinical and MRI outcomes, while SVF had lower effects on the final stage OA. Later, the same group began to process subcutaneous fat with an analogous technique. They treated knee OA in elderly patients with arthroscopic lavage and SVF-PRP injection [ 65 ]: clinical improvement was obtained both at 12 and 24 months, and positive findings were reported at second look evaluation. Moreover, SVF injections significantly improved the benefits of high tibial osteotomy (HTO) for symptomatic varus knee, compared to control (HTO and PRP-only), both at clinical and second look evaluation [ 66 ].

Michalek et al. [ 67 ] administered single-dose SVF injections to the largest available group of patients, reporting no treatment-related adverse events and gradual clinical improvement between 3 and 12 months, with a slower recovery for obese and higher OA degrees.

Finally, the group of Koh also investigated SVF use in the ankle joint: Kim et al. injected SVF after marrow stimulation in two comparative studies, and observed higher clinical and MRI improvement both for ankle OA [ 68 ] or OLTs [ 69 ], compared to surgery alone. The benefit was greater for younger patients with smaller lesions, but the treatment was effective even in older patients.

Surgical delivery

Koh et al. [ 70 ] reported a significant clinical improvement 2 years after a scaffold-free SVF implantation for focal chondral lesions in OA knees, but abnormal repair tissue was observed in most cases at second look evaluation. In a subsequent study, the association with FG as scaffold significantly improved tissue quality, even though clinical results remained similar to SVF alone [ 71 ]. Later, they reported positive short-term results and correlation with MRI findings after SVF-FG implantation for OA [ 72 ]. Furthermore, a larger prospective study confirmed good/excellent results in 75 % patients at 24 months [ 73 ]. Interestingly, older age, higher BMI, and larger defect size were negative predictors in all these studies. SVF-FG augmentation also improved the outcome versus MF alone in an RCT, despite comparable histology findings [ 74 ].

Finally, a study on matched-paired groups found comparable clinical results but better ICRS macroscopic scores at 12 months for SVF surgical implantation versus injective delivery, whereas at the further follow-up, a significant clinical superiority was also obtained for surgical SVF delivery [ 75 ].

SDSCs are a promising source of stem cells for cartilage tissue engineering, thanks to the greatest chondrogenic and lowest osteogenic potential among MSCs [ 57 ]. Sekiya et al. [ 76 ] reported promising results up to mid-term follow-up using SDMSCs scaffold-free implantation into single knee cartilage defects, with ¾ biopsies showing hyaline cartilage.

Comparative studies

Skowronski et al. [ 77 ] performed the only clinical comparative study among stem cell types showing superior results with PBSCs rather than BMC under a collagen membrane for OLKs at 5-year follow-up.

This systematic research highlighted that the use of mesenchymal precursors as a biological approach to treat cartilage lesions and OA has widely increased (Fig.  2 ), as confirmed by the growing number of clinical trials published on this topic. In addition to an intensive preclinical research, the use of these procedures has recently broken down the barriers towards clinical application, with more than half of the available papers published in the last 3 years. Different sources have been investigated for clinical application, especially targeting knee or ankle cartilage disease. Among them, the most exploited cell types are those derived from bone marrow and adipose tissue. Cells have been used either after culture expansion or simply concentrated for one-step procedures: in particular, adipose cells have been applied mainly through cell concentration, and cells derived from bone marrow are currently applied both after expansion or concentration, while PBSCs and SDSCs can be only exploited through in vitro expansion due to their low number.

Regardless of cell source and manipulation, cells have being administered either surgically or through i.a. injection, to target focal lesions as well as degenerative joint disease.

Overall, despite the increasing literature on this topic, there is still limited evidence about the use of MSCs for the treatment of articular cartilage, in particular as far as high-level studies are concerned: in fact, most of the available papers are case series, while only few papers reported RCTs. Moreover, the few high level studies do not allow to clearly prove the effective potential of MSCs, due to the limited number of patients treated and to the presence of several confounding factors (PRP concomitant use, cell use in combination with scaffolds, etc.). To this regard, while several studies applied cells in association with PRP, with the rationale to provide both cells and growth factors at the same time, there is no evidence that adding platelet-derived growth factors provides any increased benefit with respect to cell administration alone, and specifically designed studies are needed in order to clarify the role of PRP with respect to MSCs and/or scaffolds in cartilage treatment. Furthermore, the tissue harvest procedure poses practical and ethical limitations which prevent from performing studies with a blinded design, therefore leaving an important bias related to the placebo effect, which is an important issue in this field of new fashionable regenerative treatments.

On the other hand, the available studies still allow to draw some indications on potential and limitations of MSCs clinical use for the treatment of cartilage lesions and OA.

First, the use of MSCs in the clinical setting can be considered safe, since no major adverse events related to the treatment nor to the cell harvest have been reported, at least from the available reports at short- to mid-term follow-up. Second, a clinical benefit of using MSCs therapies has been reported in most of the studies, regardless of cell source, indication, or administration method. This effectiveness has been reflected by clinical improvement but also positive MRI and macroscopic findings, whereas histologic features gave more controversial results among different studies. Third, different studies also gave a few indications regarding the patients who might benefit more from MSCs treatment: young age, lower BMI, smaller lesion size for focal lesions, and earlier stages of OA joints have been shown to correlate with better outcomes, even though the available data strength does not allow to define clear cutoff values.

The systematic analysis of the literature also allowed to underline other interesting findings that deserve to be discussed. Definite trends can be observed with regard to the delivery method: while different combinations of products and delivery methods have been investigated over the years, currently cultured cells are mostly being administered by i.a. injection, while one-step surgical implantation is preferred for cell concentrates. The different trends observed in this field are explained both by the controversial preclinical and clinical findings, which still leaves space for clinical investigations in opposite direction, but also by practical considerations, both in terms of economical, ethical, and regulatory limitations [ 6 ]. Many aspects are taken in consideration for the treatment choice, with physicians and researchers exploring different strategies, each one presenting potential advantages and possible drawbacks. To this regard, while culture expansion guarantees a selected MSC lineage to be delivered, but presenting high costs and some contamination risks related to cell manipulation, cell concentration offers a lower number of MSCs, in a heterogeneous cell population, and can be performed in one step, thus simplifying the procedure, reducing costs, and increasing patient compliance. To date, no clear evidence of superior outcome between the two cell manipulations is available, and also their most effective delivery method remains to be defined, with only a single retrospective study reporting better results for surgical delivery compared to i.a. SVF injection in a matched-paired analysis of two groups treated for single focal defects in knee OA [ 75 ]. Regarding surgical implantation, the use of solid scaffolds has been shown to be beneficial for SVF implantation [ 71 ], and it is the gold standard for the application of BMC [ 37 – 41 , 43 , 47 , 48 , 50 , 51 ]. The good results obtained with scaffolds implanted with BMC have been compared with chondrocyte-based surgical techniques, showing similar outcomes, but with the advantage of the one-step approach [ 42 , 43 , 47 ].

Finally, regardless of cell source, manipulation and delivery method, the optimal cell dose is still under investigation. After a first preliminary study reported no complications related to high dose of cultured ADSCs [ 59 ], only a single clinical study specifically focused on this aspect, suggesting benefits and absence of side effects by using higher dose of BMSCs for the treatment of post-meniscectomized knees [ 25 ]. However, the lack of standardization and the heterogeneity of the studies reported in the current literature do not allow to extend these findings to the several proposed MSCs treatment strategies.

The clinical application of MSCs for the treatment of articular cartilage defects and OA shows promising results, but too many questions still remain open. Even though no complications have been reported, longer follow-ups on broader patient population are needed to confirm the safety of these procedures. Likewise, while promising results have been shown, the potential of these treatments should be confirmed by reliable clinical data through double-blind, controlled, prospective, and multicenter studies with longer follow-up. In addition, specific studies should be designed to identify the best cell sources, manipulation, and delivery techniques, as well as pathology and disease phase indications, with the aim of optimizing the outcome for a treatment focused on focal chondral defects or joint degeneration.

This systematic review revealed a high interest of researchers in the clinical use of MSCs for cartilage and OA treatment, as testified by the increasing number of reports published over time. Whereas the lack of contraindication and generally promising clinical outcomes have been reported, the prevalence of low-quality studies, with many variables, shows several aspects that still need to be optimized, such as the best cell source and the most appropriate processing method, the most effective dose and delivery procedure. On the other hand, the first hints on the kind of patients who might benefit more from these procedures are being drawn. High-level studies with large number of patients and long-term follow-up are mandatory to evaluate the real potential of this biological approach for cartilage repair.

Abbreviations

mesenchymal stem cells derived from adipose tissue

bone marrow concentrate

bone marrow expanded stem cells

fibrin glue

hyaluronic acid

high tibial osteotomy

intra-articular

matrix-assisted stem cells transplantation

microfractures

mesenchymal stem cells

osteoarthritis

osteochondral knee lesion

osteochondral lesions of the talus

stem cells derived from peripheral blood

pulsed electromagnetic fields

randomized controlled trial

mesenchymal stem cells derived from synovial tissue

stromal vascular fraction

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This project received institutional support by the Italian Ministry of Health Ricerca Finalizzata (RF-2011-02352638).

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Giuseppe Filardo, Francesco Perdisa, Maurilio Marcacci & Elizaveta Kon

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Filardo, G., Perdisa, F., Roffi, A. et al. Stem cells in articular cartilage regeneration. J Orthop Surg Res 11 , 42 (2016). https://doi.org/10.1186/s13018-016-0378-x

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Current concepts and perspectives for articular cartilage regeneration

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Articular cartilage injuries are common in the population. The increment in the elderly people and active life results in an increasing demand for new technologies and good outcomes to satisfy longer and healthier life expectancies. However, because of cartilage's low regenerative capacity, finding an efficacious treatment is still challenging for orthopedics.

Since the pioneering studies based on autologous cell transplantation, regenerative medicine has opened new approaches for cartilage lesion treatment.

Tissue engineering combines cells, biomaterials, and biological factors to regenerate damaged tissues, overcoming conventional therapeutic strategies. Cells synthesize matrix structural components, maintain tissue homeostasis by modulating metabolic, inflammatory, and immunologic pathways. Scaffolds are well acknowledged by clinicians in regenerative applications since they provide the appropriate environment for cells, can be easily implanted, reduce surgical morbidity, allow enhanced cell proliferation, maturation, and an efficient and complete integration with surrounding articular cartilage. Growth factors are molecules that facilitate tissue healing and regeneration by stimulating cell signal pathways.

To date, different cell sources and a wide range of natural and synthetic scaffolds have been used both in pre-clinical and clinical studies with the aim to find the suitable solution for recapitulating cartilage microenvironment and inducing the formation of a new tissue with the biochemical and mechanical properties of the native one. Here, we describe the current concepts for articular cartilage regeneration, highlighting the key actors of this process trying to identify the best perspectives.

Introduction

Articular cartilage covers the bony ends of diarthrodial joints; it is a smooth thin hyaline tissue with friction-reducing and load-bearing functions. It lacks blood vessels, lymphatics, and nerves [ 1 ]. As for connective tissues, an extracellular matrix (ECM) composed of water, collagens, and proteoglycans surrounds the cells. Chondrocytes are mature secretory scarcely distributed cells located in spaces termed lacunae. They are spread among superficial, middle, deep, and calcified four zones. Those zones display different cell shapes, secretory patterns, and fiber orientation [ 2 ]. The layer of bone below the hyaline cartilage is known as subchondral bone. It has a structural and mechanical function (shock absorber) and can be involved in the etiology or effects of cartilage damages or diseases [ 3 ].

Chondral (affecting articular cartilage) and osteochondral (affecting cartilage and the underlying bone) lesions are very common and can appear at any age [ 4 , 5 ]. In the young population, they are most of traumatic origin (sport or accident), often with ligament and meniscal injuries. Some conditions like osteochondritis dissecans may lead to articular surface disruption and release of intra-articular bodies composed of cartilage or cartilage and bones [ 6 ]. In the elderly population, lesions are associated with rheumatic diseases (i.e., Osteoarthritis-OA) or derived from wear and tear due to excessive use (occupational injury) or age [ 7 , 8 ].

In general, repair of full-thickness cartilage lesions mainly depend on the patient’s age, defect size, and location. Small full-thickness defects may adjust through the formation of hyaline cartilage. In contrast, large osteochondral defects repair by forming a scar, fibrous tissue, or fibrocartilage in which the predominant component is collagen type I [ 2 , 9 ].

This kind of cartilage could be functionally active for a short period, but the tissue does not present the mechanical and strength characteristics of normal cartilage, thus not ensuring the healing of the defect nor the symptom remission. This situation could favor, over time, the development of OA [ 10 ].

Conventional cartilage treating procedures are palliative and reparative. Palliative strategies usually represent the first-line treatment to decrease symptoms without addressing the causes. Reparative approaches are abrasions drilling and microfracture. They include a subchondral bone penetration allowing stem cells to migrate from bone marrow to the injury site and form, as explained before, mostly a tissue with fibrous features.

Regenerative methods have emerged as an alternative to tissue repair or replacement to regrow or restore diseased cells, tissues, or organs. Osteochondral grafting represents a possible solution for creating a hyaline-like tissue in the affected area. However, it has shown some drawbacks like donor site morbidity, graft failure (autograft), or possible disease transmission (allograft) [ 11 ]. Lately, different therapeutic solutions in cartilage regenerative medicine have emerged.

With this manuscript, we would like to report the main therapeutic strategies in this field, focusing on recent approaches in tissue engineering and particularly on up-to-date knowledge’s on cells, growth factors, and scaffolds.

• Tissue engineering

Tissue engineering has represented a new fascinating, and innovative approach to regenerating articular tissue [ 12 ]. As reported above, this therapeutic strategy involves the use of cells, scaffolds, and growth factors (GF), trying to mimic the complex three-dimensional microenvironment of the joint that requires the interaction between these different components. Reproducing such complexity is critical, and many issues are still open concerning the ideal cell population, the use of GF and the suitable scaffolds [ 1 , 13 ].

Cells can be administered as therapeutic agents to rebuild damaged cartilage in joints. The leading cell types used in treating chondral and osteochondral defects are chondrocytes and mesenchymal stromal cells from various sources [ 14 ]. The technique requires that cells are isolated and then expanded ex vivo in a monolayer culture before the implant. In terms of legislation, expanded cells belong to Advanced Therapy Medicinal Products (ATMPs) and must follow specific rules already encoded for conventional drugs and known as Good Manufacturing Practices (GMPs). GMPs entail the standardization and control of medicinal manufacturing, ensuring their safety and reducing contaminations [ 15 ].

Autologous chondrocyte implantation

Autologous Chondrocyte Implantation (ACI) is a two-step procedure that has been used in the clinic for many years [ 16 ]. In the original ACI technique (first-generation technique), the first step consisted of surgically removing small biopsies of normal cartilage from non-weight-bearing areas of the knee. Chondrocytes were then enzymatically isolated from the biopsies, expanded ex vivo in monolayer culture condition, and, after several weeks, harvested as a cell suspension. In the second step, surgeons injected the cell suspension under a periosteal flap harvested from the proximal medial tibia and previously sutured over the cartilage. Chondrocyte expansion was deemed necessary due to cartilage cell scarcity [ 17 ].

ACI that uses suspended cultured chondrocytes with a covering of collagen type I/III membrane is considered a second-generation. Third-generation ACI comprises those procedures that deliver autologous cultured chondrocytes using cell carriers or cell-seeded scaffolds. These second and third generation modifications are also known as autologous chondrocyte implantation using collagen membrane (C-ACI), membrane-associated autologous chondrocyte implantation (MACI), and scaffold-based ACI [ 13 , 18 ]. These procedures were introduced in the clinical practice one decade ago, showing similar results while at the same time overcoming most of the concerns related to the first-generation ACI. The use of scaffolds to create a cartilage-like tissue in a three-dimensional culture system allows for the optimization of the procedure from both the biological and surgical points of view [ 19 , 20 ].

Although good clinical radiological and histological outcomes of the different ACI procedures, one of the main drawbacks is the need for a cell expansion phase which is long-lasting, complicated, and expensive primarily due to GMPs requirements. Moreover, the need for two hospitalizations increases the risk for the patients and the costs for the public health system. For all these reasons, investigations have been moving towards different cell populations as reported below [ 9 , 21 , 22 ].

Mesenchymal stromal cells (MSC)

Stromal cells from various sources are currently available for cartilage regeneration. This is due to their ability to proliferate in culture and directionally differentiate by synthesizing structural and functional hyaline ECM molecules. Moreover, they can release many anti-inflammatory, anti-apoptotic, and immuno-modulatory factors favoring the healing process [ 23 ]. Many studies have reported benefits in treating cartilage injuries with adult bone marrow-derived MSC [ 23 ]. Adipose-derived stem cells (ASC) have also drawn attention for their analogy with bone marrow ones, but with easier harvesting, a higher cell density, and proliferation. Other sources of stem cells investigated for cartilage repair include muscle, synovial membrane, trabecular bone, dermis, blood, umbilical cord blood, and periosteum [ 23 ]. Although various successful applications in cartilage regeneration, several problems remain, like stem cell heterogeneity and premature differentiation during in vitro expansion [ 24 ].

Induced pluripotent stem cells (iPSCs) have a promising potential for cartilage regeneration. Besides, they allow overcoming limitations associated with current cell sources since large numbers of cells can derive from small starting populations. However, issues related to genomic modifications still need addressing [ 25 ].

Genetically modified cells showed the ability to potentiate cartilage regeneration. Transfected genes inducing chondrogenic differentiation, synthesis of a hyaline matrix, and release of pro-inflammatory factors differentiation are feasible. Gene transfection may be systemic or local, ex vivo or in vivo. Because cartilage injuries are not life-threatening, it is critical to ensure a safe procedure [ 26 ].

MSC, as a pure cell population, require the selective elimination of cells that do not express their typical markers. Recently, new insight turned into the role of the surrounding MSC microenvironment (or “niche”) that also encloses ECM, accessory cells, adhesion molecules, growth factors, cytokines, and chemokines. Stem cell activity is not only the expression of intrinsic capabilities but also the result of the interactions with the “niche”. It is the whole “niche” that can support the healing process. No cell selection and expansion in the laboratory are necessary, and a single operative procedure is effective [ 27 , 28 , 29 ].

In recent years, also articular cartilage regeneration research moved towards the use of the stem cell “niche” in the form of concentrates such as Bone Marrow Concentrate (BMC) and Stromal Vascular Fraction (SVF) from adipose tissue. Both concentrates are obtained with minimal manipulation (no expansion in culture) and provide a less invasive (one step-surgery) and less expensive (no GMPs) alternative to cultured cells.

Our laboratory investigated the behavior of BMC cells within a hyaluronan-based scaffold. Histological immunohistochemical and molecular results showed the formation of a cartilage-like ECM [ 30 ]. We also evaluated BMC chondrogenic and osteogenic potential on a bi-layered scaffold mimicking the osteochondral compartment (collagen and hydroxyapatite). The obtained data demonstrated the ability to reproduce the native osteochondral compartment by generating two separated cartilage and bone zones [ 31 , 32 ].

SVF obtained from lipoaspirate contains several cell types like ASCs, ECM fibroblasts, and white and red blood cells. After washing passages, the obtained SVF can be combined with scaffold and soluble factors and administered into the joint. Compared to BMC, SVF ensure easier accessibility and the availability of an increased number of stem cells per gram of tissue [ 33 ].

Cell free products

In the early stages, it seems that the ability of MSC to differentiate into various cell types played the main therapeutic effect. Later, it emerged that their capacity to release some GF and chemokines play a role (secretome). MSCs secrete bioactive molecules inhibiting apoptosis and the formation of fibrosis or scarring at the injury site; stimulate angiogenesis and blood supply, and mitosis of tissue-specific progenitors. They also secrete immunomodulatory agents that deactivate the T cells surveillance and chronic inflammatory processes. Therefore, the secretome use for tissue regeneration increased, based on its composition of trophic factors (chemokines, cytokines, hormones, and lipid mediators) with paracrine effects on the cells of the local microenvironment [ 34 ].

The soluble factors of the secretome can initiate regenerative signaling events also without the use of cells. The therapeutic effect of this biological product in musculoskeletal diseases is a frontier of regenerative medicine. The secretome could overcome the negative aspects of cell use and help concentrate paracrine factors at physiological levels at the injury site.

Although many studies provide strong evidence for the potency of MSC-secreted factors in mediating tissue repair and regeneration, the precise mechanisms of action are still not fully understood [ 35 ].

The paracrine action of MSC is not limited to the production of soluble factors but also of many extracellular vesicles (EVs). EVs [ 36 ] are involved in intercellular communication by releasing mRNAs and proteins. Besides, they have anti-apoptotic, antifibrotic, pro-angiogenic, and anti-inflammatory effects. EVs released from tissue-damaged cells can re-program stem cells' phenotype by releasing specific mRNAs or microRNAs. EVs produced by circulation-recruited or resident MSCs can re-program tissue-damaged cells by inducing de-differentiation, production of soluble paracrine mediators, and initiation of the cell cycle of these cells, thus promoting tissue regeneration [ 37 ].

• Growth factors

Biologic agents represent an emerging treatment for several musculo-skeletal pathologies. These agents are mainly represented by natural GF and anti-inflammatory mediators that can accelerate tissue healing and regeneration. They can act through various mechanisms, including matrix synthesis and remodeling, cell recruitment and modulation of inflammatory markers and metalloproteinases.

Moreover, GF may influence protein synthesis and cellular interactions, controlling stem cell differentiation. Bone Morphogenic Protein-2 (BMP-2) regulates osteogenesis, Vascular Endothelial Growth Factor (VEGF) angiogenesis, and Transforming Growth Factor-β1 (TGF-β1) chondrogenesis. The possible role played by GF in pian reduction and tissue regeneration has generated a growing interest in their possible therapeutic use in patients with musculo-skeletal injuries.

Recently, discoveries, combined with knowledge of the importance and role of growth factors for tissue engineering, have been further developed and deepened [ 38 ].GF facilitate and promote the regeneration of new tissues by interaction with specific transmembrane receptors and regulating protein synthesis within cells. Binding to the specific growth factor receptor specifically stimulates cell signal transduction pathways that trigger cell migration, survival, adhesion, proliferation, growth, and differentiation.

Although GF have great potential to stimulate cartilage repair, only a limited number of treatments have been approved by government regulatory agencies for clinical use [ 39 ].

Platelet-rich plasma (PRP) represents an economical source for obtaining many GF in physiological proportions and has already been widely applied in various fields of medicine for its property of promoting tissue regeneration [ 12 , 40 ]. PRP can be defined as a blood derivative product in which platelets are present in high concentration. Platelets have demonstrated regenerative properties because they are rich in important GF.

In particular, α platelet granules contain and release numerous GF including PDGF, TGF-β1, VEGF, Epidermal Growth factor (EGF), Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF).

In recent years, PRP has achieved great success in clinical practice, thanks to its safety and simply preparation technique, which allows exploiting its biologically active content.

PRP has been used successfully in several surgical techniques, obtaining good results in association with microfractures or scaffolds for the treatment of cartilage lesions [ 41 ]. The most significant evidence on PRP is instead for its intra-articular use in the treatment of osteoarthritis, especially in the knee. Despite this, the most suitable type of PRP remains debated, with different preparation methods available that can give products with different composition and properties [ 42 ].

Scaffolds are support sustaining three-dimensional (3D) tissue development. They differ in material composition, structure, and status. An ideal scaffold should be biomimetic, biocompatible, biodegradable, and non-immunogenic; induce cell attachment, growth, and differentiation. Once implanted, it should integrate into the lesion site and support the healing process. It should also be easy to handle by surgeons, and cost-effective Scaffolds for cartilage regeneration may be natural, or synthetic [ 43 ].

Natural materials possess high biocompatibility and bioactivity. However, show poor mechanical stability because of their rapid hydrolysis. A list of the most known natural materials with the principal advantages and disadvantages is reported in Table 1 .

Synthetic polymers like poly(ethylene glycol) (PEG), polycaprolactone (PCL), polylactic acid (PLA), polyurethane, poly(glycolic acid) (PGA), polyethersulfone (PES), and polysulfone provide cell attachment, and good mechanical, physical, and chemical properties. Moreover, the mechanical properties and degradation time can be controlled by combining them as copolymers or blends. Disadvantages consist of the lack of biological properties and the host organism's side effects in response to metabolite production, mainly concerning acids that can be toxic or induce an inflammatory response [ 43 ].

Hybrid scaffolds, such as a combination of collagen-chitosan- PLA, merge the advantages of synthetic and natural materials, allowing biocompatible membranes with defined mechanical properties and tunable degradation necessary for cartilage regeneration [ 43 ].

Studies highlighted the in vitro and in vivo interaction of cells with scaffolds [ 44 ]. Our group had the opportunity to evaluate some natural scaffolds based on collagen or hyaluronan. We highlighted that scaffold presence allows the re-creation of physiological-like conditions whereby cells interact with the biomaterial and produce a new ECM by the secretion of anabolic, anti-inflammatory, and anti-apoptotic factors [ 45 , 46 , 47 , 48 , 49 , 50 ].

A challenge in the design and fabrication of scaffolds is the reproduction of the osteochondral compartment. To this end, composite bilayer or gradient scaffolds mimicking the osteochondral tissue have been developed and evaluated in association with cells. The data obtained demonstrated cell ability to zonally interact and reproduce the native osteochondral compartment by generating separated cartilage- and bone-like zones [ 5 ].

Another challenge is the cell seeding onto the scaffold. Conventional method involves the manual/static or the automated/dynamic seeding of cells onto previously fabricated scaffolds. The static seeding allows an uneven cell distribution into the width of the biomaterial. The dynamic seeding carried out with bioreactors (for instance perfusion) favor a more homogenous cell distribution [ 24 ]. The recent approach of bioprinting foresees that cells and biomaterial are released together in order to produce a construct. Such options allow a better cell encapsulation and spatial distribution [ 51 ].

Future directions

In the next decades we will assist to important steps forwards the repair of articular cartilage lesions. The use of iPSCs and or stem cell derivatives such as secretome, EVs could contribute to improve tissue regeneration.

Emerging technologies like Additive Manufacturing three-dimensional (3D) printing should allow for a further improvement of the treatment. 3D printing replicates the damaged tissue shape starting from a patient medical image. It creates scaffolds through the progressive addition of material layer by layer until reaching the desired shape. The technology can mimic cartilage organization, ECM composition, and functional and mechanical properties [ 52 , 53 , 54 , 55 ].

Indeed, the identification of the ideal cell population, cell-free products, clinical grade growth factors and customized scaffolds could contribute to ameliorate the technique, reducing the time for surgery and enhancing patient recovery.

Chondral and osteochondral damages remain a tough challenge for clinicians. Tissue engineering-based strategies have proven feasible for cartilage regeneration with good results on patients' quality of life. More research needs to find the best combinations of cells, bioactive factors, and scaffolds. More clinical trials should confirm the obtained results. There is also the demand to develop minimally invasive and cost-effective strategies which do not require long-lasting hospitalization.

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Acknowledgements

We acknowledge Italian Ministry of Health for the funds of the Project “Medicina rigenerativa e riparativa personalizzata per le patologie dei tessuti muscolo-scheletrici e la chirurgia ricostruttiva ortopedica" 5x1000 2019 (redditi 2018).

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Roseti, L., Grigolo, B. Current concepts and perspectives for articular cartilage regeneration. J EXP ORTOP 9 , 61 (2022). https://doi.org/10.1186/s40634-022-00498-4

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Knee osteoarthritis is the most common joint disease. It causes pain and suffering for affected patients and is the source of major economic costs for healthcare systems. Despite ongoing research, there is a lack of knowledge regarding disease mechanisms, biomarkers, and possible cures. Current treatments do not fulfill patients’ long-term needs, and it often requires invasive surgical procedures with subsequent long periods of rehabilitation. Researchers and companies worldwide are working to find a suitable cell source to engineer or regenerate a functional and healthy articular cartilage tissue to implant in the damaged area. Potential cell sources to accomplish this goal include embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The differentiation of stem cells into different tissue types is complex, and a suitable concentration range of specific growth factors is vital. The cellular microenvironment during early embryonic development provides crucial information regarding concentrations of signaling molecules and morphogen gradients as these are essential inducers for tissue development. Thus, morphogen gradients implemented in developmental protocols aimed to engineer functional cartilage tissue can potentially generate cells comparable to those within native cartilage. In this review, we have summarized the problems with current treatments, potential cell sources for cell therapy, reviewed the progress of new treatments within the regenerative cartilage field, and highlighted the importance of cell quality, characterization assays, and chemically defined protocols.

Osteoarthritis (OA) is the most common form of chronic joint disease, affecting all joints in the body, resulting in progressive cartilage degeneration. Risk factors associated with OA include age, obesity, family history, or trauma that has caused damage to the cartilage (Haq et al., 2003 ). Physical inactivity has also been shown to lead to cartilage degradation as joints require mechanical load and motion to maintain healthy cartilage structure and function (Sophia Fox et al., 2009 ). As cartilage is an avascular tissue with sparse cell density, it has poor regenerative capacity. Due to this, OA results in pain, dysfunction, and substantial healthcare costs (Hudetz et al., 2017 ; Hiligsmann & Reginster, 2013 ). In addition to these direct effects, the disease leads to an indirect economic burden for societies due to decreased productivity and premature disability (Hiligsmann & Reginster, 2013 ). Since age is a substantial risk factor (Haq et al., 2003 ), and that the global life expectancy continues to increase, OA-related costs will also increase with time. Therefore, the potential cost savings provided by a cure, or other better alleviation methods, will also be substantial given the high prevalence of people suffering from the disease worldwide.

Recent reviews discuss cell-based treatments of OA and cartilage defects with a different focus. Both Agarwal et al. and Wiggers et al. dive deep into clinical studies of cellular therapies for improved knee function and decreased pain (Agarwal et al., 2021 ; Wiggers et al., 2021 ). In their respective meta-analyses, Agarwal et al. show that such treatment may be effective, while Wiggers et al. concluded that there is limited evidence for a qualitative effect. The future of stem cell therapy is dependent on high-quality cartilage to repair damage to a greater extent than is possible today. Kamaraj et al. reviewed studies that used induced pluripotent stem cells (iPSCs) to produce high-quality cartilage and tested the effect in vivo (Kamaraj et al., 2021 ). They concluded that iPSCs offer a valuable source of cartilage for effective cell-based therapy and that comparability of study findings is of utmost importance, in line with the focus areas of this review. This review will present an overview of current and possible future strategies for cell-based treatments for OA and cartilage defects. It will address current progress within the regenerative medicine field. It will also address the need for robust protocols for generating stem cell-derived chondroprogenitors or chondrocytes and valid characterizations used in stem cell therapies.

Articular cartilage is a highly specialized and avascular tissue that is the most common type of cartilage covering the surface of articular joints (Schmutzer & Aszodi, 2017 ). It consists primarily of water (65–80% of wet weight), collagen fibers (10–20% of wet weight and 60% of dry weight, where type II collagens represent 90–95% of the collagen fibers), and proteoglycans (10–15% of wet weight). It also contains smaller amounts of other molecules such as glycoproteins, hyaluronan, and various elastic fibers, which form a dense extracellular matrix (ECM) network (Sophia Fox et al., 2009 ). The specialized cell type within adult cartilage, chondrocytes, is non-proliferating, and the cell density is relatively low. Only about 2% of cartilage consists of chondrocytes, where matrix proteins constitute the rest of the dry weight (Sophia Fox et al., 2009 ). The main function of chondrocytes is metabolic regulation, i.e., synthesis and degradation of ECM proteins, mainly collagen type II and aggrecan (Frazer et al., 1994 ). There are two alternative RNA splicing of collagen type II, one long-chained (collagen type IIA) characteristically expressed in pre-chondrocytes, and one short-chained (collagen type IIB) expressed in mature chondrocytes (Nah et al., 2001 ). Aggrecan is the most abundant proteoglycan within cartilage, and it is essential to maintain structure and function in this tissue. Due to its linkage to hyaluronan, aggrecan provides a hydrated gel structure necessary for biochemical and mechanical function. Aggrecan synthesis and degradation are regulated and, therefore, not constant throughout life. The degradation is directly linked to cartilage erosion and diseases such as OA (Song et al., 2007 ).

Current treatments for OA

Despite much ongoing research regarding OA, there is a lack of knowledge regarding disease-related biomarkers, disease mechanisms, and drug targets (Zhang et al., 2016 ). There is no existing drug-based disease-modifying therapy on the market, although potential drugs are currently under investigation. There is also no specific treatment for halting cartilage degradation (Fosang et al., 2003 ). Current treatments are patient-specific and depend on the levels of pain a patient experience. Treatments are focused on lifestyle modifications such as diet and physical activity, pain and inflammation-reducing drugs, interarticular drug treatments, cell transplantations, and if needed, entire joint replacements (Zhang et al., 2016 ). However, pain-relieving and anti-inflammatory medications do not prevent the progression of the disease. Since surgery is an invasive procedure followed by long rehabilitation periods, it is normally only recommended for patients with a severe pain history.

Surgical methods

Limb malalignment induces stress on articular cartilage, and when present in early OA, such malalignment causes further loss of articular cartilage. Unloading osteotomies can be used to realign the limb, reduce stress on degenerative cartilage, and to slow down disease progression. Osteotomies are used primarily for the knees, and can be used as a preservative tool for the joint (Mina et al., 2008 ). Joint distraction is a more recent technique where the bones are pulled apart to increase space, and the distracted area is fixed using pins combined with an external frame. It temporarily unloads the degenerative region, and the method has been used for OA conditions in the ankle and knee. Joint distraction appears to give patients short-term clinical and structural benefits with sustained effect up to 9 years (Goh et al., 2019 ).

Painful subchondral cysts in OA can be treated by subchondral plasty, filling the cysts with calcium phosphates and/or bone marrow concentrates (Szwedowski et al., 2020 ).

Joint replacements are a standard procedure with a limited lifespan that is used as late as possible in OA treatment. These procedures incur large costs for patients, hospitals, and healthcare systems. According to Chen et al., the money spent on joint replacements in the US alone has increased from ca 7 billion dollars in 1997 to over 22 billion dollars in 2004, with no sign of slowing down (Chen et al., 2012 ). With this in mind, we will focus on future alternative ways to treat OA.

Autologous Chondrocyte Implantation (ACI)

Brittberg et al. developed an alternative method to treat local cartilage defects in the knee joints; autologous chondrocyte implantation (ACI). It involves harvesting the patient’s cells from a healthy and non-weight-bearing donor site, the isolation of chondrocytes, i.e., ECM removal, and cell expansion ex vivo to a sufficient number of cells. The cells are then implanted into the damaged area as a cell suspension, covered with a periosteal flap harvested from the patient’s tibia (Brittberg et al., 1994 ). It is, therefore, a two-step surgical procedure. The first trial was performed in humans in 1987 with good clinical outcomes and long-term follow-up (Peterson et al., 2010 ). Today, the method is widespread and used by surgeons worldwide (Ogura et al., 2017 ). This first-generation approach has evolved, first by the replacement of the periosteal flap with a collagen membrane (generation II) and then later to cells being grown on a cell carrier (generation III) such as matrix-assisted chondrocyte implantation (MACI) (Brittberg et al., 2018 ), or in a porous scaffold such as Hyalograft (Tognana et al., 2007 ). After culture, the cell-seeded scaffold is implanted into the defect (Gille et al., 2016 ). Scaffolds for tissue-engineered cartilage defects are commonly generated from biodegradable natural or synthetic biopolymers. Examples of scaffold materials for this purpose include cellulose, polycaprolactone, hyaluronan, collagens, as well as hydrogels such as agarose and alginate (Nguyen et al., 2011 ; El-Sherbiny & Yacoub, 2013 ). One strategy is to mix gels with rigid materials to create a more rigid scaffold. Liu et al. created a polycaprolactone/gelatin surrounded scaffold to enhance chondrogenesis of mouse iPSCs in vitro and in vivo with a promising outcome (Liu et al., 2014 ). Several studies have independently reported successful clinical outcomes of the cell-seeded implant approach using arthroscopy for implantation. (Gille et al., 2016 ; Basad et al., 2015 ). Although positive clinical outcomes are evident, the two-step surgical implantation process both involves the risk of limited access to autologous chondrocytes, as well as and their harvesting at a healthy donor site resulting in additional injury. Due to the cell expansion in monolayers, chondrocytes tend to dedifferentiate and change phenotype, which affects the synthesis of cartilage-specific matrix proteins essential for regeneration of the implanted chondrocytes (Watt, 1988 ).

One-stage 4th generation ACI techniques are emerging, and they are increasingly being implemented. Examples include mixing directly isolated chondrocytes with either directly isolated autologous bone marrow stem cells or allogeneic stem cells (Słynarski et al., 2020 ; de Windt et al., 2017 ). Particulated or fragmented autologous or allogeneic cartilage as a source for chondrocytes is also regarded as a 4th generation ACI. From crushed cartilage, the most active chondrocytes may migrate out into a surrounding supportive scaffold, gel, or similar (Cole et al., 2011 ; Grawe et al., 2017 ).

Microfracture

An alternative surgical technique for treating local cartilage lesions uses bone marrow stimulations (BMS) such as microfracture. This arthroscopic technique creates small microfractures in the bone under the cartilage defect to trigger a regenerative response from mesenchymal stem cells (MSCs) in the bone marrow. The method is best suited for smaller defects created by trauma and not for OA (Lee et al., 2013 ). Additionally, younger patients (30–40 years old) have shown better outcomes than older patients (Knutsen et al., 2004 ). This procedure is relatively quick and cost-effective, as well as less invasive than ACI or joint replacements. However, the quality of the repaired MSC-derived cartilage exhibits variations between individuals.

Moreover, high-quality collagen type II-rich hyaline cartilage seems difficult to achieve, and a collagen type I-rich fibrous or hypertrophic cartilage is more likely to be generated (Saris et al., 2008 ). Despite this, the fibrocartilage might decrease symptoms in the affected joint and reduce pain for the patient. Local chondral and osteochondral lesions are mostly of traumatic origin, while osteoarthritis is an organ disease. A local cartilage lesion, if not treated, may increase in size and lead to OA. For local cartilage lesions, the choice of treatment is mainly based on the size of the lesion. A suggested cartilage lesion local treatment choice is presented here (Brittberg, 2021 ):

BMS for small defects 0.5 cm 2 .

Augmented BMS for small-medium sized defect 0.6–2 cm 2 .

Augmentation is also an alternative for re-operations in such defects if a simple BMS has been done previously.

Cell based treatments for large defects >2 cm 2 .

Cell based treatments for re-operations >1 cm 2 .

Osteochondral Allografts for extra-large defects.

The drawbacks and possibilities of stem cell origins

The quality of the cells involved is one drawback of current surgical methods. Researchers are exploring other suitable cell sources that overcome the drawbacks of using autologous-derived chondrocytes to create a functional and healthy hyaline cartilage. Embryonic stem cells (ES-C), MSCs, and iPSC are potential cell sources for understanding OA disease mechanisms and use in a cell therapy-based treatment. ESCs are pluripotent and can divide infinitely (Takahashi & Yamanaka, 2006 ). However, problems such as the formation of teratomas and immune rejection have been reported. Such issues complicate the use of ESCs in regenerative medicine. Adult stem cell sources, such as MSCs that can be found in, e.g., bone marrow and adipose tissue, also have the potential to differentiate into several types of tissue (De Bari et al., 2001 ). The use of MSCs does not require immunosuppression, making them suitable for allogeneic cell banking as well as an off-the-shelf product (Huaman et al., 2019 ). They are also relatively easy to culture in vitro , as they do not tend to dedifferentiate like chondrocytes (Tallheden et al., 2004 ). However, MSCs have shown differing proliferation and differentiation capacity, depending on their tissue and molecular microenvironment origin (Maleki et al., 2014 ). Although MSCs have shown to be safe and efficient in pre-clinical studies, they have a tendency to form hypertrophic chondrocytes and bone instead of hyaline cartilage during chondrogenic differentiation, resulting in impaired biomechanical properties. A genetic discrepancy between articular and MSC-derived chondrocytes has also been detected. It was shown that MSC-derived chondrocytes resulted in a differing cartilage phenotype, and it was concluded that articular chondrocytes and MSCs differentiate along different pathways (Karlsson et al., 2007 ).

As MSCs are multipotent and can only differentiate into cells within the mesodermal lineage (Pittenger et al., 1999 ), an alternative cell origin is pluripotent stem cells. Like ESCs, iPSCs are pluripotent, have similar morphology and gene expression profiles, and can be divided infinitely (Takahashi & Yamanaka, 2006 ; Liu et al., 2010 ). iPSCs are a possible cell source with great potential within regenerative medicine and the treatment of cartilage defects and diseases such as OA. The use of iPSCs would overcome any present ethical issues surrounding ESCs, as they can be derived from a minimal number of easily accessible non-invasively harvested somatic cells. Mouse embryonic or adult fibroblasts were first induced to have pluripotency by Takahashi and Yamanaka in 2006 by using retroviruses. Since then, the required factors, Oct3/4, Sox2, Klf4, and c-Myc, have been used to induce pluripotency. These factors combined are known as the Yamanaka factors (Takahashi & Yamanaka, 2006 ). An adult cell can thus be reprogrammed back into the pluripotency developmental stage and be differentiated into any mature cell. This makes iPSCs useful in tissue engineering, regenerative medicine, drug screening, toxicity testing, and disease modeling.

One of the Yamanaka factors, C-Myc, is also a known oncogene, which is critical to consider when using iPSCs in clinical applications (Miller et al., 2012 ). Okita et al. showed how mouse fibroblasts were reprogrammed into iPSCs using the Yamanaka factors. They also discovered tumor generation in chimeric mice after cell transplantation due to the reactivation of the c-Myc gene (Takahashi et al., 2007 ; Nakagawa et al., 2010 ). Moreover, due to the pluripotency, there is a possibility that transplanted iPSCs form teratomas in vivo . Therefore, it is essential that no undifferentiated stem cells remain in the transplanted area (Liao et al., 2018 ). As mentioned, Yamanaka factors were first used to induce pluripotency via retroviruses. By using retroviral reprogramming, the virus’ RNA is converted to DNA and integrates with the donor cells’ cellular genome, which induces genomic change that can lead to unwanted gene transcription and increase the risk for tumor formation. Therefore, silencing the expression of Oct3/4, Sox2, Klf4, and c-Myc after reprogramming is essential to avoid harmful gene expressions. The use of retroviruses to induce pluripotency and the integrations with the cell genome makes this method unsuitable for human clinical applications (Takahashi & Yamanaka, 2006 ; Okita et al., 2007 ). To improve the reprogramming method, Okita et al. developed a viral-free method circumventing some of the above-mentioned side effects (Okita et al., 2008 ). A similar footprint-free method has been used to obtain large quantities of fully differentiated astrocytes from iPSCs (Mormone et al., 2014 ). Concurrently, Boreström, Simonsson et al. have shown that it is possible to eliminate the risk of genomic integrations or aberrations using a genetic footprint-free mRNA delivery system to induce iPSCs chondrogenic redifferentiation (Boreström et al., 2014 ). This discovery provides a significant step in the procedure to find a suitable cell source for regenerative medicine to treat, e.g., cartilage defects and OA.

The type and source of stem cells are only some aspects to consider when regenerating new and healthy hyaline cartilage. The cell microenvironment, biomolecular signaling, and other aspects of the differentiation process are equally significant issues that must be addressed. During early embryonic development, concentrations and signaling molecules in the cellular microenvironment are crucial, and morphogen gradients are essential inducers for all tissue development, including cartilage (Zecca et al., 1996 ; Dee et al., 2002 ; Jullien & Gurdon, 2005 ; Peret & Murphy, 2008 ). Differentiation into different tissue types can be complex, and the suitable concentration range of specific growth factors is critical (Dakhore et al., 2018 ). The morphogen gradients involved in the developmental process to engineer functional cartilage may be a potential tool for generating cartilage comparable to the function and strength of native cartilage. Using gradients as such a tool will be discussed further later in this review.

One controversial, due to the mentioned safety issues, question has been raised, especially applicable for cartilage regeneration; whether direct transplantation of iPSCs or committed cells at a certain differentiation stage would achieve better outcomes. While developmental immaturity of iPSC-derived cells can be a challenge for tissues like muscle and brain, Lee et al. demonstrate that it can be advantageous for cartilage (Lee et al., 2017a ). This idea arises from the fact that particulated juvenile allograft cartilage (PJAC) transplantation has shown better long-term efficacy compared with, e.g., microfractures (Zhang et al., 2021 ; Adkisson et al., 2010 ). Nakayama et al. explore the possibility to treat cartilage lesions with iPSCs differentiated into juvenile chondrocytes, aiming to avoid the safety issues but letting the final differentiation to fully mature cells take place after transplantation (Nakayama et al., 2021 ).

3D Bioprinting as scaffolds for local repair

An upcoming strategy to improve the repair of local cartilage lesions is to use 3D bioprinting to generate a cartilage-like scaffold for the cells. Nguyen et al. concluded that a nanofibrillated cellulose composite bioink combined with alginate printed with human iPSCs and co-cultured with irradiated human chondrocytes was well suitable for bioprinting. This combination generated a cartilage-mimicking construct with cells expressing collagen II (Nguyen et al., 2017 ). One important goal that has yet to be reached with various scaffolds is to replicate the structural and biomechanical properties of native cartilage. 3D bioprinted MSC-containing hydrogels were used as constructs in an in vivo study in mice showing high structural integrity and good mechanical properties (Möller et al., 2017 ). Trials are also conducted ex vivo, where chondrocytes are 3D bioprinted in situ with promising results (Gatenholm et al., 2020 ). 3D-bioprinting is a hot topic and is discussed further in other recent reviews (Askari et al., 2021 ; Wu et al., 2021 ).

Chondrocyte characterization and validation

To use stem cell-derived chondrocytes for cartilage regeneration in vivo , the characteristics of chondrocytes must be well-established. Different kinds of experimental setups such as immunoassays, histological assays, microarrays, quantitative polymerase chain reaction (qPCR), and fluorescent-activated cell sorting (FACS) are commonly used in combination with well-known chondrocyte markers such as collagen type II, SOX9, and aggrecan (Tallheden et al., 2004 ; Lach et al., 2019 ; Suchorska et al., 2017a ). We reviewed articles featuring where ESCs, MSCs, or iPSCs differentiated into the chondrogenic lineage, as well as native chondrocytes, to understand how different research groups characterize chondrocytes and chondroprogenitors. Table 1 shows the four most commonly used experimental methods in the reviewed articles. Other methods used to a minor extent in the publications have been excluded from Table 1 . Based on the reviewed articles, the most commonly used methods to characterize chondrocytes are qPCR, immunostaining, and histological staining that were often used in combination. FACS was used less than the other three assays in the studied articles to obtain supporting data or detect a study-specific marker.

Many of the publications describe new or improved protocols for chondrogenic differentiation of stem cells. Some compare the level of gene expressions with adult chondrocytes (Lach et al., 2019 ; Suchorska et al., 2017a ; Weissenberger et al., 2020 ; Suchorska et al., 2017b ; Diederichs et al., 2019 ; Adkar et al., 2019 ; Koyama et al., 2013 ) . Others choose to compare the increase and decrease of markers within the study samples (Cheng et al., 2014 ; Oldershaw et al., 2010 ; Wang et al., 2019 ; Nejadnik et al., 2015 ). A high presence of the chondrogenic markers SOX9, COL2, and aggrecan is associated with high-quality articular cartilage regeneration. While the fibro- and hypertrophic cartilage markers, COL1A1, and COL10A1, respectively, should be low (Kamaraj et al., 2021 ). Also, SOX5, SOX6, COL9, and COL11 are well-known chondrogenic markers. Proteoglycans (Safranin O-staining), Glycosaminoglycans (Alcian Blue-staining), and immunohistochemistry staining for Collagen II are supportive in describing functional cartilage tissue. Other markers mentioned give additional supportive data, e.g., CD44 indicates normal chondrocyte function via connection to hyaluronic acid (Ishida et al., 1997 ), Hematoxylin and Eosin to visualize tissue cell structures, chondroitin sulfate is a chemical building block of cartilage, and lubricin and COMP indicates a functioning cartilage matrix (Flowers et al., 2017 ). During the differentiation process, the decrease in expression of pluripotency markers such as OCT4, Nanog, and SOX2 must be measured to ensure the absence of teratoma (Kamaraj et al., 2021 ).

Tissue engineering projects creating structures that should support the differentiation process can be evaluated using the same markers (Nguyen et al., 2017 ; Meng et al., 2016 ; Lu et al., 2017 ). The markers can also be used when comparing different cell origins after reprogramming them into iPSCs, and then differentiation towards chondrocytes (Rim et al., 2018 ; Wei et al., 2012 ). Additional uses are assessing the chondrogenic potential of cells isolated from patients, e.g., for ACI treatment (Tallheden et al., 2004 ; Naranda et al., 2017 ), and when studying the signaling pathways of chondrocytes (Enochson et al., 2014 ).

The characterization and validation are of significant importance to ensure cell specificity and quality. Obtaining high-quality cartilage repairing cells may be possible with an optimized protocol with defined cartilage-specific markers that can provide tight control over the resulting cell populations. There are advantages and drawbacks to consider depending on the choice of cell source (ESCs, MSCs, iPSCs, and chondrocytes), but all have a high potential for cartilage regeneration. We have reviewed different cell-based products for cartilage regeneration to summarize their current market status and ongoing clinical trials with current methods and problems in mind.

Commercialization of new therapies

Worldwide, companies are focused on developing cell-based products that repair or regenerate cartilage to amend defects caused by, e.g., OA or trauma. Different strategies have been applied to accomplish this. The well-known ACI method has evolved to include a supporting matrix or scaffold product, aka matrix-associated autologous chondrocyte implantation (MACI). Recently, products that involved the administration of autologous or allogeneic stems cells through intra-articular injection have emerged, either with or without a supporting matrix. Another strategy is to surgically implant 3D biocompatible cell-seeded scaffolds, as described earlier in the review. However, cell-based therapies have been subject to strict regulation by authorities (Reisman & Adams, 2014 ) as well as logistical and production challenges. In Table 2 , cell-based products that are currently approved or within clinical development for the treatment of cartilage damage are summarized.

The earliest approved cell-based products of those reviewed are autologous chondrocyte implantation products. Of the chondrocyte-based products currently approved or in development, the majority are matrix-associated ACI products (JACC, MACI, Ortho-ACI, Spherox, Novocart 3D, Cartlife) where arthroscopically harvested chondrocytes are seeded within a matrix or scaffold material before implantation during a second procedure. These products have largely replaced previous product generations, which involved a liquid cell suspension and the use of a peristomal flap or a collagen membrane, such as Carticel and ChondroCelect (European Medicin Agency, 2017 ), both having been withdrawn from the market. Recent advances in this area have led to MACI products where cells are cultured to become more cartilage-like and include extracellular components. One example of this is Spherox, which was approved in the EU in 2017 (European Medicin Agency, 2021 ) following a Phase III clinical trial (NCT01222559) (Clinicaltrials.gov, 2010b ). In this product, patient chondrocytes are condensed into spheroids, that is, spherical aggregates of ex vivo expanded chondrocytes with self-synthesized cartilage-specific extracellular matrix (Eschen et al., 2020 ). Also utilizing the ECM is Cartilife, which is approved in South Korea (Ministry of Food and Drug Safety, 2019 ) and is currently undergoing a Phase II clinical trial in the US (NCT04744402) (Clinicaltrials.gov, 2021 ). Cartilife uses costal-derived autologous chondrocytes, which are harvested, expanded, and then undergo a 3-dimensional pellet culture where cells form small beads with immature hyaline cartilage-like ECM (Lee et al., 2017b ).

Of the reviewed chondrocyte-based products, there was only one that utilized an allogeneic cell source. Invossa is an intra-articular injection comprised of a combination of juvenile chondrocytes and cells transduced to express TGF- ß used in knee osteoarthritis. Recently, animal model studies into the potentially disease-modifying mechanisms behind the clinical results showed the treatment in rats caused an increase in anti-inflammatory cytokine IL-10 (Lee et al., 2020 ). The researchers suggest that the treatment improved OA through the structural improvement and analgesic effects of an anti-inflammatory microenvironment promoted by M2 macrophages, which are known to exhibit immunosuppressive properties within the knee joint (Lee et al., 2020 ). The product was approved in South Korea in 2017 but withdrawn in 2019. A Phase III Study is underway in the US (NCT03203330) (Clinicaltrials.gov, 2017 ).

Stem cell-based products

An increasing number of products are emerging using stem cells such as MSCs and other progenitor cell types. In contrast to ACI, which more often focuses on focal defects, all the reviewed products are indicated for OA. For autologous stem cell products, common cell sources for MSCs are adipose tissue, bone marrow, and peripheral blood. The autologous adipose-derived MSC product JOINTSTEM recently completed a phase III trial (NCT03990805) (Clinicaltrials.gov, 2019d ) in South Korea and is conducting a Phase II/III trial in the US (NCT04368806) (Clinicaltrials.gov, 2020b ). Additionally, four companies currently are conducting Phase I or Phase II trials (Clinicaltrials.gov, 2020c ; Clinicaltrials.gov, 2013b ; Clinicaltrials.gov, 2015 ; Clinicaltrials.gov, 2019e )(NCT04448106, NCT01809769, NCT04043819, NCT02544802). A recent systematic review of randomized controlled trials (RCTs) for autologous stem cell therapy in knee osteoarthritis reviewed 14 RCTs and found a positive effect on patient-reported outcomes. However, they also reported a high risk of bias and low certainty of evidence (Wiggers et al., 2021 ).

Compared to the autologous stem cell products reviewed, a larger number of products in commercial clinical development were allogeneic. Allogeneic cell sources in Table 2 include adipose tissue, bone marrow, umbilical cord blood, and induced pluripotent stem cells. Allogeneic therapies have the advantage of being “off-the-shelf” as opposed to needing to source, transport, and process cells from a patient’s bone marrow or adipose tissue in the case of autologous therapy. As mentioned, it is generally accepted that MSCs can be used for allogeneic transplantations without the need for immunosuppression since the MSCs do not display immunogenic properties, which is a key advantage of using MSCs (Huaman et al., 2019 ).

The first allogeneic MSC product for cartilage injury, Cartistem, was launched in South Korea in 2012 (Ministry of Food and Drug safety, 2016 ), has conducted a Phase I/II trial in the US (NCT01733186) (Clinicaltrials.gov, 2012 ). The product combines allogeneic umbilical cord blood-derived MSCs and a hyaluronic acid hydrogel (Park et al., 2017 ). Unlike all the other reviewed stem cell products, which are intra-articular injections, Cartistem is administered through arthrotomy or arthroscopy with drilling (Park et al., 2017 ). Medipost, Cartistem’s developer, is currently developing a new generation product, an injectable MSC product, SMUP-IA-01, which has completed Phase I clinical trial in Korea (NCT04037345) (Clinicaltrials.gov, 2019c ).

Cynata is currently conducting a Phase III study in Australia for CYP-004, an iPSC-derived MSC product (ACTRN12620000870954) (Australian New Zealand Clinical Trial Registry, 2020 ). Uniquely, CYP-004 is manufactured from iPSC cells through the intermediate step mesenchymoangioblasts (MCAs). iPSCs, as a cell source for cartilage regeneration, have some biosafety issues regarding the use in vivo discussed above. Eight other allogeneic stem cell products have completed or are undergoing Phase I or II studies, see Table 2 .

The importance of gradients in tissue-mimicking for stem cell therapy development

For decades, researchers have known about the importance of gradients in developmental biology (Zecca et al., 1996 ; Dee et al., 2002 ; Jullien & Gurdon, 2005 ; Peret & Murphy, 2008 ). Gradients are present in a wide range of biological processes in vivo , including development, inflammation, wound healing, and cancer metastasis. These processes can be studied in vitro using quantifiable and controllable gradients to mimic those present in vivo . In stem cell differentiation and development, the gradients are essential inducers of tissue structure generation and functionality (Zecca et al., 1996 ; Dee et al., 2002 ; Jullien & Gurdon, 2005 ; Peret & Murphy, 2008 ). The local gradients, consisting of biomolecules such as morphogens or growth factors, or physical characteristics such as stiffness gradients, are involved in cell regulation and the inducement of developmental processes (Zecca et al., 1996 ; Dakhore et al., 2018 ; Gurdon et al., 1994 ; Gurdon et al., 1998 ; Joaquin et al., 2016 ). Only a few articles have managed to visualize morphogen gradients in vivo or in vitro (Teleman & Cohen, 2000 ; Lagunas et al., 2013 ). However, as technology develops, different gradient setups have been increasingly employed to study stem cells. As gradient-regulated processes are present in various signaling systems throughout the cell surroundings, there are different approaches to how they are used depending on the aim of the study. It is also important to consider the scale and the level of precision available, from a macro scale down to influencing cells on a nano- or molecular level. The most studied gradual cell environment factors are stiffness, chemical/cell attachment, and biomolecular (e.g., morphogens, growth factors). Such studies aim to study migration, differentiation, cell proliferation, and growth optimization. The choice of approach varies and can overlap. Examples of types of gradients are hydrogels, microfluidics, nano-gradients, and plasma-treated polymer surfaces. Table 3 summarizes the literature on these approaches. There are drawbacks and benefits with all strategies, and in some cases combining techniques may be a successful alternative, depending on the aim of the study.

Regarding stem cell differentiation towards chondrocytes, little research is published around biomolecular gradients and their influence on differentiation despite the evident importance during tissue development (Jullien & Gurdon, 2005 ; Gurdon & Bourillot, 2001 ). The primary focus has been stiffness gradients based on mimicking the complex zonal microstructure of cartilage tissue. According to Idazec et al., current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure resulting in the regenerated tissue lacking long-term stability (Idaszek et al., 2019 ). The study used a microfluidic printing device to shape gradients of chemical, mechanical, and biological factors into a layered cartilage-like structure in which MSCs and chondrocytes were co-cultured (Idaszek et al., 2019 ). This layered structure approach has been investigated and created in multiple ways using microfluidics, hydrogels, electrospun fibrous meshes, and cell sheets (Nguyen et al., 2011 ; Jin et al., 2019 ; Shi et al., 2013 ). Hydrogel stiffness gradients have also been used for investigating favorable stiffness ranges for induction of differentiation into specific cell types (Oh et al., 2016 ). All these techniques have their respective benefits and drawbacks depending on their use. However, they all aim to demonstrate how mechanical cues and loads control stem cell differentiation and tissue regeneration. Such studies are of great importance as it has been found that externally applied mechanical forces can stimulate stem cells to promote tissue regeneration (Enochson et al., 2014 ).

Nano-gradient technology offers a platform with an extensive range of biomolecule binding possibilities, providing a broad potential to gain knowledge of differentiation and cell-protein interactions. Moreover, the technology provides new opportunities to elucidate dose-dependent events, such as inducing migratory behavior. The nano-gradients are gradients of activator molecules bound to gold nanoparticles precisely distributed on a surface. They provide a unique chemically and physically defined substrate for controlled culture systems with a highly reproducible capacity (Andreasson et al., 2020a ; Andreasson et al., 2020b ; Lundgren et al., 2014 ; Evenbratt et al., 2020 ). One purpose of using gold nanoparticles is to present growth factors in a controlled manner to the cells. As the cells are immobilized on a surface, stimulations are comparable to in vivo conditions with matrix-bound cells, where local concentrations influence them (Fig. 1 ). These precise and stable molecular gradients enable dictating cell responses during differentiation because of the defined surface composition, density, and slope on a nano-level (Andreasson et al., 2020a ; Andreasson et al., 2020b ; Lundgren et al., 2014 ; Evenbratt et al., 2020 ).

The figure is a schematic image of the use of gradient nanotechnology in cell differentiation. The cells are seeded on a gradient surface (left), and the differentiation process reveals an optimal cell population (middle). A specific molecular density surface provides optimal, homogenous cell populations (right), a possibility owed to the information gathered on the gradient

The nano-gradients also allow the opportunity to combine factors, e.g., a growth factor with an ECM protein, further mimicking in vivo conditions, combining other materials and technologies, and forming a step in the differentiation protocol from where the cells can be removed and further cultured. The nano-gradient technology allows for screening an optimal growth factor density providing a robust differentiation protocol due to a precise and controlled stimulation, compared to, e.g., a solution-based gradient where growth factors are constantly moving (Minchiotti et al., 2006 ). Involving the gradient in differentiation protocols to generate chondrocyte progenitors could improve the ability to yield a defined cell population for differentiation before implantation into a damaged cartilage area (Andreasson et al., 2020a ). However, further research is needed.

All stem cell research and therapeutic applications, such as tissue regeneration, require defined and stable protocols to precisely control the cells during differentiation, but also to maintain required cellular properties and simultaneously mimic in vivo conditions. Stem cell cultures for therapy require high cell quality and a homogeneous cell population; however, traditional 2D cultures provide limited expansion and differentiation capacity (Zhang et al., 2004 ). As mentioned, concentration gradients in vivo enable regulation of cell responses, which are necessary for the function and structure during tissue generation in embryonic development (Zecca et al., 1996 ; Peret & Murphy, 2008 ). Such gradients are essential inducers of many developmental and articular cartilage-generating processes.

Current treatments of local cartilage lesions and OA focus on reducing pain and inflammation with insufficient long-term results. Today, no treatment is focused on disease-modifying mechanisms, and cell-based therapies struggle to generate high-quality cartilage. MSCs have become a commonly used cell source in developing approved and generally accepted stem cell therapy. Many companies have ongoing or completed clinical trials with promising results despite possible drawbacks, such as MSCs tending to form hypertrophic chondrocytes and bone instead of high-quality hyaline cartilage during chondrogenic differentiation. iPSC-derived chondrocytes have emerged as a potential alternative to MSCs, overcoming many of their drawbacks. However, issues, such as safety, have not been fully investigated to successfully commercialize iPSC-based treatments. To our knowledge, only one iPSC-based therapy for OA is in the clinical phase, currently undergoing a significant phase III trial. Biomolecular gradients are a potential aid to overcome problems with the differentiation of iPSCs. Gradients are essential in embryonic development. By utilizing gradients in the differentiation protocols, it is possible to provide a defined molecular stimulation to the cells and increase robustness compared to earlier protocols. A stable and more robust gradient would theoretically aid in generating a defined cell population for implantation into the damaged cartilage area. Further research, however, is required to accomplish this. Nonetheless, the research and development in this area are rapidly evolving in the quest to use stem cell-based therapies to treat cartilage damage.

Availability of data and materials

Not applicable.

Abbreviations

• Osteoarthritis

Induced pluripotent stem cells

Extracellular matrix

Autologous chondrocyte implantation

Matrix-assisted chondrocyte implantation

Three-dimensional

Mesenchymal stem cells

Embryonic stem cells

Bone marrow stimulation

Particulated juvenile allograft cartilage

Quantitative polymerase chain reaction

Fluorescent-activated cell sorting

Randomized controlled trials

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Most funding by Cline Scientific AB and partly funded by the European Union’s Horizon 2020 research and innovation program under grant agreement RESTORE no 814558 and AUTOCRAT no 874671.

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H. Evenbratt, L. Andreasson, V. Bicknell & R. Mobini

Institute of Biomedicine at Sahlgrenska Academy, Department of Clinical Chemistry and Transfusion Medicine, University of Gothenburg, SE-413 45, Gothenburg, Sweden

L. Andreasson & S. Simonsson

Cartilage Research Unit, University of Gothenburg, Region Halland Orthopaedics, Kungsbacka Hospital, S-434 80, Kungsbacka, Sweden

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HE and LA were major contributors in writing the manuscript. VB analyzed the companies focused on cell-based therapies for cartilage regeneration that have entered clinical trials. RM and SS contributed with cell expertise, SS specifically on cartilage regeneration. MB provided clinical expertise and mechanistic information on treatments. All authors read and approved the final manuscript.

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Correspondence to H. Evenbratt .

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HE, LA, VB, RM are employed by Cline Scientific AB, and HE also holds stock in the company. The authors have no additional competing financial interests.

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Evenbratt, H., Andreasson, L., Bicknell, V. et al. Insights into the present and future of cartilage regeneration and joint repair. Cell Regen 11 , 3 (2022). https://doi.org/10.1186/s13619-021-00104-5

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• Articular Cartilage
• Joint treatments
• Stem cell therapy
• Differentiation
• Chondrocyte characterization

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• Published: 11 October 2018

Trends in clinical trials for articular cartilage repair by cell therapy

• Takaharu Negoro 1 ,
• Yuri Takagaki 2 ,
• Hanayuki Okura 1 &
• Akifumi Matsuyama 1 , 3  

npj Regenerative Medicine volume  3 , Article number:  17 ( 2018 ) Cite this article

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• Adult stem cells
• Stem-cell research

Focal and degenerative lesions of articular cartilage greatly reduce the patient’s quality of life. Various therapies including surgical treatment have been developed, but a definitive therapy is not yet known. Several cell therapy products have already been developed and are available in the market. In this study, we examined the clinical research trends related to cell therapy products in the cartilage repair field based on data obtained from the ClinicalTrial.gov website. Although this website does not provide comprehensive results of clinical trials, it offers information on prospective clinical trials, including work in progress, and thus allows for chronological analysis of the data. We selected 203 studies related to the field of cartilage regeneration from ClinicalTrial.gov. The results showed a shift in the clinical translational trend in utilized cells from cartilage- and bone marrow- to adipose tissue-based cells. Whereas the studies that used cartilage as the cell source included many phase III trials, fewer studies using bone marrow and adipose tissue cells progressed to phase III, suggesting that most clinical developments using the latter sources have not been successful so far. One product covered the entire period from the start of phase I to the completion of phase III, with a time to completion of more than 100 months. Translational trends in autologous chondrocyte implantation were also discussed. The use of ClinicalTrials.gov as the sole data source can yield a perspective view of the global clinical translational trends, which has been difficult to observe up to this point.

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Introduction

ClinicalTrials.gov 1 is the clinical trial registration database of the United States, which provides information on the implementation status of more than 260 000 clinical trials from over 200 countries and is the world’s largest clinical trial registration site. Although ClinicalTrials.gov does not provide comprehensive results of clinical trials, it is a database of the plans for individual trials and provides information about target diseases, sponsors/principal investigators, planned schedule, and protocols of the clinical trials and enrollment of the subjects. Furthermore, since the database provides comprehensive information on the details of the content of the planned clinical trial, one can perform various targeted analyses by extracting and tagging attribute data from each clinical study plan.

Focal and degenerative lesions of articular cartilage greatly reduce a patient’s quality of life. Various therapies including surgical treatment have been developed, but a definitive therapy is not yet known. Investigators have attempted to repair cartilage defects by using cell therapy since the end of the last century. In fact, there are already several early cell therapy commercial products on the market in the United States, Europe, Korea, and Japan. 2 , 3 , 4 , 5 , 6 , 7 Several excellent reviews have been published of products developed in this field so far. 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 However, to the best of our knowledge, there are no scientific reports that have comprehensively analyzed and examined the clinical research trends on cell therapy for articular cartilage regeneration based on the ClinicalTrials.gov data registry. In this article, focusing on cell therapy products for cartilage repair, which require manufacturing and marketing approval by national authorities, based on the data obtained from ClinicalTrials.gov, we aimed to grasp a big picture of the global translational trend, which has thus far been difficult to decipher.

We surveyed the website ClinicalTrials.gov and selected 203 studies on regenerative cartilage repair. Using the retrieved data, we then analyzed the translational trends described in these studies. First, we classified the entire list of studies by the cell source organ used. The results are shown in Fig. 1a . The major organs used were as follows: bone marrow (31%), cartilage (28%), adipose tissue (25%), and umbilical cord (12%).

Analysis of projects in ClinicalTrials.gov according to the cell source organ used for cell therapy and cartilage repair. “Others” include studies that are using multiple cell sources for combination or comparison. a Percentage of each cell source relative to the total number of studies. b Comparison of number of clinical trials on cartilage repair according to countries of origin. Each color-coded part of the bar depicts the corresponding cell-source organ by country. The top 12 countries are shown in this graph. c Each study was color-coded by the corresponding cell source organ and displayed from the start year to the (planned) completion year, sorted by start year in chronological order. Shaded column: current year (2018) to 2025. Since 2018, a trial bar displays if the trial is registered. Please note that we could not show if the trial continued or was halted prematurely. Red-dashed column: 2014–15. Vertical-striped bar indicates “suspended,” “terminated,” or “withdrawn” study

Figure 1b shows the analyzed results by country. The United States, which manages ClinicalTrials.gov, had 79 studies, ranking at the top. In second place was Korea, followed by China, Germany, France, Iran, and Spain. In the same graph, each color-coded bar depicts the corresponding source of cells by country. The US studies used mainly cartilage, bone marrow, and adipose tissues at a rate of approximately 1:1:1 as the cell source. This rate varied among the listed countries. For example, Korea and Germany used mainly cartilage, while Iran, Spain, and Australia used bone marrow only.

To analyze the clinical research trends described in these projects chronologically, we arranged all studies by order of the corresponding start year, plotted them from the start year to the completion year, and color-coded them according to the organs of cell origin (Fig. 1c ). This analysis showed that cartilage was used as a source of cells from the beginning of 1995 to the present, but the rate of use decreased from an average of 45% (2006–2012) to 10–15% (after 2013). On the other hand, bone marrow has been used since 2009, followed by adipose tissue since 2012. The total number of studies using umbilical cord is small, but this source of cells has been implemented since 2009. The above trends have continued to date. Interestingly, a clinical trend of cell-derived tissues shifting from cartilage and bone marrow to adipose tissue has been observed since 2014–2015. Collectively, these findings indicate that the clinical application of cartilage repair began in the mid-1990s with the use of cartilage tissue as the cell source, and bone marrow has also been studied since the mid-2000s, but in the mid-2010s, both were replaced mainly with adipose tissue.

In the next step, the origin (autologous or allogeneic) of each cell source was analyzed (Fig. 2a ). Studies that used cells of allogeneic origin comprised approximately one-third of the entire database. Overall, no specific chronological trend was observed for either origin (Fig. 2b ).

Analysis of origin of cells (autologous or allogeneic) used for cell therapy and cartilage repair in clinical trials registered in ClinicalTrials.gov. a Percentage of each origin of cells relative to the total number of studies. b Each study is color-coded by corresponding origin of cells and displayed from the start year to the (planned) completion year, sorted by start year in chronological order. Shaded column: current year (2018) to 2025. Since 2018, a trial bar displays if the trial is registered. Please note that we could not show if the trial continued or was halted prematurely. Vertical-striped bar indicates “suspended,” “terminated,” or “withdrawn” study

Table 1 provides a detailed list of the cell therapy products designed for cartilage repair that are approved by the regulatory authorities of various countries and are currently available in the market. Briefly, Carticel® 2 was a first-generation autologous chondrocyte implantation (ACI) product, which required open arthrotomy implantation of in vitro-cultured autologous chondrocytes beneath an autologous periosteal cover. ChondroCelect® 8 was also a first-generation ACI product utilizing a proprietary genetic marker profile score that optimizes the likelihood of a hyaline phenotype and its associated biological, cartilage-forming capability. MACI® 3 , 4 , 8 , 9 is a type I/III collagen membrane seeded with expanded autologous chondrocytes. Spherox (chondrosphere® 16 ) consists of small spheroids of neocartilage composed of expanded autologous chondrocytes and their associated matrix. Chondron™ 5 is an autologous chondrocyte-pre-seeded fibrin three-dimensional matrix gel. CARTISTEM® 6 17 is a composite of allogeneic umbilical blood mesenchymal stem cells (MSCs) and hyaluronic acid hydrogel. Invossa™ 18 (TissueGene-C) is a gene therapy implant that includes modified transforming growth factor-β (TGF-β)-expressing allogeneic chondrocytes. 19 JACC is cultured autologous chondrocytes embedded in atelocollagen gel. 7 Although MACI® was initially approved by European Medicines Agency (EMA), it was suspended in the European Union (EU) in 2014 because of a manufacturing site closure in Europe. 20 Furthermore, ChondroCelect® was withdrawn from the market in 2016 because of a reimbursement problem in the EU. 21 Carticel® was phased out because of the new approval of MACI® in the United States. 22 On the other hand, two new products, Spherox 16 and Invossa™ 18 were recently approved in the EU and Korea, respectively. Both are derived from cartilage as the cell sources. As shown in Table 1 , most of the globally marketed products for cartilage repair (with the exception of CARTISTEM® 6 ) are derived from cartilage. Unfortunately, the available data on the registered studies in ClinicalTrials.gov do not allow for direct estimation of the market share of each type of product.

The origin of the cell source (autologous or allogeneic) and the clinical stage were analyzed chronologically to determine progress in testing new products derived from each of the four main cell sources (bone marrow, cartilage, adipose tissue, and umbilical cord). Although not shown in Fig. 3a because of the lack of description of the phase of trial, the earliest cartilage cell therapy trials in ClinicalTrials.gov started in 1995 (Fig. 1c ). As shown, cartilage has been examined as a material for cell therapy for cartilage repair for a long time. The start of phase III clinical trials on a newly developed product reflects positive results in phase II studies with regard to its effectiveness. Interestingly, a large proportion (15%) of phase III clinical trials for cartilage repair registered in the ClinicalTrials.gov database was included in this group, most of which used autologous cells (12/15). Therefore, our findings indicate that several earlier clinical studies using autologous cartilage as the cell source have shown encouraging results to warrant phase III clinical trials for cartilage repair. On the other hand, allogeneic cartilage cells were used in 11 studies. Only 4 of these were registered as phase III, but all of them were studies on TissueGene-C (NCT02072070, NCT03203330, NCT03291470, and NCT03383471). TissueGene-C was approved by the Korean authorities in July 2017 and marketed as Invossa™ (Table 1 ).

Chronological display (sorted by start year) of cartilage repair trials in which a cartilage, b bone marrow, c adipose tissue, or d umbilical cord 7 was used as the cell source. Each study was plotted from start year to completion year as a color-coded bar showing the origin of the cell source (autologous or allogeneic) and the corresponding clinical stage, as shown in examples in the frame below d . For convenience, trackable products with multiple trials were linked with colored lines and arrows as follows; a blue: Chondron, green: NeoCart, yellow: TissueGene-C, black: Chondrosphere, and purple: Novocart; b blue: NeoFuse and yellow: Chondrogen; c blue: JointStem and yellow: StroMed; d black: CARTISTEM. e Chronological display (sorted by start year) of the clinical trials of each generation of ACI products. Each study was plotted from the start to the completion year as a color-coded bar, which indicates the generation of ACI and corresponding clinical phases, as shown in examples in the frame below e . We could not find any phase I and II studies corresponding to the first ACI in ClinicalTrials.gov. Shaded column: current year (2018) to 2025. Since 2018, a trial bar displays if the trial is registered. Please note that we could not show if the trial continued or was halted prematurely. Vertical-striped bar indicates “suspended,” “terminated,” or “withdrawn” study

Six of the phase III clinical trials using autologous cartilage cells are currently being conducted in 2018. They are examining three products (NeoCart 8 (NCT01066702), chondrosphere® (NCT01222559), and Novocart 3D 8 (including 3D plus, and Inject plus) (NCT01656902, NCT01957722, NCT03219307, NCT03319797, and NCT03383471)), all of which are classified as ACI. Among them, Chondrosphere® and Novocart 3D are cell therapies that have already been used clinically under the hospital exemption (HE) scheme in Germany. The HE is a European-specific scheme that grants approval for use of medical products on an experimental basis in specific hospitals, even though the effectiveness of such products remains to be confirmed. 23 Chondrosphere® was also approved by EMA in July 2017 and marketed as Spherox (Table 1 ).

Since Wakitani et al. 24 reported the first case of treatment of cartilage defects with autologous MSCs in 2004, bone marrow has often been used as a source of MSCs. Figure 3b shows the results of studies to repair cartilage defect using bone marrow as the cell source. The earliest study was conducted in 2006. Research employing bone marrow as the source for chondrocytes became active around 2009. Two phase II/III trials (NCT00891501 and NCT01873625) were conducted in the early years (2006 and 2009, respectively), although no information is available regarding their approval. Interestingly, no phase III trials were conducted for several years after the above two studies. One phase III trial using an autologous cell source in 2015 was found, but this study (NCT02848027) was not relevant to our analysis because it was for a 361 HCT/P product, which does not require the approval of the US Food and Drug Administration (FDA). 25

Although many studies using allogeneic cell sources were also examined in the initial stages, no phase III studies were registered in the database until 2014. While degenerative disc disease (DDD) is not an articular cartilage disease, one phase III clinical trial using rexlemestrocel-L (NeoFuse™) 26 for DDD in the United States and Australia (NCT02412735) was registered in 2015. In summary, there are no approved cartilage repair products based on clinical trials that used bone marrow-derived cells registered in ClinicalTrials.gov, and the rate of progression to phase III is low (3.2%). Among these studies, there is only one allogeneic product for DDD that is currently under development in a phase III clinical trial.

Adipose tissue has also been used as a source for MSCs. Figure 3c shows the analysis of the projects that used adipose tissue as the cell source. The earliest study was conducted in 2008, but such studies became more common after 2012. There is only one phase III trial among these studies (NCT03467919). Although this study used MSCs extracted from adipose tissue, any earlier corresponding trials were not found in ClinicalTrials.gov. On the other hand, the use of allogeneic cell sources was low (3.9%). Aggressive use of adipose tissue as the cell source for cartilage repair began around 2012 and has been actively studied, but other phase III trials were not found.

The results of the analysis of studies using cells originating from the umbilical cord are shown in Fig. 3d . The data shown in this figure also include studies using cells from Wharton’s jelly, placenta, and amniotic membrane/fluid. All registered studies were conducted after 2008 and included two phase III trials in the early years for CARTISTEM® (NCT01041001 and NCT01626677). A phase II/III trial using amniotic fluid started last year, although we could not find any earlier corresponding trials in ClinicalTrials.gov. All other studies using cell sources classified in this category remain in phase II or earlier phases to date.

We also focused on studies on ACIs that were translationally successful among all clinical trials. Figure 3e includes information on ACI studies that were analyzed for clinical development trends in chronological order. The ACI studies were classified into three generations based on the method described by Harris et al. 8 Studies on the first-generation ACI were completed by 2010, while the second-generation ACI has been actively studied since 2006 to the present. Furthermore, the third generation was also studied at almost the same time as the second generation. Interestingly, the proportion of phase III trials relative to all trials for each generation was high, suggesting successful development of the technology. Thus, our analysis suggests that the major current trend in clinical development is the second- and third-generation ACI, while the first-generation ACI is superseded technology.

Next, we analyzed the time required for clinical development in this field. For this purpose, we analyzed all products (including candidates) that were used in phase I to phase III and that could be traced by product name or development code. Specifically, we analyzed the time required for a series of studies from phase I to phase III trials. Only two products/three research projects that covered phases I–III were identified in the ClinicalTrials.gov database (Table 2 ). Among the three projects that were entirely trackable (from the start of phase I to the completion of phase III) in ClinicalTrials.gov, only one project completed in practice was for TissueGene-C in Korea, and the time required to complete this entire project was 103 months. Since the remaining two projects were incomplete at this time (June 2018), it is necessary to be aware that these are projected periods. With regard to the clinical trial on the use of NeoFuse™ for DDD, the estimated time for the completion of phase I–phase III is 150 months.

Alternatively, to examine whether there is any tendency in the period required for each trial depending on the combination of cell source and origin, we extracted all the completed trials in practice and classified them according to their cell source and origin, and calculated the actual period taken to complete each study. Studies using autologous cartilage cells reached completion at a median of 74 months, while those using allogeneic cartilage cells were completed within a median of about 26 months, and the difference between these two types was significant. On the other hand, the use of allogeneic bone marrow cells and adipose tissue cells was associated with slightly longer times (median 46 and 36 months, respectively) than those using corresponding autologous cells (median 34 and 32 months), although the difference in these times was not significant. Interestingly, compared to the use of autologous chondrocytes, both the use of autologous bone marrow and autologous adipose tissue was significantly shorter, with a median of 34 and 32 months, respectively.

Finally, to examine the time required for each phase in more detail, the completed studies registered as phase I, phase II, or phase III were extracted, and we analyzed them by the time required for individual studies using the completion year, instead of the start year. Figure 4b shows the median time required for completion of individual studies completed by 2017 by each phase as boxplots. In the entire period, the phase I trials were completed in a median of 24 months, the phase II trials in a median of 36 months, and the phase III trials in a median of 41 months. Moreover, significant differences were observed in the required times between the phase I and phase III studies, and a difference in those between phase I and phase II studies was nearly significant ( p  = 0.0563). The simple summation of the median time required to complete each phase was 102 months. As few corresponding studies were completed by 2008, the period from 2009 to 2017 was divided into three sections, which are shown in the figure, to reveal the transition every 3 years. Although statistical analysis was impossible because of the small number of samples, it was found that the median duration of phase I studies was 15–31 months, 14–35 months for phase II studies, and 35–61 months for phase III studies.

a Box-plots show the comparison of periods required for studies using cell sources derived from autologous or allogeneic origin. Blue boxes with whiskers: autologous cells; orange boxes with whiskers: allogeneic cells. Pink-colored dots: period required for individual trials. As Shapiro-Wilk tests revealed that three of six specimens were not normally distributed, Steel-Dwass’ tests were conducted to test the difference between the six specimens. b Box-plots show the transition of time required for completion of individual studies completed until 2017 by each phase. The data in the entire period from 2006 to 2017 (underlined) are shown on the left side of this figure, and the transition of the required time by each 3-year period is indicated in the rest. Blue boxes with whiskers: phase I; orange boxes with whiskers: phase II; green boxes with whiskers: phase III. Pink-colored dots: periods required for individual trials. As Shapiro-Wilk tests revealed that one of three specimens was not normally distributed, Steel-Dwass’ tests were conducted to test the difference between the three specimens

There are already two interesting studies that provided comprehensive analysis relevant to regenerative medicine based on data from ClinicalTrials.gov. Monserrat et al. 27 analyzed the entire disease field with a special focus on stem cells and provided comprehensive information on the global trend of translational research, but unfortunately, their research did not include any detailed information on the individual fields. On the other hand, Fung et al. 28 provided a comprehensive assessment of the extent to which the publication of results of clinical trials of innovative cell-based interventions reflects the best practice guidelines of the International Society for Stem Cell Research and discussed various ethical considerations. Apart from these studies, to date, there are no clear reports on comprehensive clinical development trends in specific fields of regenerative medicine using the clinical trial registry. In this study, we focused on cell therapy applied so far for cartilage repair and used data available on the ClinicalTrials.gov registry as the primary source to conduct comprehensive and chronological research, classification, and analysis of clinical trials registered in this field, including world research trends on cell therapy.

The reason why ClinicalTrial.gov is not used for research as a sole information source is because this database does not provide comprehensive results of clinical trials, and it is impossible to analyze the results of the trials by themselves. That is, in order to review and analyze clinical development in a certain field, it is necessary to obtain the result data from another information source. The above two papers could avoid this drawback. This study aimed to comprehensively analyze cell therapy in the cartilage repair field, but unlike ordinary reviews, we decided to use it as a database of the trial plan, ignoring results in ClinicalTrials.gov. In other words, we utilized this planning database of clinical trials to capture the big trends in translational studies in this field by comprehensive chronological analysis. By performing attribute analysis using chronological display, we were able to obtain a perspective view (Figs. 1c , 2b , and 3a–e ). The increase in the number of registered studies since the mid-2000s is thought to be due to the International Committee of Medical Journal Editors (ICMJE) and/or the FDA Amendments Act of 2007, which promoted registration. The data shown in Fig. 1c indicate that cartilage repair therapy started originally using autologous cartilage tissue as the cell source. This was followed by the use of cells from the bone marrow and adipose tissue, as well as other tissues. Based on this analysis, the results showed a shift in the selected tissue from cartilage and bone marrow to adipose tissue in 2014–2015.

In clinical trials registered in ClinicalTrials.gov, bone marrow was the most popular cell source in this field (Fig. 1a, c ), but there are no products on the market at present that utilize these cells. Our investigation revealed that most of the commercial products were derived from cartilage (Table 1 ). In the field of cartilage repair, any cell therapy products prepared from cells other than cartilage and umbilical cord have not yet been approved by any national authorities. Comparison of the use of each cell source at the clinical translational stage showed a high proportion of phase III using cartilage as the cell source (Fig. 3a ), compared with a low rate of those of bone marrow-derived cells (Fig. 3b ). On the other hand, the registry contained only one phase III study using adipose tissue (Fig. 3c ). Worth nothing, the start year of translational studies using cartilage as a cell source was almost more than 10 years earlier than the others. In 2004, Wakitani’s article triggered further research on bone marrow as a cell source. Also, adipose tissue has been used as a source of MSCs since 2008. In other words, since the start of the translational study for both bone marrow and adipose tissue was substantially slower than that of cartilage (Fig. 3a–c ), the time might have been insufficient to reach phase III. Even in the trials using cartilage, phase III trials using autologous cells were quite popular, while those using allogeneic cells were not. One reason is due to the difference in start year of the translational research, as described above. To use autologous cartilage, it is necessary to collect cartilage for expanding culture beforehand, but the first operation is not necessary for using allogeneic cells. Despite such benefits, highly invasive surgical techniques such as conventional microfractures have been established as a standard therapy using autologous cartilage so far, and on the extended line of such procedures, cell therapy requiring autologous cartilage collection might have been relatively easily to accept. Using autologous cartilage, two new products (Neocart and Novocart) were examined in phase III. On the other hand, despite the benefits described above, allogeneic products need to be expanded in culture even more than autologous cells and require more strict quality control. It is important that TissueGene-C, which is the only approved product using allogeneic cartilage, is a transgene product expressing TGF-β.

According to Dewan et al., 14 cartilaginous tissues regenerated from bone marrow MSCs are fibrocartilaginous and inferior to the original cartilage. On the other hand, a recent review reported that the quality of the regenerated tissue varies according to the clinical trial. 29 With regard to Provenge® (sipuleucel-T), which is a product for immunotherapy, Galipeau 30 reported a lack of activity of bone marrow MSCs in a large clinical trial because of the heterogeneity of donors and senescence due to long cultivation. Martin et al. 31 pointed out that the vision of producing MSCs based on a unique standard is not yet scientifically achievable. Thus, there seems to be certain difficulties in large-scale clinical trials using bone marrow MSCs. In the case using autologous cells, it is concerning that there might be a large influence of inter-individual differences between donors as cell sources. On the other hand, allogeneic products are advantageous because one can choose good cell source(s) among donor candidates. Especially, umbilical cord blood-derived products are more advantageous because of younger cell source(s). Although the first study using umbilical cord cells was found in ClinicalTrials.gov on 2009 (Fig. 3d ), this was a phase III study in Korea of CARTISTEM®, which has already been marketed in Korea since 2012. We thought that these factors described above, in addition to the positive attitude toward cell therapy from the Korean government at an early stage, may have contributed the translational success of CARTISTEM® in Korea.

In the present study, statistical analysis of the time required for the development of products (from the start of phase I to the completion of phase III) based on the data from the ClinicalTrials.gov registry (Table 2 ) was not possible because of the small number of samples. However, the obtained data showed that TissueGene-C development was completed in 103 months (about 8.5 years). Regarding the time period required to complete the relevant clinical trial, Kaitin and DiMasi 32 reported that the time from Investigational New Drug filing to New Drug Application/Biologic License Application submission for new drugs was 6.5 years on average. In comparison, our data, though limited, suggest that a longer time may be required to develop cell therapy products for cartilage repair. In this regard, approval of TissueGene-C, which can be potentially used for gene therapy, may require a longer time to pass various regulatory bodies. These kinds of hurdles are unavoidable, especially for the leading runners in this new field.

Analysis of the time required for individual studies based on cell source and cellular origin (autologous or allogeneic) revealed that the studies using autologous cartilage tissue took more than a twofold-longer period than the studies using the other cell sources and allogeneic cells, as shown in Fig. 4a . Using autologous cartilage, the proportion of phase III trials is higher than other cell sources (compare Fig. 3a with Fig. 3b–d ) and the period required for phase III trials is longer than that of phases I and II (from Fig. 4b ). Thus, we thought that these were the reasons why a relatively longer period was needed for completing trials using autologous cartilage as a cell source.

Figure 4b shows the transition of the time required to complete an individual study, summarized in the year each study was completed, not the year in which each the study was initiated. Since only the completed studies were analyzed, the number of studies was not sufficient for statistical analysis when they were divided by every 3 years. However, in the whole period (2006–2017), the number was sufficient to try statistical analysis, and the trend of time required for each individual phase (phase I < phase II < phase III) was considered reasonable. The results for the entire period showed that simple summation of the median time of each phase resulted in 102 months to complete all phases. This is equivalent to 8.5 years and is in accordance with the period observed for the clinical development of TissueGene-C in Korea (103 months) shown in Table 2 . In the results for 2015–2017, a 6-month decrease was observed. This is a favorable trend for clinical development, although it is necessary to keep in mind that the completion of each phase still required more than 90 months (7.5 years).

The data shown in Fig. 3e suggest that the major trend in the clinical development of products at present is the use of the second- and third-generation ACI after the replacement of the first-generation ACI. Harris et al. 8 reported that complications, reoperations, and failures were common after first-generation ACI. In this context, TiGenix withdrew ChondroCelect® from the European market in November 2016. 21 To date, many developers have focused on obtaining regulatory approval. For this reason, it was surprising that TiGenix abandoned the first approval obtained from the EMA. In the case of ChondroCelect®, since it received the first approval from the EMA, TiGenix was able to market the product for the next 7 years. However, as mentioned earlier, the first-generation ACI has already been replaced by newer technology. Thus, because ChondroCelect® was approved as the earliest ATMP in Europe and was on the market for 7 years thereafter, it is thought that ChondroCelect® was successful as a leading cell-therapy product and might have ended its historical role in cartilage regeneration. In addition, since Vericel received FDA approval of MACI® in 2016, another first ACI product, Carticel®, was phased out in 2017. 22

The problem here is determining whether or not the marketing period of 7 years (as in the case of ChondroCelect®) was long enough in terms of the total investment. As mentioned above, the average time required for the completion of the entire project (i.e., from the start of phase I to the end of phase III) was 103 months for TissueGene-C and 150 months for NeoFuse™. For conventional drug research and development, the time period from basic research to preclinical studies, and the time from the completion of phase III to approval must be added. Because no sales can be made during the research period, it is necessary to estimate prospective profit after the product is given the approval for marketing, taking into consideration the cost of running the entire project.

In essence, it is crucial to shorten the time required for clinical development to accelerate the development of regenerative medicine products. In this sense, conditional approval, as typified by the PMD Act in Japan, 33 can reduce at least some of the time burden in the clinical development of regenerative medicine products. Based on the PMD Act, if the efficacy is presumed and the safety is confirmed, conditional, and time-limited marketing approval (5 years, conducting post-marketing efficacy studies) can be obtained. 34

In conclusion, our analysis of the clinical trials registered at the ClinicalTrials.gov website showed that the clinical trend in the use of cells in research has shifted from cartilage- and bone marrow- to adipose tissue-based cells. Whereas studies using cartilage as the cell source included many phase III trials, fewer studies using bone marrow and adipose tissue cells progressed to phase III, suggesting that most clinical developments using the latter sources have not been successful so far. Furthermore, all products approved by the authorities have been those that used cartilage as the cell source, except for one product that used cells from umbilical cord blood.

The time required for the development of such products (from start of phase I to completion of phase III) was more than 100 months. No doubt this period of time is long and may deter future investment in the manufacturing of such products. We believe that the conditional approval system legislated through the Japan PMD Act can help reduce, at least in part, the development time burden and encourage investment in future research and development. The attribute analysis based on the chronological display used in this study seems useful in providing supportive perspective viewpoints.

We searched the entire database at ClinicalTrials.gov on 8 May 2018 using the following search terms: “stem cell” OR “regenerative” OR “cell therapy” OR “implant” OR “transplant osteoarthritis” OR “cartilage injury” OR “cartilage repair” OR “Osteochondral Defects” OR “Articular Cartilage” OR “Traumatic Arthritis” OR “cartilage disease” OR “cartilage defect” OR “chondrocyte”. Among the identified 749 studies, we excluded studies using only surgical procedures, low-molecular-weight drugs, protein drugs, or scaffolds by carefully reading the descriptions of the individual studies, and selected 181 studies corresponding to cell therapy, which administered cells to humans to examine their safety and efficacy. Furthermore, the relevant studies were re-surveyed using the product name, development code, and/or sponsor’s name described in the 181 studies as search terms and were selected manually. Twenty-two studies were found to be incorporated into the above previous studies. Accordingly, we selected 203 studies and used their content for analysis.

We recorded the cell source organ, product name (if any), and country where the clinical study was performed. We did not include protocols on the registry that contained minimal information or incomplete data. The cell sources used were classified as cartilage, bone marrow, adipose tissue, umbilical cord, and others. Unspecified mesenchymal progenitor cells and MSCs were included in the “bone marrow” set according to the general usage. “Adipose tissue” included all materials described as adipose and fat. The “umbilical cord” set contained stem cells derived from umbilical cord blood, Wharton’s jelly, placenta, and amniotic membrane/fluid. All cell sources classified as umbilical cord were regarded as “allogeneic”.

To estimate the time of clinical development, we chose projects that were traceable with product name or development code, and calculated the time required for clinical trials using these individual products.

Normality tests were conducted using Shapiro-Wilk test in IBM SPSS Statistics v23. Steel-Dwass’ tests, a nonparametric multiple comparison method, were performed using pSDCFlig in NSM3 package of R v3.4.4. A p -value (asymptotic) <0.05 was considered to be statistically significant.

Data availability

The data that support the findings of this study are available from https://doi.org/10.6084/m9.figshare.6964715 .

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Acknowledgements

This work was supported by the Highway Program for Realization of Regenerative Medicine of The Japan Agency for Medical Research and Development (AMED). Supported by AMED under Grant Number JP17bm0504009

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Yuri Takagaki

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T.N., H.O., and A.M. conceived of the work; T.N. and Y.T. processed and analyzed the data; T.N., Y.T., and H.O. wrote the paper. All authors contributed to manuscript editing.

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Negoro, T., Takagaki, Y., Okura, H. et al. Trends in clinical trials for articular cartilage repair by cell therapy. npj Regen Med 3 , 17 (2018). https://doi.org/10.1038/s41536-018-0055-2

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New way to generate human cartilage

University of Montana researchers and their partners have found a new method to generate human cartilage of the head and neck.

Mark Grimes, a biology professor in UM's Division of Biological Sciences, said they have induced stem cells to become the cell type that normally makes up human craniofacial cartilage. Stem cells can replicate themselves and also develop into different types of cells.

"The cells that normally give rise to this type of cartilage are called neural crest cells," Grimes said. "We found a novel method for generating craniofacial organoids from neural crest cells."

Organoids are a simplified, miniature version of an organ that mimic the architecture and gene expression of the organ. "Organoids are a good model for certain human tissues that we can study in ways that are not possible using tissue from human beings," he said.

Grimes said there is a critical unmet need for new methods to regenerate human cartilage for the 230,000 children born annually in the U.S. with craniofacial defects. Growing cartilage in the laboratory also could lead to effective treatments to repair craniofacial cartilage damage due to injuries.

The researchers studied gene expression data at the RNA and protein level to reveal how cartilage cells arise from stem cells. They revealed that stem cells communicate in the early stages to become elastic cartilage, which makes up human ears.

To accomplish this, the team used extensive analysis of biological markers and machine-learning pattern-recognition techniques to understand the cell signaling pathways involved when cells differentiate into cartilage.

It is difficult to reconstruct natural features such as a person's ears, nose or larynx with current plastic surgery techniques, and transplanted tissue is often rejected without immunosuppressants.

"To use patient-derived stem cells to generate craniofacial cartilage in the laboratory, you need to understand the human-specific differentiation mechanisms," Grimes said. "Our aim is to develop a protocol for craniofacial cartilage generation for transplantation using human stem cells."

The research was published in the journal iScience . Besides Grimes, contributing UM authors include Lauren Foltz, Nagashree Avabhrath and Jean-Marc Lanchy. Other authors are Bradly Peterson of Missoula's Pathology Consultants of Western Montana and Tyler Levy, Anthony Possemato and Majd Ariss of Cell Signaling Technology of Danvers, Massachusetts.

• Joint Health
• Human Biology
• Skin Cancer
• Prostate Cancer
• Immune System
• Medical Topics
• Head injury
• Osteoarthritis
• Human skeleton
• Stem cell treatments
• Adult stem cell

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Materials provided by The University of Montana . Note: Content may be edited for style and length.

Journal Reference :

• Lauren Foltz, Nagashree Avabhrath, Jean-Marc Lanchy, Tyler Levy, Anthony Possemato, Majd Ariss, Bradley Peterson, Mark Grimes. Craniofacial Chondrogenesis in Organoids from Human Stem Cell-Derived Neural Crest Cells . iScience , 2024; 109585 DOI: 10.1016/j.isci.2024.109585

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Sex-dependent variation in cartilage adaptation: from degeneration to regeneration

• Jhanvee Patel 1 ,
• Song Chen 2 ,
• Torey Katzmeyer 1 ,
• Yixuan Amy Pei 1 , 3 &
• Ming Pei   ORCID: orcid.org/0000-0001-5710-3578 1 , 4  

Biology of Sex Differences volume  14 , Article number:  17 ( 2023 ) Cite this article

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Despite acknowledgement in the scientific community of sex-based differences in cartilage biology, the implications for study design remain unclear, with many studies continuing to arbitrarily assign demographics. Clinically, it has been well-established that males and females differ in cartilage degeneration, and accumulating evidence points to the importance of sex differences in the field of cartilage repair. However, a comprehensive review of the mechanisms behind this trend and the influence of sex on cartilage regeneration has not yet been presented. This paper aims to summarize current findings regarding sex-dependent variation in knee anatomy, sex hormones’ effect on cartilage, and cartilaginous degeneration and regeneration, with a focus on stem cell therapies. Findings suggest that the stem cells themselves, as well as their surrounding microenvironment, contribute to sex-based differences. Accordingly, this paper underscores the contribution of both stem cell donor and recipient sex to sex-related differences in treatment efficacy. Cartilage regeneration is a field that needs more research to optimize strategies for better clinical results; taking sex into account could be a big factor in developing more effective and personalized treatments. The compilation of this information emphasizes the importance of investing further research in sex differences in cartilage biology.

Evidence indicates males and females respond differently to cartilage degeneration and regeneration.

Few review articles available provide comprehensive information referencing clinical and laboratory research.

This review offers an update on sex-dependent variation in knee anatomy and sex hormones’ effect on cartilage degeneration and regeneration as well as potential application in stem cell therapy.

Understanding sex differences in cartilage biology contributes to personalized treatment in cartilage diseases.

Introduction

Adult cartilage defects present a challenge in orthopaedic medicine, as cartilage possesses limited intrinsic healing capacity. Onset of cartilage degeneration increases with age, leading to prevalent diseases such as osteoarthritis (OA) in the elderly. Cartilage degeneration is accompanied by pain and discomfort, hindering activities of daily living. These difficulties have prompted a mass accumulation of research in cartilage repair and regeneration. Stem cell-based therapy becomes a promising approach to healing cartilage in which patient-derived stem cells are rejuvenated and grown in vitro to be injected or implanted into cartilage defects. This therapy has the potential to reduce morbidity, mortality, and economic costs from complications of traditional surgical techniques [ 1 ].

Increasing evidence indicates that males and females differ in cartilage characteristics and risk of degeneration through sex-dependent gene expression [ 2 ], which necessitates consideration of sex differences when designing preclinical studies and clinical trials for cartilage repair [ 3 ]. For example, males have thicker articular cartilage and greater knee cartilage volume than females [ 4 , 5 ]. Accordingly, females have a higher chance of developing OA than males, specifically in the knee after menopause [ 6 ]. Females also tend to experience more severe cases of knee arthritis and are more than three times more likely to be candidates for total knee or hip replacement surgery than males [ 5 ]. Despite increased scientific awareness of sex differences in cartilage development, degeneration, and repair, many studies continue to assign sex arbitrarily, as a comprehensive review of the differences in male versus female cartilage adaptation has not been presented. This review focuses on sex-dependent variation in cartilage degeneration and regeneration with an emphasis on stem cell-based therapies.

Chondrocyte biology overview

This section provides a brief introduction of articular cartilage development and maintenance as well as associated signaling pathways.

Development of articular cartilage

During fetal development and early neonatal life, articular cartilage does not have the same properties as adult cartilage and must progress through additional developmental stages. Early articular cartilage consists of matrix-poor tissue with an irregular and uneven distribution of cells; however, by 6 weeks of age, it increases in thickness through both chondrocyte hypertrophy and increases in matrix secretion [ 7 ]. As articular cartilage develops, it also establishes zonal organization consisting of the surface zone, medial zone, and deep zone. The surface zone consists of flat articular chondrocytes that produce and secrete lubricating proteoglycans encoded by the proteoglycan 4 (PRG4) gene for joint protection during movement. Both the medial and deep zones contain large, round, vertically oriented chondrocytes that excrete extracellular matrices. There is some uncertainty regarding the exact mechanisms underlying chondrocyte expansion in the neonate, but some studies suggest that PRG4-positive cells in the surface zone are responsible for cartilage growth and transformation [ 7 ]. Growth and differentiation factor 5 (GDF5), Wingless/integrated gene 9a (WNT9A), Doublecortin (DCX), SRY Box transcription factor 9 (SOX9), and ETS-related gene (ERG) are all genetic markers of cells in the interzone, which is the primitive joint during fetal development. The interzone consists of flat mesenchymal stem cells (MSCs) required for joint formation. By regulating expression of the above genes and others, the fate of interzone cells is determined [ 7 ]. For example, SOX9 is expressed throughout the interzone cells during early development, but by day 14, expression is limited only to the outer regions of the interzone and the flanking outer chondrocytes; the intermediate zone, on the other hand, ceases expression of type II collagen (COL2A1), leading to its involvement in the formation of cruciate ligaments. Finally, transforming growth factor beta (TGFβ) receptor 2 (TGFBR2)-expressing interzone cells are found only in the dorsal and ventral regions of joints in mature cartilage, indicating that expression is either induced in these regions or deleted in other regions to form adult articular cartilage [ 8 ]. GDF5 is expressed in all of these cells and, therefore, is a broad marker associated with multiple cell types within the interzone that give rise to not only articular cartilage, but also synovial tissue and intra-joint ligaments [ 7 ]. However, interzone cells alone are not sufficient for normal articular cartilage development. Interzone cells were found to be mitotically quiescent, while flanking cells of non-interzone origin were mitotically active, indicating that the underlying proliferative cells that get recruited by interzone cells to the flank area play a major role in the thickening of developing articular cartilage [ 7 ].

Maintenance and repair of articular cartilage

Articular cartilage in the adult is considered a permanent tissue, meaning it has little-to-no turnover throughout adult life. This property differs from cartilage in other locations, such as growth plate cartilage. ERG expression persists only in the superficial cells of the articular cartilage after 6 months of age and is responsible for the permanent quality of articular cartilage. In the case of ERG deficiency, Friend leukemia integration 1 ( FLI1 ), another transcription factor, provides redundancy to maintain the permanent nature of articular cartilage [ 7 ]. Although articular cartilage exhibits limited capacity for cell turnover and regeneration, there are progenitor cells that exist within fully developed articular cartilage; however, under normal conditions, these cells do not proliferate in the case of injury or degeneration, such as in OA [ 7 ]. These progenitor cells have been targeted as a mechanism of OA treatment through the surgical movement of these cells to the cartilage surface and induction of their proliferation. Several proteins and hormones have been indicated in the maintenance and protection of articular cartilage, including parathyroid hormone (PTH), PTH related protein, and PRG4; in addition, the Hedgehog signaling pathway as well as A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and metalloproteinase have been indicated in cartilage pathogenesis, as ablation and elimination have shown increased resistance to OA development in mice [ 7 ].

Signaling pathways involved in articular cartilage anabolism and/or anti-catabolism include TGFβ1, insulin-like growth factor I (IGFI), hypoxia-inducible factor (HIF) 1 alpha (HIF1α), and bone morphogenetic protein 7 (BMP7) [ 9 ]. Catabolic signals involve interleukin 1 (IL1), IL6, HIF2α, and fibronectin fragments. Conflicting information exists regarding the role of fibroblast growth factor 2 (FGF2) in articular cartilage maintenance. These mechanisms are highlighted throughout the discussions in the “ Inflammatory biomarkers ” and “ Molecular mechanisms ” sections [ 9 ].

Sex differences in knee anatomy and cartilage degeneration

There are observed anatomical differences in cartilage growth and knee structure between sexes, which could contribute to the sex-related differences in cartilage degeneration [ 10 ]. The rate of cartilaginous degeneration in an individual is multifactorial, and several mechanisms influencing risk of OA and cartilage injury are sex-dependent in nature [ 10 ].

Knee anatomy and articular cartilage thickness and degeneration

In the distal femur and proximal tibia, the mean aspect ratio (mediolateral distance versus anteroposterior distance) of the male femora is larger than the female femora [ 11 ]; the plateau aspect ratio (tibial medial anteroposterior dimension versus tibial mediolateral dimension) of the male is also larger than the female [ 12 ]. With a high degree of variation between individuals, sex-specific designs of total/unicompartmental knee arthroplasty have been developed to accommodate anatomical differences between sexes [ 11 , 13 ].

In healthy children, cartilage thickness in the knee differs significantly between sexes, with girls having thinner cartilage than boys [ 14 , 15 ]. Weight, height, and body mass index (BMI) contribute to cartilage thickness, but age is the leading contributor among school age children [ 14 ]. Physical activity benefits tibial cartilage volume [ 16 ]. In adults, males have substantially higher cartilage thickness and volume than females [ 17 ]. Both sexes exhibit a clear decrease in cartilage with increasing age, but the decrease is more drastic for females [ 15 , 18 ]. Females have a higher incidence rate of obesity in most countries, which has been linked to increased risk of OA through increased weight on joints [ 19 ]. A high BMI was closely related to both knee and hand OA rather than hip OA [ 20 ]. Weight reduction becomes an important part of OA treatment [ 21 ]. Similar to obesity, ethnicity is also a co-variable that influences OA incidence and severity. Elderly Chinese women in Beijing have a higher knee OA incidence than women in Framingham, Massachusetts; however, the incidence in men was comparable [ 22 ]. African–Americans have a higher prevalence of knee OA than Caucasians in the U.S., but this rate may vary according to sex [ 23 ]. Despite the progress in some racial differences in OA incidence and severity, more attention should be put on under-studied racial and ethnic groups and joint groups (e.g., foot, spine), emphasizing potential analytical factors, including but not limited to genetic, anatomical, environmental, and biomechanical features [ 23 ].

Inflammatory biomarkers

In both healthy individuals and OA patients, females have higher levels of inflammatory biomarkers [ 24 ]. For example, healthy women tend to have higher serum levels of leptin, an adipokine with pro-inflammatory properties [ 25 ]. As an essential hormone for bone development, high leptin levels in older adults are associated with lower cartilage volumes, and therefore, this hormone is thought to contribute to the increased OA susceptibility in women [ 25 , 26 ]. Extreme obesity caused by impaired leptin signaling leads to changes in subchondral bone morphology, but does not increase the incidence of knee OA, suggesting that obesity due to leptin-impaired signaling is insufficient to induce systemic inflammation and knee OA in female C57BL/6J mice [ 27 ]. As a link between obesity and OA [ 28 ], the use of leptin might be a potential approach for therapy in bone and joint diseases [ 29 ], especially for obese patients. In OA patients, pro-inflammatory chemokine CC chemokine ligand 3-like-1 (CCL3L1) is also found in higher levels in females than males, along with several inflammatory cytokines [ 30 ]. Interestingly, females with OA also tend to have higher serum levels of anti-inflammatory adipokines, such as apelin and adiponectin [ 31 ], suggesting that sex differences in cartilage degeneration are not perfectly aligned with the level of inflammation in the joint.

When comparing the synovial fluid composition in male and female OA patients, women had less 25-hydroxyvitamin D3 [25(OH)D3], a metabolite of Vitamin D that protects against cartilage degeneration [ 32 ], which may explain why men with OA typically have lower inflammatory markers. Dehydroepiandrosterone (DHEA), the precursor for both estrogen and testosterone, is one agent with promising anti-inflammatory effects for both sexes. DHEA injection into arthritic joints of male and female rabbits resulted in decreased expression of inflammatory response elements, such as matrix metalloproteinase 3 (MMP3) and increased tissue inhibitor of matrix metalloproteinase 1 (TIMP1), indicating that DHEA treatment may protect against further cartilage degradation in both men and women by reducing inflammation [ 33 ]. Interestingly, male rats have higher sensitivity to anti-inflammatory agents, such as parecoxib and dexamethasone, while female rats exhibited higher levels of IL6 after dexamethasone treatment, which likely explains the limited response of females to anti-inflammatory treatment [ 34 ]. IL1, another catabolic pro-inflammatory cytokine, may be upregulated by FGF2, which is correlated with expression of estrogen receptors (ERs) [ 35 , 36 ]. According to a study published in Clinical and Experimental Rheumatology, the articular cartilage of female rats was found to be more sensitive to IL1-mediated inhibition of proteoglycan synthesis, increasing risk of cartilage damage [ 35 ].

Extracellular matrix biomarkers

It has been observed that there are differences between sexes on extracellular matrix (ECM) biomarker expression following the onset of cartilage degeneration. Variations in ECM composition can significantly influence cell behavior. In healthy females, ECM of articular cartilage is likely to have higher spontaneous loss of glycosaminoglycans (GAGs) along with reduced levels of proteoglycan and collagen when compared to males [ 35 ]. Structural change to ECM proteins is a classic indicator of early OA, supporting the clinical findings of female predisposition to OA development [ 37 ]. Another sex disparity in OA is the rate of collagen turnover. Female OA patients tend to have genetic upregulation of type I collagen, indicating that OA females lose collagen at a faster rate than OA males and, therefore, require more collagen transcription to compensate [ 38 ]. Female chondrocytes are also more susceptible to the inhibition of proteoglycan production by IL1, indicating that females are less likely to synthesize new proteoglycan than males [ 35 ].

Another OA biomarker found in synovial fluid, cartilage acidic protein 1 (CRTAC1), is induced by inflammatory cytokines, such as IL1 [ 39 ]. As female chondrocytes are more sensitive to IL1, CRTAC1 tends to exist at higher levels in the female population than in males [ 39 ]. Therapeutic research using murine models is beginning to look into the feasibility of CRTAC1 gene knockout in the prevention of female OA development [ 39 ].

When comparing men and women with clinical OA, C-terminal telopeptide of type I collagen (CTX-I) and, to a lesser degree, CTX-II, are both found in higher serum concentrations in females [ 40 ]. CTX is a product of cartilage degradation, so higher serum CTX levels indicate increased damage to articular cartilage; in addition, levels of CTX-II are associated with pain intensity, suggesting that OA females likely perceive more pain related to their cartilage loss than males with the same condition [ 41 ]. Increased serum CTX also illustrates the predisposition to OA susceptibility in females found in clinical studies.

Serum values of cartilage oligomeric matrix protein (COMP) also vary based on sex, with healthy Caucasian males having higher serum COMP on average compared to healthy Caucasian females [ 42 ]. COMP expression also varies by ethnicity, as COMP levels were found to be higher in African–American women than Caucasian women with corresponding ages; no significant COMP differences were found between African–American men and women or between Caucasian and African–American men [ 42 ]. In all groups, increasing age and BMI were directly associated with elevated serum COMP [ 42 ]. Increased COMP levels predict subsequent cartilage loss, but the degree of association is only around 60 percent; nevertheless, elevated serum COMP is a mild predictor of cartilage degradation, so this finding contrasts from the majority of other degenerative biomarkers in which women typically present with higher levels [ 43 ]. There is some contradictory evidence, however, regarding the sex differences of COMP levels. COMP levels increase acutely with physical activity, and women with thinner anterior femoral cartilage have greater resting COMP levels [ 44 ].

Protective effect of sex hormones on cartilage degradation

Cartilage growth regulation is complex. Several factors act on chondrocytes during their proliferation and differentiation, such as IGF-I and -II, FGF2, TGFβ, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and sex hormones (estrogens and androgens) [ 45 ]. Estrogens promote chondrocyte proliferation, and androgens affect chondrocyte proliferation via conversion to estrogen by aromatase [ 45 ]. Male and female differences in these factors, especially sex hormones, could be crucial for developing specific treatments targeting cartilage degradation (Table 1 ).

Effect of estrogen on cartilage

Estrogen is a steroid hormone existing in the body in three different subtypes: estrone (E1), estradiol (E2), and estriol (E3) [ 46 ]. E2 is the most prevalent and has the highest bioactivity. These estrogen subtypes signal through three main ERs: nuclear ERα and ERβ, and membrane G-protein coupled ER (GPER/GPR30) [ 46 ]. Availability of aromatase, the enzyme responsible for conversion of testosterone to estrogen, directly corresponds to estrogen levels in both males and females. Granulosa cells in the female ovary contain aromatase, as well as bone, breast, brain, and adipose tissue in both sexes. Therefore, obesity increases the availability of estrogen in both sexes. Consideration of aromatase availability must be taken into consideration while evaluating the impact of estrogen on cartilage [ 47 ].

Estrogen receptors and cartilage

In 1999, Ushiyama et al. identified the gene expression of both ERα and ERβ in human articular chondrocytes [ 48 ]. They found that, despite the fact that women have higher estrogen levels, men show a significantly higher level of gene expression for both ER paralogs than women. All women in the study were postmenopausal and had never had estrogen replacement therapy (ERT), which is linked to decreased estrogen levels. Expression of ERα in articular cartilage decreases with age, which can be linked to cartilage degeneration and increased OA severity [ 49 ]. Selective ER modulators have clinical utility in the context of osteoporosis, ER + breast cancer, and other estrogen-related pathologies to mediate stimulation and/or antagonism of site-specific ERs in the body. Raloxifene, often prescribed for osteoporosis, has been observed to activate ERs and the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in human chondrocytes, preventing tumor necrosis factor alpha (TNFα)-induced caspase-3-dependent apoptosis [ 50 ]. In addition, estrogen via an ERβ-dependent mechanism inhibits cell proliferation and ERα expression, while estrogen via an ERβ-independent mechanism regulates chondrogenesis [ 51 ]. ERβ deficiency has been documented to increase condylar growth in female mice by inhibiting the turnover of fibrocartilage [ 52 ]. Overall, studies point to increased ERα expression having both a chondroprotective effect and an inhibitory role in ERβ expression during chondrogenesis.

The estrogen‑related receptor (ERR) family of orphan nuclear receptors related to ERα, composed of ERRα, ERRβ, and ERRγ, has been shown to potentially play a significant role in OA pathogenesis [ 53 , 54 ]. Despite sharing sequence homology to the ERα and ERβ, ERRα is unable to bind estrogen [ 53 ]. ERRα could have dual contrasting roles in the induction and progression of OA. ERRα is essential for cartilage formation by regulating its target gene SOX9 expression [ 54 ]. One study has shown, similar to its effect on ERα, 17β-E2 increased mRNA and subsequent protein expression of ERRα, which in turn led to an increase in SOX9 , GDF5 , and CYP19A1 (aromatase) during in vitro mandibular condylar chondrocyte cultivation [ 55 ]. SOX9 and GDF5 contribute to chondrocyte proliferation, differentiation, and maturation. When XCT790, a synthetic inverse agonist of ERRα, was used to inhibit ERRα expression, the proliferative capacity of the mandibular condylar chondrocytes was reduced [ 55 ]. Furthermore, knockdown of ERRα has resulted in impaired expression of genes including SOX5 / SOX6 / SOX9 , COL2A1 / COL10A1 , and RUNX2 (runt-related transcription factor 2) and ectopic expression of SOX9 rescued defective formation of cartilage, indicating that ERRα is involved in chondrocyte growth by regulating SOX9 expression [ 56 ]. However, ERRα-mediated degradation of cartilage has also been observed. Increased expression of ERRα has been linked with IL1β treatment in human OA chondrocytes via the PGE2 (prostaglandin E2)/cAMP (cyclic Adenosine 3′,5'-monophosphate)/PKA (protein kinase A) signaling pathway [ 57 ]. ERRα could upregulate IL1-induced MMP13 expression in OA chondrocytes. In addition, XCT790 decreased MMP13 gene level [ 57 ]. These results point to ERRα involvement in IL1β-mediated OA cartilage degradation and loss.

Of the ERR subtypes, ERRγ has significantly increased expression in humans and various models of mouse OA cartilage. Overexpression of ERRγ in cartilage is connected to chondrodysplasia and reduced chondrocyte proliferation [ 57 ]. ERRγ contributes to cartilage destruction through the IL6-mediated mitogen-activated protein kinases (MAPK), such as the ERK1/2 pathway [ 58 ]. As a downstream transcription factor of ERK1/2, upregulation of ERRγ leads to ECM degradation and angiogenesis in osteoarthritic temporomandibular joints (TMJ) [ 59 ]. Overexpression of ERRγ in chondrocytes directly upregulates MMP3 and MMP13 expression. Moreover, GSK5182, a small-molecule ERRγ inverse agonist, has promising therapeutic potential by inhibiting pro-inflammatory cytokine-induced catabolic factors [ 60 ]. As ERRγ is involved in the catabolic modulation of OA pathogenesis, it has strong potential to be a therapeutic target for OA [ 61 ].

During early puberty, GPER1/GPR30 positively regulates chondrocyte proliferation at the growth plate, contributing to the longitudinal growth of long bones [ 62 ]. GPER1/GPR30 has been observed to alleviate mechanical stress-mediated apoptosis of chondrocytes in OA through suppression of Piezo1, a mechanosensitive ion channel ubiquitously expressed in adipose tissue that, when upregulated, has been linked to chondrocyte apoptosis [ 63 ]. E2 works with GPER1/GPR30 to suppress acid-sensing ion channel 1a (ASIC1a) and ASIC1a can overregulate intracellular calcium levels, resulting in articular chondrocyte damage [ 64 ]. E2’s suppression of the channel protects rat cartilage with adjuvant arthritis from acidosis-mediated injury and autophagy [ 64 ].

Estrogen and cartilage

Estrogen is an attractive candidate for cartilage engineering due to evidence that local estrogen production is crucial for chondrocyte proliferation and protection from spontaneous cell death [ 65 ]. Chondrocytes have been observed to be capable of both in vivo and in vitro estrogen synthesis [ 66 ], which appears to increase COL2A1 gene expression [ 67 ].

Growth factors, such as TGFβ, BMP, FGF, PDGF, growth hormone (GH), and IGF-I, are integral for cartilage development and healing. Observations suggest that E2 may interact with the synthesis and secretion of these growth factors, specifically TGFβ and IGF-I. TGFβ1 has been observed to be a modulator in 17β-E2 activity on costochondral chondrocytes from female rats in a sex-specific manner [ 68 ]. IGF promotes the production of matrix as well as the proliferation and inhibition of apoptosis in chondrocytes [ 69 ]. IGF regulation could be influenced by estrogen action [ 70 ]. Research demonstrates that E2 has an indirect effect of priming IGF-I activity in cartilage metabolism [ 71 ]. The interaction between the IGF-I receptor and ERα has been observed to promote proliferation and suppress inflammation in nucleus pulposus cells [ 72 ].

Catabolic cytokines such as IL1 have detrimental effects on the composition and mechanical properties of articular cartilage. Research suggests estrogen deficiency can increase cytokine receptor numbers and cofactors of cytokine action, which enhances cell response to cytokines [ 73 ]. β-Ecdysterone, an estrogen analog, has demonstrated anti-apoptosis and anti-inflammation ability in IL1β-induced rat chondrocytes [ 74 ].

Estrogen has been documented to suppress MMPs. MMP1, MMP3, and MMP13 are intimately involved in the process of articular cartilage degeneration [ 75 ]. One study observed 17β-E2 suppressed MMP13 expression in human articular chondrocytes [ 76 ]. The use of 17β-E2 in physiological doses can improve the MMP and TIMP imbalance in articular chondrocytes, suggesting a potential chondroprotective effect of hormone replacement therapy [ 77 , 78 ]. Current findings demonstrate that estrogen can be modulated to reduce the effect of reactive oxygen species (ROS). In rat nucleus pulposus cells, the interaction between E2 and ER has interfered with the ROS/nuclear factor kappa-B (NF-κB) pathway, reducing TNFα-induced premature senescence [ 79 ]. Pretreatment with 17β-E2 not only decreased acid-induced damage, it also inhibited apoptosis and helped to restore mitochondrial function. Specifically, studies have shown that 17β-E2 is capable of decreasing levels of ASIC1a through the ERα and the autophagy–lysosomal pathway [ 80 ].

While estrogen can play a protective role in cartilage through the many factors discussed above, it has been documented to have detrimental effects as well. 17β-E2 stimulation has also resulted in the loss of ECM and increased expression of TNFα, IL1, HIF2α and its downstream OA-related cytokines [MMP13, vascular endothelial growth factor (VEGF), and type X collagen] in primary condylar chondrocytes via ERβ [ 81 ]. Estrogen has been reported to be chondrodestructive in animal models; specifically, increased activity of ERs has been suggested as a factor in initiating osteoarthritic changes in a rabbit model [ 82 ]. High E2 concentration has been linked to increased IL1β stimulated proteoglycan degradation and MMP production in chondrocytes [ 83 ]. Interestingly, E2 has been observed to reduce nerve growth factor (NGF) expression in chondrocytes significantly, even after stimulation by TGFβ1 or IL1β, indicating estrogen can play a role in regulating NGF, which is integral to the development of OA pain, suggesting that E2 is associated with decreased OA pain [ 84 ]. The contradictory results make it difficult to understand the role estrogen plays in cartilage degradation and further demonstrates more research is needed.

Protective effect of androgens on cartilage

Androgens are steroid hormones with the most prevalent being testosterone, dihydrotestosterone (DHT), and androstenedione. In addition, androgens are the precursors for estrogen. Like estrogen, research suggests that androgens play a role in cartilage protection [ 85 ]. Androgen levels are high in males and low in females, which could be a reason why males have less risk of OA. However, 17β-E2 has been observed to have a greater impact in chondrocyte functionality and gene expression profiles, which is particularly apparent in chondrocytes from females [ 86 ]. Regardless, understanding the relationship between chondrocytes and androgens is a crucial step in determining OA risk differences between males and females.

Androgen receptors and cartilage

Androgen receptor (AR) is a steroid hormone receptor that influences the transcription of androgen-responsive genes by binding their respective DNA sequences [ 87 ]. Moreover, AR can affect cell physiological activities, such as proliferation, apoptosis, and migration [ 88 ]. AR is expressed in human primary articular chondrocytes [ 89 ]. Testosterone receptors have been discovered in rat chondrocytes from growth zone and resting zone cartilage in both sexes [ 90 ]. Additionally, in rabbits, AR overexpression has resulted in a reduced apoptosis rate and has maintained the phenotype of chondrocytes through inhibition of the mammalian target of rapamycin (mTOR) pathway to improve autophagy [ 88 ].

Androgen and cartilage

Androgens are related to cartilage tissue maintenance. Testosterone at physiological concentrations increases chondrogenic potential of chondrogenic progenitor cells in male arthritic tissue in vitro [ 91 ]. In male intervertebral disc (IVD) cells, testosterone has effectively enhanced chondrogenesis in vitro but does not affect female IVD cells or mesenchymal stem cells (MSCs) from either sex similarly [ 92 ]. In chondrocytes of mice and rabbits, testosterone stimulates growth and local production of IGF-I [ 71 , 93 ], suggesting that testosterone has an indirect priming effect on the response of chondrocytes to IGF-I.

Studies have also shown DHEA has a protective role against OA, specifically with inflammation [ 33 , 94 , 95 ]. A rabbit study concluded that DHEA has a cartilage-protecting effect during OA development following bilateral anterior cruciate ligament transection [ 96 ]. In male and female mice, DHEA treatment has demonstrated the ability to delay onset and decrease the severity of collagen-induced arthritis [ 97 ]. In human osteoarthritic knee chondrocytes, DHEA treatment has been shown to significantly reduce MMP1 but increase TIMP1 gene expression and protein levels [ 98 ]. In rats with synovial arthritis, DHT treatment has reduced TNFα and MMP2 levels [ 99 ]. These data indicate that DHEA is associated with reduced inflammation and modulation of collagen breakdown in both rats and humans.

Androgens could play a role in cartilage inflammation through cytokines. Androgens stimulate articular cartilage integration. Low concentrations of IL1β could influence this effect favorably [ 100 ]. In female animals, testosterone has been shown to have a protective influence on IL1-induced cartilage breakdown [ 101 ]. Moreover, testosterone has decreased the effect of IL1 on both proteoglycan loss and synthesis, which are crucial parts of cartilage ECM. Androgens also have demonstrated a protective role in the development of adolescent idiopathic scoliosis, potentially by inhibiting IL6-induced abnormal chondrocyte development [ 102 ]. The mechanisms behind the association between androgens and cytokines could be numerous, involving direct immunomodulatory effects and interaction of glucocorticoid response to inflammation [ 100 ].

Effect of sex hormone on subchondral bone and adjacent synovium

Strong evidence supports a connection between subchondral bone changes and cartilage damage and loss [ 103 ]. Estrogen has been investigated as an influence in this relationship, because it has been documented to increase cartilage alteration [ 104 ]. In OA, increased production of synovial fluid results in swelling of the synovium [ 105 ]. Understanding the influence of sex hormones on these structures is crucial to understanding the differences in male and female OA prevalence.

Protective effect of sex hormones on subchondral bone

Multiple studies point to estrogen modulating OA by increasing subchondral bone structure. Bone remodeling has been connected to estrogen depletion by ovariectomy, affecting the subchondral trabecular bone of joints [ 106 , 107 ]. Estrogen deficiency led to subchondral bone resorption and articular cartilage degeneration in an ovariectomized (OVX) rat model of postmenopausal OA [ 108 ]. Estrogen replacement treatment in a cynomolgus macaque model protects subchondral bone mass from remodeling [ 109 ]. Estrogen treatment in elderly women has been observed to lessen subchondral bone weakening in the OA knee [ 110 ]. ERs are also commonplace in bone tissue and help to regulate bone turnover, which is relevant to OA pathophysiology. In murine models, ERα knockout has led to development of larger osteophytes and a thinner lateral subchondral plate [ 111 , 112 ]. No studies currently exist examining the influence of androgen on subchondral bone.

Effect of sex hormones on adjacent synovium/synovial fluid

Synovial fluid is made up of a myriad of cellular metabolites, one of which are extracellular vesicles (EVs). Exosomes are 40–100 nm diameter packaged vesicles comprised of lipid, protein, and small RNA [ 113 ]. Synovial fluid-derived EVs have demonstrated the ability to change microRNA (miRNA) cargo with sex-specific alterations. Within the synovial fluid of OA patients, exosomal miRNA content can be altered, and in females, there exist some estrogen responsive miRNAs that are capable of targeting toll-like receptor (TLR) pathways [ 114 ]. In female OA EVs, studies have observed increases in haptoglobin, orosomucoid, and ceruloplasmin levels and a decrease in apolipoprotein; in male OA EVs, β-2-glycoprotein and complement component 5 proteins have been observed to increase, and Spt-Ada-Gcn5 acetyltransferase (SAGA)-associated factor 29 has decreased [ 115 ]. The sex-specific alteration in synovial fluid EV protein content with OA patients could be a mechanism behind the high OA prevalence and severity in women.

In the TMJ, fibroblast-like type B synoviocytes could be affected by expression of ERα-immunoreaction, indicating that TMJ can be influenced by estrogen and resulting in higher prevalence of temporomandibular disorders in females than males [ 116 ]. Interestingly, compared to ERα, normal human synovia regularly expresses large amounts of ERβ [ 117 ]. Arthritic synovium also demonstrates expression of ERs, linking estrogen as a modulator in synovial inflammation [ 118 ]. Estrogens have been shown to aggravate TMJ inflammation and pain, potentially by amplifying the expression of cadherin-11 and release of pro-inflammatory cytokines in synoviocytes [ 119 ]. It has also been suggested that E2 aggravates TMJ inflammation by the NF-κB pathway, which similarly results in pro-inflammatory cytokine release [ 120 ]. In a rat study, male TMJ particularly was found to be an estrogen target especially for ERα [ 121 ]. Additionally, estrogen regulates the IGF system and cytokines, which act in the synovial fluid [ 70 ]. In addition to the prophylactic impact estrogen has through inhibiting the synovial inflammation and articular cartilage degeneration seen in OA, estrogens are suspected to also partially regulate sensory neuropeptide expression in the synovium of experimental OA models (anterior cruciate ligament transection rat models), for instance substance P and calcitonin gene-related peptide [ 122 ].

Chondrocytes from human OA synovium and intra-articular injection in rabbit OA knee joints have demonstrated suppressed MMP3 expression and enhanced TIMP1 level when treated with DHEA, indicating a protective role of androgens in cartilage degradation and synovial inflammation [ 33 , 98 ]. DHEA has also been observed to have a protective effect in the synovial tissue of TMJ, potentially through increasing fibromodulin formation, which could prevent IL1β-induced inflammation and TGFβ1-induced hyperplasia of fibrous tissue [ 123 ].

Molecular mechanisms

Joint tissue biology is heavily influenced by estrogen, which helps to regulate the expression of key signaling molecules and their activity in several distinct pathways. Molecularly, the ERs are transcription factors that bind to the DNA either directly or indirectly; they are then able to signal through one of four pathways, three ligand-dependent and one ligand-independent [ 124 , 125 ]. Increasing evidence indicates that interaction between estrogen and ERs as well as related signaling pathways contributes to sex-dependent variation in cartilage adaptation (Fig.  1 ).

Main signaling pathways of sex hormones on chondrocytes. Wnt signaling pathway ( A ): Wnt14 binding inhibits the phosphorylation of β-catenin by GSK3, and the unphosphorylated β-catenin then travels into the chondrocyte nucleus to act as an OA phenotype transcription factor [ 9 ]. E2 upregulates the expression of SOST , which codes for an inhibitor of Wnt14 called sclerostin [ 130 ]. DHEA decreased the expression of β-catenin, resulting in upregulation of MMP13 and downregulation of TIMP1 and COL2A1 [ 131 ]. TGF-β signaling pathway ( B ): E2 upregulates the expression of ALK5 receptors, promoting ACAN and COL2A1 production and inhibiting COL10A1 , MMP13 , VEGF , OPN , BGLAP , and ALP expression [ 9 , 135 , 136 ]. Cellular energy and survival related pathways ( C ): PI3K/AKT signaling pathway is downregulated in human cartilage tissues with OA or in OA-like chondrocytes exposed to IL1, TNFα [ 153 , 154 ]. E2 could function through the PI3K/AKT/NF-κB pathway by inhibiting chondrocyte apoptosis [ 229 ], through the PI3K/AKT/FOXO3 pathway by downregulating MMP 3 expression and preventing ECM degradation [ 156 ]. Upregulated PI3K/AKT/mTOR in OA cartilage is linked to decreased expression of autophagy-related genes [ 158 ]. Overexpression of androgen has been shown to promote chondrogenesis and prevent degradation and apoptosis, potentially through mTOR-related signaling inhibition [ 88 ]. In addition, E2 inhibited autophagy upregulation to protect chondrocytes via the SIRT1-mediated AMPK/mTOR pathway [ 165 ]. Acid environment and cellular inflammation related pathways ( D ): E2 can increase the mRNA and protein expression levels of ERRα , which in turn led to an increase in SOX9 , GDF5 , and CYP19A1. Through the ERRα–AMPK–ULK1 signaling pathway, E2 could support autophagy–lysosome pathway-dependent ASIC1a protein degradation and defend against acidosis-induced cytotoxicity [ 168 ]. IL1/6 and TNFα activate NF-κB signaling pathways through receptor binding and ultimately help to upregulate expression of ASIC1a. Activation of ASIC1a could aggravate the effects of IL1/6 and TNFα on ECM metabolism by increasing MMP3 / 13 and ADAMTS5 mRNA expression in articular chondrocytes [ 166 ]. Extracellular acidification activates ASIC1a, which ultimately leads to the autophagy of articular chondrocytes [ 167 ]. Low levels of E2 have been observed to inhibit IL1-induced proteoglycan degradation, downregulating cartilage degeneration [ 83 ]. DHEA has been shown to play its protective role against cartilage degeneration through regulation of MMP3 , TIMP1 , IL1, COX2 , and iNOS gene expression [ 33 , 169 ]. MicroRNA related pathways ( E ): IL1 stimulation increased miR-203 expression [ 171 ]. MiR-203 directly targets ERα, followed by downregulation of ACAN and COL2A1 [ 170 ] . The estrogen/ER/miR-140 pathway inhibited IL1-induced cartilage matrix degradation [ 76 ]. LRP low density lipoprotein receptor-related protein, FZD frizzled receptor, GSK3 glycogen synthase kinase 3, DHEA dehydroepiandrosterone, RUNX2 runt-related transcription factor 2, MMP13 matrix metalloproteinase 13, TIMP1 tissue inhibitor of matrix metalloproteinase 1, COL2A1 type II collagen, E2 estradiol, ER estrogen receptor, TGFβ transforming growth factor beta, ALK5 activin-like kinase 5, SOX9 sry-type high-mobility-group box transcription factor 9, ACAN aggrecan, VEGF vascular endothelial growth factor, OPN osteopontin, OC osteocalcin, ALP alkaline phosphatase, SIRT1 silencing information regulator 2 related enzyme 1, AMPK AMP-activated protein kinase, mTOR mammalian target of rapamycin, ULK unc-51-like kinase, ERRα estrogen‑related receptor α, GDF5 growth and differentiation factor 5, GPER1/GPR30 G-protein coupled estrogen receptor, PI3K phosphatidylinositol 3-kinase, AKT protein kinase B, NF-κB nuclear factor kappa-B, FOXO3 forkhead box O-3, IL1 interleukin 1, ASIC1a acid-sensing ion channel 1a, ADAMTS5 a disintegrin and metalloproteinase with thrombospondin motifs 5, COX2 cyclooxygenase-2, iNOS inducible nitric oxide synthase, TNFα tumor necrosis factor α, MAPK mitogen-activated protein kinases, miR-203 microRNA-203

Wnt signaling pathway

During cartilage development and regeneration, Wnt14 is upregulated [ 126 ]. By binding to cell surface frizzled receptors (FZD) and low density lipoprotein receptor-related protein (LRP) co-receptors [ 9 ], Wnt14 inhibits β-catenin phosphorylation by glycogen synthase kinase 3 (GSK3). The unphosphorylated β-catenin then travels into the chondrocyte nucleus to act as a transcription factor [ 9 ], stimulating tissue breakdown typically consistent with the OA phenotype [ 127 , 128 , 129 ]. Since β-catenin is found in higher concentrations in OA tissue, the promotion of β-catenin degradation protects cartilage from damage [ 127 , 128 ]. Interestingly, E2 upregulates the expression of the SOST gene, which codes for an inhibitor of Wnt14 called sclerostin [ 130 ]. By inhibiting Wnt14, β-catenin gets phosphorylated and tagged for degradation. Though E2 promotes sclerostin expression, this Wnt inhibitor is still found in higher concentrations in males than females, which may be a factor in the increased clinical incidence of OA in females [ 60 ]. Evidence indicates that DHEA decreases the expression of β-catenin [ 131 ] and inhibits MMP13 expression and increases TIMP1 and COL2A1 expression in IL1β-induced rabbit chondrocytes [ 95 ]. Following treatment with DHEA after the transfection of β-catenin, rabbit chondrocytes showed significantly elevated expression of MMP13 and depressed expression of TIMP1 and COL2A1 ; meanwhile, after inactivating Wnt/β-catenin signaling with DKK1, the expression of MMP3 , MMP13 , and TIMP1 were suggestive of enhanced protective effects of DHEA [ 131 ].

The β-catenin signaling pathway is activated by chronic dysregulation of circadian rhythm due to downregulation of brain and muscle ARNT-Like 1 (BMAL1) [ 132 ]. BMAL1 is a protein that helps generate circadian rhythms in cartilage; dysfunctional BMAL1 in OA results in increased expression of β-catenin, MMP3, MMP13, ADAMTS4, and subsequent cartilage degeneration [ 132 ]. Circadian rhythm dysregulation is also associated with dysregulation of TGFβ signaling in chondrocytes, which is an essential signaling pathway for cartilage homeostasis [ 133 ]. Though the exact mechanism is unknown, murine castration studies indicate that estradiol is an important factor for the modification of circadian rhythms during development in both sexes [ 134 ]. Therefore, alterations in estrogen level are a likely influence of OA development due to circadian rhythm dysregulation, especially during menopause when estrogen levels decrease significantly [ 134 ].

TGFβ signaling pathway

Like the Wnt pathway, cartilage development and maintenance are highly regulated by TGFβ signals. TGFβ signals through type II receptors that recruit and subsequently activate type I receptors. Two main types of type I receptors, activin-like kinase 1 (ALK1) and ALK5, exist in cartilage and result in contrasting outcomes. Activation of ALK1 leads to the stimulation of terminal hypertrophic differentiation, characterized by increased production of COL10A1 , MMP13 , VEGF , OPN (osteopontin), BGLAP (osteocalcin), and ALP (alkaline phosphatase). On the other hand, E2 upregulating the expression of ALK5 results in the inhibition of hypertrophic differentiation and type II collagen and aggrecan production and maintains the quiescent stage of chondrocytes [ 9 , 135 , 136 ], indicating that E2 promotes chondrocyte development and homeostasis through the TGFβ pathway [ 75 ]. In OA, cartilage typically has a dramatic reduction of ALK5 receptors, indicating the protective role of E2 against cartilage degradation [ 9 , 136 ].

This signaling pathway interacts with other signaling molecules such as BMPs and transcription factors such as HIFs. BMP7 exhibits both anabolic and anti-catabolic effects on cartilage. For example, BMP7 induces the production of ECM to help protect against damage from IL1, IL6, and fibronectin fragments; BMP7 is also involved in the preservation of chondrogenic potential [ 9 ]. On average, males consistently have higher BMP7 expression in cartilage, which is consistent with the lower incidence of OA in males versus females [ 137 ]. BMP2 exhibits both anabolic and catabolic effects on cartilage by inducing type II collagen and aggrecan production, promoting proteoglycan synthesis, and elevating MMP13 expression. BMP2 is typically elevated in cartilage injury or OA, suggesting that its primary role is likely regulation of MMP13. HIF1α and HIF2α are also elevated in degenerative conditions, but the two transcription factors have contradicting functions. Elevated HIF1α plays a compensatory role in damaged cartilage, as it promotes transcription of type II collagen and aggrecan. HIF2a, contrarily, increases the expression of MMP13 and ADAMTS4 for a catabolic effect. E2 downregulates HIF1a , RUNX2 , and BMP2 , all of which help maintain cartilage integrity [ 138 , 139 , 140 ]. In pathologic conditions, such as OA, the usual hypoxic conditions of articular cartilage are exacerbated, which stimulates the increased expression of HIFs [ 140 ]. Therefore, HIF1a is overexpressed in OA cartilage, leading to discoordination of type II collagen and aggrecan production, since HIF1α indirectly regulates COL2A1 transcription [ 140 ]. E2 helps limit this discoordinate expression by reducing the expression of HIF1a in pathologically hypoxic conditions [ 140 ]. Similarly, the downregulation of BMP2 limits overexpression of COL2A1 and ACAN genes while also reducing BMP2-induced expression of MMP13 [ 9 ]. RUNX2 coordinating with TGFβ signaling via ALK1 phosphorylation is typically upregulated in OA, so limiting its expression also reduces levels of MMP13 , COL10A1 , and VEGF , indicating hypertrophic differentiation and cartilage damage [ 9 , 139 ].

Notch signaling

Notch signaling pathways, highly conserved in mammals, require careful regulation for maintenance of healthy cartilage. Downstream, Notch upregulates expression of MMP13 while downregulating the synthesis of type II collagen. Therefore, Notch signaling is chondrodestructive in nature. This mechanism was demonstrated by an experimental mouse model in which one group received treatment with a γ-secretase inhibitor, an inhibitor of Notch, while the other group did not. In the group that did not receive the γ-secretase inhibitor, the mice exhibited greater severity of articular cartilage degeneration due to upregulated MMP13 and decreased synthesis of type II collagen [ 141 ]. Estrogen has been shown to upregulate Notch1, indicating a connection between female sex hormones and a chondrodestructive signaling pathway [ 142 ]. Further exploration of the relationship between biological sex and cartilage-specific Notch signaling is needed to better determine the specific mechanisms and interactions, as four types of Notch proteins exist, each with unique functions.

Fibroblast growth factor

FGFs are important growth factors involved in the development and maintenance of articular cartilage. FGF18 serves an anabolic role in cartilage, acting as an inducer of chondrocyte proliferation and ECM synthesis. In a murine model, injection of FGF18 in OA joints resulted in increased cartilage formation [ 143 ]. Unlike FGF18, the exact role of FGF2 in cartilage maintenance is unclear; FGF2 seems to serve a combination of anabolic and catabolic functions. In the presence of existing cartilage defects, FGF2 was found to have a regenerative effect much like FGF18 [ 144 ]. However, while FGF2 effectively induced proliferation in these studies, it failed to induce ECM production [ 145 ]. This growth factor was also found to upregulate several metalloproteinases, such as MMP13, ADAMTS4, and ADAMTS5, leading to the stimulation of pro-inflammatory cytokines and the inhibition of anabolic molecules, such as BMP7 [ 9 ]. Since BMP7 is typically expressed at higher concentrations in males, male cartilage may be better equipped to override the inhibitory effects of FGF2 [ 137 ]. While limited data exist regarding the relationship between sex hormones and FGFs in cartilage, a study of bovine ovarian tissue concluded that FGF18 decreased the synthesis of estrogen and progesterone production, indicating a negative correlation between female sex hormones and FGF18 in female reproductive organs [ 146 ]. Whether this effect is limited to ovarian tissue warrants further exploration.

Retinoic acid

Retinoic acid is well-known to influence chondrocyte-specific gene expression through interaction with retinoic acid receptors (RARs) in the nucleus [ 147 ]. Vitamin A metabolites act as ligands for RARs and are found to be elevated in OA patients [ 148 ]. As observed in a recent in vitro study, activated RARs have an inhibitory effect on chondrogenesis, especially RARα and RARγ [ 147 ]. Specificity protein 1 is a transcription factor present in chondrocytes that can form complexes with ERs, inducing the activation of RARα [ 125 ]. Therefore, the presence of ERs in chondrocytes directly relates to inhibition of chondrogenesis [ 125 ].

Cellular energy and survival related pathway

E2 loss correlates with the increased incidence of knee and hip OA. Interestingly, in rat OA model chondrocytes, E2 mediated activation of PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B) markedly stimulates cell proliferation [ 149 ]. Through this signaling, administration of E2 also elevates proliferation and viability of ATDC5 chondrocytes [ 150 ]. High expression levels of GPER1/GPR30 in the hypertrophic zone have been indicated as regulating longitudinal bone growth as the expression decreases during puberty [ 151 ]. GPER1/GPR30 also plays a role in chondrocyte proliferation and has been demonstrated as required for a normal estrogenic response in the growth plate [ 152 ].

Typically involved in ECM synthesis, the PI3K/AKT pathway is downregulated in human OA cartilage or in OA-like chondrocytes exposed to IL1 or TNFα [ 153 , 154 ]. Chondrocyte apoptosis is negatively regulated by PI3K/AKT signaling through inhibition of NF-κB [ 155 ]. 17β-E2 could function through the PI3K/AKT/FOXO3 (forkhead box O-3) pathway by downregulating MMP3 expression and preventing ECM degradation [ 156 ]. A key suppressor of autophagy, the mTOR pathway is regulated through upstream PI3K/AKT and AMPK (adenosine 5′-monophosphate-activated protein kinase) pathways [ 157 ]. In OA cartilage, upregulated mTOR signaling has been shown to lead to increased chondrocyte apoptosis and decreased autophagy-related gene expression; as such, cartilage-knockdown of mTOR reduces apoptosis and upregulates autophagy, shifting cartilage homeostasis in mice [ 158 ]. Furthermore, mTOR-regulated autophagy is linked to the inflammatory response, serving as an integral link between inflammation and autophagy in OA pathogenesis.

AMPK is critical to maintaining cell energy metabolism and survival. Given their reciprocal enhancement of each other’s activity, silencing information regulator 2 related enzyme 1 (SIRT1) and AMPK interact closely to regulate energy, metabolism, and aging [ 159 ]. In OA chondrocytes, pharmacologic AMPK activation not only improved mitochondrial biogenesis and function but also stimulated SIRT1-peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC1α) signaling, ultimately delaying chondrocyte aging [ 160 ]. One study showed that SIRT1 and mTOR play a role in regulating cell aging by adjusting the autophagy function [ 161 ]. Specifically, SIRT1 restored oxidative stress-induced autophagy impairment and improved embryonic stem cell (ESC) survival by blocking the mTOR pathway [ 162 ]. In contrast, SIRT1 inhibition activates the mTOR pathway, resulting in autophagy injury [ 163 ]. In a mouse OA model, E2 inhibited the mTOR pathway via activating ERK, promoting chondrocyte autophagy to protect AMPK mutant mice from OA [ 164 ]. In vitro , E2 given to ATDC5 chondrocytes at a pharmacological concentration is capable of inducing SIRT1 expression through the AMPK/mTOR pathway in mitophagy [ 165 ]. These data culminated in a new appreciation for 17β-E2 signaling in OA and point to the SIRT1-mediated AMPK/mTOR signaling pathway as a potential target for OA therapy.

Acidic environment and cellular inflammation related pathways

In vivo, there is increased expression of ASIC1a co-localized with NF-κB expression in articular cartilage of rat adjuvant arthritis [ 166 ]. NF-κB signaling pathways are activated by IL6, IL1β, and TNFα signaling, leading to ASIC1a upregulation. Extracellular acid activation of ASIC1a could exacerbate the TNFα- and IL1β-mediated impact on ECM metabolism by increasing MMP3 / MMP13 and ADAMTS5 mRNA expression in articular chondrocytes [ 166 ]. Extracellular acidification also increases intracellular Ca 2+ influx, culminating in articular chondrocyte apoptosis, autophagy, pyroptosis, and necroptosis [ 167 ].

17β-E2 can increase the mRNA and protein expression levels of ERRα, which in turn led to an increase in SOX9 , GDF5 , and CYP19A1 during in vitro mandibular condylar chondrocyte cultivation [ 55 ]. Additionally, estrogen treatment activates the AMPK/ULK1 (unc-51-like kinase 1) signaling pathway, which was abrogated by ERRα-silencing. 17β-E2 has been linked to ASIC1a protein degradation through the ERRα, resulting in protection against acidosis-induced cytotoxicity in chondrocytes [ 80 ]. The estrogen-mediated downregulation of ASIC1a expression was mitigated by methyl-piperidino-pyrazole, an inhibitor of ERRα, suggestive of the involvement of ERRα in the estrogen regulating expression of ASIC1a. AMPK–ULK1 signaling activation promotes protein degradation of ASIC1a by the autophagy–lysosome pathway [ 168 ]. DHEA reduced COX2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) gene expressions [ 169 ]. These findings all point to the role of estrogen in promoting the autophagy–lysosome pathway-dependent degradation of ASIC1a and protecting against acidosis-induced cytotoxicity, which is thought to be influenced by the ERRα–AMPK–ULK1 signaling pathway [ 168 ].

MicroRNA related signaling pathway

In the postmenopausal rat model, miR-203 has been suggested to have critical involvement in OA onset and worsening, indicating that inhibiting the miRNA may reduce cartilage degradation [ 170 ]. MiR-203 has been shown to increase cellular inflammation and injury. IL1β stimulation increased miR-203 expression, which inhibited ERα expression and decreased ACAN and COL2A1 [ 171 ]. This finding indicates that miR-203 is especially critical in estrogen deficiency and ERα instability-induced OA [ 171 ]. Interestingly, estrogen treatment increased miR-140 level and suppressed MMP13 expression in human articular chondrocytes; miR-140 knockdown mitigated this inhibitory influence of estrogen; further, the estrogen/ER/miR-140 pathway inhibited IL1β-induced cartilage matrix degradation [ 76 ]. In idiopathic condylar resorption (ICR), an aggressive form of OA in adolescent female patients, the E2-miRNA-101-3p–HAS2 pathway has been considered important. It has been theorized that E2 targets miRNA-101-3p in synovial fibroblasts of ICR patients, which regulates HAS2 expression [ 172 ]. The connection between estrogen and miRNAs presents a sex-related mechanism that could provide a more specific treatment approach for OA.

Regeneration and prevention

While sexual dimorphism has been well-documented in cartilage degeneration, cartilage regeneration may also exhibit variations between males and females. Sex-dependent differences in stem cell regenerative capacity must be considered when evaluating the effectiveness of OA treatment options. This section will review current findings on how sex influences the outcomes of cartilage regeneration therapy.

Surgical methods

Currently, the most common form of cartilage repair is surgical intervention. While there are advantages to utilizing surgical methods, such as microfracture to treat cartilage defects, these practices are invasive and often lead to poor clinical responses, such as fibrocartilage formation [ 173 ]. Interestingly, current responses to surgical techniques for cartilage repair exhibit observable sex differences; however, further investigation is still needed to best apply this information in practice.

Microfracture is a procedure in which holes are drilled into areas of a cartilage defect in an attempt to circulate the healthy MSCs from deep within the cartilage to the surface, leading to cartilage regeneration [ 174 ]. While this procedure is considered less invasive than traditional surgical implantation, it can only be performed for small defects and thus has limited applicability [ 174 ]. Notably, microfracture does have some success in alleviating chondral degradation [ 174 ]. Males tend to have better microfracture outcomes than females, scoring higher on assessments for symptom relief and functional improvement [ 175 ]. Compared to female patients, more males also undergo microfracture treatments, along with chondroplasty and osteochondral allografts [ 176 ].

Autologous chondrocyte implantation (ACI) is a technique that harvests chondrocytes from a patient’s intact articular cartilage to be implanted at the patient’s site of defect or degeneration. This procedure is not ideal, as it disturbs healthy cartilage and may lead to donor site morbidity. However, some positive outcomes have been recorded, including pain reduction, tissue repair, and improvement of function [ 177 ]. Studies show that males typically display better responses to ACI treatment than females [ 177 , 178 , 179 ]. Compared to males, females who underwent ACI were found to have a higher rate of postoperative revision or arthroplasty [ 178 , 180 ]. In a study that followed patients after ACI surgery, results revealed that defect location affects regeneration in females but not in males; males with defects on the femoral condyles had much better postoperative results than females with defects in the patellofemoral compartment [ 181 ]. Despite the documented sex differences in procedure results, there is still dispute regarding the significance of these disparities. Several studies suggest a lack of significant difference in ACI outcomes between males and females [ 182 , 183 ]. These studies indicate that ACI has the potential to benefit both sexes indiscriminately and that sex does not influence the success of ACI [ 184 ].

Autologous matrix-induced chondrogenesis (AMIC) is a cartilage defect treatment that combines techniques of both ACI and microfracture, inserting a collagen scaffold and fibrin glue over the hole produced by microfracture [ 185 ]. This sort of treatment results in better cartilage regeneration for males than females [ 185 ]. However, recent studies reported conflicting results in which no significant differences in postsurgical prognosis were found between sexes, suggesting that both males and females have the same potential for successful AMIC outcomes [ 186 , 187 ]. Since females tend to have worse preliminary defects, sex-related repair outcomes may falsely appear to have observable differences [ 184 ]. However, when compared over a long-term postoperative period, males and females exhibit similar levels of healing, especially when comparing younger populations [ 184 ].

Sex hormone treatment

Since cartilage degeneration is closely linked to hormonal influences, as previously mentioned, sex hormone therapy has the potential to help prevent degeneration and enhance regeneration (Table 2 ). Studies have shown that estrogen and estrogen receptors may play a functional role in chondrocytes [ 188 , 189 ]. ERs on female chondrocytes have higher estrogen affinity than those on male chondrocytes [ 190 ]. It is theorized that this observation may contribute to the increased female incidence of OA compared to males. Levels of estrogen decrease after menopause, and accordingly, OA becomes more prominent in postmenopausal females. As such, modulation of ERs may help treat collagen degradation [ 6 ].

Kinney et al. found that chondrocytes from healthy females respond to E2 treatment; however, a lack of response to E2 is observed in males [ 190 ]. Another study done on a murine model similarly confirmed estrogen’s chondroprotective effect in female mice but not in male mice [ 111 ]. Oral and transdermal E2 treatment resulted in a decrease in levels of urine CTX-I and CTX-II in women; since urine CTX is a product of cartilage degradation, this finding supports estrogen therapy as a promising treatment for women in the field of cartilage repair and regeneration [ 191 ]. Females with low serum estrogen concentrations are more likely to develop OA [ 192 ]. Therefore, menopausal women have a higher risk of cartilage degeneration because of estrogen decline [ 6 ]. In postmenopausal women, ERT seems to have a benefit in increasing cartilage thickness [ 193 ]. In an in vivo study, postmenopausal women having taken ERT for more than 5 years were observed to have more articular tibial cartilage than women without hormone replacement [ 193 ]. The Framingham Study also observed preventative and protective results against cartilage degradation in postmenopausal women on ERT; participants on ERT experienced less OA progression compared to the control, indicating cartilage protection resulted from estrogen supplementation [ 194 ]. ERT was also found to increase expression of IGF binding protein 2, proteoglycans, and collagen in articular cartilage, demonstrating some of the chondroprotective mechanisms of this therapy [ 189 ]. However, risks of hormone replacement may outweigh the benefits [ 195 ]. Therefore, additional therapies should continue to be considered for OA in postmenopausal women.

Sex steroids may help enhance the chondrogenic potential of human chondrogenic progenitor cells (CPCs) in a sex-dependent manner, improving the regeneration capacity of late-stage OA tissue [ 91 ]. Koelling and Miosge found that SOX9 was highly expressed in the CPCs from women treated with E2 and CPCs from men treated with testosterone; CPCs from women responded better to steroid treatment, which may be explained by the higher concentration of sex hormone receptors in females than males [ 91 ].

Unlike the strong body of research on ERT, not as much information exists on sex variation regarding testosterone replacement therapy. One study assessed the association between OA pain progression and total serum testosterone levels among patients who had undergone total knee replacement surgery and found that both sexes reported less pain with higher testosterone levels; interestingly, all females that reported less disability had higher levels of serum testosterone [ 196 ]. These findings suggest that testosterone levels are positively correlated with OA improvement in both sexes. However, a study led by Alessandro Bertolo concluded that male IVD cells exposed to testosterone experience an increase in expression of aggrecan and types I and II collagen, while the female cells experience no effect, suggesting that testosterone only influences chondrogenesis for male cells [ 92 ]. Another in vitro experiment in which male and female rat chondrocytes were treated with testosterone yielded similar results; testosterone treatment increased collagen production in male cells but not in female cells [ 197 ]. Current evidence points to testosterone being positively associated with cartilage health in males, but its role in females is less certain. More research investigating the potential of testosterone treatment is needed to fully understand if it can play a meaningful role in cartilage repair.

An alternative approach to sex hormone therapy is using isoflavone, a soy phytoestrogen from legumes. Soybean isoflovane can have estrogenic effects on tissues [ 198 ]. In OVX rats, soybean isoflavone has been observed to limit cartilage degeneration [ 199 ]. Another study observed arctigenin, a dietary phytoestrogen, worked as a cartilage protector in human chondrocytes and mouse OA models [ 200 ]. Similarly, another phytoestrogen, daidzein, had anti-inflammatory and anti-oxidant effects in a rat OA model, especially when in combination with hyaluronic acid therapy [ 201 ]. In human chondrocytes, daidzein has had comparable results, having positive effects on ECM formation and pheotype regulation [ 202 ]. These studies demonstrate using phytoestrogen as a therapy method could be a promising approach to treat OA.

• Stem cell therapy

Stem cell-based tissue engineering is a promising approach for repairing adult cartilage defects. Specifically, MSCs are at the forefront amongst stem cells for cartilage regeneration and repair given their high proliferation rate and chondrogenic potential [ 1 ]. MSCs can be found in different areas of the body, such as bone marrow, adipose tissue, synovium, and muscle, and can be differentiated into chondrocytes and other types of cells (Fig.  2 ) [ 203 ]. MSCs display sex differences in proliferation and differentiation [ 204 ], indicating that stem cell therapy can be enhanced by taking these differences into account.

Origin and differentiation of MSCs including ASCs, MDSCs, SDSCs, and BMSCs. Upper hemisphere shows MSC origin and lower hemisphere shows MSC differentiation

Optimization of stem cell therapy

Given the integral role sex hormones play in cartilage maintenance and regulation, testosterone, DHT, and E2 have the ability to influence stem cell proliferation and differentiation and may be used to optimize stem cell therapy as a cartilage regeneration method. Studies on the influence of testosterone on stem cell proliferation have mixed results, indicating a need for further experimentation [ 205 ]. In a castration study, elimination of testosterone suppressed DNA synthesis of male bone-marrow-derived stem cells (BMSCs), implying testosterone has the capacity to stimulate proliferation [ 206 ]. However, testosterone was found to have the opposite effect in vitro , limiting the cell count of male chondrocytes [ 197 ]. In contrast, a study on ESCs concluded that testosterone had neither a positive nor negative influence on proliferation [ 207 ]. These mixed results indicate that cell type is a probable factor in the influence of testosterone on cell expansion. The same study also suggests that DHT is a more influential hormone on proliferation than testosterone, as DHT exposure demonstrated marginal inhibitory effects on stem cell proliferation [ 207 ]. The impact of testosterone on differentiation is more clearly established, with both in vivo and in vitro experiments concluding that testosterone exposure helps induce chondrogenic differentiation of BMSCs [ 197 ]. Furthermore, testosterone has been observed to increase ECM deposition in male IVD cells during differentiation, further supporting its role in promoting chondrogenesis [ 92 ]. While testosterone may stimulate chondrogenesis, sex of the stem cell donor must be considered, as testosterone likely has greater capacity to influence cells derived from male donors [ 197 ]. Further evaluation is needed to conclusively determine the impacts of androgens on proliferation and differentiation, but DHT may encourage proliferation, while testosterone promotes chondrogenic differentiation of BMSCs.

Contrary to male sex hormones, female sex hormones have remarkable evidence supporting their effects on stem cells, overall promoting proliferation and inhibiting differentiation. An in vitro study concluded that E2 promotes proliferation of ESCs by increasing cyclin D1, cyclin E, cyclin dependent kinase 4 (CDK4), and CDK2, therefore, promoting cell cycle progression [ 208 ]. Like ESCs, the proliferation rate of other stem cell types is also promoted by estrogen. The proliferation of BMSCs is mediated by estrogen in a concentration-dependent and sex-dependent manner [ 209 ]. Peak proliferation rate was produced by exposure of E2 at 10 –12  M in female BMSCs and between 10 –8 and 10 –12  M in male BMSCs [ 209 , 210 ]. Mouse adipose-derived stem cells (ASCs) also respond to E2 treatment by increasing proliferation rate through ERα [ 211 ]. In vivo, both BMSCs and ASCs isolated from female donors divide more rapidly compared to those from male donors, indicating that the effect of estrogen on proliferation is greater than that of testosterone at physiologic concentrations [ 212 , 213 ]. Since menopause reduces estrogen production, resulting decreases in proliferative capacity of BMSCs contributes to the rise in incidences of OA and osteoporosis [ 214 ]. Estrogen hormone therapies may be considered for treatment options, but only for females, as only chondrocytes from female donors respond to E2 [ 190 ]. While estrogen stimulates stem cell proliferation, it has the opposite effect on differentiation. In vitro studies concluded that E2 has an inhibitory effect on chondrogenic differentiation of both ASCs and BMSCs [ 215 , 216 ].

Similar to endogenous hormones, exogenous factors such as dietary metabolites also influence properties of stem cell growth and may be used to optimize stem cell therapies. Metabolites of Vitamin D impact differentiation, with 24,25-(OH)2D3 increasing the size of the hypertrophic zone of cartilage at the growth plate in both sexes [ 217 ]. 1-Alpha,25(OH)2D3 was found to induce expression of E2 in female, but not male, chondrocytes, which may be partially responsible for the chondroprotective role of Vitamin D in females [ 32 ]. While Vitamin D is generally chondroprotective in both sexes, estrogen promotes Vitamin D accumulation and upregulation of Vitamin D receptors, indicating it may have a greater impact in females [ 218 ]. Another metabolite impacting chondrogenic differentiation and proliferation is maternal β-hydroxy-β-methyl butyrate (HMB), a product of leucine metabolism. In vivo, prenatal HMB treatment of pregnant sows increased proteoglycan content in articular cartilage, especially in female offspring [ 219 ]. HMB treatment also increased IGF-I concentration, proliferation, and survival in both sexes of offspring. In HMB-pretreated female offspring, the temperature at which collagen denatured was observed to be significantly higher compared to that in the control group, suggesting that prenatal HMB treatment may yield female offspring with stronger, more resistant cartilage [ 219 ]. Male cartilage has a higher abundance of cross-linking proteins, such as lysyl oxidase-like protein 2 and fibulins, which likely contributes to the greater impact of HMB treatment on female offspring compared to males [ 220 ].

Other treatments yield sex-specific responses in stem cells as well, such as exposure to hypoxia and ROS. After undergoing lipopolysaccharide dose treatment followed by hypoxia treatment for 1 h, female-derived MSCs consistently produced more VEGF than male-derived cells and also exhibited a decrease in TNFα expression, while male donor cells did not [ 221 ]. These findings further support the notion that male and female stem cell properties differ and can be enhanced with more specialized cultivation techniques to advance stem cell therapy.

Sex differences in stem cell therapy

Currently, few studies distinctly focus on the potential differences between sexes in MSC cartilage regeneration. This review aims to summarize the overall sex-dependent trends and implications they have for future clinical applications. Human BMSCs have been identified as a good source for articular cartilage repair because of their great chondrogenic capacity [ 222 ]. Increasing research supports that human BMSCs display sexual dimorphisms in regenerative ability. Specifically, studies have pointed to male-derived BMSCs exhibiting greater chondrogenic potential than female cells [ 223 ]; male BMSCs decrease in chondrogenic potential with age, while female BMSCs do not [ 224 ]. However, a recent study found no substantial differences in adipogenic and chondrogenic differentiation potential of human BMSCs from healthy male donors as compared to healthy female donors [ 225 ].

Another promising cell candidate for stem cell-based therapy is muscle-derived stem cells (MDSCs). MDSCs display good chondrogenic potential and cartilage regeneration properties [ 226 ]. Like BMSCs, this cell type also exhibits variation in regenerative ability between male and female donors. An in vitro study revealed that human and murine MDSCs from male donors might have a greater chondrogenic potential than that of female donors [ 227 ]. Sex not only influences MDSC proliferation and rejuvenation potential, but also that of ASCs; ASCs from male donors also demonstrate greater chondrogenic potential compared to female donors [ 223 , 228 ]. Overall, current studies point to donor sex as a major determinant of stem cell properties, conclusively establishing the need to disclose donor sex in all future studies. With this measure in place, sexual dimorphisms of stem cells will not be a confounding variable.

Interestingly, the sex of the treatment recipient has also been shown to influence treatment response. Some experiments have demonstrated that males respond better to stem cell treatment. For example, an in vivo experiment implanted BMSCs into human subjects with articular cartilage defects in the knee and found that outcomes were sex-dependent. Results revealed significant differences between sexes up to 10 years postoperatively, with males experiencing greater response to the method in terms of physical ability, repair, and healing after implantation [ 177 , 179 ]. This finding may relate to the high incidence of OA in postmenopausal women with low estrogen levels which is linked to increased OA susceptibility and may hinder their ability to respond to treatment. Women also tend to develop worse cases of OA compared to men, which could also contribute to decreased responses to treatment [ 184 ].

Discussion/conclusion

This review aims to highlight and summarize the sex differences in the field of cartilage repair and regeneration, specifically in regard to cartilage degeneration treatment with stem cell-based therapy. It is well-known that women have a higher likelihood of developing OA compared to men, but the mechanisms underlying this clinical finding are still being investigated. A decrease in estrogen following menopause seems to be an underlying trend in women with OA, leading to the conclusion that estrogen is a big factor in cartilage degradation and can be supplemented for treatment and prevention of OA in women. However, men may also develop cartilage degeneration. Though males do not respond to estrogen treatment like women, males have responded to testosterone treatment, indicating that androgens also play a role in OA. These distinctions in sex-based response to hormone therapies indicate the presence of variation in treatment options between sexes.

Biomarkers also are integral to the prevention of cartilage degeneration. Increasing evidence points to sex-dependent differences between cartilage biomarkers, suggesting that signs of OA in males and females present differently. Understanding the sex differences between biomarkers may lead to earlier diagnosis and the development of sex-specific treatment options.

In regard to stem cell therapies, the role of donor sex in MSC-based treatments is still being investigated. Currently, there is contradictory evidence, meaning further experimentation must be done to accurately determine the influence of donor sex on the regenerative potential of MSCs. Some studies conclude that male donors have healthier MSCs with more chondrogenic potential, making them more ideal candidates for stem cell treatment. However, other studies found no significant difference in the quality of MSCs between healthy male and healthy female donors.

The impact of recipient sex on cartilage repair method efficacy is also up for debate. In the case of BMSCs for stem cell treatment, males seem to experience better postoperative results than females. The difference in male and female responses to receiving stem cell treatment has not been extensively studied and needs more research. ACI treatment also seems to benefit males more, with females having less successful post-ACI outcomes. However, this finding may be skewed by the increased likelihood of women to have more severe cartilage defects than men going into these procedures, as there are some studies that show a lack of significant differences in the outcomes of surgical treatments.

This study focuses on sex differences at the molecular level and the implications on cartilage health and therapeutic strategies. Obesity and ethnicity factors in sex-related differences are not heavily discussed as they can be influenced by lifestyle and economic elements. However, there is merit for studying these factors, and future studies should focus on such elements to truly optimize clinical strategies for cartilage health.

In conclusion, there are clear sex-based variances in cartilage degeneration and regeneration, and the underlying mechanisms and exact effects still need further exploration. Additional research is critical to fully understand the role of sex in cartilage repair. Currently, most studies do not adjust their designs for sex variability and comparisons, resulting in a lack of statistical evidence on the influence of sex [ 2 ]. By fully understanding the sex differences in cartilage degeneration and regeneration, future studies can more intentionally select a target demographic. The development of sex-specific approaches to cartilage tissue engineering aims to provide more personalized and effective clinical treatments.

Availability of data and materials

Not applicable.

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Acknowledgements

The authors thank Suzanne Danley for editing the manuscript.

This work was supported by Research Grants from the National Institutes of Health (1R01AR067747 and 1R56AR078846) to M.P., and Health Commission of Sichuan Province (18PJ008), General project of The General Hospital of Western Theater Command (2021-XZYG-B07), and Science & Technology Department of Sichuan Province (2019YFS0267) to S.C.

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Authors and affiliations.

Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, 64 Medical Center Drive, PO Box 9196, Morgantown, WV, 26506-9196, USA

Jhanvee Patel, Torey Katzmeyer, Yixuan Amy Pei & Ming Pei

Department of Orthopaedics, The General Hospital of Western Theater Command, Chengdu, 610083, Sichuan, China

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

Yixuan Amy Pei

WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, 26506, USA

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Patel, J., Chen, S., Katzmeyer, T. et al. Sex-dependent variation in cartilage adaptation: from degeneration to regeneration. Biol Sex Differ 14 , 17 (2023). https://doi.org/10.1186/s13293-023-00500-3

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DOI : https://doi.org/10.1186/s13293-023-00500-3

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• J Exp Orthop
• v.9; 2022 Dec

Current concepts and perspectives for articular cartilage regeneration

Livia roseti.

IRCCS Istituto Ortopedico Rizzoli Bologna, Bologna, Italy

Brunella Grigolo

Articular cartilage injuries are common in the population. The increment in the elderly people and active life results in an increasing demand for new technologies and good outcomes to satisfy longer and healthier life expectancies. However, because of cartilage's low regenerative capacity, finding an efficacious treatment is still challenging for orthopedics.

Since the pioneering studies based on autologous cell transplantation, regenerative medicine has opened new approaches for cartilage lesion treatment.

Tissue engineering combines cells, biomaterials, and biological factors to regenerate damaged tissues, overcoming conventional therapeutic strategies. Cells synthesize matrix structural components, maintain tissue homeostasis by modulating metabolic, inflammatory, and immunologic pathways. Scaffolds are well acknowledged by clinicians in regenerative applications since they provide the appropriate environment for cells, can be easily implanted, reduce surgical morbidity, allow enhanced cell proliferation, maturation, and an efficient and complete integration with surrounding articular cartilage. Growth factors are molecules that facilitate tissue healing and regeneration by stimulating cell signal pathways.

To date, different cell sources and a wide range of natural and synthetic scaffolds have been used both in pre-clinical and clinical studies with the aim to find the suitable solution for recapitulating cartilage microenvironment and inducing the formation of a new tissue with the biochemical and mechanical properties of the native one. Here, we describe the current concepts for articular cartilage regeneration, highlighting the key actors of this process trying to identify the best perspectives.

Introduction

Articular cartilage covers the bony ends of diarthrodial joints; it is a smooth thin hyaline tissue with friction-reducing and load-bearing functions. It lacks blood vessels, lymphatics, and nerves [ 1 ]. As for connective tissues, an extracellular matrix (ECM) composed of water, collagens, and proteoglycans surrounds the cells. Chondrocytes are mature secretory scarcely distributed cells located in spaces termed lacunae. They are spread among superficial, middle, deep, and calcified four zones. Those zones display different cell shapes, secretory patterns, and fiber orientation [ 2 ]. The layer of bone below the hyaline cartilage is known as subchondral bone. It has a structural and mechanical function (shock absorber) and can be involved in the etiology or effects of cartilage damages or diseases [ 3 ].

Chondral (affecting articular cartilage) and osteochondral (affecting cartilage and the underlying bone) lesions are very common and can appear at any age [ 4 , 5 ]. In the young population, they are most of traumatic origin (sport or accident), often with ligament and meniscal injuries. Some conditions like osteochondritis dissecans may lead to articular surface disruption and release of intra-articular bodies composed of cartilage or cartilage and bones [ 6 ]. In the elderly population, lesions are associated with rheumatic diseases (i.e., Osteoarthritis-OA) or derived from wear and tear due to excessive use (occupational injury) or age [ 7 , 8 ].

In general, repair of full-thickness cartilage lesions mainly depend on the patient’s age, defect size, and location. Small full-thickness defects may adjust through the formation of hyaline cartilage. In contrast, large osteochondral defects repair by forming a scar, fibrous tissue, or fibrocartilage in which the predominant component is collagen type I [ 2 , 9 ].

This kind of cartilage could be functionally active for a short period, but the tissue does not present the mechanical and strength characteristics of normal cartilage, thus not ensuring the healing of the defect nor the symptom remission. This situation could favor, over time, the development of OA [ 10 ].

Conventional cartilage treating procedures are palliative and reparative. Palliative strategies usually represent the first-line treatment to decrease symptoms without addressing the causes. Reparative approaches are abrasions drilling and microfracture. They include a subchondral bone penetration allowing stem cells to migrate from bone marrow to the injury site and form, as explained before, mostly a tissue with fibrous features.

Regenerative methods have emerged as an alternative to tissue repair or replacement to regrow or restore diseased cells, tissues, or organs. Osteochondral grafting represents a possible solution for creating a hyaline-like tissue in the affected area. However, it has shown some drawbacks like donor site morbidity, graft failure (autograft), or possible disease transmission (allograft) [ 11 ]. Lately, different therapeutic solutions in cartilage regenerative medicine have emerged.

With this manuscript, we would like to report the main therapeutic strategies in this field, focusing on recent approaches in tissue engineering and particularly on up-to-date knowledge’s on cells, growth factors, and scaffolds.

Tissue engineering

Tissue engineering has represented a new fascinating, and innovative approach to regenerating articular tissue [ 12 ]. As reported above, this therapeutic strategy involves the use of cells, scaffolds, and growth factors (GF), trying to mimic the complex three-dimensional microenvironment of the joint that requires the interaction between these different components. Reproducing such complexity is critical, and many issues are still open concerning the ideal cell population, the use of GF and the suitable scaffolds [ 1 , 13 ].

Cells can be administered as therapeutic agents to rebuild damaged cartilage in joints. The leading cell types used in treating chondral and osteochondral defects are chondrocytes and mesenchymal stromal cells from various sources [ 14 ]. The technique requires that cells are isolated and then expanded ex vivo in a monolayer culture before the implant. In terms of legislation, expanded cells belong to Advanced Therapy Medicinal Products (ATMPs) and must follow specific rules already encoded for conventional drugs and known as Good Manufacturing Practices (GMPs). GMPs entail the standardization and control of medicinal manufacturing, ensuring their safety and reducing contaminations [ 15 ].

Autologous chondrocyte implantation

Autologous Chondrocyte Implantation (ACI) is a two-step procedure that has been used in the clinic for many years [ 16 ]. In the original ACI technique (first-generation technique), the first step consisted of surgically removing small biopsies of normal cartilage from non-weight-bearing areas of the knee. Chondrocytes were then enzymatically isolated from the biopsies, expanded ex vivo in monolayer culture condition, and, after several weeks, harvested as a cell suspension. In the second step, surgeons injected the cell suspension under a periosteal flap harvested from the proximal medial tibia and previously sutured over the cartilage. Chondrocyte expansion was deemed necessary due to cartilage cell scarcity [ 17 ].

ACI that uses suspended cultured chondrocytes with a covering of collagen type I/III membrane is considered a second-generation. Third-generation ACI comprises those procedures that deliver autologous cultured chondrocytes using cell carriers or cell-seeded scaffolds. These second and third generation modifications are also known as autologous chondrocyte implantation using collagen membrane (C-ACI), membrane-associated autologous chondrocyte implantation (MACI), and scaffold-based ACI [ 13 , 18 ]. These procedures were introduced in the clinical practice one decade ago, showing similar results while at the same time overcoming most of the concerns related to the first-generation ACI. The use of scaffolds to create a cartilage-like tissue in a three-dimensional culture system allows for the optimization of the procedure from both the biological and surgical points of view [ 19 , 20 ].

Although good clinical radiological and histological outcomes of the different ACI procedures, one of the main drawbacks is the need for a cell expansion phase which is long-lasting, complicated, and expensive primarily due to GMPs requirements. Moreover, the need for two hospitalizations increases the risk for the patients and the costs for the public health system. For all these reasons, investigations have been moving towards different cell populations as reported below [ 9 , 21 , 22 ].

Mesenchymal stromal cells (MSC)

Stromal cells from various sources are currently available for cartilage regeneration. This is due to their ability to proliferate in culture and directionally differentiate by synthesizing structural and functional hyaline ECM molecules. Moreover, they can release many anti-inflammatory, anti-apoptotic, and immuno-modulatory factors favoring the healing process [ 23 ]. Many studies have reported benefits in treating cartilage injuries with adult bone marrow-derived MSC [ 23 ]. Adipose-derived stem cells (ASC) have also drawn attention for their analogy with bone marrow ones, but with easier harvesting, a higher cell density, and proliferation. Other sources of stem cells investigated for cartilage repair include muscle, synovial membrane, trabecular bone, dermis, blood, umbilical cord blood, and periosteum [ 23 ]. Although various successful applications in cartilage regeneration, several problems remain, like stem cell heterogeneity and premature differentiation during in vitro expansion [ 24 ].

Induced pluripotent stem cells (iPSCs) have a promising potential for cartilage regeneration. Besides, they allow overcoming limitations associated with current cell sources since large numbers of cells can derive from small starting populations. However, issues related to genomic modifications still need addressing [ 25 ].

Genetically modified cells showed the ability to potentiate cartilage regeneration. Transfected genes inducing chondrogenic differentiation, synthesis of a hyaline matrix, and release of pro-inflammatory factors differentiation are feasible. Gene transfection may be systemic or local, ex vivo or in vivo. Because cartilage injuries are not life-threatening, it is critical to ensure a safe procedure [ 26 ].

MSC, as a pure cell population, require the selective elimination of cells that do not express their typical markers. Recently, new insight turned into the role of the surrounding MSC microenvironment (or “niche”) that also encloses ECM, accessory cells, adhesion molecules, growth factors, cytokines, and chemokines. Stem cell activity is not only the expression of intrinsic capabilities but also the result of the interactions with the “niche”. It is the whole “niche” that can support the healing process. No cell selection and expansion in the laboratory are necessary, and a single operative procedure is effective [ 27 – 29 ].

In recent years, also articular cartilage regeneration research moved towards the use of the stem cell “niche” in the form of concentrates such as Bone Marrow Concentrate (BMC) and Stromal Vascular Fraction (SVF) from adipose tissue. Both concentrates are obtained with minimal manipulation (no expansion in culture) and provide a less invasive (one step-surgery) and less expensive (no GMPs) alternative to cultured cells.

Our laboratory investigated the behavior of BMC cells within a hyaluronan-based scaffold. Histological immunohistochemical and molecular results showed the formation of a cartilage-like ECM [ 30 ]. We also evaluated BMC chondrogenic and osteogenic potential on a bi-layered scaffold mimicking the osteochondral compartment (collagen and hydroxyapatite). The obtained data demonstrated the ability to reproduce the native osteochondral compartment by generating two separated cartilage and bone zones [ 31 , 32 ].

SVF obtained from lipoaspirate contains several cell types like ASCs, ECM fibroblasts, and white and red blood cells. After washing passages, the obtained SVF can be combined with scaffold and soluble factors and administered into the joint. Compared to BMC, SVF ensure easier accessibility and the availability of an increased number of stem cells per gram of tissue [ 33 ].

Cell free products

In the early stages, it seems that the ability of MSC to differentiate into various cell types played the main therapeutic effect. Later, it emerged that their capacity to release some GF and chemokines play a role (secretome). MSCs secrete bioactive molecules inhibiting apoptosis and the formation of fibrosis or scarring at the injury site; stimulate angiogenesis and blood supply, and mitosis of tissue-specific progenitors. They also secrete immunomodulatory agents that deactivate the T cells surveillance and chronic inflammatory processes. Therefore, the secretome use for tissue regeneration increased, based on its composition of trophic factors (chemokines, cytokines, hormones, and lipid mediators) with paracrine effects on the cells of the local microenvironment [ 34 ].

The soluble factors of the secretome can initiate regenerative signaling events also without the use of cells. The therapeutic effect of this biological product in musculoskeletal diseases is a frontier of regenerative medicine. The secretome could overcome the negative aspects of cell use and help concentrate paracrine factors at physiological levels at the injury site.

Although many studies provide strong evidence for the potency of MSC-secreted factors in mediating tissue repair and regeneration, the precise mechanisms of action are still not fully understood [ 35 ].

The paracrine action of MSC is not limited to the production of soluble factors but also of many extracellular vesicles (EVs). EVs [ 36 ] are involved in intercellular communication by releasing mRNAs and proteins. Besides, they have anti-apoptotic, antifibrotic, pro-angiogenic, and anti-inflammatory effects. EVs released from tissue-damaged cells can re-program stem cells' phenotype by releasing specific mRNAs or microRNAs. EVs produced by circulation-recruited or resident MSCs can re-program tissue-damaged cells by inducing de-differentiation, production of soluble paracrine mediators, and initiation of the cell cycle of these cells, thus promoting tissue regeneration [ 37 ].

Growth factors

Biologic agents represent an emerging treatment for several musculo-skeletal pathologies. These agents are mainly represented by natural GF and anti-inflammatory mediators that can accelerate tissue healing and regeneration. They can act through various mechanisms, including matrix synthesis and remodeling, cell recruitment and modulation of inflammatory markers and metalloproteinases.

Moreover, GF may influence protein synthesis and cellular interactions, controlling stem cell differentiation. Bone Morphogenic Protein-2 (BMP-2) regulates osteogenesis, Vascular Endothelial Growth Factor (VEGF) angiogenesis, and Transforming Growth Factor-β1 (TGF-β1) chondrogenesis. The possible role played by GF in pian reduction and tissue regeneration has generated a growing interest in their possible therapeutic use in patients with musculo-skeletal injuries.

Recently, discoveries, combined with knowledge of the importance and role of growth factors for tissue engineering, have been further developed and deepened [ 38 ].GF facilitate and promote the regeneration of new tissues by interaction with specific transmembrane receptors and regulating protein synthesis within cells. Binding to the specific growth factor receptor specifically stimulates cell signal transduction pathways that trigger cell migration, survival, adhesion, proliferation, growth, and differentiation.

Although GF have great potential to stimulate cartilage repair, only a limited number of treatments have been approved by government regulatory agencies for clinical use [ 39 ].

Platelet-rich plasma (PRP) represents an economical source for obtaining many GF in physiological proportions and has already been widely applied in various fields of medicine for its property of promoting tissue regeneration [ 12 , 40 ]. PRP can be defined as a blood derivative product in which platelets are present in high concentration. Platelets have demonstrated regenerative properties because they are rich in important GF.

In particular, α platelet granules contain and release numerous GF including PDGF, TGF-β1, VEGF, Epidermal Growth factor (EGF), Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF).

In recent years, PRP has achieved great success in clinical practice, thanks to its safety and simply preparation technique, which allows exploiting its biologically active content.

PRP has been used successfully in several surgical techniques, obtaining good results in association with microfractures or scaffolds for the treatment of cartilage lesions [ 41 ]. The most significant evidence on PRP is instead for its intra-articular use in the treatment of osteoarthritis, especially in the knee. Despite this, the most suitable type of PRP remains debated, with different preparation methods available that can give products with different composition and properties [ 42 ].

Scaffolds are support sustaining three-dimensional (3D) tissue development. They differ in material composition, structure, and status. An ideal scaffold should be biomimetic, biocompatible, biodegradable, and non-immunogenic; induce cell attachment, growth, and differentiation. Once implanted, it should integrate into the lesion site and support the healing process. It should also be easy to handle by surgeons, and cost-effective Scaffolds for cartilage regeneration may be natural, or synthetic [ 43 ].

Natural materials possess high biocompatibility and bioactivity. However, show poor mechanical stability because of their rapid hydrolysis. A list of the most known natural materials with the principal advantages and disadvantages is reported in Table ​ Table1 1 .

Natural materials for cartilage tissue engineering. Advantages and disadvantages

Synthetic polymers like poly(ethylene glycol) (PEG), polycaprolactone (PCL), polylactic acid (PLA), polyurethane, poly(glycolic acid) (PGA), polyethersulfone (PES), and polysulfone provide cell attachment, and good mechanical, physical, and chemical properties. Moreover, the mechanical properties and degradation time can be controlled by combining them as copolymers or blends. Disadvantages consist of the lack of biological properties and the host organism's side effects in response to metabolite production, mainly concerning acids that can be toxic or induce an inflammatory response [ 43 ].

Hybrid scaffolds, such as a combination of collagen-chitosan- PLA, merge the advantages of synthetic and natural materials, allowing biocompatible membranes with defined mechanical properties and tunable degradation necessary for cartilage regeneration [ 43 ].

Studies highlighted the in vitro and in vivo interaction of cells with scaffolds [ 44 ]. Our group had the opportunity to evaluate some natural scaffolds based on collagen or hyaluronan. We highlighted that scaffold presence allows the re-creation of physiological-like conditions whereby cells interact with the biomaterial and produce a new ECM by the secretion of anabolic, anti-inflammatory, and anti-apoptotic factors [ 45 – 50 ].

A challenge in the design and fabrication of scaffolds is the reproduction of the osteochondral compartment. To this end, composite bilayer or gradient scaffolds mimicking the osteochondral tissue have been developed and evaluated in association with cells. The data obtained demonstrated cell ability to zonally interact and reproduce the native osteochondral compartment by generating separated cartilage- and bone-like zones [ 5 ].

Another challenge is the cell seeding onto the scaffold. Conventional method involves the manual/static or the automated/dynamic seeding of cells onto previously fabricated scaffolds. The static seeding allows an uneven cell distribution into the width of the biomaterial. The dynamic seeding carried out with bioreactors (for instance perfusion) favor a more homogenous cell distribution [ 24 ]. The recent approach of bioprinting foresees that cells and biomaterial are released together in order to produce a construct. Such options allow a better cell encapsulation and spatial distribution [ 51 ].

Future directions

In the next decades we will assist to important steps forwards the repair of articular cartilage lesions. The use of iPSCs and or stem cell derivatives such as secretome, EVs could contribute to improve tissue regeneration.

Emerging technologies like Additive Manufacturing three-dimensional (3D) printing should allow for a further improvement of the treatment. 3D printing replicates the damaged tissue shape starting from a patient medical image. It creates scaffolds through the progressive addition of material layer by layer until reaching the desired shape. The technology can mimic cartilage organization, ECM composition, and functional and mechanical properties [ 52 – 55 ].

Indeed, the identification of the ideal cell population, cell-free products, clinical grade growth factors and customized scaffolds could contribute to ameliorate the technique, reducing the time for surgery and enhancing patient recovery.

Chondral and osteochondral damages remain a tough challenge for clinicians. Tissue engineering-based strategies have proven feasible for cartilage regeneration with good results on patients' quality of life. More research needs to find the best combinations of cells, bioactive factors, and scaffolds. More clinical trials should confirm the obtained results. There is also the demand to develop minimally invasive and cost-effective strategies which do not require long-lasting hospitalization.

Acknowledgements

We acknowledge Italian Ministry of Health for the funds of the Project “Medicina rigenerativa e riparativa personalizzata per le patologie dei tessuti muscolo-scheletrici e la chirurgia ricostruttiva ortopedica" 5x1000 2019 (redditi 2018).

Authors’ contributions

Dr. Roseti wrote the paper and Dr. Grigolo advised and edited. The authors read and approved the final manuscript.

Declarations

The authors declare non conflict of interest.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.