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Cellular therapy and tissue engineering for cartilage repair

  • Author Footnotes
    a Equal author contribution.
    A. Zelinka
    Footnotes
    a Equal author contribution.
    Affiliations
    Lunenfeld Tanenbaum Research Institute, Sinai Health, Dept. Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
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  • Author Footnotes
    a Equal author contribution.
    A.J. Roelofs
    Footnotes
    a Equal author contribution.
    Affiliations
    Arthritis and Regenerative Medicine Laboratory, Aberdeen Centre for Arthritis and Musculoskeletal Health, University of Aberdeen, Aberdeen, UK
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  • R.A. Kandel
    Correspondence
    Address correspondence and reprint requests to: R.A. Kandel, Mt. Sinai Hospital, 600 University Ave, Toronto M5G 1X5, Canada. Tel: 1-416-5868516.
    Affiliations
    Lunenfeld Tanenbaum Research Institute, Sinai Health, Dept. Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
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  • C. De Bari
    Correspondence
    Address correspondence and reprint requests to: C. De Bari, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. Tel: 44-1224-437477.
    Affiliations
    Arthritis and Regenerative Medicine Laboratory, Aberdeen Centre for Arthritis and Musculoskeletal Health, University of Aberdeen, Aberdeen, UK
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  • Author Footnotes
    a Equal author contribution.
Open AccessPublished:September 19, 2022DOI:https://doi.org/10.1016/j.joca.2022.07.012

      Summary

      Articular cartilage (AC) has limited capacity for repair. The first attempt to repair cartilage using tissue engineering was reported in 1977. Since then, cell-based interventions have entered clinical practice in orthopaedics, and several tissue engineering approaches to repair cartilage are in the translational pipeline towards clinical application. Classically, these involve a scaffold, substrate or matrix to provide structure, and cells such as chondrocytes or mesenchymal stromal cells to generate the tissue. We discuss the advantages and drawbacks of the use of various cell types, natural and synthetic scaffolds, multiphasic or gradient-based scaffolds, and self-organizing or self-assembling scaffold-free systems, for the engineering of cartilage constructs. Several challenges persist including achieving zonal tissue organization and integration with the surrounding tissue upon implantation. Approaches to improve cartilage thickness, organization and mechanical properties include mechanical stimulation, culture under hypoxic conditions, and stimulation with growth factors or other macromolecules. In addition, advanced technologies such as bioreactors, biosensors and 3D bioprinting are actively being explored. Understanding the underlying mechanisms of action of cell therapy and tissue engineering approaches will help improve and refine therapy development. Finally, we discuss recent studies of the intrinsic cellular and molecular mechanisms of cartilage repair that have identified novel signals and targets and are inspiring the development of molecular therapies to enhance the recruitment and cartilage reparative activity of joint-resident stem and progenitor cells. A one-fits-all solution is unrealistic, and identifying patients who will respond to a specific targeted treatment will be critical.

      Keywords

      Introduction

      Articular cartilage (AC) has limited capacity for repair, in part due to its intrinsic properties. It is a hypocellular tissue
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      . The tissue is avascular, aneural, and alymphatic
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      . When repair does occur, the tissue formed is often fibrocartilage, which is compositionally different to AC and thus biomechanically inferior.
      Since 1977, when to our knowledge the first tissue engineering approach was described for AC repair
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      , cellular therapies and tissue engineering strategies have been extensively pursued as treatment options for cartilage repair. The goal is to repair or regenerate damaged AC by restoring structure, zonal architecture, and function of the damaged tissue
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      Cartilage tissue engineering is advantageous over current surgical practices which use auto/allografts. Osteochondral autograft transfer is not optimal, because osteoarthritis (OA) can develop at the harvest sites, and the size of the defect that can be repaired is limited. Procedures involving transplant of fresh osteochondral allografts (FOCAs) are limited by availability of donor tissue
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      , often result in inadequate integration with surrounding cartilage, and can transmit disease. Importantly, tissue engineered cartilage constructs can be personalized to fit individual joint shapes and defect sizes.
      To be successful, cartilage produced by cellular therapy or tissue engineering must have the characteristics of native AC. That is, regenerated cartilage must contain appropriate mechanical, compositional, and structural anisotropies
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      Tissue engineering for OA treatment introduces different considerations as compared to repair of focal defects. OA often involves larger and more diffuse involvement of articular surfaces and greater alteration of joint homoeostasis
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      .
      In this review, we discuss the regenerative medicine and tissue engineering approaches to cartilage repair (Fig. 1). The review is not exhaustive, and we apologize to those whose work was not cited because of space constraints.
      Fig. 1
      Fig. 1Components involved in cellular therapy and tissue engineering for cartilage repair. Therapies for cartilage repair require any combination of cells, biomaterials, mechanical loading, and/or bioactive effectors. The light blue ring shows examples of these four major components whereas the dark blue innermost ring represents the ways in which they can be utilized for cartilage tissue engineering as discussed in this review.

      Cell therapy for the repair of joint surface defects

      Autologous chondrocyte implantation (ACI) has pioneered cell therapy for the repair of symptomatic, full-thickness AC defects
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      • Microfracture
      Surgical technique and rehabilitation to treat chondral defects.
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      . Results from up to 20 years follow-up have demonstrated that ACI is an effective and durable solution for the treatment of large cartilage defects in the knee
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      , and ACI has entered routine clinical practice in some countries. Positive predictors of good outcome include age, location of defect, early intervention (<3 years), and no radiographical signs of OA
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      . However, chondrocytes dedifferentiate in culture
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      , limiting their expandability and number of cells available for transplantation. In addition, tissue overgrowth especially when using a periosteal flap is not uncommon and may necessitate another surgery
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      Use of a type I/III bilayer collagen membrane decreases reoperation rates for symptomatic hypertrophy after autologous chondrocyte implantation.
      .
      Mesenchymal stromal cells (MSCs) from various tissues are an alternative cell source as they are easy to expand in culture. Preclinical studies have shown promising results when adopting MSCs for osteochondral repair
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      , although advancement of the bone front at the expense of the overlaying AC is not uncommon
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      Observations of subchondral plate advancement during osteochondral repair: a histomorphometric and mechanical study in the rabbit femoral condyle.
      . Studies in humans have reported variable structural outcome ranging from hyaline-like cartilage to fibrous tissue
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      . Autologous bone marrow MSCs were non-inferior to chondrocytes in clinical outcomes at 24 months in an ACI-like procedure
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      , but longer-term follow-up will be essential to support their use in routine clinical practice. Allogeneic MSCs have shown an acceptable safety profile
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      , and their production could be upscaled to generate large batches of cells ready for use, which would increase consistency and decrease cost of cell therapy.
      MSCs for cell therapy can be derived from various tissues, including bone marrow
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      , periosteum
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      Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age.
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      , synovium
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      Multipotent mesenchymal stem cells from adult human synovial membrane.
      ,
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      Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane.
      , or adipose tissue
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      Human adipose tissue is a source of multipotent stem cells.
      . Bone marrow MSCs are most used but may not be ideal for the repair of AC due to their propensity to undergo chondrocyte hypertrophy, perhaps as an integral part of their endochondral bone formation programme
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      Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering.
      ,
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      A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.
      . Adipose-derived MSCs, while attractive due to their ease of harvesting, tend to be poorly chondrogenic
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      Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source.
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      Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans.
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      • Nimura A.
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      Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit.
      , possibly due to their lack of expression of TGF-β type I receptor and low expression of BMPs
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      Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6.
      . MSCs from synovium displayed superior cartilage-forming potency compared to MSCs from bone marrow, subcutaneous adipose tissue, and periosteum
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      • Yagishita K.
      • Muneta T.
      Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source.
      ,
      • Mochizuki T.
      • Muneta T.
      • Sakaguchi Y.
      • Nimura A.
      • Yokoyama A.
      • Koga H.
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      Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans.
      , and have shown promise in preclinical and clinical studies
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      • Koga H.
      Arthroscopic transplantation of synovial stem cells improves clinical outcomes in knees with cartilage defects.
      ,
      • Ozeki N.
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      • et al.
      Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats.
      . AC and synovium have a common developmental origin from the embryonic joint interzone
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      A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.
      ,
      • Roelofs A.J.
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      • Ansboro S.
      • White N.
      • et al.
      Joint morphogenetic cells in the adult mammalian synovium.
      . It is therefore fascinating to contemplate how potency including morphogenetic tissue repair ability may be imprinted in the MSCs based on their ontogeny.

      Cell therapy for osteoarthritis

      Intra-articular MSC therapy was pioneered with a study that showed regeneration of the medial meniscus and reduced secondary OA in goats in response to intra-articular injection of bone marrow MSCs after medial meniscectomy and anterior cruciate ligament resection
      • Murphy J.M.
      • Fink D.J.
      • Hunziker E.B.
      • Barry F.P.
      Stem cell therapy in a caprine model of osteoarthritis.
      , paving the way to clinical studies in patients with knee OA
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      • Murphy M.
      Mesenchymal stem cells in joint disease and repair.
      . Recent systematic reviews of phase I/II clinical trials (not always controlled or blinded) concluded that intra-articular injection of MSCs, typically from bone marrow or adipose tissue, into the knee is overall safe and well tolerated. Furthermore, MSCs can decrease pain and improve function of the knee, with histological data indicating that hyaline-like cartilage repair can be achieved
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      Mesenchymal stem cells in joint disease and repair.
      ,
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      ,
      • McIntyre J.A.
      • Jones I.A.
      • Han B.
      • Vangsness C.T.
      Intra-articular mesenchymal stem cell therapy for the human joint: a systematic review.
      . A meta-analysis of 11 trials of MSC therapy for knee OA, including a total of 582 patients, reported improvements across a range of clinical outcome measures
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      • Bo L.
      • Lin C.
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      . While most studies have used autologous cells, allogeneic MSCs appear to have an acceptable safety profile
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      Treatment of knee osteoarthritis with allogeneic bone marrow mesenchymal stem cells: a randomized controlled trial.
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      Efficacy and safety of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (Stempeucel®): preclinical and clinical trial in osteoarthritis of the knee joint.
      . However, large, controlled trials, as well as standardisation of cell product manufacturing, optimal delivery, and definition of target patient populations through stratification are needed to ascertain efficacy and allow comparisons of clinical study outcomes.
      The mechanisms of action of MSC therapy in OA remain unclear, and there is limited evidence to support direct contribution of the injected MSCs to repair tissue. MSC-derived extracellular vesicles (EVs) can promote cartilage repair and protect against OA-induced cartilage degeneration
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      • Zhang S.
      • Chuah S.J.
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      • Lim S.K.
      • Toh W.S.
      MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity.
      , supporting the notion that MSCs could mediate tissue repair via release of EVs and other paracrine signals.

      Bioengineering cartilage tissue implants

      Tissue engineering techniques for cartilage repair aim to create tissues which effectively mimic native AC and restore joint function
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      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      . Tissue engineering requires the use of 1) a scaffold, substrate or matrix to provide structure, 2) cells to generate the tissue, and/or 3) signalling in the form of chemical or physical cues to promote a cartilage or bone phenotype
      • Hollister S.J.
      • Murphy W.L.
      Scaffold translation: barriers between concept and clinic.
      . Implanted constructs must be sufficiently porous to allow for nutrient transport and waste removal, contain or promote formation of a mature zonal organization with a biochemically appropriate composition, and must integrate with the surrounding tissue to enable smooth articulation and transfer and dissipation of joint loads
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      • et al.
      Application of bone and cartilage extracellular matrices in articular cartilage regeneration.
      . Constructs must also be biocompatible, customizable in shape and size to fill defects or to replace an entire joint, and be easy to place and secure in the defect during surgery
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      • Chen G.
      • Xu X.
      • Abdou P.
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      • et al.
      Advances of injectable hydrogel-based scaffolds for cartilage regeneration.
      .
      Scaffolds are composed of natural or synthetic materials and may be coupled with bioactive molecules such as growth factors, drugs, or deoxyribonucleic acid (DNA). They can be used either seeded with cells, or without cells to support cell ingrowth following implantation. Scaffolds can differ in charge, wettability, material, microstructure (porosity, pore size, pore shape), and stiffness, each of which influence cell phenotype, proliferation, differentiation, migration, and extracellular matrix (ECM) production
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      Scaffolding strategies for tissue engineering and regenerative medicine applications.
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      • Yang Z.
      The influence of scaffold microstructure on chondrogenic differentiation of mesenchymal stem cells.
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      • Detamore M.S.
      Strategies and applications for incorporating physical and chemical signal gradients in tissue engineering.
      . Scaffolds can influence tissue formation by activating intracellular signalling pathways via interaction with cell adhesion molecules, such as integrin-mediated mechanotransduction, and/or via release of soluble factors
      • Tamaddon M.
      • Liu C.
      Enhancing biological and biomechanical fixation of osteochondral scaffold: a grand challenge.
      . Thus, determining the optimal scaffold characteristics that induce and maintain articular chondrocyte phenotypes that produce cartilage tissue with a zonal architecture is critical.
      While cartilage engineering scaffolds have been extensively studied, consensus on the optimal material, fabrication technique, or structure has not yet been reached
      • Smith B.D.
      • Grande D.A.
      The current state of scaffolds for musculoskeletal regenerative applications.
      . However, certain scaffold characteristics have been identified
      • Hollister S.J.
      • Murphy W.L.
      Scaffold translation: barriers between concept and clinic.
      . Scaffolds must be biocompatible and biomimetic (if not derived from natural substances) to support chondrogenesis by promoting cell adhesion, cell proliferation, and ECM production
      • Gao C.
      • Peng S.
      • Feng P.
      • Shuai C.
      Bone biomaterials and interactions with stem cells.
      . Scaffolds and their degradation products should not produce immunological reactions following implantation
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      • Wang L.
      • Fast L.
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      Growth, differentiation, transplantation and survival of human skeletal myofibers on biodegradable scaffolds.
      . They must be processable into different shapes and sizes
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      • Foster E.J.
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      , and allow integration with native tissue. The scaffold-containing construct must be mechanically strong and resistant to an applied load
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      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      . As scaffolds biodegrade, degradation rate must match tissue formation rate
      • Camarero-Espinosa S.
      • Rothen-Rutishauser B.
      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      to ensure sufficient load bearing function, and not generate cytotoxic by-products nor induce a fibrotic response.
      There are many different methods for making scaffolds, including 3D printing, hydrogels
      • Vega S.L.
      • Kwon M.Y.
      • Burdick J.A.
      Recent advances in hydrogels for cartilage tissue engineering.
      , supercritical fluid technology
      • García-González C.A.
      • Concheiro A.
      • Alvarez-Lorenzo C.
      Processing of materials for regenerative medicine using supercritical fluid technology.
      , electrospinning
      • Ding H.
      • Cheng Y.
      • Niu X.
      • Hu Y.
      Application of electrospun nanofibers in bone, cartilage and osteochondral tissue engineering.
      ,
      • Lopa S.
      • Mondadori C.
      • Mainardi V.L.
      • Talò G.
      • Costantini M.
      • Candrian C.
      • et al.
      Translational application of microfluidics and bioprinting for stem cell-based cartilage repair.
      and weaving. 3D printing allows precise cell and biomolecule positioning in scaffolds consisting of different materials, and predefined designs and geometries, and can be combined with microfluidics to enhance cell seeding
      • Pina S.
      • Ribeiro V.P.
      • Marques C.F.
      • Maia F.R.
      • Silva T.H.
      • Reis R.L.
      • et al.
      Scaffolding strategies for tissue engineering and regenerative medicine applications.
      ,
      • Lopa S.
      • Mondadori C.
      • Mainardi V.L.
      • Talò G.
      • Costantini M.
      • Candrian C.
      • et al.
      Translational application of microfluidics and bioprinting for stem cell-based cartilage repair.
      .

      Tissue engineering approaches using natural scaffolds

      Natural scaffolds are highly biocompatible, biodegradable, and have multiple cell attachment sites due to their similarity with native ECM
      • Pina S.
      • Ribeiro V.P.
      • Marques C.F.
      • Maia F.R.
      • Silva T.H.
      • Reis R.L.
      • et al.
      Scaffolding strategies for tissue engineering and regenerative medicine applications.
      ,
      • Smith B.D.
      • Grande D.A.
      The current state of scaffolds for musculoskeletal regenerative applications.
      . Degradation of this type of scaffold is usually enzymatic, and consequently, degradation products should not result in immunological reactions
      • Pina S.
      • Ribeiro V.P.
      • Marques C.F.
      • Maia F.R.
      • Silva T.H.
      • Reis R.L.
      • et al.
      Scaffolding strategies for tissue engineering and regenerative medicine applications.
      . Natural scaffolds that have been evaluated include proteins (i.e., silk fibroin, collagen, gelatin, keratin, fibrinogen, elastin), polysaccharides (i.e., chitosan, chitin, alginate, gellan gum), and glycosaminoglycans (i.e., hyaluronic acid) [Fig. 2(A) and (B)]. Structural proteins (elastin, fibrin, silk) may have an added benefit as they are suitable as well for drug delivery
      • Nair L.S.
      • Laurencin C.T.
      Biodegradable polymers as biomaterials.
      ,
      • Malafaya P.B.
      • Silva G.A.
      • Reis R.L.
      Natural–origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications.
      . Natural scaffold limitations include poor shape customizability, batch to batch differences in degradation rate, and difficulties in functionalization
      • Camarero-Espinosa S.
      • Rothen-Rutishauser B.
      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      . Most of these scaffolds have been evaluated in small animals pre-clinically, and there have been some clinical trials, although most of these are single-arm trials. One clinical trial using nasal chondrocytes and collagen scaffold (Chondro-Guide) implanted in a post-traumatic cartilage defect in the knee after 2 weeks in culture resulted in improved symptomatology. While there was variable fill of the defect as visualized by magnetic resonance imaging (MRI), glycosaminoglycan content of the repair tissue significantly increased between six and 24 months after the procedure, as determined by delayed gadolinium-enhanced MRI
      • Mumme M.
      • Barbero A.
      • Miot S.
      • Wixmerten A.
      • Feliciano S.
      • Wolf F.
      • et al.
      Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.
      .
      Fig. 2
      Fig. 2Examples of cartilage tissue engineering. (A) Cartilage tissue formed by chondrocytes within a scaffold of denatured or native collagen. Composite image is reproduced from Ref. 
      • Jiang L.B.
      • Su D.H.
      • Liu P.
      • Ma Y.Q.
      • Shao Z.Z.
      • Dong J.
      Shape-memory collagen scaffold for enhanced cartilage regeneration: native collagen versus denatured collagen.
      . (B) Cartilage tissue formed by chondrocytes within an agarose, alginate, collagen, fibrin or PGA scaffold. Shown are interior sections stained with Safranin O after culture for 20 days, with articular cartilage for comparison. Image is reproduced from Ref. 
      • Mouw J.K.
      • Case N.D.
      • Guldberg R.E.
      • Plaas A.H.
      • Levenston M.E.
      Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering.
      . (C) Cartilage tissue formed on the top surface of a substrate (porous polyphosphate, left) or scaffold (3D-printed polycaprolactone, right). PGA: polyglycolic acid.
      Another type of natural scaffold is tissue that has been decellularized to generate cartilage-derived matrix that preserves tissue macromolecules and structure
      • Crapo P.M.
      • Gilbert T.W.
      • Badylak S.F.
      An overview of tissue and whole organ decellularization processes.
      . Decellularization procedures include physical, chemical, and enzymatic treatments
      • Dai L.
      • He Z.
      • Jiang Y.
      • Zhang X.
      • Ren S.
      • Zhu J.
      • et al.
      One-step strategy for cartilage repair using acellular bone matrix scaffold based in situ tissue engineering technique in a preclinical minipig model.
      • Hardingham T.
      Extracellular matrix and pathogenic mechanisms in osteoarthritis.
      • Kim B.S.
      • Kim H.
      • Gao G.
      • Jang J.
      • Cho D.-W.
      Decellularized extracellular matrix: a step towards the next generation source for bioink manufacturing.
      • Gilbert T.W.
      • Sellaro T.L.
      • Badylak S.F.
      Decellularization of tissues and organs.
      , but optimal decellularization has been difficult to achieve as there is often a trade-off between DNA removal and glycosaminoglycan loss
      • Somers P.
      • de Somer F.
      • Cornelissen M.
      • Thierens H.
      • Nooten G.V.
      Decellularization of heart valve matrices: search for the ideal balance.
      ,
      • Kheir E.
      • Stapleton T.
      • Shaw D.
      • Jin Z.
      • Fisher J.
      • Ingham E.
      Development and characterization of an acellular porcine cartilage bone matrix for use in tissue engineering.
      . Advantages of decellularized scaffolds include preservation of zonal architecture and growth factor distribution
      • Haghwerdi F.
      • Khozaei Ravari M.
      • Taghiyar L.
      • Shamekhi M.A.
      • Jahangir S.
      • Haririan I.
      • et al.
      Application of bone and cartilage extracellular matrices in articular cartilage regeneration.
      ,
      • Sun Y.
      • Yan L.
      • Chen S.
      • Pei M.
      Functionality of decellularized matrix in cartilage regeneration: a comparison of tissue versus cell sources.
      , potential for successful interface integration, provision of a cartilage-mimetic environment, and facilitation of differentiation of cells seeded into the matrix
      • Xia C.
      • Mei S.
      • Gu C.
      • Zheng L.
      • Fang C.
      • Shi Y.
      • et al.
      Decellularized cartilage as a prospective scaffold for cartilage repair.
      . Limitations include poor characterization of decellularized scaffold composition unless analysed using proteomic analysis
      • Haghwerdi F.
      • Khozaei Ravari M.
      • Taghiyar L.
      • Shamekhi M.A.
      • Jahangir S.
      • Haririan I.
      • et al.
      Application of bone and cartilage extracellular matrices in articular cartilage regeneration.
      , and poor mechanical properties
      • Haghwerdi F.
      • Khozaei Ravari M.
      • Taghiyar L.
      • Shamekhi M.A.
      • Jahangir S.
      • Haririan I.
      • et al.
      Application of bone and cartilage extracellular matrices in articular cartilage regeneration.
      . The success of decellularized scaffolds in cartilage tissue engineering may be improved by recellularization of the scaffold prior to implantation
      • Statham P.
      • Jones E.
      • Jennings L.M.
      • Fermor H.L.
      Reproducing the biomechanical environment of the chondrocyte for cartilage tissue engineering.
      . Additionally, decellularized ECM can be used as bioink for 3D bioprinting
      • Haghwerdi F.
      • Khozaei Ravari M.
      • Taghiyar L.
      • Shamekhi M.A.
      • Jahangir S.
      • Haririan I.
      • et al.
      Application of bone and cartilage extracellular matrices in articular cartilage regeneration.
      ,
      • Toprakhisar B.
      • Nadernezhad A.
      • Bakirci E.
      • Khani N.
      • Skvortsov G.A.
      • Koc B.
      Development of bioink from decellularized tendon extracellular matrix for 3D bioprinting.
      ,
      • Kim B.S.
      • Das S.
      • Jang J.
      • Cho D.-W.
      Decellularized extracellular matrix-based bioinks for engineering tissue- and organ-specific microenvironments.
      , or reinforced with hydrogels
      • Xia C.
      • Mei S.
      • Gu C.
      • Zheng L.
      • Fang C.
      • Shi Y.
      • et al.
      Decellularized cartilage as a prospective scaffold for cartilage repair.
      ,
      • Pinheiro A.
      • Cooley A.
      • Liao J.
      • Prabhu R.
      • Elder S.
      Comparison of natural crosslinking agents for the stabilization of xenogenic articular cartilage.
      ,
      • McGann M.E.
      • Bonitsky C.M.
      • Jackson M.L.
      • Ovaert T.C.
      • Trippel S.B.
      • Wagner D.R.
      Genipin crosslinking of cartilage enhances resistance to biochemical degradation and mechanical wear.
      or synthetic polymers. A recent study in pigs in which decellularized allogenic cartilage was implanted into knee defects had promising results at 6-month follow-up
      • Nie X.
      • Chuah Y.J.
      • Zhu W.
      • He P.
      • Peck Y.
      • Wang D.A.
      Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair.
      .

      Tissue engineering approaches using synthetic scaffolds

      Synthetic scaffolds can be manufactured with highly predictable properties
      • Smith B.D.
      • Grande D.A.
      The current state of scaffolds for musculoskeletal regenerative applications.
      , allowing for precise manipulation of construct mechanical characteristics
      • Statham P.
      • Jones E.
      • Jennings L.M.
      • Fermor H.L.
      Reproducing the biomechanical environment of the chondrocyte for cartilage tissue engineering.
      . Their advantages include potential to specify composition, reproducibility, ease of processing and preservation of sterility, and control of degradation times. Drawbacks include lack of natural binding motifs for cell attachment, insufficient biological activity, variable hydration, hydrophobic nature depending on the material, potential for inflammation, mismatch between degradation rate and tissue formation leading to tissue collapse in vivo, and failure to recapitulate zonal architecture of cartilage
      • Statham P.
      • Jones E.
      • Jennings L.M.
      • Fermor H.L.
      Reproducing the biomechanical environment of the chondrocyte for cartilage tissue engineering.
      ,
      • Lu Y.
      • Zhang W.
      • Wang J.
      • Yang G.
      • Yin S.
      • Tang T.
      • et al.
      Recent advances in cell sheet technology for bone and cartilage regeneration: from preparation to application.
      . Some polymers have limited use as they generate acidic degradation products
      • Camarero-Espinosa S.
      • Rothen-Rutishauser B.
      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      . Examples of synthetic scaffolds include poly(alpha-esters) such as polyglycolic acid, polylactic acid, and their copolymers, polycaprolactone
      • Moutos F.T.
      • Guilak F.
      Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering.
      , biodegradable polyurethanes, and polyethylene glycol [Fig. 2(B)]
      • Camarero-Espinosa S.
      • Rothen-Rutishauser B.
      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      .
      There are numerous evaluation studies of scaffold implants in animal models, as demonstrated by the publication of 334 papers over the past 5 years in Pub Med which were identified by the search terms scaffold and cartilage repair. They describe variable outcomes, and many are short-term studies that do not address durability of repair
      • Friedman J.M.
      • Sennett M.L.
      • Bonadio M.B.
      • Orji K.O.
      • Neuwirth A.L.
      • Keah N.
      • et al.
      Comparison of fixation techniques of 3D-woven poly(-caprolactone) scaffolds for cartilage repair in a weightbearing porcine large animal model.
      . For example, in one study using minipigs, polycaprolactone woven scaffold was anchored into a focal full thickness chondral defect (4 mm) in the knee joint. At two months, the scaffold was retained and there was fibrovascular ingrowth of the scaffold which suggested that this scaffold had promise to be effective for cartilage repair. However, the 12-month results were poor as repair was impaired, the tissue that developed had lower mechanical properties than AC and the implant had subsided into bone and induced extensive remodelling
      • Friedman J.M.
      • Sennett M.L.
      • Bonadio M.B.
      • Orji K.O.
      • Neuwirth A.L.
      • Keah N.
      • et al.
      Comparison of fixation techniques of 3D-woven poly(-caprolactone) scaffolds for cartilage repair in a weightbearing porcine large animal model.
      ,
      • Sennett M.L.
      • Friedman J.M.
      • Ashley B.S.
      • Stoeckl B.D.
      • Patel J.M.
      • Alini M.
      • et al.
      Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.
      . This emphasizes the need for longer term studies (6 months or greater depending on the species) to better assess the utility of an implant and the extent of remodelling over time. Of note, a 6-month study in dogs using a modification of this woven implant did not show bone resorption. This raises the question as to whether the animal model used to evaluate implants may itself influence outcome
      • Estes B.T.
      • Enomoto M.
      • Moutos F.T.
      • Carson M.A.
      • Toth J.M.
      • Eggert P.
      • et al.
      Biological resurfacing in a canine model of hip osteoarthritis.
      .
      There have been very few clinical trials using synthetic scaffolds, and they have been used for focal defect repair. An example of one of these trials was placement of an acellular scaffold composed of photoreactive chondroitin-sulfate/polyethylene glycol hydrogel in a post-traumatic defect in the femoral condyle following microfracture
      • Wolf M.T.
      • Zhang H.
      • Sharma B.
      • Marcus N.A.
      • Pietzner U.
      • Fickert S.
      • et al.
      Two-year follow-up and remodeling kinetics of ChonDux hydrogel for full-thickness cartilage defect repair in the knee.
      . Variable repair by MRI was observed at 24-month follow-up, with five out of 18 patients showing cartilage delamination and four showing cartilage overgrowth. These complications are not uncommonly seen in scaffold-based implants.

      Scaffold-free tissue engineering approaches

      Another approach to cartilage repair is scaffold-free cartilage tissue engineering, whereby cells are induced to produce ECM and form a tissue in vitro prior to implantation [Fig. 2(C)]. This approach aims to mimic, in a short time period in vitro, developmental, mechanical, structural, and cellular changes which occur over several years during the development and maturation of native AC
      • Camarero-Espinosa S.
      • Rothen-Rutishauser B.
      • Foster E.J.
      • Weder C.
      Articular cartilage: from formation to tissue engineering.
      . Scaffold-free tissue engineering includes self-organizing and self-assembling approaches. Self-assembly occurs in closed systems where cells undergo condensation, proliferation, differentiation, ECM production, and tissue maturation
      • Athanasiou K.A.
      • Eswaramoorthy R.
      • Hadidi P.
      • Hu J.C.
      Self-organization and the self-assembling process in tissue engineering.
      . This is likely driven by differential cell adhesion and interfacial tension
      • Brodland G.W.
      The differential interfacial tension hypothesis (DITH): a comprehensive theory for the self-rearrangement of embryonic cells and tissues.
      • Krieg M.
      • Arboleda-Estudillo Y.
      • Puech P.H.
      • Käfer J.
      • Graner F.
      • Müller D.J.
      • et al.
      Tensile forces govern germ-layer organization in zebrafish.
      • Manning M.L.
      • Foty R.A.
      • Steinberg M.S.
      • Schoetz E.-M.
      Coaction of intercellular adhesion and cortical tension specifies tissue surface tension.
      . For example, deep zone articular chondrocytes can be grown scaffold-free in vitro and produce biphasic tissue rich in proteoglycans
      • Yu H.
      • Grynpas M.
      • Kandel R.A.
      Composition of cartilagenous tissue with mineralized and non-mineralized zones formed in vitro.
      with a localized calcified layer, similar to in vivo calcified cartilage
      • Kandel R.
      • Hurtig M.
      • Grynpas M.
      Characterization of the mineral in calcified articular cartilagenous tissue formed in vitro.
      . Self-organizing culture systems require exogenous input of energy
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      and include pellet culture, aggregate culture, cell sheets, or high-density cell culture on the top surface of a substrate. To generate cell/tissue sheets, cells are expanded in monolayer to high confluency and released as a sheet from mechanically or temperature-responsive substrate systems
      • Lu Y.
      • Zhang W.
      • Wang J.
      • Yang G.
      • Yin S.
      • Tang T.
      • et al.
      Recent advances in cell sheet technology for bone and cartilage regeneration: from preparation to application.
      , and released sheets are rolled, layered, or applied to molds
      • Sato M.
      • Yamato M.
      • Hamahashi K.
      • Okano T.
      • Mochida J.
      Articular cartilage regeneration using cell sheet technology.
      to generate thick tissues
      • Shimizu T.
      • Sekine H.
      • Yang J.
      • Isoi Y.
      • Yamato M.
      • Kikuchi A.
      • et al.
      Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.
      . Aggregate culture involves subjecting cells to rotational culture in the presence of growth factors
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      . The cells that can be used in these approaches are chondrocytes, MSCs from various tissue sources, induced pluripotent stem (iPS) cells and embryonic stem cells.
      Scaffold-free systems circumvent some of the limitations of scaffolds
      • Huey D.J.
      • Hu J.C.
      • Athanasiou K.A.
      Unlike bone, cartilage regeneration remains elusive.
      • Vunjak-Novakovic G.
      • Martin I.
      • Obradovic B.
      • Treppo S.
      • Grodzinsky A.J.
      • Langer R.
      • et al.
      Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.
      • Avula M.N.
      • Rao A.N.
      • McGill L.D.
      • Grainger D.W.
      • Solzbacher F.
      Foreign body response to subcutaneous biomaterial implants in a mast cell-deficient Kit(w-Sh) murine model.
      . Scaffold-free systems may work by decreasing stress shielding
      • Hu J.C.
      • Athanasiou K.A.
      A self-assembling process in articular cartilage tissue engineering.
      , altering mechanotransduction
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      , enhancing matrix deposition
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      , promoting a rounded chondrocyte phenotype
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      ,
      • Huey D.J.
      • Hu J.C.
      • Athanasiou K.A.
      Unlike bone, cartilage regeneration remains elusive.
      and/or enhancing integration with native AC due to increased cell numbers at tissue edges
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      ,
      • Athens A.A.
      • Makris E.A.
      • Hu J.C.
      Induced collagen cross-links enhance cartilage integration.
      . Limitations of scaffold-free systems include the large number of cells required
      • Lee W.D.
      • Gawri R.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.
      , limited tissue thickness and potential for necrosis in the core
      • Lu Y.
      • Zhang W.
      • Wang J.
      • Yang G.
      • Yin S.
      • Tang T.
      • et al.
      Recent advances in cell sheet technology for bone and cartilage regeneration: from preparation to application.
      ,
      • De Pieri A.
      • Rochev Y.
      • Zeugolis D.I.
      Scaffold-free cell-based tissue engineering therapies: advances, shortfalls and forecast.
      , need for longer culture times
      • De Pieri A.
      • Rochev Y.
      • Zeugolis D.I.
      Scaffold-free cell-based tissue engineering therapies: advances, shortfalls and forecast.
      , and poor tissue mechanical properties
      • Lu Y.
      • Zhang W.
      • Wang J.
      • Yang G.
      • Yin S.
      • Tang T.
      • et al.
      Recent advances in cell sheet technology for bone and cartilage regeneration: from preparation to application.
      . Scaffold-free constructs have been used to successfully repair focal defects in pigs
      • Shimomura K.
      • Ando W.
      • Tateishi K.
      • Nansai R.
      • Fujie H.
      • Hart D.A.
      • et al.
      The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model.
      and sheep
      • Kandel R.A.
      • Grynpas M.
      • Pilliar R.
      • Lee J.
      • Wang J.
      • Waldman S.
      • et al.
      Repair of osteochondral defects with biphasic cartilage-calcium polyphosphate constructs in a sheep model.
      . One study using cartilage tissue sheets to treat focal defects in humans (n = 5) resulted in symptom relief at up to 2 years follow-up and showed repair tissue (biopsy) at 48 weeks that resembled hyaline cartilage, suggesting that this approach may have clinical utility
      • Shimomura K.
      • Yasui Y.
      • Koizumi K.
      • Chijimatsu R.
      • Hart D.A.
      • Yonetani Y.
      • et al.
      First-in-human pilot study of implantation of a scaffold-free tissue-engineered construct generated from autologous synovial mesenchymal stem cells for repair of knee chondral lesions.
      .

      Multiphasic or gradient-based tissue engineering constructs

      Tissue-engineered constructs can be multiphasic or gradient-based
      • Wei W.
      • Dai H.
      Articular cartilage and osteochondral tissue engineering techniques: recent advances and challenges.
      , and this is being pursued so the construct better resembles the joint surface, with a zonal architecture consisting of non-mineralized cartilage, calcified cartilage, and subchondral bone. Incorporation of a zone of calcified cartilage would help to maintain construct integrity by regulating force transmission across the interface and preventing cell migration between layers
      • Da H.
      • Jia S.-J.
      • Meng G.-L.
      • Cheng J.-H.
      • Zhou W.
      • Xiong Z.
      • et al.
      The impact of compact layer in biphasic scaffold on osteochondral tissue engineering.
      . This can be accomplished for example by using mechanical cues to direct cell differentiation, i.e., scaffold stiffness and topography can be modified to influence cell fate
      • Lutolf M.P.
      • Gilbert P.M.
      • Blau H.M.
      Designing materials to direct stem-cell fate.
      ,
      • Watt F.M.
      • Huck W.T.S.
      Role of the extracellular matrix in regulating stem cell fate.
      . Soft matrices favour cartilage formation, while stiff matrices favour chondrocyte hypertrophy and osteogenesis, driven by nuclear transduction of mechanical cues involving Yes-associated protein (YAP) and WW-domain-containing transcription regulator protein 1 (WWTR1, also known as transcriptional co-activator with PDZ binding motif (TAZ))
      • Dupont S.
      • Morsut L.
      • Aragona M.
      • Enzo E.
      • Giulitti S.
      • Cordenonsi M.
      • et al.
      Role of YAP/TAZ in mechanotransduction.
      • Yang C.
      • Tibbitt M.W.
      • Basta L.
      • Anseth K.S.
      Mechanical memory and dosing influence stem cell fate.
      • Karystinou A.
      • Roelofs A.J.
      • Neve A.
      • Cantatore F.P.
      • Wackerhage H.
      • De Bari C.
      Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells.
      • Kania K.
      • Colella F.
      • Riemen A.H.K.
      • Wang H.
      • Howard K.A.
      • Aigner T.
      • et al.
      Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.
      • Lee J.
      • Jeon O.
      • Kong M.
      • Abdeen A.A.
      • Shin J.-Y.
      • Lee H.N.
      • et al.
      Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.
      .
      Biphasic scaffolds, consisting of a soft zone and a hard zone that may or may not include calcium, have been evaluated clinically for the repair of focal cartilage defects. An example of this is a BiCRI (polylactic-co-glycolic acid (PLGA) and PLGA plus β-tricalcium phosphate) construct which is currently in clinical trial

      BiPhasic Cartilage Repair Implant (BiCRI) IDE Clinical Trial – Taiwan. https://clinicaltrials.gov/ct2/show/NCT01477008.

      . While biphasic scaffolds with an apatite-containing inferior layer have been created, the presence of a calcified cartilage interface was not confirmed
      • Khanarian N.T.
      • Jiang J.
      • Wan L.Q.
      • Mow V.C.
      • Lu H.H.
      A hydrogel-mineral composite scaffold for osteochondral interface tissue engineering.
      . To our knowledge, this has only been shown when cartilage was formed on the top surface of a substrate
      • Lee W.D.
      • Hurtig M.B.
      • Pilliar R.M.
      • Stanford W.L.
      • Kandel R.A.
      Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.
      . Layers and gradients may differ in terms of composition (cellular and scaffold), fabrication technique, and structural characteristics, which can create transitional or stepwise depth-dependent differences in composition, arrangement, distribution, dimensions, orientations, and interfaces of the tissues
      • Wei W.
      • Dai H.
      Articular cartilage and osteochondral tissue engineering techniques: recent advances and challenges.
      . These gradient-type constructs have not been tested clinically as yet.

      Cyclic loading to improve the mechanical properties of engineered cartilage

      Tissue engineering approaches for cartilage repair commonly result in tissue that is less mechanically robust than native cartilage. Application of mechanical loading, either cyclic, hydrostatic and/or shear, under the appropriate conditions, during tissue formation in vitro has been successful in increasing matrix content. However, it is important to identify the optimal parameters for a specific tissue engineering methodology as these applied forces, if excessive, can induce tissue degradation
      • Vaca-González J.J.
      • Guevara J.M.
      • Moncayo M.A.
      • Castro-Abril H.
      • Hata Y.
      • Garzón-Alvarado D.A.
      Biophysical stimuli: a review of electrical and mechanical stimulation in hyaline cartilage.
      . Factors to consider in the determination of the load include type and amount of load, and frequency, duration and timing of application. Identification of optimal conditions from the literature is hampered by the use of different methods to apply load and the variability in metrics that are assessed in different studies, making comparisons difficult. However, there are a series of experiments using one type of scaffold-free self-assembly tissue engineering approach and one type of instrument to apply the load that demonstrate the importance of selecting the right parameters. For example, one application of cyclic compression for 30 min, 1 day after cell seeding in 3D culture, resulted in an increase in dry weight of the tissue, higher collagen and proteoglycan content, and just over double the maximum equilibrium stress and equilibrium modulus of the tissue 4 weeks later. In contrast, the same force applied 8 or 14 days later had either no or a negative effect on matrix synthesis
      • Waldman S.D.
      • Couto D.C.
      • Grynpas M.D.
      • Pilliar R.M.
      • Kandel R.A.
      A single application of cyclic loading can accelerate matrix deposition and enhance the properties of tissue-engineered cartilage.
      . In another study, cyclic compression after 4 weeks of culture could increase tissue formation, but a larger force was required
      • Waldman S.D.
      • Spiteri C.G.
      • Grynpas M.D.
      • Pilliar R.M.
      • Kandel R.A.
      Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.
      . Interestingly, cyclic compression applied after cartilage had formed, for as little as 6 min every other day for 4 weeks, was sufficient to induce a stimulatory effect
      • Waldman S.D.
      • Spiteri C.G.
      • Grynpas M.D.
      • Pilliar R.M.
      • Kandel R.A.
      Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.
      . This series of studies highlights the need for further rigorous standardized studies to investigate the use of mechanical stimulation to improve cartilage tissue formation. It should be noted that there are very few in-vitro-formed cartilage tissues that attain mechanical properties approaching those of native cartilage even in the presence of mechanical loading. However, this goal may not be necessary, as the loading that occurs with use post-implantation could lead to improved mechanical properties, as was shown to occur in a biphasic implant (cartilage integrated with a porous biomaterial) in a sheep model
      • Kandel R.A.
      • Grynpas M.
      • Pilliar R.
      • Lee J.
      • Wang J.
      • Waldman S.
      • et al.
      Repair of osteochondral defects with biphasic cartilage-calcium polyphosphate constructs in a sheep model.
      . At present, it is not known what mechanical properties are required of bioengineered cartilage to be able to withstand the complex forces experienced by the human joint during daily acts of living (ranging from 7 to 23 MPa of compressive strength and 5–15 MPa tensile modulus
      • Tamaddon M.
      • Liu C.
      Enhancing biological and biomechanical fixation of osteochondral scaffold: a grand challenge.
      ).

      Other approaches to improve engineered cartilage constructs

      To improve cartilage thickness, organization and mechanical properties, constructs can be grown in a bioreactor, such as a perfusion, spinner or rotating vessel, to enhance nutrient diffusion and/or to apply loading
      • Fu L.
      • Li P.
      • Li H.
      • Gao C.
      • Yang Z.
      • Zhao T.
      • et al.
      The application of bioreactors for cartilage tissue engineering: advances, limitations, and future perspectives.
      . Culture under hypoxic conditions to more closely mimic in vivo conditions where the O2 can go as low as 1% could improve cartilage tissue development
      • Fu L.
      • Zhang L.
      • Zhang X.
      • Chen L.
      • Cai Q.
      • Yang X.
      Roles of oxygen level and hypoxia-inducible factor signaling pathway in cartilage, bone and osteochondral tissue engineering.
      . Culture media supplementation with naturally occurring macromolecules, such as polyphosphate
      • St-Pierre J.-P.
      • Wang Q.
      • Li S.Q.
      • Pilliar R.M.
      • Kandel R.A.
      Inorganic polyphosphate stimulates cartilage tissue formation.
      , link N
      • Antoniou J.
      • Epure L.M.
      • Grant M.P.
      • Richard H.
      • Sampalis J.
      • Roughley P.J.
      • et al.
      Short link N acts as a disease modifying osteoarthritis drug.
      , and platelet-rich plasma
      • Chona D.V.
      • Kha S.T.
      • Minetos P.D.
      • LaPrade C.M.
      • Chu C.R.
      • Abrams G.D.
      • et al.
      Biologic augmentation for the operative treatment of osteochondral defects of the knee: a systematic review.
      ,
      • Sermer C.
      • Devitt B.
      • Chahal J.
      • Kandel R.
      • Theodoropoulos J.
      The addition of platelet-rich plasma to scaffolds used for cartilage repair: a review of human and animal studies.
      , have also been shown to enhance cartilage tissue formation. Additionally, there have been many studies exploring the use of proteins, particularly growth factors
      • Shah S.S.
      • Mithoefer K.
      Current applications of growth factors for knee cartilage repair and osteoarthritis treatment.
      . The major signalling molecules and pathways controlling the process of joint repair are similar to those involved in joint morphogenesis during embryonic development, including transforming growth factor (TGF)-β superfamily, Wnt fibroblast growth factor (FGF), hedgehog, parathyroid hormone (PTH)/PTH-related protein (PTHrP), Wnt, and NOTCH signalling
      • Luyten F.P.
      • De Bari C.
      • Dell'Accio F.
      . Targeting these signalling pathways can offer opportunities to enhance cartilage formation, but fine-tuning of intensity, duration, and downstream signalling cascades will be essential. Indeed, excessive or sustained activation of TGF-β signalling can lead to cartilage degradation and OA
      • van Beuningen H.M.
      • Glansbeek H.L.
      • van der Kraan P.M.
      • van den Berg W.B.
      Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections.
      ,
      • Blaney Davidson E.N.
      • van der Kraan P.M.
      • van den Berg W.B.
      TGF-beta and osteoarthritis.
      , while inhibition of TGF-β signalling protects cartilage integrity in models of OA
      • Zhen G.
      • Wen C.
      • Jia X.
      • Li Y.
      • Crane J.L.
      • Mears S.C.
      • et al.
      Inhibition of TGF-β signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis.
      • Chen R.
      • Mian M.
      • Fu M.
      • Zhao J.Y.
      • Yang L.
      • Li Y.
      • et al.
      Attenuation of the progression of articular cartilage degeneration by inhibition of TGF-β1 signaling in a mouse model of osteoarthritis.
      • Xie L.
      • Tintani F.
      • Wang X.
      • Li F.
      • Zhen G.
      • Qiu T.
      • et al.
      Systemic neutralization of TGF-β attenuates osteoarthritis.
      . Similarly, excessive or sustained activation of Wnt/β-catenin signalling can be detrimental
      • Loughlin J.
      • Dowling B.
      • Chapman K.
      • Marcelline L.
      • Mustafa Z.
      • Southam L.
      • et al.
      Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females.
      • Lories R.J.U.
      • Peeters J.
      • Bakker A.
      • Tylzanowski P.
      • Derese I.
      • Schrooten J.
      • et al.
      Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice.
      • Zhu M.
      • Tang D.
      • Wu Q.
      • Hao S.
      • Chen M.
      • Xie C.
      • et al.
      Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice.
      • Yuasa T.
      • Kondo N.
      • Yasuhara R.
      • Shimono K.
      • Mackem S.
      • Pacifici M.
      • et al.
      Transient activation of Wnt/{beta}-catenin signaling induces abnormal growth plate closure and articular cartilage thickening in postnatal mice.
      . Optimal growth factor stimulation may also require sequential exposure to multiple growth factors. Current investigations are focussed on the effects of spatial and temporal release of growth factors from scaffolds on cartilage and bone formation
      • He W.
      • Reaume M.
      • Hennenfent M.
      • Lee B.P.
      • Rajachar R.
      Biomimetic hydrogels with spatial- and temporal-controlled chemical cues for tissue engineering.
      .
      Other approaches to improve tissue formation in engineered cartilage include use of exosomes
      • Kim Y.G.
      • Choi J.
      • Kim K.
      Mesenchymal stem cell-derived exosomes for effective cartilage tissue repair and treatment of osteoarthritis.
      , microRNA
      • Lolli A.
      • Colella F.
      • De Bari C.
      • van Osch G.J.V.M.
      Targeting anti-chondrogenic factors for the stimulation of chondrogenesis: a new paradigm in cartilage repair.
      , anti-inflammatory M2 macrophages
      • Wu C.L.
      • Harasymowicz N.S.
      • Klimak M.A.
      • Collins K.H.
      • Guilak F.
      The role of macrophages in osteoarthritis and cartilage repair.
      , and modified cells using clustered regularly interspaced short palindromic repeats-based gene editing
      • Dicks A.
      • Wu C.L.
      • Steward N.
      • Adkar S.S.
      • Gersbach C.A.
      • Guilak F.
      Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.
      , but these are still in the experimental stage.
      Interestingly, a recent study suggested that increased temperature, as occurs with mechanical loading (thermomechanical stimulation), can enhance chondrogenic gene expression in chondroprogenitor cells. It was postulated that this could lead to better cartilage formation by these cells
      • Nasrollahzadeh N.
      • Karami P.
      • Wang J.
      • Bagheri L.
      • Guo Y.
      • Abdel-Sayed P.
      • et al.
      Temperature evolution following joint loading promotes chondrogenesis by synergistic cues via calcium signaling.
      . Finally, identifying ways to establish and maintain the superficial zone chondrocyte phenotype, and their expression of joint lubricating factors such as Prg4/lubricin that protect against the development of OA
      • Rhee D.K.
      • Marcelino J.
      • Baker M.
      • Gong Y.
      • Smits P.
      • Lefebvre V.
      • et al.
      The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth.
      ,
      • Ruan M.Z.C.
      • Erez A.
      • Guse K.
      • Dawson B.
      • Bertin T.
      • Chen Y.
      • et al.
      Proteoglycan 4 expression protects against the development of osteoarthritis.
      , is an important goal. A recent study showed that the transcription factor Creb5 is selectively expressed in the superficial zone and augments TGF-β and epidermal growth factor receptor-induced expression of Prg4/lubricin in superficial zone chondrocytes
      • Zhang C.-H.
      • Gao Y.
      • Jadhav U.
      • Hung H.-H.
      • Holton K.M.
      • Grodzinsky A.J.
      • et al.
      Creb5 establishes the competence for Prg4 expression in articular cartilage.
      . In addition, YAP and TAZ have been shown to regulate expression of Prg4 and tenascin C in superficial zone chondrocytes
      • Delve E.
      • Co V.
      • Regmi S.C.
      • Parreno J.
      • Schmidt T.A.
      • Kandel R.A.
      YAP/TAZ regulates the expression of proteoglycan 4 and tenascin C in superficial-zone chondrocytes.
      , linking mechanosensing to joint lubrication.

      Clinical studies employing tissue engineering approaches for osteoarthritic cartilage repair

      There have been very few clinical studies evaluating the efficacy of tissue-engineered cartilage in the treatment of OA. A review of studies registered on clinicaltrials.gov (February 2022) did not identify any. There are papers describing clinical evaluation but they are usually small studies and not always controlled. One study described the use of ChonDux hydrogel (chondroitin sulfate (CS)/polyethylene glycol (PEG)), with or without microfracture, to repair full-thickness cartilage defects in individuals with no or early OA. At 2 years, there was significantly increased defect fill and less pain compared to microfracture alone
      • Wolf M.T.
      • Zhang H.
      • Sharma B.
      • Marcus N.A.
      • Pietzner U.
      • Fickert S.
      • et al.
      Two-year follow-up and remodeling kinetics of ChonDux hydrogel for full-thickness cartilage defect repair in the knee.
      , although 30% of treated individuals had dropped out of the study. In another study, adipose-derived MSCs were loaded into a fibrin scaffold and used to treat knee OA. Outcome was compared to adipose-derived MSCs alone at an average follow-up of 28.6 months (minimal followup – 24 months). There was an increase in activity scores in both groups and the fibrin scaffold group had better International Cartilage Repair Society macroscopic scores at second-look arthroscopy
      • Kim Y.S.
      • Choi Y.J.
      • Suh D.S.
      • Heo D.B.
      • Kim Y.I.
      • Ryu J.S.
      • et al.
      Mesenchymal stem cell implantation in osteoarthritic knees: is fibrin glue effective as a scaffold?.
      . A cell-free aragonite-based scaffold was evaluated in individuals with mild to moderate knee OA who had at most three discrete cartilage lesions
      • Kon E.
      • Di Matteo B.
      • Verdonk P.
      • Drobnic M.
      • Dulic O.
      • Gavrilovic G.
      • et al.
      Aragonite-based scaffold for the treatment of joint surface lesions in mild to moderate osteoarthritic knees: results of a 2-year multicenter prospective study.
      . Two-year follow-up showed symptom improvement and variable fill as determined by MRI, but there was no control group. Hollander et al. showed that implantation of an esterified hyaluronic acid scaffold (Hyalograft
      • Vaca-González J.J.
      • Guevara J.M.
      • Moncayo M.A.
      • Castro-Abril H.
      • Hata Y.
      • Garzón-Alvarado D.A.
      Biophysical stimuli: a review of electrical and mechanical stimulation in hyaline cartilage.
      ) seeded with passaged chondrocytes in nine patients with OA resulted in formation of hyaline-like cartilage in some of these individuals at 14 months follow-up
      • Hollander A.P.
      • Dickinson S.C.
      • Sims T.J.
      • Brun P.
      • Cortivo R.
      • Kon E.
      • et al.
      Maturation of tissue engineered cartilage implanted in injured and osteoarthritic human knees.
      . These studies raise the possibility that biological repair of cartilage using tissue engineering approaches in knee OA is possible.

      Enhancing endogenous repair by joint-resident stem and progenitor cells

      An exciting prospect would be to promote endogenous repair using pharmaceuticals. Investigations of joint-resident stem and progenitor cells, and their molecular regulation, will generate the knowledge base that is essential for targeted molecular interventions aiming to activate and modulate intrinsic repair mechanisms. In recent years, genetic cell-lineage tracing and cell transplant studies have provided insight into the stem and progenitor cells that form, maintain and repair skeletal tissues. The most-well studied are skeletal stem cells (SSCs) in bone, which are heterogeneous and enriched in the perivascular bone marrow niche
      • Morikawa S.
      • Mabuchi Y.
      • Kubota Y.
      • Nagai Y.
      • Niibe K.
      • Hiratsu E.
      • et al.
      Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.
      • Méndez-Ferrer S.
      • Michurina T.V.
      • Ferraro F.
      • Mazloom A.R.
      • Macarthur B.D.
      • Lira S.A.
      • et al.
      Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.
      • Ding L.
      • Saunders T.L.
      • Enikolopov G.
      • Morrison S.J.
      Endothelial and perivascular cells maintain haematopoietic stem cells.
      • Isern J.
      • García-García A.
      • Martín A.M.
      • Arranz L.
      • Martín-Pérez D.
      • Torroja C.
      • et al.
      The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.
      • Zhou B.O.
      • Yue R.
      • Murphy M.M.
      • Peyer J.G.
      • Morrison S.J.
      Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.
      and growth plate region
      • Worthley D.L.
      • Churchill M.
      • Compton J.T.
      • Tailor Y.
      • Rao M.
      • Si Y.
      • et al.
      Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.
      • Chan C.K.F.
      • Seo E.Y.
      • Chen J.Y.
      • Lo D.
      • McArdle A.
      • Sinha R.
      • et al.
      Identification and specification of the mouse skeletal stem cell.
      • Chan C.K.F.
      • Gulati G.S.
      • Sinha R.
      • Tompkins J.V.
      • Lopez M.
      • Carter A.C.
      • et al.
      Identification of the human skeletal stem cell.
      . SSCs in mice can contribute to repair of osteochondral lesions that extend into the underlying marrow
      • Zhou B.O.
      • Yue R.
      • Murphy M.M.
      • Peyer J.G.
      • Morrison S.J.
      Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.
      ,
      • Murphy M.P.
      • Koepke L.S.
      • Lopez M.T.
      • Tong X.
      • Ambrosi T.H.
      • Gulati G.S.
      • et al.
      Articular cartilage regeneration by activated skeletal stem cells.
      , and activation of SSCs in subchondral bone marrow is considered to be at the basis of microfracture therapy. However, microfracture typically results in fibrocartilage repair tissue in both mice
      • Murphy M.P.
      • Koepke L.S.
      • Lopez M.T.
      • Tong X.
      • Ambrosi T.H.
      • Gulati G.S.
      • et al.
      Articular cartilage regeneration by activated skeletal stem cells.
      and humans
      • Saris D.B.F.
      • Vanlauwe J.
      • Victor J.
      • Haspl M.
      • Bohnsack M.
      • Fortems Y.
      • et al.
      Characterized chondrocyte implantation results in better structural repair when treating symptomatic cartilage defects of the knee in a randomized controlled trial versus microfracture.
      . Stem and progenitor cells are also present in the superficial zone of the AC
      • Williams R.
      • Khan I.M.
      • Richardson K.
      • Nelson L.
      • McCarthy H.E.
      • Analbelsi T.
      • et al.
      Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.
      • Kozhemyakina E.
      • Zhang M.
      • Ionescu A.
      • Ayturk U.M.
      • Ono N.
      • Kobayashi A.
      • et al.
      Identification of a Prg4-expressing articular cartilage progenitor cell population in mice.
      • Li L.
      • Newton P.T.
      • Bouderlique T.
      • Sejnohova M.
      • Zikmund T.
      • Kozhemyakina E.
      • et al.
      Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.
      • Decker R.S.
      • Um H.-B.
      • Dyment N.A.
      • Cottingham N.
      • Usami Y.
      • Enomoto-Iwamoto M.
      • et al.
      Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.
      , synovium
      • De Bari C.
      • Dell'Accio F.
      • Tylzanowski P.
      • Luyten F.P.
      Multipotent mesenchymal stem cells from adult human synovial membrane.
      ,
      • Roelofs A.J.
      • Zupan J.
      • Riemen A.H.K.
      • Kania K.
      • Ansboro S.
      • White N.
      • et al.
      Joint morphogenetic cells in the adult mammalian synovium.
      ,
      • Kurth T.B.
      • Dell'accio F.
      • Crouch V.
      • Augello A.
      • Sharpe P.T.
      • De Bari C.
      Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo.
      and periosteum
      • De Bari C.
      • Dell'Accio F.
      • Luyten F.P.
      Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age.
      ,
      • Debnath S.
      • Yallowitz A.R.
      • McCormick J.
      • Lalani S.
      • Zhang T.
      • Xu R.
      • et al.
      Discovery of a periosteal stem cell mediating intramembranous bone formation.
      ,
      • Ortinau L.C.
      • Wang H.
      • Lei K.
      • Deveza L.
      • Jeong Y.
      • Hara Y.
      • et al.
      Identification of functionally distinct Mx1+αSMA+ periosteal skeletal stem cells.
      , and these could all potentially contribute to the repair and remodelling of joints throughout life.
      Traditionally, stem cells are identified by the tissue they reside in. However, stem and progenitor cells within the same tissue are ontogenetically and functionally diverse, while stem and progenitor cells that reside in different tissues can share a common ontogeny. Perivascular SSCs in bone marrow derive, at least in part, from the neural crest
      • Nagoshi N.
      • Shibata S.
      • Kubota Y.
      • Nakamura M.
      • Nagai Y.
      • Satoh E.
      • et al.
      Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad.
      ,
      • Morikawa S.
      • Mabuchi Y.
      • Niibe K.
      • Suzuki S.
      • Nagoshi N.
      • Sunabori T.
      • et al.
      Development of mesenchymal stem cells partially originate from the neural crest.
      , and play an important role in the regulation of haematopoietic stem cells
      • Méndez-Ferrer S.
      • Michurina T.V.
      • Ferraro F.
      • Mazloom A.R.
      • Macarthur B.D.
      • Lira S.A.
      • et al.
      Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.
      ,
      • Isern J.
      • García-García A.
      • Martín A.M.
      • Arranz L.
      • Martín-Pérez D.
      • Torroja C.
      • et al.
      The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.
      . Perivascular cells expressing SSC markers are also present in synovium and periosteum, but their functions are less clear. They do not appear to directly contribute to cartilage repair after injury
      • Roelofs A.J.
      • Zupan J.
      • Riemen A.H.K.
      • Kania K.
      • Ansboro S.
      • White N.
      • et al.
      Joint morphogenetic cells in the adult mammalian synovium.
      or osteophyte formation in OA
      • Roelofs A.J.
      • Kania K.
      • Rafipay A.J.
      • Sambale M.
      • Kuwahara S.T.
      • Collins F.L.
      • et al.
      Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.
      . Instead, these processes are largely mediated by Gdf5-lineage cells, mesodermally derived cells that are progeny of the Gdf5-expressing joint interzone cells in the embryo that form the synovial joints during development
      • Koyama E.
      • Shibukawa Y.
      • Nagayama M.
      • Sugito H.
      • Young B.
      • Yuasa T.
      • et al.
      A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.
      ,
      • Rountree R.B.
      • Schoor M.
      • Chen H.
      • Marks M.E.
      • Harley V.
      • Mishina Y.
      • et al.
      BMP receptor signaling is required for postnatal maintenance of articular cartilage.
      . Gdf5-lineage cells in the adult mouse knee respond to acute cartilage injury by proliferation, homing to the site of injury, and chondrogenic differentiation to repair the defect
      • Roelofs A.J.
      • Zupan J.
      • Riemen A.H.K.
      • Kania K.
      • Ansboro S.
      • White N.
      • et al.
      Joint morphogenetic cells in the adult mammalian synovium.
      , while they respond to chronic injury resulting from joint destabilisation by forming osteophytes
      • Roelofs A.J.
      • Kania K.
      • Rafipay A.J.
      • Sambale M.
      • Kuwahara S.T.
      • Collins F.L.
      • et al.
      Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.
      .
      The adult Gdf5-lineage cell population is not specific to any one tissue in the joint and contains several progenitor populations that could contribute to repair of the AC after injury (Fig. 3). There may be cooperation of different progenitor populations, as observed during osteophyte formation in experimental OA in mice, which is mediated by Sox9-expressing progenitors in periosteum and Prg4-expressing progenitors in synovial lining
      • Roelofs A.J.
      • Kania K.
      • Rafipay A.J.
      • Sambale M.
      • Kuwahara S.T.
      • Collins F.L.
      • et al.
      Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.
      . Prg4-expressing synovial lining cells may also be involved in AC repair
      • Decker R.S.
      • Um H.-B.
      • Dyment N.A.
      • Cottingham N.
      • Usami Y.
      • Enomoto-Iwamoto M.
      • et al.
      Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.
      , which could involve direct synovial attachment to the defect
      • Decker R.S.
      • Um H.-B.
      • Dyment N.A.
      • Cottingham N.
      • Usami Y.
      • Enomoto-Iwamoto M.
      • et al.
      Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.
      , or migration of synovial cells along the cartilage surface
      • Hunziker E.B.
      • Rosenberg L.C.
      Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane.
      or via synovial fluid
      • Jones E.A.
      • Crawford A.
      • English A.
      • Henshaw K.
      • Mundy J.
      • Corscadden D.
      • et al.
      Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level.
      ,
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      • et al.
      Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.
      . Adverse environmental conditions could make the cells ineffective and unable to repair damaged cartilage, highlighting the need to understand the context-specific regulation of stem and progenitor cells in their own environment.
      Fig. 3
      Fig. 3Joint-resident stem and progenitor cells. Cells with progenitor activity are present in multiple joint tissues, including synovium, periosteum, cartilage, and subchondral bone marrow. The main joint-reparative cells are found in the Gdf5-lineage cell population that descends from the joint interzone, the embryonic tissue that gives rise to the synovial joint during development. Gdf5-lineage progenitor cells include Prg4-expressing cells in the superficial zone of articular cartilage, Prg4-expressing cells in synovial lining, and Sox9-expressing cells at the periosteal surface. Gdf5-lineage cells are also present in synovial sub-lining and perivascular and endosteal niches in subchondral bone marrow. Other stromal cells in synovium, periosteum and subchondral bone marrow may contribute to repair. Pericytes, including cells expressing SSC markers such as Nestin or Leptin receptor, as well as macrophage-like synoviocytes in synovial lining and other immune cells, may contribute to regulating the reparative response. However, there is little evidence of a direct contribution of pericytes to joint surface repair. FLS: fibroblast-like synoviocyte; MLS: macrophage-like synoviocyte.
      Recent studies have focussed on the identification and manipulation of molecular signals that can promote endogenous stem cell recruitment and their differentiation into a stable chondrocyte phenotype. Suppression of canonical β-catenin signalling and activation of the CaMKII/CREB pathway by the proteoglycan Agrin was shown to enhance recruitment of endogenous Gdf5-lineage progenitor cells to an osteochondral defect, and to improve osteochondral repair in mice and sheep
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      Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis.
      . Other studies have investigated molecular signals related to the avascular nature of cartilage. In mice, physically preventing vascular invasion during femoral fracture healing, or blocking vascular endothelial growth factor (VEGF) signalling in a renal capsule implant model of bone marrow SSCs, favoured chondrogenic over osteogenic differentiation
      • Chan C.K.F.
      • Seo E.Y.
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      • Sinha R.
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      Identification and specification of the mouse skeletal stem cell.
      ,
      • van Gastel N.
      • Stegen S.
      • Eelen G.
      • Schoors S.
      • Carlier A.
      • Daniëls V.W.
      • et al.
      Lipid availability determines fate of skeletal progenitor cells via SOX9.
      . Delivery of PEG hydrogels loaded with BMP2 together with a VEGF inhibitor in osteochondral defects that were created in OA mouse knees induced the formation of a cartilage repair tissue with biomechanical properties similar to native cartilage
      • Murphy M.P.
      • Koepke L.S.
      • Lopez M.T.
      • Tong X.
      • Ambrosi T.H.
      • Gulati G.S.
      • et al.
      Articular cartilage regeneration by activated skeletal stem cells.
      . The promotion of chondrogenesis in an avascular environment may be driven by hypoxia-induced upregulation of hypoxia-inducible factor (HIF)-1α and HIF-2α, which bind to the Sox9 promoter
      • Thoms B.L.
      • Dudek K.A.
      • Lafont J.E.
      • Murphy C.L.
      Hypoxia promotes the production and inhibits the destruction of human articular cartilage.
      ,
      • Bouaziz W.
      • Sigaux J.
      • Modrowski D.
      • Devignes C.-S.
      • Funck-Brentano T.
      • Richette P.
      • et al.
      Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice.
      . In addition, limited nutrient supply, and specifically lipid scarcity, regulates chondrogenesis in skeletal progenitors via activation of FoxO transcription factors that bind to and activate the SOX9 promoter
      • van Gastel N.
      • Stegen S.
      • Eelen G.
      • Schoors S.
      • Carlier A.
      • Daniëls V.W.
      • et al.
      Lipid availability determines fate of skeletal progenitor cells via SOX9.
      . Thus, the avascular nature of cartilage is intricately linked to the molecular signals that regulate its formation and maintenance, and manipulation of these signals could induce formation of more stable cartilage by SSCs. Whether this will be sufficient to induce durable repair in synovial joints by stem or progenitor cells not ontogenetically derived from the joint interzone, or whether Gdf5-lineage cells remain the best candidate cells to target for the enhancement of endogenous repair, remains to be clarified.

      Conclusions

      Regenerative interventions have entered clinical practice in orthopaedics, with potential for long-term and possibly life-long benefit to patients, and a multitude of tissue engineering approaches to cartilage repair are in the translational pipeline towards clinical application (Fig. 4). While cellular products for cartilage repair have pioneered the field of tissue engineering, a common challenge is the standardization of processing and manufacture to obtain a consistent product of defined identity and known potency to patient benefit. The use of cell-free biomaterials and/or bioactive molecules that activate endogenous reparative processes might render the regulatory pathway more straightforward, but their utility has yet to be shown in clinical trials. The use of biomaterials and bioactive molecules, not only in combination with seeded exogenous cells but also as acellular functionalised scaffolds to promote intrinsic repair mechanisms, is an active area of investigation. Identifying the ideal scaffold and the ideal spatio-temporal delivery of bioactive molecules remain extremely challenging tasks. As our understanding of the intrinsic cellular and molecular mechanisms of tissue repair advances, new signals and targets will be identified that will inspire the development of molecular therapies that are more in line with classical pharmacological interventions. While these will target small cartilage lesions and will possibly lead to the long-awaited disease-modifying OA drugs (DMOADs), more comprehensive approaches relying on exogenous cells and/or combination tissue engineering products will still be needed for the repair of larger defects. The engineering of biological spare parts or even custom-made prostheses could be achieved through the coordinated design of consistent, fully controlled, and upscalable manufacturing processes using advanced technologies such as bioreactors, biosensors and 3D bioprinting. Efforts should be devoted to understanding the underlying mechanisms of action of cell therapy and tissue engineering approaches, not only to enhance our scientific knowledge and fulfil the regulatory requirements, but also, and most importantly, to help improve and refine therapy development over the years. Finally, properly designed, randomised, controlled clinical studies are required to define an evidence-based treatment algorithm for selection of patients with cartilage defects and/or OA who will respond to the treatment. Additionally, appropriate rehabilitation programs will need to be developed. A one-fits-all solution is unrealistic, and stratification of patients will be necessary for targeted treatments to be successfully delivered to the right patient group at the right time.
      Fig. 4
      Fig. 4Schematic summary of methods for cartilage repair. All approaches shown can be enhanced with, for example, bioactive effectors or mechanical loading.

      Contributors

      All authors contributed to drafting, editing and approving the manuscript.

      Conflict of interest

      CDB and AJR have received research grant funding from Biosplice Therapeutics (formerly Samumed LLC).

      Funding sources

      The authors are grateful to the Medical Research Council (grant numbers MR/L020211/1 and MR/L022893/1 ; CDB, AJR), Versus Arthritis (formerly Arthritis Research UK , grant numbers 20050 , 20775 , 20865 , 21156 , and 21800 ; CDB, AJR), Biosplice Therapeutics (CDB, AJR), and the Canadian Institute of Health Research ( CIHR PJT 159722 ; AZ, RAK) for supporting their research.

      Acknowledgements

      We would like to thank Drs. M Mozafari, Sang Jin and Anthony Atala for providing the right-hand image in Fig. 2(C).

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