Exploiting Stem Cell-Extracellular Matrix Interactions ...

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Exploiting Stem Cell-Extracellular Matrix Interactions for Cartilage Regeneration: A Focus on Basement Membrane Molecules Wei Seong Toh1,2,*, Casper Bindzus Foldager3, James Hoi Po Hui2,4, Bjorn Reino Olsen5, Myron Spector6,7 1

Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083, Singapore; 2Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 27 Medical Drive, Singapore 117510, Singapore; 3Orthopaedic Research Laboratory, Aarhus University Hospital, Noerrebrogade 44, building 1A, 8000 Aarhus C, Denmark; 4Cartilage Repair Program, Therapeutic Tissue Engineering Laboratory, Department of Orthopaedic Surgery, National University Health System, National University of Singapore, 1E Kent Ridge Road, Singapore 119288, Singapore; 5Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts 02115; 6Tissue Engineering Laboratories, VA Boston Healthcare System, MS 151, Boston, Massachusetts 02130; 7Department of Orthopaedic Surgery, Brigham and Women’s Hospital,Harvard Medical School, Boston, Massachusetts 02115 Abstract: The extracellular matrix (ECM) is a complex network of proteins and glycosaminoglycans which surrounds cells and serves a critical role in directing cell fate and functions, as well as imparting the necessary mechanical behaviour to the tissue. To achieve successful cartilage regeneration, stem cells and/or progenitor cells have to be able to undergo an orderly spatiotemporal differentiation process, along with specific changes in the ECM expression and deposition, to form a cartilage tissue with the defined hierarchical matrix organization. In the last decade, significant advances have been made in our understanding of the role of the ECM during chondrogenesis and in cartilage homeostasis following differentiation, with some unexpected findings. This review will survey the major ECM components and their interactions with relevant stem cell populations for the regeneration of cartilage. Future therapies will likely benefit from a better understanding and a more precise control of stem cell-ECM interactions implicated in the regenerative response.

Keywords: Basement membrane, cartilage, biomaterials, extracellular matrix, stem cells, pericellular matrix, tissue regeneration. 1. INTRODUCTION Articular cartilage is a unique hypocellular, aneural, alymphatic, and avascular load-bearing tissue, supported by the underlying subchondral bone [1]. Due to the lack of vascularization, articular cartilage has limited capacity for regeneration upon injury. Articular cartilage injuries have a high incidence and henceforth a high socio-economic impact that cannot be underestimated [2]. In the knee joint alone, approximately 60% of patients who underwent arthroscopy displayed cartilage lesions [2]. Articular cartilage lesions can result in potentially crippling symptoms including activityrelated pain, swelling and impaired mobility. When left untreated, these lesions can lead to osteoarthritis (OA), an inflammatory and degenerative joint disease characterized by the degradation of the meniscus, ligaments, and subchondral bone as well as the articular cartilage, and the formation of painful osteophytes [3, 4]. OA is the most common form of arthritis affecting numerous joints including the knee joint *Address correspondence to this author at the Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083; Tel: +65 6779 5555 ext. 1619; Fax: +65 6778 5742; E-mail: [email protected] 1574-888X/16 $58.00+.00

and the temporomandibular joint (TMJ), and is the leading cause of disability worldwide [4]. Treatment options for articular cartilage injuries, currently employed in the clinic, include: arthroscopic lavage and debridement; microfracture; osteochondral autografting / allografting; injection of blood-derived products such as platelet-rich plasma (PRP) into the joint; and injection of cultured autologous chondrocytes under an autologous periosteal flap or an off-the-shelf collagen membrane sutured over the defect [5, 6]. Symptomatic relief in some patients has been reported for 5 years or more for microfracture and for autologous chondrocyte implantation [7] Biopsies from some of these patients have demonstrated that the reparative tissue is principally composed of fibrocartilage and hyaline cartilage that lacks the make-up and organization of articular cartilage [8], which is likely the reason why the relief of symptoms is temporary in most patients [5]. Therefore, there is a need for development of regenerative strategies as effective, sustainable and long-term treatment options for cartilage repair. The potential of stem cells in cartilage tissue engineering and regenerative medicine has been widely recognized [911]. Several stem cell sources, including mesenchymal stem © 2016 Bentham Science Publishers

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cells (MSCs) and embryonic stem cells (ESCs), have emerged and yielded promising results for these applications [12-15]. Notably, human bone marrow-derived MSCs have been applied in clinical cartilage repair [14, 15]. Notwithstanding the initial positive results of applying stem cells for cartilage repair, some limitations have come to light [9, 10]. For instance, the proliferation and differentiation of adult MSCs are negatively affected by increasing donor age [16, 17]. On the other hand, the induction of ESC differentiation to the chondrogenic lineage is still a major challenge [10]. Comprehensive reviews focused on the use of stem cells for cartilage tissue engineering and regeneration can be found elsewhere [9-11]. Stem cell self-renewal and differentiation are controlled by both intrinsic gene regulatory machinery and extrinsic microenvironment or niche, which collectively influence the cell fate and functions [18]. Extracellular matrix (ECM), soluble bioactive factors and neighbouring support cells constitute the stem cell microenvironment that controls stem cell behaviour [18, 19]. The important regulatory function of the ECM has renewed interest in investigations focused on elucidating the native articular cartilage ECM composition and complex cell-ECM interactions in attempt to apply this knowledge to influence stem cells towards cartilage regeneration. This review will examine our current understanding of the cartilage ECM, pertinent stem cell-ECM interactions, and the recent advances in harnessing these cell-ECM interactions towards cartilage regeneration. 2. ARTICULAR CARTILAGE STRUCTURE AND EXTRACELLULAR MATRIX Despite its uniform macroscopic appearance, articular cartilage has a very distinct anisotropy and polarity at the microscopic level. The chondrocytes of the superficial zone secrete the principal lubricating glycoprotein of the body, superficial zone protein (SZP) which is also known as lubricin, and the cells near the surface are of a flattened morphology [20, 21]. The middle zone chondrocytes secrete type II collagen and aggrecan. The deep zone of articular cartilage is continuous with an underlying calcified cartilage layer comprising mineralized ECM with a distinct tidemark that separates it from the subchondral bone. Generally, the cartilage ECM is composed of a hydrated network of type II collagen fibrils, which are specifically arranged architecturally in the three zones of articular cartilage, and enforced with water-retaining aggrecan molecules linked to the hyaluronic acid (HA). The fibrillar type II collagen network is further stabilized by other collagen types, IX and XI, in the territorial / interterritorial matrix, and biglycan, decorin, matrilins, type VI and XVIII/endostatin collagens in the pericellular matrix (PCM). The territorial organization of matrix surrounding the chondrocyte is illustrated in Fig. (1). More recently, and to some extent unexpectedly, proteins normally associated with basement membrane, including type IV collagen, laminin, perlecan and nidogens, have also been found prominently in the PCM surrounding the chondrocytes [2126]. This distinct ultrastructural feature in cartilage, the PCM which surrounds chondrocytes, comprising laminin and type IV collagen appears to be unique, different in dimensions, location, and functions from the other principal type IV col-

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lagen/laminin-containing structure: the basement membrane/ basal lamina.

Fig. (1). Matrix organization surrounding the chondrocytes in the articular cartilage. Reproduced with permission from [58].

3. PERICELLULAR MATRIX AND COMPONENTS Although the PCM was first described as a distinct feature of articular cartilage several decades ago [27, 28], it is only in recent years that there is an emerging paradigm in the field to decipher the components of the PCM and their functional roles in chondrocyte biology and chondrogenesis [29, 30]. PCM is a specialized, thin layer of the ECM that immediately surrounds the chondrocyte, and serves as a transducer for both biochemical and biomechanical signals to the chondrocyte [29-31]. The chondrocyte and PCM together constitute the chondron. Therefore, the PCM plays an important role in maintaining the phenotype and integrity of the chondrocytes [29-31], as shown below in: 1) biochemical interactions with regulatory proteins (viz., growth factors); 2) regulating the mechanical environment of the chondrocyte; and 3) generally maintaining the microenvironment of the chondrocyte and controlling (preventing) interaction of the chondrocyte with the ECM molecules of the territorial matrix. The importance of PCM components is probably best evident in developmental studies [32, 33]. For example, reduced perlecan in mice results in a skeletal chondrodysplasia resembling Schwartz-Jampel syndrome (SJS) in humans [33, 34]. Similarly, several studies have reported the role of PCM in regulating the growth factor presentation and signalling transduction to chondrocytes [35-37] For instance, fibroblast growth factor (FGF)-2 participates in mechanical signal transduction and is co-localized with perlecan in the PCM [35]. Additionally, it was found that perlecan in the cartilage PCM plays a critical role in endochondral ossification by promoting angiogenesis essential for cartilage matrix remodelling and subsequent endochondral bone formation [37]. Several studies have also indicated the important biomechanical role of PCM in cartilage homeostasis, in protecting the individual chondrocytes from excessive mechanical stress (strain) [31, 32, 35]. While there are significant zonal differences in ECM modulus through the depth of the tissue, it is noted that the PCM exhibited zonal uniformity in mechanical properties, thus protecting individual chondrocytes from excessive mechanical strains at the cellular level [31, 38]. Notably, in type VI collagen knockout mice, the PCM

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exhibited reduced mechanical properties which predisposed to accelerated development of OA [32], likely due to the increase in strain experienced by the chondrocytes. Additionally, it has been suggested that perturbation of the PCM affects the chondrocyte’s interaction with the fibrillar collagens (e.g. type II collagen) and this could contribute to disease [39]. In in vitro studies in which isolated chondrocytes and chondrons were treated with monoiodoacetate, commonly used to induce apoptosis of chondrocytes and OA in animal studies [40, 41], re-establishment of type VI collagen in the PCM effectively prevented chondrocytes from monoiodoacetate-induced cell death [42], supporting the idea that type VI collagen has a protective role in the PCM. Recently, we have demonstrated differential expression of laminin and type IV collagen in normal healthy and OA human articular cartilage tissues [26]. In the study [26], the basement membrane proteins were localized in the PCM surrounding chondrocytes in normal healthy articular cartilage, but only type IV collagen and not laminin was found pericellularly in the osteoarthritic cartilage tissues (Fig. 2). This suggests that laminin is a marker of cartilage degeneration, although it remains yet to be determined if selective loss of laminin would also lead to reduced mechanical properties of the PCM.

Fig. (2). Deposition of basement membrane proteins in articular cartilage. Pericellular stain for type IV collagen was seen in both normal and degenerated cartilage. Laminin positive pericellular stain was only seen in normal articular cartilage. Scale bars: large image = 200 μm, small image = 20 μm. Reproduced with permission from [26].

While the functions of individual matrix components are not fully deciphered, several studies have supported an important functional role of ECM in the regulation of the mechanical and physiochemical environments, and regulating chondrocyte metabolism, cartilage homeostasis, and overall joint health [29-31]. Looking forward, understanding the cartilage ECM, particularly the PCM components and their roles in chondrogenesis and cartilage metabolism and homeostasis are likely to advance the innovation of new strategies in stem cell differentiation and cartilage tissue engineering.

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4. EXTRACELLULAR MATRIX DEPOSITION DURING CHONDROGENESIS Chondrogenesis, the process of cartilage formation, is an instructive system for studying the spatiotemporal changes in ECM expression at defined stages of mesenchymal proliferation, condensation and differentiation [43]. Indeed, this process is regulated by several growth factors and signalling pathways, prominently involving FGF, bone morphogenetic protein (BMP) and hedgehog pathways. These growth factors interact with the surrounding ECM to initiate cellular signalling pathways and transcription of specific genes in a well-concerted manner, and coordinated expression of ECM components towards cartilage formation [43, 44]. During embryogenesis, the prechondrogenic mesenchymal progenitors within the limb bud rearrange into cell aggregates, of increased cell number density, in a process known as “condensation” [45] with attendant changes in cell shape (from spread to rounded) and enhanced cell-cell interactions [43]. Notably, the condensation process depends on the coordinated expression of different factors including cell adhesion proteins - N-cadherin and neural cell adhesion molecule (NCAM), and ECM proteins - type I collagen, fibronectin, hyaluronic acid (HA) and chondroitin sulfate, as reported in studies performed using limb bud cultures [43, 46, 47]. At the initial phase of condensation, cell adhesion is ensured through accumulation of HA and upregulated expression of N-cadherin and NCAM. This is accompanied by the deposition of pre-cartilaginous ECM rich in type I collagen and fibronectin, with the latter rapidly upregulated as the mesenchymal cells condensed. As the differentiation proceeds, a matrix that is rich in type I collagen and fibronectin is replaced with one that composed mainly of type II collagen and glycosaminoglycans (GAGs) including the aggrecan [47]. Dynamic changes in the growth factor and integrin receptor expression also occur during this process, mediating specific cell-matrix interactions and signalling that contribute to progression of chondrogenesis. Early studies employing antibody-blocking experiments confirmed the importance of integrin-matrix interactions in the induction of chondrogenesis [48, 49]. For instance, blocking of 1-integrins in limb bud cultures has been shown to inhibit chondrogenesis [48] To dissect the molecular mechanisms of chondrogenesis in vitro, high-density micromass and pellet culture systems have been widely used to recapitulate the stages of in vivo chondrogenesis [50-52]. Using this approach, various cell types including ESCs and MSCs can be induced to undergo chondrogenesis [51, 52] (Fig. 3), and recapitulate the stages of in vivo differentiation, which includes the condensed prechondrogenic mesenchymal progenitors in the limb bud. Although members of transforming growth factor (TGF)- have been commonly used to induce in vitro chondrogenic differentiation of cells, it has been suggested in recent studies that continuous treatment of TGF- may not be an optimal treatment condition [53, 54]. This may be due to dynamic changes in growth factor receptors during MSC chondrogenesis, where differentiating MSCs may have varying growth factor requirements depending on their stage of differentiation [54]. Similarly, the expression of integrins is highly upregulated during MSC chondrogenesis and they


have been suggested to play critical roles in the induction and maintenance of chondrocytic phenotype during chondrogenesis [55]. While integrins have been reported to bind to a wide range of ECM components, including collagens, fibronectin and laminin, their individual functions are not completely understood [55]. Looking ahead, deciphering the changes in growth factor receptor and integrin expression and pertinent cell-matrix interactions during differentiation, would be necessary to further improve strategies based on stem cell differentiation and to fully recapitulate in vivo chondrogenesis.

Fig. (3). Deposition of extracellular matrix proteins during mesenchymal stem cell chondrogenesis. Bone marrow-derived MSCs were induced to undergo chondrogenesis in pellet culture for a period of 14 days in chondrogenic medium supplemented with TGF1. Type II collagen and GAGs accumulate in the territorial / interterritorial matrix during chondrogenesis. The deposition of type IV collagen and laminin followed an orderly spatiotemporal shift in pattern from a diffuse territorial and interterritorial distribution to a defined pericellular localization. Scale bar = 50 μm.

5. BASEMENT MEMBRANE MOLECULES: TYPE IV COLLAGEN AND LAMININ Basement membrane molecules (viz. type IV collagen and laminin) may serve distinct functions in MSC chondrogenesis. It has been recently reported that deposition of type IV collagen and laminin in MSC pellet cultures followed an orderly spatiotemporal shift in pattern during chondrogeneses, from a diffuse territorial and interterritorial distribution to a defined pericellular localization, as seen in normal articular cartilage [26, 56]. These findings are in agreement with immunohistochemical studies performed on mouse cartilage tissues, where several of these PCM components including type VI collagen [57], laminin, type IV collagen, nidogen and perlecan are widespread in the territorial and interterritorial matrix of cartilage from newborn mice, but transit to localization in the narrow pericellular zone surrounding the chondrocytes in mature cartilage of adult mice [57, 58]. However, the underlying mechanisms by which these components are organized and assembled in relation-

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ship to the receptors and signalling pathways involved, remain to be elucidated. To date, the regulation of chondrogenesis and cartilage tissue histogenesis to generate different types of cartilage (hyaline, fibro- and elastic) is still poorly understood. It is likely that the different types of cartilage tissues exhibit not only differences in territorial / interterritorial ECM composition and organization and mechanical properties, but also differences in the PCM make-up. For instance, it was found that the hyaline articular cartilage expressed a thicker band of type IV collagen and laminin in the PCM than observed in the fibrocartilage of meniscus and in calcified articular cartilage. Furthermore, there was a notable differential expression of the two molecules where normal healthy calcified articular cartilage expressed laminin but not type IV collagen, while articular hyaline cartilage expressed both basement membrane proteins in the PCM [26]. Similarly, type XVIII collagen/endostatin was found in the PCM of articular chondrocytes and meniscal fibrochondrocytes, but absent in hypertrophic chondrocytes of the growth plate [24]. This differential expression of type XVIII collagen/endostatin relates to the anti-angiogenic property of the protein in maintaining the avascularity of articular cartilage while permitting vascular invasion of hypertrophic cartilage and bone growth [59]. Notably, many of these ECM and PCM components that already exist endogenously during chondrogenesis are in turn also being investigated for use as substrates for stem cell propagation and differentiation [60-63], and even as scaffolds for cartilage tissue engineering [64-67]. However, it is important to note that many of these ECM proteins, including specific isoforms are regulated in a spatiotemporal manner during chondrogenesis. As the ECM is modified during chondrogenesis, the cellular response to inductive signals may be altered. Therefore, optimal stem cell chondrogenesis towards generation of different types of cartilage is likely to require a spatiotemporal control of specific stem cell-ECM interactions and growth factor signalling at defined stages of chondrogenesis. 6. HARNESSING STEM CELL-ECM INTERACTIONS FOR CARTILAGE TISSUE ENGINEERING AND REGENERATION ECM is critically important as the stem cell physical microenvironment, providing signalling cues that regulate cellular behaviour, including adhesion, migration, proliferation, and differentiation [19]. To recapitulate the native cartilage ECM environment, early studies have focused on the use of type I and II collagens, and GAGs as the major ECM constituents to provide the microenvironment for stem cell differentiation to chondrocytes [64-66]. For instance, it was found that the chondrogenic response of MSCs in type II collagen hydrogels was higher than those seeded in alginate and type I collagen hydrogels [66]. Notably, type II collagen hydrogels have the potential to induce and maintain MSC chondrogenesis, independent of TGF-1 treatment [66]. This finding correlates with an earlier study showing that while the majority of cultured canine chondrocytes seeded in type I collagen had a fibroblastic morphology, the majority of cells seeded in type II collagen matrices displayed a chondrocytic morphology with specific increases in GAGs and type II

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collagen [68]. Similarly, when cultured on type II collagen substrates, human ESC-derived chondrogenic cells displayed enhanced chondrogenesis with increased deposition of type II collagen and GAGs in the cartilaginous pellets [62]. While type II collagen-bearing biomaterials have yielded positive results in various in vitro and in vivo cartilage tissue engineering and repair studies, a commonly used autoimmune mouse model of rheumatoid arthritis involves immunization with an emulsion of complete Freund’s adjuvant and type II collagen [69]. The collagen-induced arthritis phenomenon in select animal models along with certain in vitro studies [70, 71] raises the question of what role type II collagen-bearing biomaterials and their degradation products may play in arthritic processes. Among the GAGs, HA has emerged in the last decade as a promising candidate for cartilage tissue engineering [12, 67]. While HA is widely used as a viscosupplement for the treatment of OA in the clinics [72], its role in that application is still questionable [73, 74]. Numerous studies have found that HA can modulate specific cell functions through interactions with surface receptors including CD44 and CD168 [7577]. For instance, HA membranes supported threedimensional (3-D) spheroid formation of human MSCs, with maintenance of the stemness markers including Oct4, Sox2 and Nanog. Upon chondrogenic induction, these spheroids showed enhanced chondrogenesis with increased levels of cartilage-specific gene expression and protein deposition [78]. Similarly, HA hydrogels supported the self-renewal and differentiation of encapsulated hESCs [79]. When applied in OA treatment, HA injection enhanced the expression of chondrogenic genes and proteins, and blocked that of matrixdegrading genes and proteins in cartilage and subchondral bone. This reparative mechanism of HA is largely mediated by interaction with CD44, since ablation of the receptor resulted in abrogation of the tissue repair [77]. Chondroitin sulfate is another group of GAGs that has gained popularity in the last decade as a promising ECM component in biomaterial scaffold design for cartilage tissue engineering and repair [80-82]. Similar to HA, chondroitin sulfate has been widely used in the clinic for treatment of OA [83], although its role in that application is still questionable [84]. In one study, it was found that exogenous chondroitin sulfate enhanced TGF-1-induced chondrogenesis of human MSCs with earlier and higher expression of type II collagen and aggrecan [85]. In another study, type II collagen-coated microbeads enhanced MSC chondrogenesis with progression towards hypertrophy, but chondroitin sulfatecoated microbeads were able to maintain the differentiated chondrocytic phenotype of MSCs [86]. With increasing evidence that PCM controls the chondrocyte microenvironment, PCM components are wellpoised as substrates / supplements for stem cell proliferation and differentiation to chondrocytes. Notably, it was found that specific matrix proteins including type IV collagen, fibronectin, and laminin-111 when used as substrates for culture enhanced self-renewal and differentiation of human bone marrow-derived MSCs, with the exception of laminin332 substrate that resulted in lack of chondrogenesis [61]. This finding agrees with early studies that exogenous laminin-332 selectively suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5 [87] and


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human MSCs [88], while it favourably induces osteogenic differentiation via an ERK 1/2 (extracellular-regulated kinase) signalling pathway [89]. Matrilins are another class of PCM proteins that exert significant effects on MSC chondrogenesis. It was observed that over-expression of matrilin1 and 3 enhanced chondrogenesis of MSCs derived from synovium, with significant improvement of proliferation of the cells at an early stage and deposition of type II collagen and GAGs at a later stage. However, the effects of matrilins on chondrogenesis require the presence of TGF-1 [90]. Although our knowledge of the diversity of PCM components and their functions is accumulating, the understanding of the effects of PCM components on stem cell proliferation and differentiation is still quite limited at this point in time. Looking ahead, refinement of differentiation protocols with emphasis on the regulation of PCM is likely to improve stem cell-based chondrogenesis towards formation of functional cartilage. The reports that basement membrane proteins are critical components of the stem cell niche in skin [91] and muscle [92], direct attention to the role that these components in cartilage PCM may play in constituting a niche-like microenvironment for regulating migration, proliferation and differentiation of chondrogenic progenitor cells [93] and, more recently, endothelial cells [94]. Recent findings have revealed that MSCs are capable of synthesizing and depositing basement membrane proteins [56, 95, 96]. While many cells, including platelets and endothelial cells, bind to basement membrane proteins [97], it is likely that expression of type IV collagen and laminin by MSCs may also serve to facilitate interactions with other cell types critical for cartilage repair. On this note, based on their in vitro work, Potapova et al. [98] concluded that MSCs “serve as trophic mediators for endothelial cells,” perhaps facilitating their migration, invasion through basement membrane, proliferation and survival. We have observed diffuse deposition of type IV collagen and laminin in the reparative tissue during cartilage repair in a goat model [99] and in tissues from human subjects [100]. While the cell source of the basement membrane molecules has not yet been identified, these proteins may serve as natural ligands for chondroprogenitor cell migration and attachment at the defect site, as part of an early repair response to help in cartilage repair. Collectively, these findings provide an impetus for further studies of basement membrane molecules in cartilage repair and regeneration. That PCM has been shown to be a growth factor depot, regulating the activation, degradation and transport of growth factors, presents a unique opportunity for targeted growth factor delivery in cartilage regeneration [101]. Among the PCM components, perlecan has been shown to bind a number of growth factors including FGF-2 [35] and FGF-18 [36], as well as vascular endothelial growth factor (VEGF) 165 [37] by means of heparan sulfate-mediated mechanisms [102]. Recently, Srinivasan et al. demonstrated that conjugation of perlecan to HA microgels is an improved formulation, compared to HA alone, in the efficient delivery of BMP-2 for stimulation of proteoglycan and cartilage matrix synthesis in a mouse OA model [103]. Another PCM protein of interest is the anti-angiogenic peptide endostatin, a proteolytic fragment of type XVIII collagen that may be of benefit in the repair of the avascular articular cartilage. No-


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tably, endostatin-producing cartilaginous constructs could be formulated by culture of transfected MSCs in type I collagen sponges [104] and hydrogels [105]. The incorporation or direct use of select PCM proteins as biomaterial scaffolds for cartilage tissue engineering has yet to be fully explored. As we begin to decipher the roles of PCM, in particular basement membrane molecules, in chondrogenesis and in cartilage homeostasis, this knowledge will undoubtedly advance our strategies in biomaterial scaffold design for cartilage repair [106].

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CONCLUSION

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Understanding the structure and components of the native cartilage ECM and their changes during development, health and injury / disease has the potential to shed light on the roles of the ECM, in particular the PCM components, in chondrogenesis and in cartilage metabolism and homeostasis. This understanding will have important implications for development of efficient stem cell-based therapies for cartilage regeneration, by means of recapitulating the pertinent stem cell-ECM interactions during cartilage development to orchestrate stem cell chondrogenesis in a well-concerted manner. CONFLICT OF INTEREST

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The authors confirm that this article content has no conflict of interest.

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ACKNOWLEDGEMENTS

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This work was supported by grants from the National University Healthcare System, National University of Singapore (R221000067133, R221000070733, R221000077733 and R221000083112) and National Medical Research Council Singapore (R221000080511).

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Revised: April 22, 2015

Accepted: July 04, 2015

DISCLAIMER: The above article has been published in Epub (ahead of print) on the basis of the materials provided by the author. The Editorial Department reserves the right to make minor modifications for further improvement of the manuscript.

PMID: 26201861