Advances in Mesenchymal Stem Cell-based ...

Stem Cell Rev and Rep DOI 10.1007/s12015-014-9526-z

Advances in Mesenchymal Stem Cell-based Strategies for Cartilage Repair and Regeneration Wei Seong Toh & Casper Bindzus Foldager & Ming Pei & James Hoi Po Hui

# Springer Science+Business Media New York 2014

Abstract Significant research efforts have been undertaken in the last decade in the development of stem cell-based therapies for cartilage repair. Among the various stem cell sources, mesenchymal stem cells (MSCs) demonstrate great promise and clinical efficacy in cartilage regeneration. With a deeper understanding of stem cell biology, new therapeutics and new bioengineering approaches have emerged and showed potential for further developments. Of note, there has been a paradigm shift in applying MSCs for tissue regeneration from the use of stem cells for transplantation to the use of stem cell-derived matrix and secretome components as therapeutic tools and agents for cartilage regeneration. In this review, we will discuss the emerging role of MSCs in cartilage regeneration and the most recent advances in development of stem cell-based therapeutics for cartilage regeneration. W. S. Toh (*) Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083, Singapore e-mail: [email protected] W. S. Toh : J. H. P. Hui Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 27 Medical Drive, Singapore 117510, Singapore C. B. Foldager Orthopaedic Research Laboratory, Aarhus University Hospital, Noerrebrogade 44, building 1A, 8000 Aarhus C, Denmark M. Pei Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, One Medical Center Drive, PO Box 9196, Morgantown, WV 26506-9196, USA J. H. P. Hui Cartilage 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

Keywords Stem cells . Mesenchymal stem cells . Secretome . Extracellular matrix . Cartilage . Biomaterials . Tissue regeneration . Tissue engineering

Introduction Articular cartilage is a unique hypocellular, avascularized and aneural load-bearing tissue, supported by the underlying vascularized subchondral bone (1). Due to the lack of vascularization and innervation, articular cartilage has limited intrinsic ability for regeneration upon injury. Articular cartilage injuries have a high incidence and a high impact on society that cannot be underestimated. In approximately 60 % of the patients who underwent knee arthroscopies, cartilage lesions were observed, commonly caused by sports and recreational activities (2). Articular cartilage lesions can give rise to potentially crippling symptoms such as activity-related pain, swelling and decreased mobility. When left untreated, these lesions can lead to osteoarthritis (OA), an inflammatory and degenerative joint disease characterized by the degradation of the joint, including the articular cartilage and subchondral bone (3, 4). OA is the most common form of arthritis and leading cause of chronic disability in many countries. Current surgical intervention by application of in vitroexpanded autologous chondrocytes transplantation procedure, also known as autologous chondrocyte implantation (ACI) (5), is associated with several problems such as donor site morbidity, loss of chondrocyte phenotype upon ex vivo expansion, inferior fibrocartilage formation at the defect site, and very high cost (6). Other clinical procedures include arthroscopic lavage and debridement, microfracture techniques (7), and autogenic and allogenic osteochondral transplantations (8). While there are some promising results, most cartilage repair techniques lead to fibrocartilage

Stem Cell Rev and Rep

formation and cartilage degeneration after a temporary relief of symptoms. Stem cells represent a promising cell source for cartilage repair and can be derived from two major sources: mesenchymal stem cells (MSCs) (9) and embryonic stem cells (ESCs) (10). Recent advances in stem cell biology have also enabled the generation of ‘personalized’ pluripotent stem cell sources from both fetal and adult fibroblasts through reprogramming by defined gene and protein factors (11). Adult MSCs isolated from various adult tissues including the bone marrow (9, 12), synovium (13), muscle (14), adipose (15) and dental tissues (16) have the potential to differentiate to various mesenchymal lineages, including chondrocytes, osteoblasts and adipocytes. Bone marrow (BM)-derived MSCs are currently undergoing trials for several clinical applications including articular cartilage repair (17, 18). In recent years, MSCs and mesenchymal progenitor cells have been differentiated and derived from human pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and demonstrated potential for differentiation to chondrocytic cells for application in cartilage tissue engineering and regeneration (19–24). Although MSCs and mesenchymal progenitors demonstrated differentiation to chondrocytes when transplanted into a cartilage defect site, there is increasing evidence that MSCs secrete a wide range of bioactive factors and matrix molecules to modulate the injured tissue environment and direct subsequent regenerative processes including cell migration, proliferation and differentiation (25, 26). Recent advances in understanding the role of MSCs in cartilage regeneration has identified new bioactive factors involved in cartilage regeneration, suggesting the potential for development of ‘cell-free’ therapeutics for cartilage regeneration. This review aims to discuss the emerging role of MSCs in cartilage regeneration and present the most recent advances in development of stem cell-based therapeutics for cartilage regeneration. Stem Cells and Articular Cartilage Repair Articular cartilage is a unique avascular, aneural and alymphatic load-bearing tissue which is supported by the underlying subchondral bone. The extracellular matrix (ECM) is composed of a hydrated network of waterretaining aggrecan molecules linked to hyaluronic acid and type II collagen fibrils with a specifically arranged architecture that provides the structural integrity of the tissue. This fibrillar network is stabilized by types IX and XI collagens, and basement membrane molecules including perlican, laminin and type IV collagen that are localized distinctly in the pericellular matrix surrounding the chondrocytes (27, 28). This combination of matrix molecules maintains the

cartilage structure and gives the tissue its unique ability to resist repetitive compressive loading in daily activities. Recent studies comparing the distribution of matrix molecules in normal and osteoarthritic cartilage tissues observed the selective loss of basement membrane molecules, laminin and type IV collagen, in degenerated cartilage tissues, implicating the role of these matrix molecules in chondrocytic differentiation (29) and cartilage homeostasis (27). Based on the depth of the focal cartilage defects, these defects can be either chondral, involving only the cartilage layer, or osteochondral, penetrating into the subchondral bone. As chondral defects do not penetrate the vascularized subchondral bone, they cannot be accessed by the host blood supply, resident macrophages and MSCs originating from bone marrow. The repair that relies on the limited mitotic activities of resident chondrocytes is rarely effective. Although resident stem cells have been isolated in the cartilage (30), effective mobilization of these cells to participate in tissue repair have yet to be demonstrated and is likely due to the low occurrence of these cells. On the contrary, the osteochondral defects are lesions that penetrate the subchondral bone, and in such cases, the bone marrow provides vascularization and MSCs to help in repair. Surgical cartilage repair techniques such as microfracture (7) and drilling to stimulate bone marrow including endogenous MSCs into the cartilage defects mostly yield fibrocartilage repair, which are often followed by the degeneration of the repaired tissue (31). This may be due to insufficient number of endogenous MSCs induced from bone marrow stimulation, which necessitates the isolation of MSCs from other tissues for transplantation. Developing MSC-based strategies for cartilage repair would need to consider the tissue environment for the regenerative process. While focal cartilage lesions with no jointcomorbidities (e.g., meniscal or ligament injuries) provide a closely homeostatic native environment for repair, OA joints or joints that are exposed to alterations in mechanical stress result in an inflammatory and degenerative environment. The inflammatory tissue environment is characterized by synovitis with secretion of inflammatory molecules into the synovial fluid (32). Increasingly, studies are indicating that the role of MSCs in tissue repair/regeneration is not merely tissue replacement through cell differentiation, but through secretion of trophic factors and matrix molecules to modulate the tissue environment and mediate the overall tissue regeneration (25, 26). These findings open up new possibilities for potential development of novel MSC-based strategies for cartilage tissue regeneration, as shown in Fig. 1. Mesenchymal Stem Cells for Direct Transplantation Among the stem cells currently being tested for cartilage repair, MSCs are the most widely used stem cells and are

Stem Cell Rev and Rep Fig. 1 Schematic representation of the potential development pathways of MSC-based strategies for cartilage regeneration. MSCs can be utilized for cartilage repair in various fashions. (a) MSCs can be used in direct transplantation. Alternatively, the cells may be pre-treated/pre-conditioned and processed to harness specific (b) secretome factors and (c) matrix molecules as stem cell-based therapeutics for the treatment

currently undergoing trials for several clinical applications, including articular cartilage repair (17). This is partly due to the ease of accessibility that MSCs can be derived from several tissue sources including bone marrow, synovium, muscle and adipose tissues, and the ability to expand to sufficient numbers in a short time. Several studies have reported the differences in MSCs derived from various tissues at genomic and proteomic levels suggesting the influence of the tissue origin (33), and functional differences in their differentiation programs (34, 35). Among the adult MSCs, synovium-derived MSCs (SDSCs) reportedly demonstrated superiority in proliferation and differentiation to chondrocytes (36), supporting the use of these cells for articular cartilage repair (13, 37). However, there are still some limitations to the use of adult MSCs. These include the limited capacity for self-renewal and with increasing donor age, proliferation and differentiation are impaired (38). The derivation of MSCs from human pluripotent stem cells represents as a promising alternative to obtain large number of batch standardized and off-the-shelf ready MSCs, and to overcome the impeding issues of cellular senescence of adult MSCs and donor tissue variability. To date, several studies have reported the derivation of MSCs and mesenchymal progenitor cells from human pluripotent stem cells including hESCs (19, 39) and iPSCs (40, 41) and demonstrated potential for differentiation to chondrocytic cells for application in cartilage regeneration (22, 23, 42). Growth Factor Modulation Although adult MSCs of different tissue origin displayed intrinsic differences in their differentiation to certain lineages,

the differentiation capacity of MSCs can be modulated by the microenvironment (43). Compared to BM-MSCs, adipose tissue-derived MSCs (AT-MSCs) demonstrated reduced chondrogenesis, which in part may be due to the reduced endogenous expression of bone morphogenetic protein (BMP)-2, −4, and −6 mRNA and absence in expression of transforming growth factor (TGF)-β-receptor-I (44). This reduced chondrogenic capacity of AT-MSCs was only rescued by BMP-6 treatment that induced TGF-β-receptor-I expression and reversed by combined application of TGF-β and BMP-6, inducing a gene expression profile similar to the differentiated BM-MSCs (44). These findings undoubtedly suggest that MSCs derived from different tissue sources are likely to express distinct growth factor and receptor repertoire that determines the differentiation capabilities and responsiveness to growth factors. With more sources of MSCs being identified and isolated from different tissues, mapping of their growth factor and receptor repertoire would pave forward optimal chondrogenesis of individual tissue-specific MSCs for cartilage repair (45). Pre-Treatment of MSCs for Transplantation Although MSCs are easily sourced, the cells do not retain their proliferative and multi-lineage differentiation capabilities after prolonged ex vivo expansion. Pre-treatment and/or preconditioning strategies are frequently required to improve the therapeutic potential of the expanded cells for subsequent cartilage formation. Among these strategies, pre-treatment of MSCs with fibroblast growth factor-2 (FGF-2) is an attractive approach and has been shown to enhance proliferation and differentiation by


diverse mechanisms including selection of a cell population with inherent chondrogenic potential and priming mechanism by regulating Sox9 gene expression (46). Hypoxic preconditioning of MSCs represents as another promising approach and has been shown in several studies to be an excellent way to expand a highly clonogenic population with superior proliferation and differentiation capacities (47, 48). However, there are also studies reporting that prior exposure of MSCs to hypoxia during expansion may be detrimental to subsequent chondrogenesis (47), and only exposure to hypoxia during differentiation would enhance chondrogenesis (49). These disparate findings are likely to be due to the cell source, state of the cells, and modes and durations of exposure to hypoxia. Further studies would need to optimize the hypoxia exposure time to maximize the MSC survival and investigate the different degrees and modes of hypoxia such as brief exposure during cell expansion on subsequent cell functions including proliferation, differentiation and matrix biosynthesis. Other biophysical approaches such as low intensity ultrasound stimulation has also demonstrated efficacy in enhancing chondrogenic potential of MSCs for subsequent cartilage formation in a subcutaneous implantation model (50). Stem Cell-Derived Secretome and Paracrine Mechanisms Although many earlier studies on the use of MSCs for tissue repair were based on the hypothesis that transplanted MSCs differentiate into target cells and replace the damaged tissue, increasing evidence is suggesting that MSCs exert the reparative/regenerative efficacy by the secretion of trophic factors to orchestrate endogenous cell response and mediate tissue regeneration (25, 26). Increasingly, there are supporting studies showing the trophic effects of MSCs in improving tissue regeneration and functionality without the need of cellular differentiation and/or long-term engraftment (51–53). Of note, MSCs have been shown to improve gain-of-functions in brain stoked rats without differentiating into any neuronal cell type (53), and stimulating vascular regeneration through paracrine secretion of angiogenic factors including FGF-2 and vascular endothelial growth factor (VEGF) (52). In the context of cartilage regeneration, MSCs have shown to differentiate to chondrocytes in the defect site, but often exhibited very low cell survival when the tissue is being repaired (54). It remains unclear if MSCs need to engraft at the site of injury or can exert their paracrine effects to mediate repair. Earlier studies utilizing co-culture of MSCs and chondrocytes have further provided evidence of the interactions between MSCs and chondrocytes, where chondrocytes induced differentiation of MSCs through secretion of morphogenetic factors that have yet to be fully characterized (55). Recent studies investigating the effects of MSC coculture on chondrocyte differentiation and matrix

synthesis have yielded conflicting results. Wu et al. demonstrated that hBM-MSCs exert trophic effects in chondrogenesis by promoting proliferation and ECM formation of chondrocytes (56), and MSCs from different sources including bone marrow, adipose tissue and synovial membrane seem to exert these trophic effects, irrespective of the tissue origins and culture conditions (57, 58). On the other hand, there are also studies describing the inhibitory effects of MSC trophic factors on chondrocytes, where MSCs significantly downregulate chondrocyte differentiation and matrix deposition (59, 60). These disparate findings suggest that the effects of MSC trophic factors are context-dependent and are likely to compose of both positive and negative factors that need to be further determined in order to achieve optimal trophic effects of MSCs for cartilage repair. Therefore, to better understand the role of MSC secretome in cartilage regeneration, in vitro tissue-specific and diseasespecific models would be helpful. Despite the fact that human urine-derived stem cells (USCs) could release trophic factors, including but not limited to growth factors and immunomodulatory factors, Pei et al. (61) found that human USCs had no ability to differentiate into chondrocytes; however, the decellularized stem cell matrix (DSCM) deposited by human USCs could recharge senescent hBM-MSCs toward chondrogenic differentiation, indicative of the distinct trophic effects from stem cell matrix on chondrogenesis. In another study, Jeong et al. showed that human umbilical cord bloodderived MSCs (hUCB-MSCs) secreted different profiles of proteins under treatment of synovial fluids harvested from OA and fracture patients. In that study, thrombospondin-2 (TSP-2) was specifically expressed by MSCs treated with OA synovial fluid and demonstrated to be a potent trophic factor of hUCBMSCs in promoting cartilage regeneration in the fullthickness osteochondral defect rabbit model (62). Looking forward, further studies exploring coculture of MSCs and chondrocytes are expected to provide novel insights into the complex interactions between MSCs and chondrocytes and the responses of MSCs in different cartilage microenvironments in both normal and injury/disease states. Optimizing MSC Secretome for Cartilage Therapeutic Applications At this juncture, it is important to note that these MSC paracrine functions are still not yet optimized in laboratory and in pre-clinical studies to ensure maximum therapeutic potential. In recent years, efforts in elucidating the paracrine mediators of MSCs have been focusing on dissecting the components of MSC secretome by proteomic approaches so as to identify the secreted signaling molecules, trophic factors and microvesicles that are mediating the immunomodulatory and regenerative properties of MSCs in tissue regeneration (63). Much of our current understanding of the MSC secretome


components is based on secretome analysis of primary MSCs that were cultured for a short time frame under serum-free or defined serum conditions. However, the secretome profiles of MSCs are highly-dependent on the microenvironment, and MSCs within different microenvironments would express unique secretome profiles. Several approaches including physiological (hypoxia) (47, 48), pharmacological (small molecules) (64, 65), cytokine/growth factors preconditioning (66, 67), cell-cell interactions (68, 69) and genetic manipulation have been employed in attempt to understand MSC response to its microenvironment and to better control the MSC secretome in its composition and sustainability following transplantation. Of note, it has been reported in several studies that MSC secretome changes significantly in inflammatory conditions that closely mimic that of the in vivo tissue environment upon injury. For example, it has been shown that under proinflammatory conditions, such as in the presence of tumor necrosis factor-α (TNF-α), hAT-MSCs exhibited upregulated expression of proteins in the secretome including interleukin6 (IL-6), IL-8 and monocyte chemotactic protein-1 (MCP-1) that are involved in migration of monocytes during inflammation (66). Besides external stimuli, recent studies indicate that MSC secretome is dynamic and changes during the course of chondrogenesis. For instance, it has been shown in equine BM-MSCs that the secretome is modulated by the state of cellular differentiation, where the level of angiogenic factors reduces when the MSCs differentiate along chondrogenic and osteogenic lineages (70). The downregulation of angiogenic factors by the MSCs during chondrogenic differentiation may be beneficial for maintaining the avascular cartilage following tissue regeneration (71). Moving ahead, it would be important to examine the dynamic secretome expression in vitro under conditions that model several relevant tissue-specific in vivo microenvironments. Findings obtained from these studies are expected to provide new insights into the role of MSCs and its secretome in cartilage regeneration which can subsequently be exploited as preconditioning and/or supplementary strategies at specific stages to improve the paracrine functioning of MSCs for cartilage repair. Positive versus Negative Effects In the context of cartilage regeneration, the array of potential therapeutic mechanisms offered by MSC secretome components spans anti-inflammatory responses (anti-fibrosis and suppression of inflammatory cells), chemoattraction and endogenous regeneration (activation of resident MSCs and cartilage stem cells), proliferation and differentiation (chondrocytes), and matrix deposition and remodeling (ECM alteration and neotissue formation). A cocktail of trophic factors would be needed to orchestrate the above

processes in a specific temporal manner in order to achieve full tissue regeneration. Indeed, the secretome of MSCs is highly-complex and intensive research is ongoing to identify the secretome components to better understand the therapeutic properties of MSCs. The secretome comprises of a wide range of trophic factors [reviewed in (25, 72)], ECM molecules (72), and microvesicles [reviewed in (73, 74)] with diverse therapeutic effects that have yet to be fully explored for cartilage regeneration. Currently, the role of MSC secretome in cartilage regeneration and the exact secretome components/factors mediating the dynamic process have yet to be fully elucidated. To date, a number of factors have been identified in the MSC secretome to be involved in cartilage regeneration including chondrogenic factors such as TGF-β (75–77), insulin growth factor-1 (IGF-1) (77, 78) and TSP-2 (62), chemotactic factors such as stromal-derived factor-1 (SDF-1) (79), immunomodulatory factors including TGF-β (80) and prostaglandin E2 (PGE-2) (81), and anti-angiogenic factors including endostatin and TSP-1 (82). The list of trophic factors is still expanding as we begin to understand the role of MSCs in cartilage repair/regeneration. Identifying the positive factors that promote tissue regeneration as opposed to those negative factors that induce inflammation and fibrosis would undoubtedly determine the quality of repair/regeneration. Therefore, identifying the key MSC-secreted factors and their functional roles in cartilage regeneration would likely advance the rational design of the next-generation stem cell-based therapeutics. Stem Cell-derived Extracellular Matrix and its Therapeutic Potential ECM represents an integral component of the native stem cell microenvironment and is involved not only in providing structural support but also controls self-renewal, proliferation and differentiation by regulation of cell-matrix interactions and growth factor signaling. Of note, decellularized ECM derived from MSCs and fibroblasts have demonstrated efficacy in maintaining the self-renewal and pluripotency of hESCs (83, 84). Separately, the decellularized ECM derived from MSCs has also recently been shown to be able to maintain the differentiation potential of MSCs during expansion and to restore the activities of aging MSCs (85). In the context of cartilage regeneration, DSCM deposited by SDSCs were able to enhance the proliferation and chondrogenic potential of SDSCs, and conferring these cells an enhanced capacity in repair of partial-thickness cartilage defects in a mini-pig model (86). Interestingly, the SDSCs cultured on DSCM displayed enhanced differentiation potential for chondrogenesis, but not for osteogenesis and adipogenesis (87). Furthermore, the DSCM has also enabled


‘rejuvenation’ of mature chondrocytes (88), and nucleus pulposus cells (89, 90) by imparting enhanced proliferation and differentiation, as well as survival under oxidative stress (91). These findings support the role of DSCM in providing an in vitro tissue-specific niche-like microenvironment for propagation of stem cells and chondrocytes whilst maintaining the potential for lineage-specific differentiation. Although the underlying mechanisms remained complex and elusive, it is likely that DSCM would augment cell-matrix interactions, sequester growth factors and enhance growth factor signaling, as well as influence the expression of growth factor receptor profiles of the cultured cells (92). Optimizing Decellularized Stem Cell ECM for Cartilage Therapeutic Applications Decellularization techniques have been used for many years to isolate ECM from cells in culture, tissues or organs (93). The goal of decellularization protocols is as always to efficiently remove the cellular materials while minimizing any adverse effect on the composition, biological activity and structural integrity of the remaining ECM. The composition and bioactivity of the DSCM have been shown to largely depend on the stem cell source and culture conditions, including the additives (e.g., ascorbic acid, dextran sulfate and Ficoll®) to induce matrix deposition (94) and oxygen tension (43, 89). Indeed, the DSCM is a complex milieu of matrix molecules, and intensive research is ongoing to identify the individual matrix components to better understand their roles in stem cell renewal and differentiation. Among them, specific matrix molecules such as the collagen IV, fibronectin and laminin-1 when used as coatings have been shown to enhance self-renewal and differentiation of hBM-MSCs, but laminin-5 suppressed subsequent chondrogenic differentiation (95). Other matrix molecules such heparan sulfate has also been shown to enhance the self-renewal and differentiation of hBM-MSCs when used as a media supplement (96). In this aspect, DSCM, being a complex milieu of matrix molecules, is likely to comprise of mixture of matrix molecules with diverse effects (positive vs. negative) on growth factor signaling and cellular processes including cell adhesion, migration, proliferation and differentiation. A recent study by Li et al. found that expansion on ECM deposited by fetal SDSCs (FE) was superior to ECM deposited by adult SDSCs (AE) in promoting cell proliferation and chondrogenic potential (97). Unique proteins in FE, such as fibrillin-2, tenascin, and versican core protein, might be responsible for the rejuvenation effect of FE while advantageous proteins in AE, such as elastin, fibulin-6, periostin, TSP-1, and TGF-β1, might contribute to differentiation more than to proliferation. Proteomics data further showed that fibrillar collagens shifted into the “insoluble” fraction for AE but not in FE; in contrast, FE contained a fair amount of clusterin but none was detected

in AE. Intriguingly, no laminin was measured in either DSCM despite plenty of fibronectin and type I collagen, which might be responsible for enhanced chondrogenic potential of DSCM expanded SDSCs (95). Identifying the specific bioactive ECM molecules that selectively promote specific functions including MSC chondrogenesis and/or activation of endogenous MSCs is likely to impact the cartilage repair. Nevertheless, it is likely that a complex milieu of matrix molecules in DSCM is needed to mimic the native stem cell niche to orchestrate cellular processes and mediate tissue regeneration following injury. Looking forward, it will be important to determine the conditions that would influence the composition of the ECM deposited from the stem cells, and optimize the condition to maximize stem cell renewal, proliferation and lineage-specific differentiation for a specific application (43). Furthermore, identifying specific therapeutic matrix molecules within the DSCM and elucidating their functional roles in cartilage regeneration would enable the translation of DSCM as an alternative stem cell-based therapeutics for cartilage regeneration (97). Stem Cell-Biomaterials Interactions for Cartilage Regeneration With the paradigm shift from direct transplantation of stem cells to the use of stem cell-derived matrix and secretome as therapeutic tools/agents, new biomaterial delivery systems are being developed to harness these unique stem cell properties (98). New design considerations would need to also consider the stimulation of MSC secretion of trophic factors and matrix molecules in addition to supporting stem cell growth and differentiation. Of note, it has been shown that material properties including substrate compliance can influence the MSC paracrine activity (99). For example, hBM-MSCs grown on hydrogel substrates with stiffness matching that of hard and soft tissue secreted disparate levels of VEGF, IL-8, and uPA for up to 14 days (99). Here we discuss how biomaterials can be used to influence MSC secretion of trophic factors and matrix molecules, which in turn, the knowledge obtained from understanding the cell-material interactions can be utilized in the rational design of the next-generation biomaterials. Stimulation of MSC Secretion of Trophic Factors In recent years, biomaterials not only serve to deliver stem cells to the site of injury, but also serve as a moldable platform for incorporation of microenvironmental cues to influence stem cell fate and functions for cartilage tissue engineering (100). Criteria for design of biomaterial scaffold for stem cell delivery has broadened from basic requirements of biocompatibility and biodegradability to ability for control of stem cell fate and functions including paracrine activity of MSCs to


augment MSC therapeutic potential for tissue regeneration. In this aspect, biomaterials are likely to have the added advantage to control MSC autocrine and paracrine functioning for tissue regeneration, by influencing the spectrum of trophic factors that are being secreted, enabling the sustained and localized release of the factors, and last but not least, facilitating the homing of the endogenous MSCs and influencing them in the same way. Apart from pre-conditioning/pre-treatment strategies that have been discussed earlier to influence the trophic functions of MSCs, biomaterials-based approaches represent an attractive alternative strategy for in situ stimulation of MSC secretion of trophic factors while serving as a delivery system for both the MSCs and their trophic factors. In this aspect, several biomaterials possess inherent properties that are likely to impact the MSC secretion of trophic factors. For example, it has been shown that the composition of hydroxyapatite/ Poly(Lactide-Co-Glycolide) PLG composite scaffolds stimulate MSC osteogenesis partly by enhancing endogenous secretion of VEGF with higher HA: PLG ratios (101). For cartilage regeneration, several bioactive natural polymers including type II collagen (102, 103) and hyaluronic acid (104, 105) have been commonly used for cartilage tissue engineering and demonstrated to enhance chondrogenesis, but any underlying effects on MSC trophic secretions remain unclear. Alternatively, specific peptides and/or growth factors can be incorporated to influence the bioactivity of the biomaterial scaffold in stimulating the MSC secretion of trophic factors. For instance, Jose et al., reported an alginate hydrogel system that was decorated with Gly-His-Lys (GHK), a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential (106). In that study, hBM-MSCs responded to GHK-modified gels by secreting increased concentrations of VEGF and FGF-2, compared to unmodified gels (106). In another study, a fibronectin-based peptide GlyArg-Gly-Asp-Ser (GRGDS) was conjugated to gellan gum hydrogel and shown to modulate the BM-MSC secretome to better support the survival and differentiation of neurons (107). Looking forward, determining the effects of cellmaterials interactions in modulation of MSC secretome during chondrogenesis would be an important design criteria of carrier systems for stem cell delivery in cartilage regeneration. Incorporation of MSC-derived ECM into Material Design Decellularized tissue matrices have been used directly as scaffolds (108, 109) or incorporated into another biomaterial (110) to functionalize and reconstruct the specific tissue microenvironment for tissue engineering and regeneration. Recent advances in decellularization techniques and material processing have enabled derivation of matrix materials from decellularized matrices that can form hydrogels under specific conditions (111, 112). As discussed in earlier section, DSCM

can potentially serve as a complex milieu of matrix molecules to mimic the native stem cell niche to orchestrate cellular processes and facilitate tissue regeneration during tissue injury. To date, DSCM have been used as substrate for stem cell expansion and as a scaffold for stem cell delivery and cartilage tissue engineering (86, 97). From the materials perspective, DSCM represents as a candidate cell-derived bioproduct that potentially can serve as a multifunctional biomaterial system for regenerative applications. However, DSCM is often soft after isolation and lacks mechanical strength needed for handling. Techniques such as electrospinning (113, 114) and various means of crosslinking can be employed to alter the matrix architecture and stiffness that can facilitate ease of handling as well as utilized as means to modulate the cell fate and functions (104). The incorporation of DSCM into another biomaterial either by blending or surface coating is another possible strategy to create a composite biomaterial system that can harness the biochemical and mechanical properties of both materials. Looking forward, through understanding of the underlying mechanisms that control the composition and bioactivity of the DSCM, more effective pre-treatment regime could be developed to maximize the therapeutic effects. Furthermore, with identification of specific therapeutic matrix molecules, a more targeted approach coupled with material-based controlled release strategies (115, 116) will likely be useful to enhance the overall therapeutic benefit. Perspectives for Stem Cell-based Therapeutics for Cartilage Regeneration The discovery of stem cells and their potential in regenerative medicine has opened new avenues and possibilities for treatment of cartilage injuries and related diseases. Notably, stem cell therapies with the use of MSCs in direct transplantation for treatment of cartilage defects have proven its clinical feasibility and efficacy (17). Moving forward, the study of MSC secretome and ECM is likely to provide new understanding of MSC-based cartilage repair and the underlying mechanisms, and identify novel factors and molecules that are mediating cartilage regeneration, thus paving the potential future development of ‘cell-free’ therapeutics. Currently, common approaches used for regulating cell secretome and ECM include preconditioning with hypoxia and/or small molecules, and enhanced cell-cell interactions through spheroid formation, as earlier described. However, such approaches often suffer lack of specificity, complexity and relevance to a specific disease. Thus, additional tissuespecific and/or disease-specific models (117) would be necessary to better interrogate the MSC response to specific tissue injury and/or disease to enable targeted pre-conditioning for maximum therapeutic benefit. As MSCs from different


sources would have inherent differences in their secretome and ECM profile and bioactivity, a systematic examination of various MSCs is required to obtain an optimal and reproducible cell source, which will be central to the harnessing of secretome and matrix factors in sufficient quantity and quality to support clinical testing and applications. Looking into the future, the biomaterials offer the potential for enhanced control of cell fate and functions including the biosynthesis of trophic factors and matrix molecules, enabling the sustained and localized release of the factors, activating the homing of the endogenous MSCs, and facilitating new tissue formation through remodeling. With the advances of new bioengineering, biochemical and genetic tools, more therapeutic factors and matrix molecules are likely to be identified and harnessed from stem cells and utilized as the next-generation stem cell-based therapeutics for treatment of injuries/diseases with greater efficacy and specificity.

Acknowledgments This work was partially supported by grants (R221000068720 and R221000070733) from National University of Singapore, National University Healthcare System, and Ministry of Education, Singapore. Disclosure The authors indicate no potential conflicts of interest.

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