Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation.

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p â€‹= â€‹0.390) and led to a higher migration score ex vivo (p â€‹< â€‹0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p â€‹= â€‹0.031) and ex vivo (p â€‹< â€‹0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p â€‹< â€‹0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used.The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application.

Senescence during early differentiation reduced the chondrogenic differentiation capacity of mesenchymal progenitor cells.

Introduction: Mesenchymal stromal/progenitor cells (MSCs) are promising for cartilage cell-based therapies due to their chondrogenic differentiation capacity. However, MSCs can become senescent during in vitro expansion, a state characterized by stable cell cycle arrest, metabolic alterations, and substantial changes in the gene expression and secretory profile of the cell. In this study, we aimed to investigate how senescence and the senescence-associated secretory phenotype (SASP) affect chondrogenic differentiation of MSCs. Methods: To study the effect of senescence, we exposed MSCs to gamma irradiation during expansion or during chondrogenic differentiation (the pellet culture). Western blot analysis was used to evaluate MSCs response to the chondrogenic inductor TGF-ß. Results: When senescence was induced during expansion or at day 7 of chondrogenic differentiation, we observed a significant reduction in the cartilage matrix. Interestingly, when senescence was induced at day 14 of differentiation, chondrogenesis was not significantly altered. Moreover, exposing chondrogenic pellets to the medium conditioned by senescent pellets had no significant effect on the expression of anabolic or catabolic cartilage markers, suggesting a neglectable paracrine effect of senescence on cartilage generation in our model. Finally, we show that senescent MSCs showed lower phosphorylated SMAD2 levels after TGFß1 stimulation than control MSCs. Conclusion: Overall, these results suggest that the occurrence of senescence in MSCs during expansion or early differentiation could be detrimental for cartilage tissue engineering.

A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

An integrated in silico-in vitro approach for identifying therapeutic targets against osteoarthritis.

BACKGROUND: Without the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes, while in silico modeling can help unravel that complexity. In this study, we aim to develop a virtual articular chondrocyte to guide experiments in order to rationalize the identification of potential drug targets via screening of combination therapies through computational modeling and simulations. RESULTS: We developed a signal transduction network model using knowledge-based and data-driven (machine learning) modeling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions potentially affecting the hypertrophic switch. A selection of promising combinations was further tested in a murine cell line and primary human chondrocytes, which notably highlighted a previously unreported synergistic effect between the protein kinase A and the fibroblast growth factor receptor 1. CONCLUSIONS: Here, we provide a virtual articular chondrocyte in the form of a signal transduction interactive knowledge base and of an executable computational model. Our in silico-in vitro strategy opens new routes for developing osteoarthritis targeting therapies by refining the early stages of drug target discovery.

WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells that restricts their expansion.

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.

Intra-articular injection of triamcinolone acetonide sustains macrophage levels and aggravates osteophytosis during degenerative joint disease in mice.

BACKGROUND AND PURPOSE: Corticosteroids such as triamcinolone acetonide (TAA) are potent drugs administered intra-articularly as an anti-inflammatory therapy to relieve pain associated with osteoarthritis (OA). However, the ability of early TAA intervention to mitigate OA progression and modulate immune cell subsets remains unclear. Here, we sought to understand the effect of early intra-articular injection of TAA on OA progression, local macrophages, and peripheral blood monocytes. EXPERIMENTAL APPROACH: Degenerative joint disease was induced by intra-articular injection of collagenase into the knee joint of male C57BL/6 mice. After 1 week, TAA or saline was injected intra-articularly. Blood was taken throughout the study to analyse monocyte subsets. Mice were killed at days 14 and 56 post-induction of collagenase-induced OA (CiOA) to examine synovial macrophages and structural OA features. KEY RESULTS: The percentage of macrophages relative to total live cells present within knee joints was increased in collagenase- compared with saline-injected knees at day 14 and was not altered by TAA treatment. However, at day 56, post-induction of CiOA, TAA-treated knees had increased levels of macrophages compared with the knees of untreated CiOA-mice. The distribution of monocyte subsets present in peripheral blood was not altered by TAA treatment during the development of CiOA. Osteophyte maturation was increased in TAA-injected knees at day 56. CONCLUSION AND IMPLICATIONS: Intra-articular injection of TAA increases long-term synovial macrophage numbers and osteophytosis. Our findings suggest that TAA accentuates the progression of osteoarthritis-associated features when applied to an acutely inflamed knee.

Effect of Inflammatory Signaling on Human Articular Chondrocyte Hypertrophy: Potential Involvement of Tissue Repair Macrophages.

OBJECTIVE: In osteoarthritis, chondrocytes tend to acquire a hypertrophic phenotype, which contributes to the modification of the extracellular matrix, resulting in permanent cartilage changes. In mouse chondrocytes, pro-inflammatory macrophages and pro-inflammatory cytokines have been shown to stimulate hypertrophy via the activation of the nuclear factor kappa B (NF-κB) pathway. Whether or not this also occurs in human chondrocytes remains unclear. We therefore aimed to investigate whether hypertrophy-like responses in human cartilage are driven mainly by intrinsic inflammatory signaling or shaped by specific macrophage populations. DESIGN: Human articular chondrocytes were cultured with pro-inflammatory cytokines or medium conditioned by defined macrophage subsets. Furthermore, the effect of inhibition of NF-κB-dependent gene expression was evaluated using the NF-κB inhibitor SC-514. Hypertrophy was assessed by measuring the transcription level of alkaline phosphatase (ALPL), type X collagen (COL10A1), Indian hedgehog (IHH), and runt-related transcription factor 2 (RUNX2). RESULTS: The expression of hypertrophic genes was not promoted in human chondrocytes by pro-inflammatory cytokines neither pro-inflammatory M(IFNγ + TNFα) macrophages. Inhibition of the NF-κB-dependent gene expression did not affect human articular chondrocyte hypertrophy. However, tissue repair M(IL4) macrophages induced hypertrophy by promoting the expression of COL10A1, RUNX2, and IHH. CONCLUSION: Intrinsic inflammatory signaling activation is not involved in the hypertrophic shift observed in human articular chondrocytes cultured in vitro. However, tissue repair macrophages may contribute to the onset of this detrimental phenotype in human osteoarthritic cartilage, given the effect observed in our experimental models.

Expansion and Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Human bone marrow-derived mesenchymal stem/stromal cells (BM-MSC) are adult multipotent progenitor cells that can be isolated from bone marrow. BM-MSCs have the ability to be expanded and differentiated into the chondrogenic lineage in vitro. Here we describe a standardized method to expand and chondrogenically differentiate human BM-MSCs, highlighting how to overcome technical challenges and indicating the most common readout parameters to evaluate the chondrogenic differentiation capacity.

Enhanced Chondrogenic Capacity of Mesenchymal Stem Cells After TNFα Pre-treatment.

Mesenchymal stem cells (MSCs) are promising cells to treat cartilage defects due to their chondrogenic differentiation potential. However, an inflammatory environment during differentiation, such as the presence of the cytokine TNFα, inhibits chondrogenesis and limits the clinical use of MSCs. On the other hand, it has been reported that exposure to TNFα during in vitro expansion can increase proliferation, migration, and the osteogenic capacity of MSCs and therefore can be beneficial for tissue regeneration. This indicates that the role of TNFα on MSCs may be dependent on the differentiation stage. To improve the chondrogenic capacity of MSCs in the presence of an inflamed environment, we aimed to determine the effect of TNFα on the chondrogenic differentiation capacity of MSCs. Here, we report that TNFα exposure during MSC expansion increased the chondrogenic differentiation capacity regardless of the presence of TNFα during chondrogenesis and that this effect of TNFα during expansion was reversed upon TNFα withdrawal. Interestingly, pre-treatment with another pro-inflammatory cytokine, IL-1ß, did not increase the chondrogenic capacity of MSCs indicating that the pro-chondrogenic effect is specific for TNFα. Finally, we show that TNFα pre-treatment increased the levels of SOX11 and active ß-catenin suggesting that these intracellular effectors may be useful targets to improve MSC-based cartilage repair. Overall, these results suggest that TNFα pre-treatment, by modulating SOX11 levels and WNT/ß-catenin signaling, could be used as a strategy to improve MSC-based cartilage repair.

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity.

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.

Identification of TGFß-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets.

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor ß (TGFß) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFß-related genes are regulated during murine OA and endochondral ossification. METHODS: A list of 580 TGFß-related genes, including TGFß signaling pathway components and TGFß-target genes, was generated. Regulation of these TGFß-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development). RESULTS: A total of 106 TGFß-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients. CONCLUSION: By focusing on TGFß-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.

Isolating Pediatric Mesenchymal Stem Cells with Enhanced Expansion and Differentiation Capabilities.

Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate pediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs, which adhere to MSC defining guidelines. After confirming immunophenotypic marker expression, we compared expansion rates, senescence, morphology, and trilineage differentiation of P-MSCs to A-MSCs for multiple donors. We found P-MSCs have faster in vitro replication, consistently show significantly lower senescence, and are capable of more reproducible multilineage differentiation than A-MSCs. We, therefore, believe P-MSCs are a promising candidate for use in research applications and potentially as part of an allogeneic therapeutic treatment.

Activin and Nodal Are Not Suitable Alternatives to TGFß for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Objective Previously, we demonstrated the importance of transforming growth factor-ß (TGFß)-activated SMAD2/3 signaling in chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). However, TGFß also signals via the SMAD1/5/9 pathway, which is known to induce terminal differentiation of BMSCs. In this study, we investigated whether other SMAD2/3-activating ligands, Activin and Nodal, can induce chondrogenic differentiation of BMSCs without inducing terminal differentiation. Design Activation of SMAD2/3 signaling and chondrogenesis were evaluated in human BMSCs ( N = 3 donors) stimulated with TGFß, Activin, or Nodal. SMAD2/3 activation was assessed by determining phosphorylated-SMAD2 (pSMAD2) protein levels and SMAD2/3-target gene expression of SERPINE1. Chondrogenesis was determined by ACAN and COL2A1 transcript analysis and histological examination of proteoglycans and collagen type II. Results Both Activin and TGFß enhanced pSMAD2 and SERPINE1 expression compared to the control condition without growth factors, demonstrating activated SMAD2/3 signaling. pSMAD2 and SERPINE1 had a higher level of expression following stimulation with TGFß than with Activin, while Nodal did not activate SMAD2/3 signaling. Of the 3 ligands tested, only TGFß induced chondrogenic differentiation as shown by strongly increased transcript levels of ACAN and COL2A1 and positive histological staining of proteoglycans and collagen type II. Conclusions Even with concentrations up to 25 times higher than that of TGFß, Activin and Nodal do not induce chondrogenic differentiation of BMSCs; thus, neither of the 2 ligands is an interesting alternative candidate for TGFß to induce chondrogenesis without terminal differentiation. To obtain stable cartilage formation by BMSCs, future studies should decipher how TGFß-induced terminal differentiation can be prevented.

Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-based cartilage repair.

The field of cartilage repair has exponentially been growing over the past decade. Here, we discuss the possibility to achieve satisfactory regeneration of articular cartilage by means of human mesenchymal stem cells (hMSCs) depleted of anti-chondrogenic factors and implanted in the site of injury. Different types of molecules including transcription factors, transcriptional co-regulators, secreted proteins, and microRNAs have recently been identified as negative modulators of chondroprogenitor differentiation and chondrocyte function. We review the current knowledge about these molecules as potential targets for gene knockdown strategies using RNA interference (RNAi) tools that allow the specific suppression of gene function. The critical issues regarding the optimization of the gene silencing approach as well as the delivery strategies are discussed. We anticipate that further development of these techniques will lead to the generation of implantable hMSCs with enhanced potential to regenerate articular cartilage damaged by injury, disease, or aging.

Dynamic Regulation of TWIST1 Expression During Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFß-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-ß (TGFß) receptor ALK5 is crucial for chondrogenesis induced by TGFß. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFß-induced chondrogenesis. Activation of TGFß signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFß signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFß-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFß-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFß-induced chondrogenesis of human BMSCs.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells.

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno-free micro-environment for the chondrogenesis of human bone marrow-derived mesenchymal stromal cells (BM-MSCs). The rhCII hydrogels were seeded with BM-MSCs and cultured in a xeno-free chondro-inductive medium for 14, 28 and 84 days. High-density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high-density pellets, a similar potential of matrix deposition was reached by the end of the 84-day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high-density pellets, but at the end of the 84-day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)-13, MMP-14 and MMP-8, i.e. extracellular collagen network-degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno-free platform for BM-MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright © 2015 John Wiley & Sons, Ltd.

Differential Effects of Small Molecule WNT Agonists on the Multilineage Differentiation Capacity of Human Mesenchymal Stem Cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies, but loss of expansion and differentiation potential in vitro limits their applicability. Recently we showed that WNT3A protein promoted MSC proliferation and enhanced their chondrogenic potential, while simultaneously suppressing the propensity of the cartilage to undergo hypertrophic maturation. Since WNT3A protein is costly and rapidly loses its activity in culture, we investigated the possibility of replacing it with cheaper commercially available WNT agonists, specifically lithium chloride (LiCl), CHIR99021 (CHIR), SKL2001, and AMBMP. Of these, we found that only CHIR and LiCl stimulated MSC proliferation. Moreover, CHIR enhanced the chondrogenic capacity of MSCs, whereas LiCl predominantly increased the osteo- and adipogenic capacity. The different WNT agonists also differentially impacted the surface marker profile of the MSCs, possibly explaining the observed differences. Moreover, CHIR suppressed the hypertrophic propensity of the MSC-derived cartilage after in vivo implantation to an extent approaching that of WNT3A protein. These results indicate that CHIR may be a promising alternative for WNT3A protein for certain applications of human bone marrow-derived MSCs.