Introduction: Mesenchymal stem cells (MSCs) are a therapeutically relevant cell source for cartilage repair. In our study, we aim to investigate the effects of biochemical niche cues and mechanical stiffness on MSC chondrogenesis. Cartilage specific extracellular matrix (ECM) molecules chondroitin sulfate (CS) and heparan sulfate (HS) were added individually at varying dosages as biochemical niche cues within 3D hydrogels while mechanical stiffness was controlled by adding varying amounts of poly-(ethylene) glycerol dimethacrylate (PEGDMA) into hydrogels. Results from this study will facilitate elucidation of the optimal microenvironment for MSC chondrogenesis, as well as fundamental understanding of the effects of micro-environmental cues on stem cell chondrogenesis.
Materials and methods: Hydrogels were designed to contain varying dosages (0.5, 2, 5% (w/v)) of either methacrylated CS or HS as biochemical niche cues. The added CS or HS will contribute to the mechanical stiffness of hydrogels, thus concentration of PEGDMA was varied to achieve similar final mechanical stiffness of ~7kPa (soft groups) or ~35kPa (stiff groups) (Fig 1). PEGDMA was chosen to control the compressive modulus due to its bio-inert nature. All materials contain methacrylate groups to facilitate UV mediated photo-crosslinking. Passage 6 human MSCs (10M/ml) were mixed with hydrogel precursor solution, pipetted into cylindrical molds and then exposed to 365nm light for 3 minutes to induce photo-crosslinking. Hydrogels were cultured in chondrogenic media containing 10ng/ml of TGF-β3 for 21 days, before harvest. Outcome analysis include gene expression, biochemical assays (DNA, sGAG and hydroxyproline) and histology.
Results: Gene expression (collagen II and aggrecan) showed CS and HS increased chondrogenesis in soft hydrogels in a dose-dependent manner. However, no significant dose response was observed in stiff hydrogels (Fig 2A, B). MMP 13, which is associated with cell-mediated degradation, was upregulated in stiffer hydrogels in which cells are more constrained, and appeared to be largely independent of biochemical niche cues (Fig 2C). Specifically, biochemical niche cues dominated cellular responses in soft hydrogels but mechanical cues dominate cell fates in stiff hydrogels. The trend of biochemical analyses (DNA, sGAG, collagen) at day 21 agrees largely with gene expression data. MSCs secreted most neocartilage in soft hydrogels containing 5% (w/v) CS (Fig 2D-F). Collagen II immunostaining showed neocartilage was constrained in pericelluar regions in all groups except the 5% (w/v) CS soft hydrogel group, in which the secreted neocartilage was homogenously distributed within the hydrogel (Fig 2G). The homogenous distribution of secreted neocartilage in this group resulted in over 500 fold increase in mechanical stiffness compared to the acellular control. In contrast, other groups resulted in minimal changes in mechanical stiffness as compared to the acellular hydrogel (Fig 3).
Discussion: We have showed positive dose responses with ECM based biochemical niche cues in soft hydrogels. In particular, our results suggests that a soft hydrogel with a high concentration of ECM may be used to maximize neocartilage deposition and to achieve homogenous distribution.
NIH R01DE024772 (F.Y); California Institute for Regenerative Medicine (Grant #TR3-05569) (F. Y.); National Science Foundation CAREER award program (CBET-1351289, F.Y.); A*STAR PhD fellowship (T.W.)