Mechanical loading inhibits hypertrophy in chondrogenically differentiating hMSCs within a biomimetic hydrogel

Three dimensional hydrogels are a promising vehicle for delivery of adult human bone-marrow derived mesenchymal stem cells (hMSCs) for cartilage tissue engineering. One of the challenges with using this cell type is the default pathway is terminal differentiation, a hypertrophic phenotype and precursor to endochondral ossification. We hypothesized that a synthetic hydrogel consisting of extracellular matrix (ECM) analogs derived from cartilage when combined with dynamic loading provides physiochemical cues for achieving a stable chondrogenic phenotype. Hydrogels were formed from crosslinked poly(ethylene glycol) as the base chemistry and to which (meth) acrylate functionalized ECM analogs of RGD (cell adhesion peptide) and chondroitin sulfate (ChS, a negatively charged glycosaminoglycan) were introduced. Bone-marrow derived hMSCs from three donors were encapsulated in the hydrogels and cultured under free swelling conditions or under dynamic compressive loading with 2.5 ng ml(-1) TGF-beta 3. hMSC differentiation was assessed by quantitative PCR and immunohistochemistry. Nine hydrogel formulations were initially screened containing 0, 0.1 or 1 mM RGD and 0, 1 or 2 wt% ChS. After 21 days, the 1% ChS and 0.1 mM RGD hydrogel had the highest collagen II gene expression, but this was accompanied by high collagen X gene expression. At the protein level, collagen II was detected in all formulations with ECM analogs, but minimally detectable in the hydrogel without ECM analogs. Collagen X protein was present in all formulations. The 0.1 mM RGD and 1% ChS formulation was selected and subjected to five loading regimes: no loading, 5% strain 0.3 Hz (1.5% s(-1)), 10% strain 0.3 Hz (3% s(-1)), 5% strain 1 Hz (5% s(-1)), and 10% strain 1 Hz (10% s(-1)). After 21 days, B70-90% of cells stained positive for collagen II protein regardless of the culture condition. On the contrary, only B20-30% of cells stained positive for collagen X protein under 3 and 5% s(-1) loading conditions, which was accompanied by minimal staining for RunX2. The other culture conditions had more cells staining positive for collagen X (40-60%) and was accompanied by positive staining for RunX2. In summary, a cartilage-like biomimetic hydrogel supports chondrogenesis of hMSCs, but dynamic loading only under select strain rates is able to inhibit hypertrophy.