Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Double-Network Hydrogel Scaffolds for Cartilage Repair

Autologous chondrocyte implantation (ACI) therapy has most recently been used to treat articular cartilage defects. A next generation of ACI therapy is now being explored using autologous mesenchymal stem cells (MSCs) derived from the patient’s bone marrow and incorporated into hydrogel biomaterials. We investigated an innovative hydrogel combination of covalently crosslinked PEGylated fibrinogen (PF) and hyaluronic acid (HA) with guest-host (GH) interactions.

Bone marrow-derived human MSCs (hMSCs) were seeded into different formulations of double-network hydrogel and maintained in 3D culture for 21 days under chondrogenic conditions. Each formulation contained 2% GH component in 8 mg/mL PF. To create a variety of viscoelastic hydrogels with different mechanical properties, we added different amounts of polyethylene glycol diacrylate (PEG-DA): 0.2%, 0.5%, 1% and 2%. We examined cell adaptability and viability by performing Live/Dead assay, using calcein/ethi-dium staining. Moreover, dimethylmethylene blue (DMMB) assay quantified sulfated-glycosaminoglycan (GAGs) content to evaluate chondrogenic differentiation.

Live/dead staining with calcein (green) and ethidium (red) showed that cells were able to create a very well-defined interconnected network, with a relatively high number of viable cells within the hydrogels. Cell differentiation process, partly indicated by GAG production, was normalized by the number of live cells. Two groups, 0.5% and 2% PEG-DA, showed an increase in the mass of GAGs as function of time. Early stage immunostaining were performed to validate this hypothesis.

Overall, the current results illustrate the cell adaptability to this state-of-the-art viscoelastic hydrogel. Along with encouraging viability results, GAG quantification indicates the starting of a successful chondrogenic differentiation pathway.