Modulating stem cell-chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels

Stem cells can contribute to cartilage repair either directly through chondrogenic differentiation or indirectly through paracrine signaling. Using a 3D co-culture model, we have recently reported that adipose-derived stem cells (ADSCs) can catalyze cartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in biomimetic hydrogels. However, how matrix cues influence such catalyzed cartilage formation remains unknown. To answer this question, ADSCs and NChons were co-encapsulated in 39 combinatorial hydrogel compositions with decoupled biochemical and mechanical properties. Methacrylated extracellular matrix (ECM) molecules including chondroitin sulfate, hyaluronic acid and heparan sulfate were incorporated at varying concentrations (0.5%, 1.25%, 2.5% and 5%) (w/v). Mechanical testing confirmed that hydrogel stiffness was largely decoupled from ECM cues (15 kPa, 40 kPa and 100 kPa). The biochemical assay and histology results showed that the type of ECM cue played a dominant role in modulating catalyzed cartilage formation, while varying hydrogel stiffness and doses of ECM led to more modest changes. Both chondroitin sulfate and hyaluronic acid led to robust articular cartilage matrix deposition, as shown by the intense staining of aggrecan and type II collagen. In soft hydrogels (15 kPa), chondroitin sulfate led to the highest amount of sulfated glycosaminoglycan deposition and increased compressive moduli. In contrast, heparan sulfate promoted type I collagen deposition, an undesirable fibrocartilage phenotype, and increasing heparan sulfate decreased cell proliferation and ECM deposition. Findings from the present study may guide the optimal scaffold design to maximize the synergistic cartilage formation using mixed cell populations.