Silk Fibroin Scaffold-Based 3D Co-Culture Model for Modulation of Chondrogenesis without Hypertrophy via Reciprocal Cross-talk and Paracrine Signaling.

In this study, the effect of cellular cross-talk on modulation of chondrogenesis and hypertrophy while minimizing the usage of articular chondrocytes (ACs) has been investigated. Herein, co-culture of ACs with adipose-derived human mesenchymal stem cells (ADhMSCs) was employed for cross-talk within silk fibroin (SF)-based three-dimensional (3D) scaffolds. The co-culture model was developed by co-culturing four different ratios of ADhMSCs to ACs: 1:0, 3:1, 1:1, and 0:1 on porous 3D SF scaffolds for 21 days. The co-culture groups were cultured in defined media without adding any exogenous growth factors except the monoculture group, ADhMSC-only controls. The co-cultured constructs indicated significantly higher cellular viability and proliferation than the control monoculture groups. The supernatants of co-culture groups indicated significantly higher levels of TGF-β1 and IL-10, which confirmed the production of the morphogens/signaling molecules by chondrocytes for induction of ADhMSCs differentiation toward the chondrogenic phenotype. Biochemical assays indicated enhanced accumulation of sulfated glycosaminoglycans, collagen, and high DNA content along with high cellularity in co-culture groups than chondrocyte-only controls. Co-culture groups revealed synergistic interactions between cells as indicated by the interaction index value ranging from 2-3. Furthermore, upregulation of putative chondrogenic markers-aggrecan, sox-9, and collagen II, and significantly reduced expression of hypertrophic genes-collagen type X and MMP-13 was revealed in co-culture constructs. Histological and immunohistochemical staining also demonstrated even distribution and deposition of ECM in co-cultured constructs. Taken together, this work presents the potential of the developed 3D co-culture model toward modulation of chondrogenesis and hypertrophy via 3D microenvironment induced by physicochemical and biological properties of SF scaffolds, synergistic interactions between cells, and paracrine signaling in the co-culture system.