STEM CELLS CATALYSE CARTILAGE FORMATION BY NEONATAL ARTICULAR CHONDROCYTES

Summary Statement This work raises the potential of utilizing stem cells to catalyze cartilage regeneration by a minimal number of neonatal chondrocytes via controlling cell distribution in 3D matrices, and may solve the challenge of scarce donor availability associated with cell-based therapy. Introduction Cartilage loss is a leading cause of disability among adults and represents a huge socio-economical burden. Allogeneic neonatal articular chondrocytes (NChons) is a promising cell source for cartilage regeneration because these cells are highly proliferative, immune-privileged, and readily produce abundant cartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinical application. Besides their ability to differentiate into different tissue types, stem cells may contribute to tissue regeneration through the secretion of paracrine factors. Here we examined the potential for using a minimal number of NChons to catalyze cartilage tissue formation by co-culturing them with adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels. Materials & Methods NChons were isolated from articular cartilage of a three-day old calf. Human adult ADSCs were expanded to passage 5. Cells were photo-encapsulated in a hydrogel consisting of 7% w/v poly(ethylene glycol diacrylate) and 3% w/v chondroitin sulfate-methacrylate. To examine the effects of different paracrine concentrations, NChons and ADSCs were co-cultured in three different co-culture models: 1) cells cultured with conditioned medium supplementation from the other cell type (CM), 2) bi-layered co-culture confining each cell type to its own layer (BI), and 3) mixed cell co-culture at different ratios (75C:25A, 50C:50A, 25C:75A, 10C:90A). Cell-hydrogel constructs were cultured for 3 weeks in chondrogenic medium with 10ng/ml TGF-β3 and analyzed for biochemical content (DNA, sulfated glycosaminoglycan (sGAG), and collagen) and immunostaining. Fluorescent cell membrane labeling was used to identify ADSCs in mixed co-culture. To quantify interaction synergy, the interaction index, defined as the measured biochemical content in the mixed co-culture normalised by the expected value based on cell ratio and the measured content in the controls, was calculated (2). Statistical significance (∗) was set to p Results At day 21, mixed co-culture with as low as 25% NChons led to higher cell number and cartilage matrix content than NChon control. ADSC control had significantly lower matrix content. In mixed co-culture, the interaction index for DNA, sGAG, and collagen increased with an increase in ADSC ratio, reaching up to 5–6 at 90% ADSCs. Immunostaining of collagen II revealed that mixed co-culture resulted in the formation of large cartilage nodules, and that nodule size increased with an increase ADSC ratio. Cell tracking showed that the labeled ADSCs always resided outside the cartilage noduless, indicating the cartilage nodules are composed entirely of NChons. Discussion & Conclusion In this study, we demonstrated the efficacy of harnessing the paracrine effects ADSCs to catalyze cartilage tissue formation by a small number of NChons in biomimetic hydrogels. The mild effects of CM and BI co-culture on cartilage tissue formation along with the increase in interaction synergy with ADSC ratio in mixed co-culture highlighted the importance of using 3D scaffolds to probe cell-cell interactions in a spatially controlled manner. Such strategy significantly reduces the number of NChons needed, which may accelerate the translation of NChon for cartilage repair by alleviating donor scarcity limitation, and may be broadly applicable to regenerating other tissue types.