Study On Growth And Differentiation Of Mesenchymal Stem Cells In Alginate Beads As Well As Coculture With Articular Chondrocytes

It is intriguing to many researchers that how chondrocytes regulate the chondrogenesis of mesenchymal stem cells (MSCs) in cartilage tissue engineering. In the present work, we aimed to develop a coculture system for studying interactions between MSCs and articular chondrocytes. To this end, first, we isolated rabbit bone marrow MSCs (rMSCs) and rabbit articular chondrocytes (rACs) by using two different methods; second, we encapsulated MSCs in alginate hydrogel beads, focusing on addressing the impact of needle gauges on bead dimension and examining cell activity with MTT assay, survival with live/dead staining, histology by H&E staining, cellular distribution using optical microscope, metabolism activity through glucose and lactate metabolic analysis and cell recovery after alginate hydrogel dissolution; third, we applied Transwell-based culture system to develop an indirect coculture system for rMSCs and rACs at a specific cell number ratio of rACs:rMSCs=1:2in order to gain an in-depth understanding of how rACs would affect the chondrogenesis of MSCs.It was found that compared to the adherence selection, Ficoll density gradient centrifugation harvested rMSCs with a higher purity. These cells displayed a typical morphology with a spindle-like shape and were able to differentiate into osetoblasts, adipocytes and chondrocytes. rACs acquired via digestion of cartilage tissues using typeⅡ collagenase had high gene expression levels of Sox9, Aggrecan and COL2A1. To fabricate cell-laden alginate beads,22#needle performed best concerning cellular activity and bead formation. During a4-week culture period, rMSCs kept a round morphology in alginate hydrogel, only a trivial fraction died, cellular activity declined to68%of that on day0and cell recovery efficiency from beads remained above55%. Most importantly, we found that chondrogenic medium with1.25mg/mL of BS A better supported cellular activity and survival than that with1.25μg/mL. While co-culture promoted gene expression of Sox9and COL2A1in rMSCs on day7, GAG secretion within a period of4weeks was not enhanced accordingly.Collectively, this work isolated rMSCs and systemically elaborated their growth profile in alginate hydrogel beads. The concentration of BSA on growth of MSCs was explored for the first time and a feasible Transwell-based co-culture system was developed, which lay the foundation for future in-depth coculture study between MSCs and chondrocytes.