Experimental study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes

Abstract

Chondrogenic microenvironments play a very important role in chondrogenesis of bone marrow stromal cells (BMSC). This study explored the feasibility of in vitro chondrogenesis by co-culture of BMSC and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSC and thus promote in vitro chondrogenesis of BMSC. Porcine BMSC and auricular chondrocytes were in vitro expanded respectively and then were mixed at a ratio of 8:2 (BMSC:chondrocyte). 200 microl mixed cells(5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 9 mm in diameter and 3 mm in thickness, as co-culture group. Chondrocytes and BMSC with the same cell number were seeded respectively onto the scaffolds as positive control (chondrocyte group) and negative control (BMSC group). 200 microl chondrocytes (1.0 x 10(7)/ml, equal to the chondrocyte number of co-culture group) alone were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results. Cells in all groups had fine adhesion to the scaffolds and could secrete extracellular matrix. In both co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed homogenous mature cartilage after 8 weeks of in vitro culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features, and abundant type II collagen was also detected by immunohistochemistry in both groups. The average wet weight and GAG content of co-culture group were both more than 80% of those of positive control group. In negative control group, however, the constructs shrunk gradually during in vitro culture and cartilage-like tissue could only be observed at the edge area of the construct. In low concentration chondrocyte group, the constructs also shrunk gradually during in vitro culture and the average wet weight was below 40% of that of the positive control group although histology showed a small amount cartilage formation. Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of BMSC and thus promote the in vitro chondrogenesis of BMSC.