249. CHONDROCYTE DIFFERENTIATION OF HUMAN MESENCHYMATOUS STEM CELLS CULTIVATED ON COLLAGEN SPONGE: PERSPECTIVES FOR GUIDED CELL THERAPY FOR CARTILAGE

Abstract

Purpose of the study: Monolayer cultures of chondrocytes multiply and rapidly lose their chondrocyte phenotype, limiting their potential for tissue engineering. Mesenchymatous stem cells can preserve their phenotypic characteristics after several monolayer passages, offering a promising alternative for cartilage repair. The purpose of this work was to study the influence of transforming growth factor beta-1 (TGF-beta1) and bone morphogenic protein-2 (BMP2) and/or culture supplements (hyaluronic acid) on matrix synthesis and chondrocyte differentiation of human mesenchymatous stem cells (MSC) cultured on collagen sponges.

Material and methods: MSC were isolated from bone marrow harvested during hip arthroplsty. At the third passage in monolayer culture, the MSC were reseeded on collagen sponges and cultured in vitro for 28 days under seven differ conditions: insulin transferrin selenium (ITS), foetal calf serum (FCS), ITS+TGFbeta1, ITS+ hyaluronate, ITS+TGFbeta1+hyaluronate, ITS+TGFbeta1+BMP2, ITS +TGFbeta1+BMP2+hyaluronate. The phenotypic evolution was followed using the expression of different genes of interest with PCRq (collagen2, collagen1, collagen3, collagen10, agrecanne, versicanne, COMP, Sox9). Synthesis of matrix material was assessed histologically and immunohistochemically.

Results: Used alone, hyaluronic acid did not trigger chondrocyte differentiation of MSC. For the additives FCS, ITS, or hyaluronate, the synthesis of matrix material in the sponge was weak and poor in major constituents of cartilage. Conversely, the other conditions in presence of TGFbeta1±BMP2 induced important expression of collagen2, agrecanne and COMP as well as increased matrix synthesis with a strong content in proteoglycans and collagen.

Discussion: The usefulness of MSC is growing due to their pluripotent characteristics. The conditions leading to their differentiation into the chondrocyte phenotype remains a subject of discussion. Our results show the particular importance of TGFbeta1 in the process of differentiation.

Conclusion: Chondrogenic differentiation of MSC cultured in collagen sponges as well as the synthesis of the cartilaginous matrix requires the presence of TGFbeta1 in the culture medium and to a lesser extent BMP2. These results suggest the perspective of using MSC for guided cell therapy targeting cartilage.