Efficient isolation and chondrogenic differentiation of adult mesenchymal stem cells with fibrin microbeads and micronized collagen sponges.

Shainer, Reut; Gaberman, Elena; Levdansky, Lilia; Gorodetsky, Raphael

BACKGROUND Mesenchymal stem cells (MSCs) have been demonstrated to potentially undergo chondrogenic differentiation. We propose a new matrix for stem cell-based chondrogenesis using dense fibrin microbeads (FMBs) combined with grounded dehydrothermally crosslinked collagen sponges (micronized collagen). METHODS In this study, MSCs were isolated from bone marrow of transgenic green fluorescent protein C57/Bl mice by FMBs in high yield. After 48 h in slowly rotating suspension culture, micronized collagen was added. RESULTS The cells on the FMBs migrated to the collagen pieces and formed aggregates that developed into cartilage-like structures. Following chondrogenic differentiation, alcian blue staining and collagen type II immunohistochemistry demonstrated the presence of chondrocytes in the 3D structures. PCR for the expression of aggrecan and collagen type II genes supported these findings. The in vitro structures that formed were used for ectopic subdermal implantation in wild-type C57/Bl mice. However, the chondrogenic markers faded relative to the pre-implant in vitro structures. CONCLUSION We propose that FMBs with micronized collagen could serve as a simple technology for MSC isolation and chondrogenesis as a basis for implantation.