Development of a 3D Type I Collagen Assay to Evaluate Chondrogenesis of Canine Bone Marrow-Derived Mesenchymal Stem Cells

Introduction: Chondrogenic differentiation of canine MSCs (cMSCs) has been described using the classic micromass technique. However, cMSCs appear to respond inconsistently using this method. The objectives of this study were (1) to develop a collagen-based 3D serum-free system to facilitate consistent cMSC chondrogenic cultures; (2) to qualitatively and quantitatively assess the effect of various chondrogenic media on cMSC chondrogenesis. Materials and Methods: cMSCs were polymerized in 100 µL of Type I Collagen gel (5 mg/mL) at 10 7 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology after 21 days in 10 various chondrogenic media. cMSCs were next cultured with four promising media and quantitatively assessed for early cytotoxicity (LDH assay) and chondro- versus osteogenesis (GAG content; qPCR for Col I, II, X, Sox 9, Aggrecan, Osteocalcin and Osterix). Results: In the presence of dexamethasone and TGF-β3, both BMP-2 and bFGF generated larger constructs although BMP-2 was required for histologic characteristics of early MSC chondrogenesis. Constructs cultured with dexamethasone, TGF-β3, BMP-2, and bFGF exhibited a significant decrease in LDH concentrations at day 3. GAG content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were temporally induced in response to dexamethasone, TGF-β3, BMP-2, and bFGF. Discussion/Conclusion: The collagen assay proved useful in assessing cMSC differentiation and holds much promise as a model system to both characterize cMSC chondrogenesis and to produce future tissue engineering constructs. Acknowledgement: Bone and Joint Fund, Texas A and M Foundation; CVM Graduate Student Training Grant.