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 107 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.
