Introduction: The objective of this study was to evaluate preexposure of canine mesenchymal stromal cells (cMSCs) to basic fibroblast growth factor (bFGF) during cell expansion on chondrogenic differentiation in a 3D serum-free collagen type-I system.
Materials and Methods: cMSCs were expanded in α-MEM and 10% FBS with or without 10 ng/mL recombinant human bFGF. At 70% confluence, 1 × 106 cells were polymerized in 5 mg/mL collagen type I (100 µL volume). Serum-free chondrogenic media with or without bFGF were provided to cultures twice weekly for 21 days. Constructs were assessed using morphometry (size and weight). Viability was determined at various time points using flow cytometry, DNA quantification, lactate dehydrogenase (LDH), and live/dead staining. Chondrogenesis was assessed with qPCR (ColI, II, X, Sox9, Aggrecan, Osteocalcin, and Osterix), glycosaminoglycan (GAG) assay, and histology/immunohistochemistry. Data were reported as mean ± SD and analyzed using ANOVA with Tukeys’s post hoc test with significance p ≤ 0.05.
Results: cMSCs expanded with bFGF generated larger and heavier constructs as compared with controls. bFGF preexposure led to constructs with improved cell viability and reduced LDH concentrations. Constructs expanded in bFGF and cultured in chondrogenic medium containing dexamethasone, TGF-β3, and BMP-2 exhibited significantly higher GAG content, chondrogenic gene expression, and improved proteoglycan and collagen II content.
Discussion/Conclusion: Expansion of cMSCs with 10 ng/mL bFGF prior to initiation of chondrogenic cultures resulted in markedly improved in vitro chondrogenesis when assessed using a variety of subjective and objective assessment tools.
Acknowledgment: No proprietary interest. Funded by the Bone & Joint Fund, Texas A&M Foundation.
