Silveira, Catrina J (2021-05). Fibroblast growth factor and its role in the chondrogenesis of canine mesenchymal stromal cells in three-dimensional collagen scaffolds. Master's Thesis.
Basic fibroblast growth factor (bFGF) is a central growth factor required for cell proliferation, survival, migration, and differentiation. bFGF enhances both the mitogenic and chondrogenic potential of human mesenchymal stromal cells (hMSCs). The effect of bFGF on canine mesenchymal stromal cells (cMSCs) during chondrogenesis remains to be determined. The aim of the present study was to determine the temporal effect of bFGF on chondrogenesis of cMSCs in a previously validated serum-free type I collagen system in preparation for future tissue engineering applications. Passage 2 bone marrow-derived cMSCs were cultured with or without 10 ng/mL of recombinant human bFGF during expansion on two-dimensional (2D) tissue culture plastic. The effect on cell proliferation and morphology was assessed. Following initial culture, cells from each group were incorporated within three-dimensional (3D) 5 mg/ml Collagen Type I scaffolds. Serum-free chondrogenic media containing one of two growth factors combinations (+/- bFGF) was supplied to these constructs resulting in four treatment groups. Constructs were cultured for 21 days to allow for chondrogenic differentiation. Constructs were assessed using morphometry (construct size and weight), viability by flow cytometry, DNA quantification, lactate dehydrogenase (LDH), live/dead staining, histology, immunofluorescence, qPCR (Col I, II, X, Sox9, aggrecan), osteocalcin, and osterix), and glycosaminoglycan (GAG) content. Expansion in the presence of bFGF subjectively resulted in reduced adhesion to tissue culture plastic and a marked spindle-shaped appearance. When assessed quantitatively, expansion in the presence of bFGF resulted in higher cMSC yields. After 21 days of 3D culture, constructs pre-exposed to bFGF were significantly larger and heavier. Canine MSCs expanded under control conditions lacking bFGF underwent construct condensation within the first 24 hours and contracted more completely than cMSCs expanded in the presence of bFGF, which exhibited a delayed/incomplete condensation response. Inclusion of bFGF in either expansion or differentiation media reduced cell cytotoxicity as evidenced by significantly reduced LDH concentrations in cultures containing bFGF. These results were supported by live/dead staining. Total number and cell viability of cMSCs within 3D constructs were higher for bFGF expanded groups at all time points. Histologically, cMSCs expanded in the presence of bFGF and cultured with serum-free chondrogenic medium lacking bFGF generated large deposits of proteoglycan that were distributed throughout the constructs. Moreover, these cMSCs adopted a spherical, chondrocyte-like phenotype. These cultures contained robust amounts of aggrecan as well as consistent deposits of pericellular type II collagen when assessed with immunofluorescence. GAG content was markedly increased in constructs containing cMSCs expanded with bFGF. While all conditions resulted in increased expression of chondrogenic and osteogenic genes, the cMSCs expanded with bFGF exhibited superior chondrogenic gene transcription. In conclusion, both quantitative and qualitative assessment of cMSC chondrogenic differentiation demonstrated that expansion of cMSCs in media containing 10 ng/ml resulted in marked improvement in chondrogenic cultures. This effect was most evident when the chondrogenic culture medium lacked bFGF during the 21 day 3D culture period. These results suggest that bFGF may be a central growth factor in optimizing canine MSC chondrogenesis, a field which remains in its infancy. Moreover, bFGF may prove useful in future endeavors to combine canine chondrogenic strategies with tissue engineering treatments for clinical application.