Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

Abstract

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.

Keywords: bone marrow MSCs; bone repair; large animal model; osteogenic differentiation.

Figure 1

Morphological changes of sheep bone marrow-derived mesenchymal stem cells (BM-MSCs) in different culture conditions. BM-MSCs were cultivated in an αMEM or osteogenic differentiation medium, both supplemented with or without BMP-2 and/or FGF-2 for 21 days. In response to both stimuli (FGF-2 and BMP-2), the cells exhibited different cellular morphology compared to the control culture conditions, i.e., αMEM or osteogenic differentiation medium without any additional cytokines (a,b). Cells treated with FGF-2 were smaller and grew more densely (c). FGF-2 and BMP-2 added to the complete αMEM medium showed an osteogenic differentiation of the cells (e), and when added to the osteogenic differentiation medium, they altered the size and density of calcium acceleration (d,f).

Figure 2

(a) Growth curves of sheep BM-MSCs treated with or without BMP-2 and/or FGF-2 assessed with the MTT assay. Compared to the cell control cultured in αMEM, the proliferation of BM-MSCs treated with BMP-2 and/or FGF-2 increased, whereas the proliferation of cells cultured in αMEM supplemented with FGF-2 was the highest. ** p ˂ 0.005, *** p ˂ 0.0001. (b) Doubling time of ovine BM-MSCs cultured in growth media supplemented with or without the cytokines FGF-2 and BMP-2 in three independent experiments performed in triplicate.

Figure 3

Flow cytometry analysis of BM-MSCs cultured in the control medium αMEM and in a medium supplemented with BMP-2 and/or FGF-2 for 21 days. The expression of the MSC-specific antigens CD73 and CD105 was detected for all cells cultured in different conditions. Cells expressing the hematopoietic markers CD34, CD45, and HLA DR were absent or detected at low levels on day 21 of observation in all culture conditions.

Figure 4

RT-PCR (reverse transcription polymerase chain reaction) analysis for CD90 gene expression of BM-MSCs cultured in the αMEM medium supplemented with or without BMP-2 and/or FGF-2 for 7, 14, and 21 days. The highest level of CD90 was observed in cells cultured in the control medium αMEM for all time points. In contrast, BM-MSCs treated with FGF-2 and BMP-2 were characterized with the lowest level of CD90. The experiment was assessed three times in duplicate. * p ˂ 0.05, ** p ˂ 0.005, *** p ˂ 0.0001.

Figure 5

Representative images for the immunofluorescence staining of osteocalcin (Ocl) (a) and collagen type I (ColI) (b), expressed by BM-MSCs cultured in an osteogenic differentiation medium with or without BMP-2 and/or FGF-2 for 21 days. The Ocl and ColI were stained with FITC in green and nuclei in blue with DAPI. Immunofluorescence staining for osteocalcin (c) and collagen type I (d) was quantified using the ImageJ software. FGF-2 and BMP-2 promoted the expression of osteogenic-related proteins, which resulted in a moderately more intense fluorescence in the treated cells compared to the control cells cultured in the osteogenic differentiation medium alone.

Figure 5

Representative images for the immunofluorescence staining of osteocalcin (Ocl) (a) and collagen type I (ColI) (b), expressed by BM-MSCs cultured in an osteogenic differentiation medium with or without BMP-2 and/or FGF-2 for 21 days. The Ocl and ColI were stained with FITC in green and nuclei in blue with DAPI. Immunofluorescence staining for osteocalcin (c) and collagen type I (d) was quantified using the ImageJ software. FGF-2 and BMP-2 promoted the expression of osteogenic-related proteins, which resulted in a moderately more intense fluorescence in the treated cells compared to the control cells cultured in the osteogenic differentiation medium alone.

Figure 6

Real-time qRT-PCR analysis for the osteogenic differentiation gene marker of BM-MSCs following treatment with BMP-2 and/or FGF-2 for 7, 14, and 21 days. mRNA for BMP-2, Runx2, osterix (Osx), and collagen type I (ColI) (a–d) characterizes the early stage of osteogenesis; osteocalcin (Ocn) and osteopontin (Opn) (e,f) characterize the expression in the late stage of differentiation into osteogenic cells. Three different experiments were performed. * p ˂ 0.05, ** p ˂ 0.005, *** p ˂ 0.0001.

Figure 7

Multilineage differentiation of sheep BM-MSCs. Osteogenic differentiation was assessed using Alizarin Red S staining, adipogenic differentiation using Oil Red O, and chondrogenic differentiation using Alcian Blue staining.

Figure 8

(a) Secretion profile of sheep BM-MSCs using the semi-quantitative C-Series Ovine (Sheep) Cytokine Array C1 Kit, depending on culture conditions. (b) Secretome of BM-MSCs treated with FGF-2 alone, or (c) FGF-2 and BMP-2 after 7, 14 and 21 days of incubation.