INTRODUCTION: Mesenchymal stem cells (MSCs) are able to differentiate into osteogenic lineage and promote bone regeneration. However, direct cells transplantation into damaged tissue is not efficient to cover large bone defects. This problem could be solved by biocompatible scaffold support. Therefore, bone tissue engineering constructs based on a biomaterial scaffold, MSCs and osteogenic cytokines are promising tool for bone regeneration . The aim of this research was to evaluate the influence of FGF-2 and BMP-2 on osteogenic potential of ovine bone marrow derived-MSCs seeded onto HA/PCL-based scaffold.
METHODOLOGY: MSCs were isolated from sheep bone marrow (n=12) and treated with 100 ng/ml BMP-2 and/or 20 ng/ml FGF-2, following 7, 14 and 21 days of incubation. The effect of cytokines on osteogenic stimulation of MSCs was investigated by qPCR for osteogenic lineage markers: BMP-2, Runx2, Osterix (Osx), Collagen I (ColI), Osteocalcin (Ocl) and Osteopontin (Opn). Cells were then cultured on the HA/PCL-based scaffold in αMEM medium (control), and αMEM supplemented with BMP-2 and/or FGF-2. The effect of cytokines on morphology of cells growing on the scaffold was observed during 21 days of incubation. MTT assay was applied for cell viability, and the osteogenic differentiation potential was examined by Alizarin Red S staining and ALP activity.
RESULTS: Osteogenic gene profile of MSCs showed that cells cultured 14 days in the medium with FGF-2 and BMP-2 exhibited a higher level of BMP-2 gene expression than those cultured in the medium with only FGF-2 (RQ 3.97 vs. 2.34; p<0.0001). The mRNA expression level of Runx2 increased over time in the MSCs treated with FGF-2 and BMP-2 (RQ 1.84 vs. 6.78; p<0.0001). The highest peak in Osx gene expression was observed on day 14 in the MSCs cultured with FGF-2 and BMP-2 (RQ 3.33; p<0.0001), whereas in ColI gene expression after 21 days of incubation with FGF-2 and BMP-2 (RQ 5.19; p<0.0001). The gene expression of the late osteogenic marker Ocl and Opn increased over time for MSCs treated with FGF-2 and BMP-2 (p<0.0001). Morphology of cells growing on the scaffold changed during 21 days of incubation. FGF-2 and BMP-2 stimulation enhanced capacity of MSCs adherence to the scaffold. Cells seeded onto scaffold and treated only with FGF-2 had grater proliferation rate than untreated cells or treated with both BMP-2 and FGF-2 (p<0.005). MSCs supported with both BMP-2 and FGF-2 and seeded on the scaffold enhance bone mineralization compare to the control as confirmed by ALP activity assay (absorbance at 405 nm 0.75 vs. 1.01; p<0.005) and Alizarin Red S staining (absorbance at 405 nm 1.40 vs. 2.56; p<0.0001).
CONCLUSIONS: FGF-2 and BMP-2 enhance osteogenic potential of ovine MSCs as confirmed by upregulation of mRNA for early (BMP-2, Runx2, Osx) and late (ColI, Ocl, Opn) osteogenic differentiation genes. MSCs proliferate well on the HA/PCL-based scaffold. FGF-2 stimulates MSCs proliferation, whereas BMP-2 improves their osteogenic potential. Thus, bone tissue engineering construct, comprised of HA/PCL-based scaffold, sheep bone marrow-derived MSCs, and cytokines may be potentially used for bone regeneration.
- Stamnitz, S., Klimczak, A., Cells 10(8), 1925 (2021)