Effect of different concentrations of concentrated growth factor on osteogenic potential of adipose-derived mesenchymal stem cells
10.3760/cma.j.cn121382-20240708-00604
- VernacularTitle:不同浓度浓缩生长因子对脂肪间充质干细胞成骨能力的影响
- Author:
Xin HUANG
1
;
Zhengjian GUO
;
Jianing LI
;
Yajing ZHANG
;
Tingting ZHANG
Author Information
1. 天津医科大学口腔医院口腔颌面外科 天津市口腔软硬组织修复再生重点实验室 天津医科大学口腔研究所，天津 300070
- Keywords:
Growth factor;
Concentrated growth factor;
Adipose-derived mesenchymal stem cells;
Ossification;
Alkaline phosphatase;
Bone morphogenetic protein 2;
Transfo
- From:
International Journal of Biomedical Engineering
2024;47(6):537-545
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To study the effect of different concentrations of concentrated growth factor (CGF) on the osteogenic potential of adipose-derived mesenchymal stem cells (ADSCs).Methods:Preparation of CGF from rat blood by differential centrifugation method, the levels of endogenous growth factors in CGF were quantified by enzyme-linked immunosorbent assay (ELISA). ADSCs were extracted from the inguinal adipose tissue of male SD rats and cultured in media containing different concentrations (5%, 20%, 50%, 80%) of CGF. ADSCs were identified by the expression of the surface marker CD44 detected by immunofluorescence staining and by the expression of CD29 and CD90 detected by flow cytometry. Cell proliferation effects was evaluated by the cell counting kit-8 (CCK-8) assay. The effect of osteogenic differentiation was detected by alkaline phosphatase (ALP) staining, and the effect of osteogenic calcification was detected by alizarin red staining. Real-time reverse transcription-PCR was used to detect the expression levels of osteogenic genes, and Western blotting was performed to detect the expression of osteogenesis-related proteins.Results:The detected level of bone morphogenetic protein 2 (BMP-2), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor-A (VEGF-A) were (33.15±0.72) pg/ml, (95.60±2.45) ng/ml, and (351.58±22.28) pg/ml in CGF, respectively. ADSCs were spindle-shaped and well-extended. Immunofluorescence staining results showed uniform green fluorescence in the cytoplasm. Flow cytometry analysis revealed that the positive rates for CD29 and CD90 were 99.65% and 92.72%, respectively. The low-concentration (5% and 20%) CGF groups showed stable proliferative activity in ADSCs, with absorbance ( A) value reaching 3.90±0.25 and 4.04±0.22 after 7 days of culture, both of them were higher than that of control group, and the differences were statistically significant (both P<0.01). The 5% CGF group showed the most intense ALP staining, with a cumulative A value of ALP (2.299×10 7±7.156×10 6), which was significantly higher than that in the control group (4.426×10 6±1.839×10 5) ( t=?4.95, P<0.01). All CGF groups showed deeper staining compared to the control group, with the 5% CGF group showing a significantly higher cumulative A value of calcium nodule (3.599×10 7±4.094×10 6) compared to that in the control group (1.413×10 7± 1.298×10 6) ( t=?5.46, P<0.01). The relative expression levels of BMP-2, β-catenin, and TGF-β1 mRNA in the 5% CGF group were 50.97±1.75, 1.84±0.53, and 1.86±0.24, respectively, all of which were significantly higher than those in the control group (26.03±1.94, 1.13±0.12, and 1.14±0.16, respectively) (all P<0.01). Similarly, the protein expression levels of BMP-2, β-catenin, and TGF-β1 in the 5% CGF group were 1 174.33±70.02, 1 337.75±152.88, and1 087.70±112.19, respectively, all of which were significantly higher than those in the control group (845.54±61.97, 670.72±70.95, and 740.70±36.51, respectively) (all P<0.01). Conclusions:Different concentrations of CGF promote ADSCs proliferation, and the lower concentrations (5% and 20%) exhibit more stable proliferation rates. Specifically, a low concentration of CGF (5%) can induce ALP activity and calcium nodule formation, and it enhanced the expression of BMP-2, β-catenin, and TGF-1 mRNA and proteins.
