Purpose: To clarify the effect of genistein(GEN) on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin (PRF) on the repair process of bone defects in obese mice.
Methods: In in vitro experiments, the effect of GEN(0, 0.1, 1, 10, 50 μmol/L) on the proliferation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was determined by CCK 8. Alkaline phosphatase(ALP) staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells; RNA and protein expression levels of ALP, osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative real-time PCR(qRT-PCR) and Western blot. Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1. To verify the feasibility of the PRF drug loading, the ultrastructure of PRF was subsequently observed under SEM. In in vivo experiments, obese C57 mouse models were established by high-fat diet feeding. On this basis, skull defect models with a diameter of 2.8 mm were established, and the prepared GEN/PRF complexes were placed into the bone defect area. The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E) staining. Statistical analysis was performed with GraphPad Prism 5.0 software package.
Results: CCK 8 results showed that 0.1, 1 μmol/L GEN promoted cell proliferation within 7 days(P＜0.05); 10 μmol/L GEN had no significant effect on the process of cell proliferation. From the second day, 50 μmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P＜0.05). These two concentrations had similar effects in promoting cellular osteogenic differentiation. SEM results showed that PRF presented a 3-dimensional network structure, providing space for loading drug molecules. In in vivo experiments, the body weight of mice in the high-fat diet (HFD) group was 27.7% greater than that in the normal diet group(P＜0.05) and had abnormal glucose tolerance (P＜0.05). Micro-CT showed that compared with the normal diet group, the number of bone trabeculae in the femur of obese mice was decreased(P＜0.05), the distance between bone trabeculae was widened(P＜0.05), and the bone density was decreased (P＜0.05). In addition, GEN (0.1, 1.0 μmol/L) loaded by PRF increased bone volume fraction in the skull of obese mice (P＜0.05). H-E results showed that GEN/PRF promoted the healing of the bone defects.
Conclusions: GEN promotes osteogenic differentiation of MC3T3-E1, and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.