| Popis: |
Objective To study the effect of the combinational use of miR-34a-functionalized Bio-Oss® bone powder with transglutaminase crosslinked gelatin (Col-Tgel) on the osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone defect healing after irradiation. Methods The experiment was approved by the Animal Ethics Committee. BMSCs were isolated from the bone marrow of 2-week-old Sprague-Dawley (SD) rats and identified. After reaching 80% confluence, BMSCs were irradiated with 2 Gy of X-ray radiation to establish a radiation-damaged BMSC model for further experimentation. 2.5 μL or 5 μL of Col-Tgel was mixed with 10 mg of Bio-Oss® (P) to prepare PG-2.5 and PG-5. The optimal proportion of Bio-Oss® (P) and Col-Tgel was determined through in vitro and in vivo experiments. Cy3-labeled agomiR-34a, agomiR-34a, or agomiR NC was mixed with lipofectamine 2000 and added to 10 mg of Bio-Oss® (P). The mixtures were lyophilized, and 2.5 μL Col-Tgel was added to each group of lyophilized Bio-Oss®/lipofectamine/miRNA complexes or to 10 mg of Bio-Oss® to obtain PG-Cy3-miR-34a, PG-miR-34a, PG-miR NC, and PG. Irradiated BMSCs were cocultured with PG-Cy3-miR-34a to evaluate cellular uptake of Cy3-agomiR-34a using confocal microscopy. Then, irradiated BMSCs were cocultured with PG-miR-34a, PG-miR NC, and PG. The expression of miR-34a was tested by RT-qPCR and cell proliferation was tested by CCK-8 assay. After 14 days of osteogenic induction, the mRNA expression of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) was tested by RT-qPCR. The bilateral tibias of 8-week-old SD rats were irradiated with a single dose of 15 Gy of X-ray radiation. Three weeks later, tibial defects with a diameter of 3 mm and a depth of 2 mm were created 2-3 mm below the epiphyseal line in the tibial metaphysis. The composite bone substitute materials of PG-miR-34a, PG-miR NC, and PG were implanted into the defect area. Eight weeks after implantation, the tibias were harvested and evaluated for bone regeneration using micro-CT analysis and HE staining. Results The results demonstrated that 2 Gy irradiation adversely affected the osteogenic differentiation capacity of BMSCs, evidenced by the decreased ALP staining and number of mineralized nodules stained with Alizarin red in the irradiated group compared to the non-irradiated group. The composite material consisting of 10 mg Bio-Oss® and 2.5 μL Col-Tgel exhibited good osteogenic induction capability and handling properties and was used for subsequent experiments. The PG-Cy3-miR-34a could deliver the loaded Cy3-agomiR-34a into irradiated BMSCs. PG-miR-34a enhanced the expression of miR-34a in irradiated BMSCs without affecting cell proliferation. PG-miR-34a significantly upregulated the expression of osteogenic-related genes, including Runx2, ALP, and OCN. In the experiment of bone defect healing in irradiated tibias, micro-CT analysis showed that PG-miR-34a group had a higher bone volume in the bone defect area compared to other groups. The HE staining results also confirmed that implantation of PG-miR-34a can promote the healing of bone defects in irradiated tibias. Conclusion The combinational use of miR-34a-functionalized Bio-Oss® bone powder with Col-Tgel could promote the osteogenic differentiation of irradiated BMSCs and enhance bone regeneration in irradiated bone defects. |
