Combinational use of miR-34a functionalized bone powder with Col-Tgel enhances bone regeneration in irradi-ated bone defects
10.12016/j.issn.2096-1456.202440025
- VernacularTitle:miR-34a骨粉复合胶原基水凝胶促进辐照区骨缺损修复
- Author:
Huan LIU
1
;
Xi WU
Author Information
1. 陆军军医大学第二附属医院口腔科,重庆(400037)
- Keywords:
bone powder;
bone marrow mesenchymal stem cells;
osteogenic differentiation;
bone repair;
miR-34a;
transglutaminase crosslinked gelatin;
radiation damage;
radiotherapy
- From:
Journal of Prevention and Treatment for Stomatological Diseases
2024;32(9):674-683
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the effect of the combinational use of miR-34a-functionalized Bio-Oss? bone pow-der 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 induc-tion,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 be-low 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 num-ber 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 af-fecting 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 al-so confirmed that implantation of PG-miR-34a can promote the healing of bone defects in irradiated tibias.Conclu-sion The combinational use of miR-34a-functionalized Bio-Oss? bone powder with Col-Tgel could promote the osteo-genic differentiation of irradiated BMSCs and enhance bone regeneration in irradiated bone defects.
