| Objectives This study aim to study the effects of graphene oxide(GO)aerogels as scaffolds on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells(bone marrow mesenchymal stem cells,BMSCs),as well as to observe the effect on osteogenic effect of GO aerogels as artificial bone substitute materials in animals.This study will provide the basic basis for the use of GO aerogels as bone tissue engineering scaffolds in regenerative medicine.Methods Two round mandibular bone fragments of healthy SPF New Zealand white rabbits(1 male and 1 female)aged 12-16 weeks were taken,each of which was about 3mm in diameter.Cells were obtained by tissue culture and cultured to the third generation.Cell reptiles were made to identify the expression of stem cell(SC)markers CD44 and BMSCs markers CD90.Cells were divided into groups: blank group: cells were seeded on96 orifice plates;group GS: cells were seeded on gelatin sponge(GS)scaffolds;GO group:the third generation cells were seeded on GO aerogel scaffolds.Cell Counting Kit-8(CCK-8)was used to detect cell proliferation and OD values on the 1st,3rd and 5th day.After 7days of osteogenic induction,the ALP activity of three groups of cells was detected by ALP kit,and the effects of GO aerogels as scaffolds on osteogenic differentiation of cells were observed.In vivo osteogenic experiment: 12 healthy SPF New Zealand white rabbits(6 males and 6 females)aged 12-16 weeks were anesthetized after weighing.Round bone defect models were made on the buccal bone walls of bilateral mandibles.Blank group: fill blood in the defect area without any other treatment;bone meal group: Bio-Oss bone powder was filled in the defect area,and the periosteum was covered in the defect area;stent group: GO aerogel scaffold was filled in the defect area,and the periosteum was covered in the defect area.After 3 months,the new bone cover was observed,and whether the defect was consistent with the defect area,and the mandible was completely separated.X-ray dental film and Cone-Beam Computed Tomography(CBCT)were taken to observe the bone formation in the defect area,and HE staining was used to observe it.Results After 7 days of cell culture by tissue block method,a small number of cells adhered to the edge of bone fragment and grew mostly in spindle,triangle or polygon shape.After passage to the third generation,the cells were spindle-shaped,more uniform and neat than the primary cells.Immunofluorescence assay showed that the expression of SC marker CD44 and BMSCs marker CD90 was positive,which confirmed that the cultured cells were BMSCs.The results of CCK-8 showed that there was no significant difference in OD values among the three groups on the first day(P>0.05);OD values of GO group and GS group on the third day were significantly higher than that of blank group(P<0.05,P<0.05),while OD values between GO group and GS group had no significant difference(P>0.05);OD values of GO group and GS group on the fifth day were significantly higher than that of blank group(P<0.05,P<0.05),while OD values between GO group and GS group had no significant difference(P>0.05).After 7 days of osteogenesis induction,the results of ALP activity test showed that OD value of GO group was significantly higher than that of GS group(P < 0.05),and there was no significant difference between GS group and blank group(P > 0.05).The local and systemic condition of the rabbit was good after operation.After 3 months,the mandible was separated completely.The periosteum of the defect area was intact and the scaffold material was not lost.New bone was formed on the surface of bone powder group,and some new bone was connected with normal bone tissue;more than half of the surface of scaffold group was covered by relatively flat new bone tissue,similar to and continuous with surrounding bone tissue;no obvious new bone tissue was found in the bone defect area of blank group.X-ray film showed that the bone powder group found granular obstruction image with low density;the stent group showed interlaced obstruction image with high density.Compared with the bone powder group,most edges of filling materials fused with the edge of bone defect;the blank group showed large area of transmission image without obvious new bone formation.CBCT images showed that some new bone formed in the bone defect area of bone powder group,which restored the thickness of the bone defect area,and the edge of the new bone was connected with the original bone;there were many new bone formation in the defect area of the scaffold group,which effectively restored the thickness of the bone defect area,and the new bone formed was consistent with the original bone;there was no obvious new bone formation in the bone defect area of the blank group,and the bone defect remained.Apparently.Three months after the operation,the defect area was implanted and the surrounding bone tissue was embedded.The tissue sections were made and stained with HE.In the blank group,a small number of fibroblasts were observed at the edge of the bone defect area,and the cells were connected with normal bone tissue,and a small amount of fibroid bone tissue was formed;in the bone powder group,more new bone formation was observed in the bone defect area,some of the bone powder edges were connected with normal bone tissue,and layered new bone structure was observed at the boundary;in the scaffold group,a large number of new bone formation was observed in the bone defect area,and occasional staining was observed in the adjacent scaffold area.Deep new bone,the scaffold is more closely connected with normal bone tissue than the multilateral edge,and the formation of layered new bone is more intensive at the boundary.Conclusions 1 GO aerogels as tissue engineering scaffold materials can promote the proliferation of BMSCs and contribute to its osteogenic differentiation.2 GO aerogel scaffolds can promote new bone formation in bone defect area.Compared with Bio-Oss bone powder GO aerogel scaffolds,the effect of bone healing on bone defect area is better.Figure 8;Table 4;Reference 102. |
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