| Objective: To repair rabbit skull defects in animal models by using bone mesenchymal stem cells(BMSCs) combined with Bio-oss. Compare the differences of the osteogenesis capacity between the combining group, the Bio-oss group, the BMSCs group and the controlled group. By look into the process and effect of Bone Tissue Engineering, we can provide a good experiment reference for its clinical application.Methods: BMSCs were isolated from rabbit bone marrow and used as seed cells. Three skull defects(d=6mm) were made on each female rabbit by ring bone drill. 32 female rabbits were randomly divided into 4 groups as A, B, C, D. A: combining group(implanted with BMSCs combined with Bio-oss), B: Bio-oss group(implanted with Bio-oss only), BMSCs group(implanted with BMSCs only), controlled group(blank). All defects were covered with Guided tissue regeneration(GTR) membrane. After 2, 4, 8, 12 weeks, there are 2 rabbits were sacrificed at each node in each group. The specimens detection: Gross observation, X-ray detection, HE staining, Masson staining, group A and group C line of polymerase chain reaction(PCR) detection.Result:1. BMSCs were obtained from bone marrow(BM) in male rabbits, and isolation by density gradient centrifugation and plastic-adherence screening. Antigen CD44(96.8%) was positive and CD45(0.8%) was negative by flow cytometry(FCM). After osteogenic induction, BMSCs differentiation into osteoblast, cell morphology changed, the size of cell increased, mast spindle cells and irregular polygonal cells were observed, and polygonal cells were gradually increased, no obvious contact inhibition was found. After osteogenic induction of three weeks, induced-cells were strong positive to ALP staining when blue-black particles and deep blue calcified nodules were found in cells, while the controlled-cells were weak positive. nodules were observed by alizarin Bordeaux in induced group, while controlled group show no calcium nodules.2. Generally: No death, no abnormal activities. No obvious swelling, inflammation and infection was found.3. Gross observation: At 2, 4, 8, 12 weeks Bio-oss were visible in group AB, and were more obvious at 2, 4, 8 weeks, and less in 12 weeks. At 2, 4 weeks, the boundary was clear, the surface was dim. At 8 weeks, the new tissue was hard with a smooth surface and a fuzzy clear boundary, at 12 weeks, the new tissue was hard, the boundary was a little dim. New tissue was visible in CD group at 2, 4 weeks, the new tissue was soft with a clear boundary and a smooth surface, The new tissue area in 4 weeks is larger than in 2 weeks. At 8 weeks, the new tissue is of medium harshness with a smooth surface and a dim boundary, and the volume is larger than 4 weeks, there are still a few defects in the central. At 12 weeks, when group C and D both have no defects, new tissue is slightly hard with a dim boundary, a smooth and slightly concave surface, group C is a bit hardder than group D. GTR membranes are not obviously moved. It’s almost complete at 2 weeks, thin at 4 weeks, only a few remained at 8 weeks, and at 12 weeks, it’s almost fully biodegraded.4. X-ray detection: In group AB, high density resistance are visible at each node. At 2 weeks, the boundary is clear, at 4 weeks, the particles are reduced, and slightly blurred border was presented, at 8 weeks, the material and autologous bone boundaries were blur. At 12 weeks, the border became more vague. The bone forming of group CD was centripetal fusion, at 2, 4, 8, 12 weeks, there are around 0.4 mm, 1 mm, 1.5 mm and 2.5 mm new tissue in group C, and around 0.4 mm and 0.8 mm, 1 mm, 2 mm in group D. At 2, 4 weeks, the density of new tissue in group CD was obviously lower than that of autologous bone, and the boundary is obvious, at 8 weeks, it was slightly lower, the boundary is fuzzy, at 12 weeks, it was also slightly lower, the boundary is dim.5. HE staining: In group AB Bio-oss were degrading while collagen fiber were proliferation. At 2 weeks, inflammatory cells infiltrated around Bio-oss, the defect area was rich in capillaries, at 4 weeks osteoid increased obviously, a few new blood vessels was observed. At 8, 12 weeks, the new tissue were mineralization. In group CD, at 2 weeks, a large number of collagen fiber proliferate, inflammati- on cells infiltrate, and the defect area was rich in capillaries. At 4 weeks, a small amount of osteoid and a few new blood vessels were shaped. At 8, 12 weeks, the new tissue were mineralization. At two weeks, group ABCD have no significant difference; at 4 weeks, group AB is better than that of group C(P < 0.05) and group D(P < 0.05); at 8 week, group A is better than that of group B(P < 0.05), and group CD(P < 0.05); At 12 weeks, group A is better than that of CD group(P < 0.05). In group ABCD, as time passes, the new bone grow, in group A, there are more new tissue in 4, 8, 12 weeks than in 2 weeks(4 weeks: P < 0.05, 8 weeks: P < 0.01, 12 weeks: P < 0.01), and more in 8 weeks than in 4 weeks(P < 0.05); in group B, here are more new tissue in 4, 12 weeks than in 2 weeks(P < 0.05); in group C, there are more new tissue in 8 weeks than in 2 weeks(P < 0.05), more in 12 weeks than in 2 weeks(P < 0.05); in group D, there are more new tissue in 8 weeks than in 2 weeks(P < 0.05), more in 12 weeks than in 2 weeks(P < 0.05).6. Masson staining: Identify bone maturity, mature bone dye red. At 2 weeks, a large number of reddish Bio-oss was fo- und in group AB, at 4 weeks, Bio-oss were gradually encompassed by fibrous connective tissue, the edge of Bio-oss gradually turned red, at 8 weeks, Bio-oss was further degradation, red area gradually increased, but were disorderly arra- nged, at 12 weeks, the red areas expanded and the color gradually deepened. In group CD, large blue area were found at 2 weeks, at 8 weeks, relatively obvious red areas began to appear, most of them are still pale reddish, at 12 weeks red areas continuely expanded.By measuring the average optical density valueaccording, we do the following statistical analysis. To the time node, at 2 weeks, there was no significant difference be- tween ABCD group ABCD; at 4 weeks, group A is better than group CD(A > C: P < 0.05; A > D: P < 0.05); At 8 weeks, group A is better than that of group BCD(A > B: P < 0.05; A > C: P < 0.05; A > D: P ≤ 0.01), group B is better t- han that of group D(P < 0.05); at 12 weeks, group A is better than that of group BCD(A > B: P < 0.05; A > C: P < 0.05; A > D: P < 0.05).According to the group ABCD, the new bone gradually increased and gradually mature and mineralization as time passes, in group A, The mean optical density is higher in 4, 8, 12 weeks than in 2 weeks(4 weeks: P < 0.05, 8 weeks: P < 0.05, 12 weeks: P < 0.01), higher in 8, 12 weeks than in 4 weeks(8 weeks: P < 0.05, 12 weeks: P < 0.05); in Group B, it’s higher in4, 8, 12 weeks than in 2 weeks(4 weeks: P < 0.05, 8 weeks: P < 0.01, 12 weeks, P < 0.01), higher in 8, 12 weeks than in 4 weeks(8 weeks: P < 0.05, 12 weeks, P < 0.05), higher in 12 weeks than in 8 weeks(P < 0.05); in Group C, it’s higher in 4, 8, 12 weeks than in 2 weeks(4 weeks: P< 0.05, eight weeks: P < 0.01, 12 weeks: P = 0.01), higher in 8, 12 weeks than in 4 weeks(8 weeks: P < 0.05, 12 weeks, P < 0.05); in Group D, it’s higher in 4, 8, 12 weeks than in 2 weeks(4 weeks: P< 0.05,8 weeks: P ≤0.01, 12 weeks: P < 0.01), higher in 8, 12 weeks than in 4 weeks(8 weeks: P < 0.05, 12 weeks, P < 0.05).According to gross observation, X-ray examination, HE and Masson staining results, the osteogenesis capacity was better in group A than in group B, C, D, and group B is better than that of group CD, group CD has no significant difference. As time passes, the new bone gradually formed and mature and mineralization.5. The Y Chromosome detection: male rabbit Y Chromosome sex-determining gene(SRY) are positive expression in group A and group C at four time nodes.Conclusion:1. BMSCs can be isolated by density gradient centrifugation and plastic-adherence screening, cells can be purified after training and extending.2. BMSCs can differentiate into osteoblast after osteogenesis inducing, it can be used as ideal seed cells in BTE.3. Stem cell’s transplantation can choose SRY as a tracer.4. The physical and chemical properties of Bio-oss are similar to natural bone, Bio-oss also have good biocompatibility, it can be used as the biological scaffold material.5. Using Bio-oss as scaffold material, composite BMSCs as seed cells, implanted the composite into rabbit skulls can obviously promote osteogenesis capacity, it provides a good method for clinical application. |
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