| Objective:Trauma,inflammation and tumor are the three main factors leading to bone defect,and the patients with bone defect in the world are increasing every year.At present,there are obvious shortages in the treatment of bone defect repair and regeneration.Autogenous bone transplantation is still defined as the "gold standard" for the repair process,however,it is often caused trauma to the donor site accompanying with hemorrhage,deformity,infection,postoperative chronic pain and movement disorders and other symptoms.Allograft bone transplantation has obvious defects,such as the difficulty of sampling and immune rejection,and there is obvious potential pathogen infection(HIV virus,hepatitis B virus,etc.).Tissue engineering bone is a new method to repair bone defect,and it has become a hot research topic at present.The core problem of tissue engineering bone is the construction of the scaffold.At preseng,not only scaffold biological materials are in the research,but are put forward from a broad perspective,additionally,the construction of the scaffold tissue matrix materials should habve good compatibility,biodegradability,plasticity,porosity,mechanical strength,conductivity and induction,at the same time,also need to load one or more growth factors and stem cells.In this experiment,we selected two kinds of materials,collagen(COL)and nano hydroxyapatite(nHAP)as scaffold matrix materials,and then load basic fibroblast growth factor(bFGF)and bone morphogenetic protein-2(BMP-2)in the construction of the scaffold.At the same time,the bone marrow mesenchymal stem cells(BMSCs)of the rabbit were selected as the stem cells from the generalized scaffold.Through the construction of a generalized composite scaffold,BMSCs/bFGF/BMP-2/nHAP/COL,we strive to explore an ideal scaffold for tissue engineering,and provide a theoretical basis for further research of bone tissue engineering.Methods: 1.To investigate the mass ratio of two kinds of materials in nHAP/COL scaffold constructed by the mechanical mixing method and freeze drying method and observe gross morphology of nHAP/COL scaffold,test the porosity,swelling ratio,water absorption,compressive strength and compression modulus.2.The degradation of nHAP/COL scaffolds was analyzed,and the degradation rate,degradation stability,morphology change,internal structure change and PH change of degradation solution were measured.3.Rat BMSCs and nHAP/COL scaffolds were co cultured to test the adhesion,proliferation and differentiation of cells,so as to determine the biological toxicity of scaffolds,that is,the biocompatibility in vitro.4.The freeze-drying method was used to load double factor bFGF and BMP-2 on nHAP/COL scaffolds.The single factor loading scaffolds were prepared by the same method,and the release of growth factors in three kinds of scaffolds were measured.5.The artificial method was used for the extraction of rat tail collagen and double factor bFGF/BMP-2/nHAP/COL composite scaffolds,two kinds of single factor bFGF/nHAP/COL and BMP-2/nHAP/COL composite scaffold,and blank nHAP/COL scaffold were prepared and blank control group was established.6.The rat BMSCs were artificially extracted,cultured and passaged,and then was measured by flow cytometry.The rat BMSCs was cultured in the 5 groups established above.Cell count,CCK-8 and ALP were used to determine the adhesion,proliferation and differentiation.7.Rabbit BMSCs was extracted,cultured and passaged,induced by osteogenic induction solution,and determined by alizarin red staining and calcium cobalt method.8.Rabbit models of bilateral mandibular critical bone defects were prepared.They were implanted with bFGF/BMP-2/nHAP/COL scaffodt,bFGF/nHAP/COL scaffold,BMP-2/nHAP/COL scaffold and nHAP/COL scaffold containing rabbit BMSCs,respectively,and blank scaffold group and blank group were set up.9.The reconstruction of rabbit mandible defect was observed generally,three dimensional CT image were observed,CT values were measured,and HE staining histological was observed.10.Statistical analysis.Results: 1.The most suitable mass ratio of two components in the nHAP/COL scaffold constructed by mechanical mixing and freeze-drying is 1:1.5.The scaffold showed a porous three-dimensional structure.The pore size of the scaffold was(130±29)m,and the porosity was(93.1±6)%.The water absorption rate was(245±5)%,the swelling rate was(33.4±0.9)%,the compression strength was(4.24±0.92)MPa,and the compression modulus(0.58±0.58)MPa.2.The biodegradability of nHAP/COL scaffold is good,and the pH value in the degradation process is maintained at a stable level,which is in line with the needs of physiological state.The degradation rate at 6 and 8 weeks was 18.47% and 21.18% respectively,and the degradation process was basically adapted to the osteogenesis process.3.BMSCs and nHAP/COL scaffolds were co cultured.The cells were detected by cell counting,CCK-8 and ALP.It was proved that scaffolds could promote cell adhesion,proliferation and differentiation.4.The freeze-drying method was used to load double factor bFGF and BMP-2 on nHAP/COL scaffolds.The single factor loading scaffolds were prepared by the same method,and the release of growth factors in three kinds of scaffolds were measured.5.Rat tail collagen was extracted using artificial method successfully,and single and double factor composite scaffold was successfully constructed by mechanical mixing method and freeze drying method.6.Rat BMSCs were extracted artificially and cultured for flow cytometry.The results confirmed the cell purity,and the 5 experimental groups were detected by cell counting,CCK-8 assay and ALP assay for cell adhesion,proliferation and differentiation.The results showed that the double factor scaffold could promote the adhesion,proliferation and differentiation of rat BMSCs.7.The rabbit BMSCs was successfully extracted by artificial method,and it was detected by alcalin red staining after osteogenic induction.The results showed that the calcium nodules were red,which could be seen clearly under microscope.8.The critical bone defect of bilateral mandible was successfully prepared by surgical procedure,and the scaffold was successfully implanted.9.The gross observation of the rabbit mandible showed that the double factor scaffold group had basically completed the bone healing.3D-CT showed the best repair of bone defect in the double factor scaffold group.The CT value also suggests that the two factor scaffold group is close to the normal bone density.HE staining showed that the composition of the bone was the most.Conclusion: 1.The most ideal mass ratio of two materials in nHAP/COL scaffolds constructed by mechanical mixing method and freeze drying method is 1:1.5.The scaffold has good physical and biodegradability,non toxicity and good biocompatibility in vitro,which meets the basic requirements of bone tissue engineering scaffold.2.The bFGF/BMP-2/nHAP/COL double factor composite scaffold prepared by freeze-drying method is ideal,which can promote the adhesion,proliferation and differentiation of rat BMSCs.3.The b FGF/BMP-2/nHAP/COL dual factor composite scaffold loaded with rabbit BMSCs can complete the repair of the critical bone defect of the rabbit mandible well.4.bFGF/BMP-2/nHAP/COL dual factor composite scaffold has good biocompatibility in vivo and in vitro.It is an ideal scaffold for bone tissue engineering. |
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