| Bone defect,as a direct and indirect violence caused by severe tissue damage,has been the emphasis and difficulty in orthopedics academic research.Especially in recent ten years,natural disasters such as earthquakes,he long bone defect caused by the patient's treatment,prognosis,and complications such as more became a problem in orthopedic surgeon.Therefore,figuring out a way to effectively promote defect become one of the orthopedic academia must face.Early in the treatment of bone defects in the process,often using traditional methods such as surgery and external fixator for the treatment of bone defect patients.Although these treatment methods for a small piece of bone defect effect is obvious,for the treatment of long bone defect effect is not ideal.With the emergence and development of tissue engineering treatment concept,to solve especially long bone defect repair bone defect treatment problem provides the opportunities of the powerful.In recent years,the bone tissue engineering research in animal experiments is made a certain achievement,but overall is still in the firm.Vascularization is a hot issue in the study of tissue engineering bone in recent.The domestic and foreign scholars have achieved gratifying results.The structure of the scaffold material factor for vascularization of tissue engineering bone has been confirmed.Have good hole connected between the pore structure is advantageous to the vascularization of tissue engineering bone.Although these results to some extent,solve the problem of the vascularization of tissue engineering bone,but is still on repairing effect is not ideal,main show is poor biomechanics properties were problems,clinical application and the gap distance.Therefore,we consider whether in bone tissue engineering research and other key problems remain to be solved? We conducted many literature reading and doing experiments and found that in normal tissue and medullary cavity there is a lot of nerve fiber distribution,neuromodulation in normal metabolism of bone and reconstruction played an important role.A fracture in the absence of innervation,despite callus growth,but compared with the normal fracture callus regenerationWith the development of biomaterial,scaffold materials as the most important part of the tissue engineering three elements,has proved its special three-dimensional conformation for osteogenesis and vascularization of tissue engineering bone has the important influence.The study found that the 3D-scaffold conformation in vitro can affect local function cell adhesion,proliferation and migration;the study found that stents with directional structure in the body material is a peripheral nerve regeneration.Therefore,we assume material 3D conformation is likely to be affected by neural function cell migration,proliferation,in turn,affect process of tissue engineering bone nerve,will ultimately affect the regeneration of the bone tissue repairTo sum up,this experiment adopts low temperature deposition technology was prepared with different 3D conformation of PLGA/TCP scaffolds.After cells adhesion,in vitro,observe which schwann cell has the proliferation nerve function and adhesion ability.Will support material implanted in the body of animals after the femoral condyles,observe nerve regeneration in growth and tissue repair,discusses different nerve of threedimensional space structure as the scaffold material.The concrete research content is as follows:Part 1 Using low temperature deposition technique to prepare different aperture of PLGA/TCP scaffoldsMethod:Using low temperature deposition technique to prepare different aperture of PLGA/TCP scaffolds.Using electronic scanning electron microscopy(scanning electron microscope,SEM)to observe and measure large and small pore size and scaffold and porosity of hydrophilic and biomechanical properties of scaffold materials for testing and comparison.Finally,compare of support material of leaching solution and normal culture medium contrast to test cytotoxicity of materials.Result: the pore size and porosity of holesof big aperture material is bigger than small.Low temperature deposition combined with 3D-printing preparation of the drug-loaded PLGA/TCP scaffolds bibulous rate to an average of 2.2% plus or minus 0.1%.Cytotoxicity test results show that the material of the leaching solution PH value changed little;Compare with the normal culture medium,cell survival rate is decreased.Conclusion: low temperature deposition technique was used to prepare different aperture of PLGA/TCP scaffolds materials basically meet the needs of the experiment.Large aperture and small aperture scaffold materials to suit the requirements of the experiment on the pore size and porosity.Good hydrophilic material for cell adhesion.PH materials of cytotoxicity test suitable for cell growth.Cell survival rate decreased is associate with the material of organic solvent 1,4 – dioxane.Part 2 The effection of the different aperture of PLGA/TCP scaffolds on Schwann Cells in vitroMethod:Extracted the original culture of Schwann cells(Schwann cell,SCs)from the sciatic and brachial plexusthe of the 1-3 days of fetal SD rats.The purified schwann cell inoculation in different aperture on PLGA/TCP scaffolds by instillation.Observing cell in different aperture PLGA/TCP scaffolds materials on the adhesion of morphology and growth by scanning electron microscope(SEM);scaffolds cell on different aperture PLGA/TCP marked by Brd U;to detect different aperture PLGA/TCP scaffolds cell proliferation by CCK-8;observing SCs secretion of nerve growth factor(NGF plays),glial cell source,nerve growth factor(GDNF)and neural cell adhesion molecule(Ncam)expression on different aperture on PLGA/TCP scaffolds by Enzyme-linked immunoassay(Elisa)and RT-PCR.Result: The SEM showed that the micro-pore on the material surface was obviously,and the cell proliferation and migration in the large aperture group were better than that of the small aperture group,however,the cell adhesion in the small diameter group was stronger.The results of Brd U stained showed that the number of cells around the pore of the large aperture group was significantly more than that of the small aperture group.The results of CCK-8 showed that there was no significant difference in the number of cell proliferation on 24hs(P > 0.05).After 48 h,the proliferation of the large aperture group was significantly better than that the small aperture group(P < 0.0001).The results of enzyme-linked immunosorbent assay(ELISA)and RT-PCR assay showed that the expression of NGF,GDNF and Ncam in the large aperture group was higher than that in the small aperture group(P < 0.05).Conclusion: After the SCs composite on 100-300 scaffold materials,the cell proliferation,migration and differentiation were stimulated at the early stage,but the cell biological behavior of SCs was restrained,which because of the limitation of the material structure.After the SCs composite on 400-600 scaffold materials,the number of neurotrophic factor and the promoting of cell proliferation,migration and differentiation ability are smaller than the small aperture group at the early stage.However,with the developing of the number of cells and the structure of the material itself,the ability to promote SCs increased gradually.Therefore,the scaffold materials with different threedimensional structures can affect the biological behavior of SCs,and the large aperture material is better to the proliferation,migration and differentiation of cells.Meanwhile,the large aperture materials have structural advantages that contribute to local vascularization and osteogenesis.This result provides a theoretical basis for the observation the neural networks ability of different three-dimensional structures scaffold materials in vivo.Part3 The repair of rabbit femoral condyle defects of neuralization with different aperture PLGA/TCP scaffolds materials in vivoMethod: Different aperture PLGA/TCP scaffolds were implantation into a dult New Zealand rabbits with femoral condyle defects.After 2w,4w,8w and 12 w of reperfusion,observing the sample by scanning electron microscopy,immunofluorescence,bielschowshy and serum ELISA method.To investigate the nerve effects of bone tissue scaffold materials with different three-dimensional structure on bone defect site.Result: All the incisions healed well.The degradation rate of the scaffolds was smaller in the large aperture group.The histological results showed that from 2w to 4w,the periosteum grow into large aperture scaffolds,the periosteum tissue in the large aperture group more than small aperture group.At 8w,the number of nerve fibers increased significantly in the small aperture group.The results of immunofluorescence showed that the expression of neurotransmitters and their receptors in the large aperture group was obvious at the early stage of the operation,but there was only a small amount of the neurotransmitter expression in the small aperture group.Until 4w,there were many neurotransmitters and their receptors n the small aperture group.During 8 to 12 w,the expression of neuropeptides and neurotrophic factor around the new bone gradually weakened in two group.Neuropeptides in serum test results showed that from 2w to 4w,the expression of P and NPY substance in large aperture group and normal group were both increased(P < 0.05),except for a small aperture group.From 4w to 8w,the expression of NPY in the large aperture group were increased(P < 0.05),there were no significant differences in small aperture group(P > 0.05),from 8w to 12 w,the expression of P and NPY substance din the large aperture group was significantly higher than other groups(P < 0.05).The rising level of large aperture group was smaller than the small aperture group at 12 weeks(P < 0.05).Conclution: The results show that PLGA/TCP scaffolds materials with different threedimensional structures in large aperture group had good degradation rate and periosteal induction.At the same time,we also prove that the higher the degree of the nerve,the more obvious the release of neurotransmitters,the faster the rate of bone formation.In addition,the large aperture group has good nerve induction,and it also has good osteogenesis.Due to the degradation of the small aperture material,the three-dimensional structure of the interior is changed,so that the nerve tissue in the material releases enough neurotransmitters.Not only that,the changes of three-dimensional space structure also directly affect the neurotrophic factors,which ultimately affect the osteogenesis.This is the more significance to construct tissue engineering of artificial bone. |
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