In Vitro Culture Of Schwann Cells On Human Allogeneic Bone Scaffolds

With in-depth study of bone tissue engineering, bone tissue engineering technologiesfor the treatment of small bone defects has basically reached the clinical treatmentrequirements; But for large chunks of bone defects treated，there is still a huge distantdistance between bone engineering technology,and clinical applications requiring. Fromthe standpoint of bionics, rebuild the innervation and blood supply of bone,maybe couldovercome the problem of poor effect of bone repair and mechanical properties and otherdefects; Previous studies have show that nerve tissue distribute in the normal bone tissueand participate the regulation of bone metabolism, therefore we believe the neurolorizedof bone tissue engineering may improve the repair effect of large bone defects, the nervecell cultures, material identification, cellular composite materials are the main researchcontent of this experiment，this is the first time to explore the Schwann cells’ biologicalcharacteristics on allogeneic bone, create conditions for the study of bonenervalization.We choose Schwann cells which is widely used in nerve regeneration study，choose allogeneic bone which is widely used in clinical as the major study contents; theimproved method of Schwann cells primary culture, the preparation and detection of allogeneic bone scaffold, toxicity testing,the growth characteristics of Schwann cells in3Dculture mode compare with2D cell culture mode; The aim of experiment is improveprimary Schwann cell culture techniques, referring to the two-dimensional trainingmode,observe the Schwann cell characteristics affected by3D structure, physical andchemical characteristics of bone scaffold.Part one：The improvement and validation of Schwann cells (SCs) primaryculture methodsThe main defections of previous SCs culture methods are: slow cell proliferation,apoptosis, short growth cycle, easily fibroblast contamination; Against above problems，we have improved many the cell medium, drawing and aspects step， purificationmethod.Method:Configurate new culture medium with Gsn、BPE、forskolin.Take the P2SCs,the experimental group use the new culture medium,the control group use thenormal culture medium,cultured8days. Detect the cell morphology, MTT method detectthe cell proliferation of SCs.Take P0generation SCs,using SCs improved purificationmethods include repeatedly and bi-differential purification method, cytarabinesegmented suppression method; Take P1SCs,use the methods of S-100+DAPIimmunofluorescence staining,the laser confocal detection, analysis image to calculatethe purity, comparing with the results from classical S-100training methods reported inliterature research. Results: The phase contrast microscope, confocal microscopy showedthat SCs have good shape, normal proliferate condition; Cell proliferation assay: first1-3d, two cell light absorption value, the difference was not statistically significant (P>0.05), the4-8d experimental group absorbance higher, statistically analyze the differencewas statistically significant (P <0.05); P1generation of SCs purity of95.89±3.45%,higher than the previous literature reports results(purity:95%), and cell purity improvedwith cell batches. Conclusion: The new value-added medium can promote SCsproliferation and purity, the new method can provide high-quality cultured nerve cells forbone nerve studies. Part two: The preparation and testing of human allogeneic bone scaffoldObjective: To prepare allogeneic bone scaffolds, detection material’s pore size,porosity and cytotoxicity; Methods: Scaffold material is preparated and stored by XijingHospital integrated bone bank; Detect morphological characteristic of Support materialwith dissecting microscope;Detect material porosity with the liquid alternative method;Analyze the scanning electron microscopy image to detect material pore size range; Extractmaterial soak liquid, the experimental group: cell culture medium contains material soakingliquid.The control group: Do not add material soaking liquid, detected the cell proliferationwith cell counting method; Results: material porosity=78.26±2.95%, materialdiameter:300-1000μm, there is not significant toxicity between material composition andSCs; Scaffold material having good dimensional pore structure; Conclusion: Allogeneicbone scaffold space structure, physical and chemical properties are conducive to the rebuildof nerve in bone.Part three:The establishment and research of Two-dimensional plane andthree-dimensional SCs training models.Objective: To study the proliferation characteristic of SCs in three-dimensionalstructure, verifiy the biocompatible between bone scaffold and SCs, summarized the lawof nerve cells cultured on bone scaffold. Methods: The preparation of rat tailcollagen;Three-dimensional culture experimental group (SCs+bone scaffold),two-dimensional culture control group (SCs+Collagen slide); Cell proliferation assay:culture1-8d, detected cell proliferation laws and depict cell growth curve by the method ofcell counting; Morphological detection: At the point of3d and7d, use scanning electronmicroscopy to detect the culture condition of SCs in two groups. Results: The overallchanges in cell growth curve is similar, the number of cells in same time in two groups usestatistical method to analyze, the difference was not statistically significant; electronmicroscopy: two cell can be normal adhesion and growth, there is no significant cellshrinkage and disintegration phenomenon, spindle cell morphology, beaded and arrangedside by side in a characteristic way, compared to the previous test results of phase contrast microscopy, immunofluorescence did not change significantly, the image analysis of3d,in experimental group,cell body length is50-100μm, was significantly longer than thecontrol group (40-50μm), in7d time point, two groups of cells have similar length(50-100μm), the experimental group showed significant cell migration capabilities.Conclusion: The biocompatible betwen scaffolds and SCs is as good as SCs and rat tailcollagen; Cell proliferation rate in two groups has no significant difference; SCs can begrown on bone scaffold welly, the SCs’capability of adhesion, proliferation andmigration is prefect.Brief summary: In this study, we use the improved method to culture primarySCs.From the aspects of morphology and cell proliferation to research the biologicalcharacteristics both material and SCs in two groups. Experimental results display: The SCsthat cultured by new method have obvious advantages in cell proliferation and cell purity;SCs and allogeneic bone scaffold have good biocompatibility;Three-dimensional scaffoldhas no significant adverse impact on SCs’capability of adhesion and proliferation.