Primary Study On Co-cultures Of Osteoblasts And Schwann Cells For Neurotization Of Tissue Engineered Bone In Rats

Objective1. SD rats bone marrow mesenchymal stem cells(BMSCs), GFP transfected BMSCs, osteoblasts, Schwann cells(SCs), sensory neurons, sympathetic neurons was cultured in vitro. Observated proliferation of GFP transfected mesenchymal stem cells cocultured with sensory neurons, sympathetic neurons, Schwann cells, Respectively. Cbservated proliferation and Mechanism of GFP transfected BMSCs and calvarial-osteoblasts cocultured with SCs.2. To explore the cytotoxicity, labeled time, marking rate and effect on cell adhesion of QD labeled rat bone marrow mesenchymal stem cells (rBMSCs) in vitro, and to confirm its feasibility for stem cells labeling and tracer means for rat.3. The research of biocompatibility of collagen-bioactiveglass materials, including hemolysis experiments, test on the proliferation of RSC96 by extracts, he mechanical strength of collagen-bioactiveglass under wet and dry, the best inoculation of liquid volume, the best way of inoculation method, research of BMSCs and proliferation of SCs adhesion to materials, material experiments on cell proliferation and differentiation into osteoblasts and promote experimental, and preliminary test on validation subcutaneous bone formation in vivo.4. Comparing stability using Kirschner wires and plates made of rapid prototyping technology to fixed 8mm rat bone, testing axial compression using in vivo biomechanical testing and dynamic observation of the fixed failure rate in vivo experiment, and explore surgical techniques, advantages and disadvantages of two kind of fixation, and explore appropriate way of fixed rat bone5. explore feasibility of using rat saphenous artery,vein and nerve bundle as exogenous factors to construct vascularization and neurotization of tissue engineered bone simultaneously, and tracing cells by using SCs labeled with QD655 GFP tranfected BMSCs to load in collagen-bioactive glass in vivoMethods1. using 2 weeks old SD rats as the experimental animal model, cells were collected by washing the femur and tibia bone marrow, using passage 3 BMSCs for trial; take 1 day old neonatal rat's skull, using collagenase and 0.25Ⅱ% trypsin to digest for 45 minutes, and then obtained pure osteoblasts by; sterile removal of fetal dorsal root ganglia and collect using 15d pregnant rats,0.25% trypsin digestion for 50min, planted into a pre-planking of the glass coverslips, cultured sensory neurons; obtained superior cervical ganglion using Id old suckling rat, digesing by 0.25% trypsin for 30min, planting and cultivation of pre-ceiling plate; take 1d suckling rat, remove the sciatic nerve and brachial plexus, Schwann cells were obtained using tissue adherent. Using P3 BMSCs differentiate to osteoblast for 2 weeks and identified; explore proliferation and differentiation mechanisms by coculturing Schwann cells, sensory neurons, sympathetic neurons with osteoblasts and BMSCs-osteoblasts for 3d and 7d cultured respectively2. A two-week-old Sprague-Dawley rat (SD rat) were washing the femur and tibia bone marrow cavity and collecting the cells, and identification of BMSCs. The third passage of BMSCs (P3) were incubated with QD for the experimental group according to the recommended concentration of the markers, Cells were not labeled by QD as negative control. The cell survival rate after various concentrations of QD labeling by trypan-blue exclusion. QD-labeled cell proliferating by MTT, as well as the impact of osteoblast differentiation by the way of alizarin bordeaux staining, alkaline phosphatase staining and real-time PCR. Detecting time and rate of labeled cells at Ow, 1w,2w,4w,6w using fluorescent microscopy and QD-labeled cell adhesion to scaffold(bioglass/collagen composite) by electron microscope3. South China University College offers collagen-BioactiveGlass,,the implementation of hemolysis test and cell proliferation using materials extraction, comparative observation of the cell (BMSCs and SCs)adhesion in the material by electron microscope on Id,3d,5d,7d; take a test on mechanical strength of collagen-bioactive glass in the dry and wet state, compare to collagen material; preparing six material of 6×8mm2, using micro-syringe, the liquid volume per 20ul dropping material, until fluid was leaking from material surface, Statistics injection of liquid volume, computing the best added liquid; syringing in 5 points, from surface Omm,2mm,4mm gradient injection to find the best injection site; choose the best injection over pre-cut 2mm sheet of material, using Cyquant reagents were inoculated 16h, 1w,2w quantitative detection; take P3 BMSCs seeded on materials and 24 plate, the part of the materials used in SD rat bone marrow mesenchymal stem cell culture medium, a piece of the material used in osteoblast medium, respectively, the cell culture medium with the same comparison, the use of real-time PCR testing after 2w, testing osteogenic differentiation. Take P3 BMSCs to differentiate into osteoblast for 2 weeks, planting in collagen-bioactiveglass, cut into thin slices subcutaneous nude rat, the observed properties of materials can format bone.4.Using rapid prototyping technology to design plastic plate, material for the import of toxic materials. Preparation of SD rat 8mm femur bone defect. A total 12 rats of 350-500g were killed, take out the femur plate fixation and intramedullary fixation, axial biomechanical strength testing, testing two fixed axial resistance to compression methods; take 24 350-500g rat were divided into group A and group B, group A with plate fixation, group B with Kirschner wire fixation, evaluate stability of two fixed way after 2w,4w,8w by X-ray Respectively, summarizes the failure rate and type, explore a the better way of fix rat femur defect.5. With nine 150-200g SD rats, exposed and freed saphenous vein,artery and nerve bundles, transplant in collagen-bioactiveglass, observated vascular network formation by the way of HE staining on 3d,7d,14d; BMSCs transfected with GFP was differentiated to osteoblast 2 weeks in vitro, using QD655 labeled of rat SCs were seeded in collagen-bioactiveglass, Test on feasibility of cell-track when the scoffold was transplanted torepar 8mm of bone defects in rats in vivo after 1 week by confocal.Result1. Primary SD rat BMSCs, OB, DRG, SCG, SCs were successfully cultured. Sensory neurons were cocultured with osteoblasts in 5d, which had significant effect on the proliferation of osteoblasts than control group, there was significant difference (corresponding F= 0.802, P= 0.007), and there had no significant difference with the other control group in sympathetic neurons in three times on osteoblast proliferation (P>0.05). Schwann cells significantly promote the proliferation of osteblasts with the control group by 96 holes Co-culture plate except for coculture for 1d (P<0.05). Schwann cells significantly promote the proliferation of osteblasts differentiated from BMSCs with the control group by 96 holes Co-culture plate except for coculture for Id and 3d(P<0.05).The expression level of OPNmRNA, Bglap mRNA, Colla 1 mRNA, Alp 1 mRNA, Bmp 2 mRNA in the experiment group was significantly lower than that in the control group at 3d,7d. The source of osteoblasts on the BMSCs in the There are highly expressed in ALP, OPN, OCN, BMP-2, Col1a mRNA 3d, ALP, Col1a mRNA expressed was low in 7d, suggesting that SCs can play a role in promoting differentiation of osteoblasts from BMSCs in osteogenic environment.2. Experimental group and control group survival rate were 90%, the difference was not statistically significant (P>0.05); training 1,3,5,7,9 d proliferation rate between the two groups showed no significant difference (P>0.05). Marked by the induction of differentiation of BMSCs for 2 weeks, alizarin red and ALP staining were positive, real-time fluorescence quantitative PCR detection by the labeled BMSCs of OPN mRNA, Bglap mRNA, Colla 1 mRNA, Alpl mRNA, Bmp2 mRNA than the control group showed highly expressed. Fluorescence microscopy showed red fluorescence under the cytoplasm, can be labeled labeling rate was 96.5±1.59%, with marked increase in time, marking a decline in Were 1w 93.30±1.51%,2w 72.40±2.90%,4w 40.10±3.60%,6w 10.00±1.70%, control group at each time point marked positive rate was 0; scanning electron microscope after the cells and material labeled good adhesion.3. With collagen-bioactive glass material, the use of scaffold materials extraction, did not produce hemolysis on rabbit blood effect on the RSC96 proliferation than the control group had no inhibition at all time; axial compression test of mechanical strength, confirmed that under the scaffold wet strength than collagen, but significantly lower than the dry scaffold; using the method gradually add liquid, add liquid to investigate the best for the 0.88ml/cm3; with 5:00 gradient of 2mm for the most good injection methodCells can be grown even to the material; materials on BMSCs play a role in inhibition of differentiation; collagen-the load of bioactive glass embedded in nude mice osteoblasts can form bone, tissue engineering bone constructed with the BMP-2, OPN, Col1a mRNA expression of normal bone, suggesting that tissue engineering of bone in a rapid bone formation.4. Kirschner wire intramedullary fixation and plate fixation surgical techniques discussed in two ways, internal fixation with plate fixation is more convenient than that, the need for a shorter time, there existed a significant difference (P <0.05). And after 2,4,8 w X-ray statistical constant failure rate, and axial compression in vitro biomechanical testing. The best way of fixed. Found in in vitro biomechanical strength test plate axial compression force suffered as 40.38±4.04, the value received by Kirschner wire compression failure force of 29.53±2.95, Kirschner suffered complete failure of the value of force of 58.49±5.85, There were significant differences among the three groups (P<0.05).Dynamic X-ray films in the observation, the Kirschner wire group,91.67% failure rate occurs (11/12), plate failure rate was 16.67%(2/12). Come to this part of the study in the rat femur bone defects, should be more desirable plate fixation.5. Saphenous artery, saphenous vein, the saphenous nerve bundle Can be used as a whole neurovascular bundle to construct vascularization and neurotization of tissue engineered bone in the rat model.Text on angiogenesis and blood vessel density by HE staining in different time, Vascular density gradually increase as time goes on BMSCs transfected with GFP and QD655 marking SCs Can be used to implant into collagen-bioactiveglass in vivo and trace seed cells.Confirmed that saphenous artery, vein, nerve bundles can be transplanted into the scoffold as a whole. Trace the two cells by confocal microscope, and it can be found bones tissue in the defect site by X-ray film in a week.Conclusion1. Sensory neurons may promote the proliferation of osteoblasts; sympathetic neurons can't promote the proliferation of osteoblasts; Schwann cells may promote the proliferation of osteoblasts; Schwann cells play an inhibiting role on differentiation of osteoblast from skull sources; Schwann cells promote the differentiation of osteoblasts differentated from marrow-derived mesenchymal stem cells.2. QD can be used as a labeling marker for rBMSCs. Rat BMSCs labeled with QD is of high efficiency and safety.3. Collagen-bioactive glass is a biocompatibles caffold thatconstruct bone tissue.4.The plate was better than intramedullary Kirschner wire fixation in the fixed rat femur bone defects5. the bundle of saphenous artery,vein and nerve can be used as exogenous factors to transplant into scoffold inside In the construction of vascularization and neurotization of tissue engineered bone...