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Primary Study on Co-cultures of Osteoblasts and Schwann Cells for Neurotization of Tissue Engineered Bone in Rats

Author: JiangXiaoRui
Tutor: PeiGuoXian
School: Southern Medical University,
Course: Orthopedics
Keywords: Bone Tissue Engineering Proliferation Nerve cells Bone marrow mesenchymal stem cells Osteoblasts Co-culture Neurotization Vascularization Bone defects Collagen - Bioactive Glass
CLC: R329
Type: PhD thesis
Year: 2010
Downloads: 131
Quote: 0
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Abstract


Objective 1. Vitro of the original generation of cultured SD rat bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs were), GFP transfer dye marrow charge quality stem cells (GFP-BMSCs were), into bone cells (osteoblasts, OB), Schwann cells (Schwann cells, SCs), sensory neurons (dorsal root ganglion, DRG), sympathetic neurons (superior cervical ganglion, SCG) cells. Observation DRG, SCG, SCs proliferation of osteoblast differentiation of GFP-BMSCs. SCs the BMSCs differentiation OB and skull sources OB proliferation mechanism. Study using fluorescent microspheres 655 (Quantum dot 655, QD655) marked the rat BMSCs vitro feasibility research. Explore the dye toxicity rat BMSCs proliferation and osteogenic differentiation, dynamic labeling rate function tracer studies in vivo and lay the research foundation. Studied collagen - the biocompatibility of bioactive glass material research, including hemolysis test, the extracts RSC96 cell lines proliferation assay, collagen - wet and dry state mechanical strength of bioactive glass, inoculation amount of liquid best inoculation methods, BMSCs and SCs adhesion to material proliferation of research materials on cell proliferation test and to promote osteogenic differentiation test, as well as the initial body subcutaneous verify osteogenesis test. Explore the use of Kirschner made of plate fixation 8 mm bone defects in rats stability and rapid prototyping technology using in vivo the axial compression biomechanical testing and in vivo dynamic observation of fixed failure rate test, explore two fixed-way , surgical techniques, advantages and disadvantages, and explore suitable fixed rat bone defects fixed. 5 to explore the feasibility of using the rat saphenous vein and saphenous nerve bundle as neurovascular bundles transplantation as exogenous factors synchronization build the neurovascular tissue engineering bone, and the use of fluorescent microspheres labeled SCs and GFP-transfected into bone the cells combined load in collagen - the biological activity of intravitreal tracer feasibility study. Method 1. 2-week-old SD rats as the experimental animal model using femur, tibia bone marrow cells were collected by flushing method to develop third-generation BMSCs for the test; using two the 1d age suckling mice, the use of 75% ethanol for 15min sterile remove the skull using the sterile ophthalmic shear built 1-2mm3 debris. Join the trypsin of 1mL0.25% 37 ° C in the bone tissue of the Shredded digestion 90min slightly from the supernatant. Added 0.2% Ⅰ collagenase 3-5mL, 37 ℃, and continuous digestion 2 times 60min. After to suspend digestion, cells were collected, the test is repeated, and the cells were collected, counting, grown in 25 cm2 culture flasks, placed in 37 ° C, 5% CO 2 incubator culture. 24h after the medium was changed after the medium was changed every 2-3d. Reached 80% -90% confluency (primary cultured for 3-5d), digested and passaged, packet culture ,2-5 substituting for the test were identified using alkaline phosphatase staining; 15D pregnant rats aseptically removed fetal rat, take the dorsal root ganglia, and 0.25% trypsin digestion 50min planting to pre-decking of the coverslip on sensory neurons in culture; a 1d Age suckling mice, take the superior cervical ganglion, 0.25% trypsin digestion 30min planting to pre- decking plates; take 1d suckling mice, remove the sciatic nerve and brachial plexus, the use of tissue adherence of SCs obtained. P3 BMSCs induced to differentiate into bone for two weeks, were identified; SCs, DRG, SCG skull sources osteoblasts and BMSCs sources osteoblasts co-cultured detection, detection of co-culture after osteoblast proliferation and differentiation mechanisms. 2 QD655 recommended concentrations in accordance with the kit, each QD reagent A and the reagent carrier B depicting 1μL mixed placed in a 1.5mL microcentrifuge tube, placed on incubated 15min Add 100gL medium, vortex 30s, by adding to the tube 0.2mL suspension Beat 106 P3 cells placed in 37 ° C in 5% CO 2 incubator for 1h, rinsed twice with complete medium detected under a fluorescence microscope. Mark 3 cells (P3) QD reagents recommended concentration for the test group, and to not use the QD-labeled cells as blank control group. Trypan blue exclusion staining marked cell viability, MTT assay was used to observe the impact of dyes on cell proliferation, Alizarin Red, alkaline phosphatase staining, real-time PCR identification of osteogenic differentiation; respectively. immediately after the tag, 1w, 2w, 4w, 6w by fluorescence microscope counts detected in time to mark the two groups and the positive rate; electron microscopy mark the attachment of the cells in the material. 3 of Materials Science and Engineering of South China University of collagen - BIOGLASS, extract material extracts, the implementation of the hemolysis test, cell proliferation assay, cell adhesion material 1d, 3d, 5d, 7d contrast electron microscopy, and SCs load with collagen - a bioactive glass using electron microscopy material adherent SCs; using collagen - bioactive glass in the wet and dry state, the mechanical strength of the collagen material in the wet state contrast; take six 6x8mm2 microinjector dropping material every 20gL amount of liquid, until the material ground liquid leaking far, amount of statistics injection liquid, calculating the optimum amount of fluid; 5:00 injection, respectively, at the surface of the material 0mm, 2mm, 4mm gradient injection seeking the best injection sites; take the P3 generation BMSCs inoculation material and 24-well plates, wherein part of the material used SD rat bone marrow mesenchymal stem cell culture medium, part material used Osteogenic, respectively thereto cells of the same medium contrast, 2w, using real-time PCR detection to detect the degree of osteogenic differentiation. Take P3 BMSCs to the osteogenic differentiation of two weeks, planted in collagen - bioactive glass, cut into the of 1ml sheet subcutaneous nude mice, will observe the material osteoinductive properties. Using rapid prototyping technology, design their own plastic bone plate, the materials imported non-toxic material. Preparation the SD rat femur 8mm bone defects. A total of 12 350-500g rats were killed, remove the femoral plate fixation, intramedullary fixation with Kirschner the test axial biomechanical strength, the detection of two fixed-way axial anti-compression capability; take 24 350-500g The rats were randomly divided into group A and group B, A group with plate fixation group B Kirschner wire fixation. X-ray film, evaluation of fixed defects stable condition after 2w, 4w, 8w, to sum up the two sets of failure rates and types, to sum up the best fixed manner suitable for rats. 5 9 150-200g SD rats, exposure and free implicit arteriovenous saphenous nerve bundles, transplanted into the pre-treatment of collagen - the bioactive glass after 3d, 7d, 14d frozen section, HE staining vascular network formation; using the GFP transfected BMSCs to induced in vitro osteoblast two weeks, using Qtracker655 marked rat SCs seeded in collagen - bioactive glass, transplantation 8mm bone defects in rats in vivo using confocal observation of two types of cells are shown removed after one week tracking feasibility. Results. The the the primary SD rat BMSCs OB, DRG, SCG, of SCs successful culture, DRG co-cultured 5d, significant effect on the proliferation of osteoblasts compared with blank control group, a significant difference (corresponding to F = 0.802, P = 0.007) with the other control group and no significant difference in the SCG each time period than in the control group had no the skull sources osteoblasts proliferation role (P gt; 0.05). 96 holes of plates and co-cultured 1d, 3d, 5d, no significant difference in co-culture 7d 9d two periods SCs significant effect on the proliferation of osteoblasts, compared with the control group showed a significant difference (P lt; 0.05). The SCs of the BMSCs sources OB co-culture 1,3 d two groups was not statistically significant, the remaining three periods were statistically significant, the test group due to the control group. SCs in 3d, 5d, 7d role of osteoblasts in ALP, OPN, OCN, BMP-2, Col1a mRNA than not co-cultured osteoblasts were low expression, indicating that SCs inhibits differentiation of osteoblasts from. While the the BMSCs source of osteoblasts in ALP, OPN, OCN, BMP-2 mRNA in Col1a in 3d is both highly expressed in 7d, ALP, Col1a showed low expression, suggesting SCs osteogenic induction culture environment BMSCs The source of osteogenic cells play a pro-differentiation role. Cell viability test group and the control group gt; 90% difference between statistical significance (P gt; 0.05) culture 1,3,5,7,9 d the two sets proliferation rate differences were not statistically significant (P gt; 0.05). After the labeled BMSCs by the induction of differentiation two weeks, Alizarin Red and ALP staining were positive, real-time fluorescence quantitative of BMSCs labeled PCR detection OPN mRNA, Bglap mRNA, Colla 1 mRNA in, Alp1 mRNA in BMP2 mRNA compared with the control group showed a high expression. The fluorescence microscopy cytoplasm under red fluorescent marker to mark the rate up to 96.5 ± 1.59%, with the marked increase in the time, marking rate 1w 93.30 ± 1.51%, of 2w 72.40 ± 2.90%, 4w 40.10 ± 3.60%, 6w10.00 ± 1.70%, marker-positive rate of the control group at each time point were 0; scanning electron microscope labeled cells and materials attached. . Collagen - a bioactive glass material, the use of scaffolds extracts of rabbit blood did not produce hemolysis of RSC96 proliferation compared with control group in each time period had no inhibitory effect; using axial compression test mechanical strength, confirmed that collagen tissue scaffold strength is higher than in the wet state, significantly lower than the dry scaffold materials; gradually add liquid approach, investigate the optimum amount of fluid 0.88ml/cm3; five o'clock gradient 2mm for the most good injection method, the cells can be uniform planting materials; material to inhibit the differentiation of BMSCs play; collagen - load bioactive glass embedded in nude mice into bone cells can form bone, build bone tissue engineering with BMP- 2, OP, Col1a mRNA than normal bone high expression prompted tissue engineered bone in a rapid osteogenesis period. Kirschner intramedullary fixation and plate fixation in two ways surgical techniques discussed Kirschner wire fixation plate fixation is more convenient than the shorter time needed, there existed a significant difference (P lt; 0.05). 2,4,8 w and postoperative X-ray statistics fixed failure rate, and axial compression in vitro biomechanical testing. Explore the best fixed. Found in vitro biomechanical strength test, the bone plate axial compression force on 40.38 ± 4.04, Kirschner compression failure values ??suffered 29.53 ± 2.95, Kirschner force on complete failure value of 58.49 ± 5.85 among the three groups were statistically significant (P lt; 0.05). Kirschner group dynamic X-ray observation failure rate of 91.67% (11/12), bone plate failure rate of 16.67% (2/12). The part of the studies in the rat femur bone defects, plate fixation better. Can be used in a rat model of saphenous artery saphenous vein, the saphenous nerve bundle as a whole neurovascular bundle transplantation build blood vessels and nerves of tissue engineered bone the HE staining material more angiogenesis and increased vascular distribution density increased with time . Can be used lentiviral GFP transfected BMSCs, and the QD655 mark SCs planting to the collagen - bioactive glass in vivo tracer. Confirmed that the saphenous artery, vein, nerve bundles can be used as a whole for transplantation, in the material can be formed more vascular tissue. Confocal microscopy two cells the tracer and successful bone formation can be found in the X-ray film of the week. Conclusion 1.DRG may play an effect on the proliferation of osteoblasts; SCG play a promoting proliferation of osteoblasts; SCs play a significant effect on the proliferation of osteoblasts; SCs skull sources osteoblasts play inhibit differentiation; boost source of mesenchymal stem cells in the bone marrow osteoblast differentiation. The 2. Qtraker655 of rat BMSCs mark time, mark rate and high safety, does not change the performance of the cells into bone is a good marker. 3. Collagen - bioactive glass is a biocompatible scaffold for bone tissue can be constructed. Rat femur bone defect fixed effect of bone plate than Kirschner intramedullary fixation. 5 built in the rat the neurovascular tissue engineering, the implicit dynamic vein saphenous nerve bundles can be transplanted as exogenous factors to the material within the building.

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