Transfect Bmscs With PcDNA3.1-VEGF To Construct Tissue Engineered Bone In Defect Repair

BackgroundBone defect is a common challenge faced by orthopaedic surgeon resulting from various events such as trauma, infection, tumor or congenital disease. Massive bone defect requires graft implant due to limited self-restoration. Current bone repairing material has been imperfect. Recent development in tissue engineering introduced promising bioactive bone engineering material combining bone substitute with progenitor cells to which signal factors are added through controlled-release. Hydroxyapatite is the primary content of human bone. Since its advent in1970s, it has been widely used in orthoplastic, orthopaedic and stomatology to repair bone defect. Conventionally produced hydroxyapatite is crisp and weak; condensed hydroxyapatite halts osteogenesis, forming unwanted occupation. Fortunately, innovative bone substitute structurally similar to natural bone, whose building block is of nano level, has proved easily recognizable to human body cells and macromolecules, hence its bioactivity, practicability and biocompatibility. To date, artificial nanohydroxyapatite product has undergone marked progress, greatly enabling its clinical application. Notwithstanding that, integrated function and structure have yet to be reached. Identified pitfalls include unconnected inconsistent porosity, unsuitable degradation rate and lower mechanical strength than natural. The dominant concept is that bone directing hydroxyapatite is not osteoinductive. Natural bone is a sort of organic/inorganic compound in light of its combination of inorganic mineral and biomacromolecules. Therefore, focused research is now on hydroxyapatite-based scaffold to construct bonemimicing material. Angiogenesis and vascularization play pivotal role in defect heal and graft survival. VEGF, also termed as vascular permeability factor(VPF), is the most efficacious growth factor known, which counts in osteogenesis and defect repair. Multiple researches converged to conclude that VEGF works by:(1) promoting endothelial proliferation, and angiogenesis,(2) influencing bone turnover directly or as a paracrine factor—it's discorved that VEGF lifts insulin-like growth factor and endothelin secretion from endothelium,(3) exciting osteoblast migration and differetiation through impect on fit-1receptor expressed on the cell. VEGF also reacts directly with fibroblast. There are experiments proving its positive action on fibroblast migration and differention, although not proliferation.ObjectiveTo incorperate hydroxyapatite and conboxymethyl chitosan(CMCTS) constructing a three-dimensioned network similar to natural cancellous bone in mechanics, geometry, and surface properties, aiming at3D nanonetwork both highly porous and mechanically competent with two-graded channels as artificial bone matrix. The material were supposed to suffice in both structure and function. Meanwhile, in accordance with the three essentials of bone tissue engineering--scaffold, seedlings, signal factors--VEGF-transfected BMSCs were implanted into the composite so that VEGF was secreted along with osteogenesis, thus a bioactive bone engineering was established to improve defect repair, buttressing research for ideal bone substitute.Method1. Preparation of nHA/CMCT scaffoldNanohydoxyapatite powder was produced through chemical deposition. Using genipin as cross-linking agent, porous nHA/CMCTS composite was successed through particle leaching and lyophilization.2. Tests on the biological safety of nHA/CMCTSAfter establishment and grouping, liver and kidney function of the animal models were tested. Bone samples were collected around nHA/CMCTS composite in experiment group and from the defect in control group. The toxicity was tested under cytomorphometry.3. Preparation the Restructuring carrier of pcDNA3.1-VEGF165 The total RNA was extracted and the VEGF165DNA fragment was synthesized reverse transcriptionally from the primer designed. The gene was specifically amplified by the specific primer and template of cDNA to VEGF165.The bacteriphage DH5a was extracted and purified. The pMD18-T and VEGF165were double digeseed and the pMD18-T and VEGF165fragments were collected respectively on Gel Extraction Kit. pcDNA3.1and VEGF165were connected and competent E.Coli was prepared. At last, the combinant carrier was amplified and purified.4. Culture of bone marrow stromal cellsIndividual nucleus cell was seperated from bone marrow and cultured in DMEM with10%fetal calf serum and in the conditional medium with10%fetal calf serum,8-10mol/L dexamethasone,0.01mol/Lp-Glycerin sodium and0.05g/L VitC.5. Transfection of BMSCs with pcDNA3.1-VEGF165plsmids (electrotransfection)6. Establishment of animal models1.8cm length of the middle part of the rabbits'both radii was removed. One foreleg was randomly selected to be implanted with pcDNA3.1-VEGF165transfected BMSCs on nHA/CMCTS scaffold or nHA/CMCTS simply, another foreleg as the empty control,respectively.7. Postoperative observation.Observation of general condition, regional inflammation, and time of heal.8. Radiography examinationThe fracture was inspected under radiography on osteogenesis and defect recovery at the end of4w,12w, and24w after operation.9.Sample collectionThe animals were sacrificed and collected from the defect through incision on the original operated sites. Slices5μm thick were aquired through decalcification, gradient dehydration, transparent and waxing.10. HE staining Observation of material degradation, new bone formation and the new blood vessels with Haematoxylin and eosin (HE) staining.11. Biomechanical testingBiomechanical testing was carried out at the end of6w,12w, and24w after the operation with the three point bending method and the loading rate was2mm/min.12. Melecular biochemestry testingMelecular biochemestry testing was carried out at the end of6w,12w, and24w after the operation to test the expression of VEGF, CD31, OPN and IGF-1.13. Statistical analysisAll analyses were performed using SPSS v13.0. Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA. P<0.05was considered statistically significant.Result1Porous nHA/CMCTS composite was prepared through particle-leeching. HA and CMCTS stayed unchanged. Carbonylmethyl group reacted with HA and Ca2+, forming rigid bond on the interface. The maixmal porosity can be as high as87%. The pores, mainly shaped spherical, ranging from a few nanometers to600nm in diameter, interconnected, are inductive to bone ingrowth and expansion.40%CMCTS content, composite/Porous agent ratio of1:1, and porosity of75%forms a ideal combination to reach a compressive strength up to21Mpa, satisfying as bone engineering scaffold. Nano level particles were detected on the pore wall under SEM analysis after immersion in the SBF solution. One theory is that osteoinductiv carbonated hydroxyapatite crystal results from chemical bond-forming reaction of ceramic material with body fluid.2Histological and biochemical test of the composite. At week4, acute and chronic inflammatory cells infiltration, absent foreign body reaction and necrotic bone were detected in the experiment group, and inflammatory congestion with some chronic inflammatory cell infiltration in the control group. At week6, slight inflammatory reaction emerged in the experiment group and the bone was primarily intact; in the control group, no sign of abnormality showed except slight inflammation. The above suggested good biocompatibility of nHA/CMCTS. nHA/CMCTS was also tested for liver or kidney toxicity. Under analysis of variance, no statistically significant difference in ALT, AST, Cr and Urea was exhibited among the four groups, indicating biotical safety of the composite. Morphological observation of rat embryonic fibroblasts cultured in the composite further proved it non-cytotoxic.3.The PcDNA-VEGF165recombinant vector was constructed successfullyVEGF165was amplified by template of its cDNA. In the subsequent electrophoresis, a distinct band of fragments of237bp appeared near the standard nucleic acid molecule weight. The product was pure and free of non-specific band disturbance, thus qualified for transfection use. pMD-18and VEGF165were double digested with EcoR I and Hind III endonucleases respectively. Specific band of the former forged around5.4kb, the latter around225kb. The sequence figure of PcDNA-VEGF165complied with established rabbit VEGF gene depicted clear peak free of double-peak disturbance.4Neither group displayed significant inflammation in gross appearance.5Radiography indicated better result in the experiment group. With the analyses of X-Way with lane-Sandhu score, we found the VEGF-nHA/CMCTS group seem to be healing faster than not only the two control groups,but also than the nHA/CMCTS, Lane-Sandhu score6The observation of HE stainingThe VEGF-nHA/CMCTS experiment group demonstrated some bone marrowgranular cells and hardly degraded implant at postoperative week4, markedly more bone marrow cells and bone growth at postoperative week12, and largely reduced implant and good newly formed bone and fibrous callus at postoperative week24. Graphical analysis of the slices revealed much higher Lane-Sandhu Histologic score in the VEGF-nHA/CMCTS experiment group than in the nHA/CMCTS group and two control groups (P<0.05). The new small vessel inductive to oseogenesis was also detected.7The analyse of biomechanical testThe biomechanical property of the newly formed bone were tested with three point bending test, with loading rate of2mm/min. The compressive strength increased with time. In VEGF-nHA/CMCTS experiment group, it burst suddenly from week4, peaking at week12. In the nHA/CMCTS experiment group, it was raised gently and peaked at week24. No obvious increasement was observed in the two control groups.8. The results of molecular biochemistry testThe expression level of VEGF, CD31, OPN and IGF was higher in VEGF-nHA/CMCTS group not only than the two control groups, but also than nHA/CMCTS group. The expression level of theses factors was higher in nHA/CMCTS group than the two control groups. There was no significant difference in the two control groups.Conclusion1The composite has adequate porosity and compressive strength to construct ideal bone engineering scaffold. nHA/CMCTS implanted on rabbit bone defect invoked no noted inflammation or bone necrosis. Its good compatibility and biotical safety were firmly validated..2. The new Bioactive bone tissue engineering which was acquired by transporting pcDNA3.1-VEGF165-transfected BMSCs in the wound area could promote the new bone formation, the defect healing and the bending ability, so it should play promotative function in the fracture healing.3.The new Bioactive bone tissue engineering with pcDNA3.1-VEGF165-transfected BMSCs shoul heal the defect by inducing osteogenesis and angiogenesis4. We constructed a new bioactive bone engineering composite, which helps experimentally and theoretically to establish ideal bone substitute for defect repair. A new idea to promote tissue repair is come up with. Background:We previously showed that nano-hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)-transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model.Methods:The nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray analysis and hematoxylin and eosin (HE) staining.Results:The microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold is less toxic, and displays a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSC bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation.Conclusions:The n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is a potentially useful tool for bone defect repair.