Study On The HC/ACBM Combination Of Vacuum Aspiration To Bone Defect Of Rhesus

BackgroundClinically, the treatment of bone defects for various reasons, especially large bonedefect, is always the orthopedic surgeon’s problems. And the number of people required toundergo bone graft repair is rising because of car accidents, cancer and so on. The repairmaterials of basically has autologous bone, allograft bone, non-degradable bone cement,ceramics, related derivative materials and so on, and these materials are unsatisfied. Bonetissue engineering is the new technology for regeneration and repair, which is the new trendof development of bone defect repair. Since the1987officially proposed and determined,bone tissue engineering have achieved many progress in seed cells, scaffolds and thetechnology of structure. But, some problems need to be resolved. Revascularization is oneof the most important problems of them. As everybody knows, the angiogenesis requiresseveral days after bone defects implanted. The early nutrition supply of the cells can onlyrely on the body tissue fluid penetration. So, the size of scaffold material is severely limited.Only as skin and cartilage that can rely on diffusion to maintain a low metabolic untilcompletion of revascularization, which is obviously far from being able to meet therequirements of clinical applications. Meanwhile, the implant material will start thecoagulation reaction, thus make the blood penetration more difficult.The common approachs to promote vascularization of bone tissue engineering,including collagen or vascular growth factors-modified scaffold, or oseogenesis seed cellsculture with the vascular endothelial cells or vessel precursor cells etc, but ineffective.Taking into account the way of vascularization of bone tissue engineering mainly rely onperipheral vascular buds born after transplantation, studies have used peripheral vascularshoot raw strategy, and puted forward the bone tissue engineering the pre-vascularizedtechnology of scaffolds: scaffolds and cells composite embeded in subcutaneous, omental,fascia or muscle bags, and then the pre-vascularization of tissue will transplant to therecipient or tissue engineered bone implant arteriovenous also can obviously promote the vascularization and osteogenic of the compound bone scaffolds. But the process is morecomplex, and even with these methods, tissue engineering bone after implantation torecipient, the blood supply, especially early blood supply remain dependent on thepenetration, not completely solve the problem of the early revascularization.The xenogeneic acellular hone matrix (ACBM) scaffold which developed by ourresearch group retains the major components and complete structure of the extracellularmatrix of bone. With layer-by-layer of self-packaging technology, heparin (HEP) andchitosan (HC) compound were coated on ACBM’s surface. The experiment showed thatslow-release and reliable anticoagulation effects were satisfied. And then combined vacuumassisted closure (VAC), allowed blood to continuous infusion at an early stage, provided thescaffolds of tissue engineered bone real-time, continuous oxygen and nutrients. Repairdefects in rabbit and pig results confirmed that the HC/ACBM (Heparin-Chotison coatedACBM) scaffold with the vacuum assisted closure make repair materials can be obtainedimmediately blood infusion after implantation. And compared with the non-negativepressure side, the blood volume, blood flow and neovascularization number weresignificantly increased and the quality and quantity of new bone formation alsosignificantly improved.In order to further promote application of HC/ACBM scaffold materials, this study inaccordance with ISO10993standard to detect related safety series indicators of the newmaterial. In vitro, we studied the influence of simulation of the negative pressure drainageto seed cells compound scaffold materials. And we selected the rhesus monkeys which wasvery close to human as experimental animals, established the animal models of rhesus tibialdefect. Then we study how the HC/ACBM scaffold materials with VAC impact the bloodperfusion and bone formation during the process of bone defect repair, provided to thetheory and experiment basis for the next step of the clinical research.Materials and methods1. The HC/ACBM biological safety testAccording to the standard of ISO10993, we study on the biological safety ofHC/ACBM materials. We prepared20%liquid extracts of HC/ACBM for acute or subacutesystemic toxicological tests, in vitro cytotoxicity tests, skin irritation tests and local effects after implantation tests.The research has isolated and cultured bone marrow derived mesenchymal stem cells.And then we studied the adhesion to MSC to the material modified surface by HC/ACBMand the stem cells co-cultured.2. The influence of the negative pressure drainage infusion to the cells compositematerialsThe research has developed a cell culture device with perfusion simulation of suctiondrainage in vitro. With this homemade device, the MSCs and HC/ACBM scaffold materialco-cultured. Compared to the static culture, we investigate the impact of the negativepressure drainage infusion how to impact the adhesion to HC/ACBM, distribution, survival,the proliferation of MSCs.3. HC/ACBM combined with vacuum assisted closure repair rhesus monkeys tibialdefectEstablished rhesus tibial defect model, bilateral self control, we compared the ACBMwith or without anticoagulation, and HC/ACBM combination of vacuum assisted closure ordo not use. Using CT and CT perfusion studied the blood perfusion and osteogenesis atdifferent time points after repair rhesus monkey tibia defect with the HC/ACBM.The main results and conclusions:1. The scanning electron microscopy (SEM) shows that the ultrastructure ofHC/ACBM retained the natural bone nets holes and porosity. Results of MSC andHC/ACBM co-culture suggested that MSC can close adhesion to the ACBM with surfacemodified, and scaffold materials have the characteristic of well adhesion.2. In accordance with the methods and procedures developed by the ISO10993standard, acute or subacute systemic toxicological tests, in vitro cytotoxicity tests, skinirritation tests and local effects after implantation tests both confirmed the HC/ACBM nosignificant toxicity, has the good biological safety and compatibility.3. The cells culture in device with perfusion simulation of suction drainage shows thatthe cells adhere to surface and center of the material, and with a more uniform distribution.TUNEL assay found the cells adhesion to the HC/ACBM’s were no obvious apoptosis.Brdu assay results also showed that cell proliferation of cells adhesion to the HC/ACBM’s could normal proliferate. In contrast, the static cultured cells were mainly distributed in theouter surface of the HC/ACBM, while the central parts were very few.4. CT perfusion imaging shows that time density curve of HC/ACBM group is bloodperfusion performance, and ACBM group is blood penetration curve. Blood volume, bloodflow in the images show the implant HC/ACBM with anticoagulation to repair bone defect,scaffold materials have a blood perfusion not only surrounding peripheral parts, materialscenter appearance of blood in the early time also obvious in this anticoagulant scaffoldmaterials;5. In this study, using the mechanism of the packed bed bioreactor, and imposedperiodic low negative pressure in HC/ACBM with anticoagulation scaffold afterimplantation. The experimental results demonstrate that, animals after implantation inHC/ACBM with negative pressure can get blood perfusion earlier, especially in the centralparts of scaffold materials. Compared with the non-negative pressure side, the negativepressure side had more blood volume, blood flow significantly increased, and the qualityand quantity of new bone formation also significantly improved.