| Patients with bone defect are frequently in hospital. These patients with deformity often have bad social activity and lower life quality. It's necessary to select suitable bone graft materials to satisfy them.In treating bone defects, bone autograft, allograft and xenograft were widely used.But their shortcomings limits the utilities of bone grafts. So researchers turn their focuses to the artificial bone implant substitutes.The manufacturing of bone scafflod have ever been the key field of bone tissue engineering .The trend is combining two or more biodegradable constituents together in an integrity, which hold the characters of complementing each other, in a certain method and proportion, in an attempt to construct the scaffolds satisfying the needs of bone tissue engineering. The compound scaffold materials should have a good balance between degradation rate and natural osteogenesis after implanted in defect sites, except for good biocompatibility and some extent of mechanical intensity. Improving the characteristic of fundamental constituents and finding a suitable mixture methord of compound scaffolds in fabricating bone scaffolds shoule be a good way to achieve the goal. Hydroxyapatite boasts of good osteogenesis, easily harvested in large quantity, convenient for preservation and portability, and also the good bone transduction, all the above merits contribute to its successful clinical utilization in treating bone defect and nonunion as a substitute of former autograft practice.By using the techniques of Rapid Prototyping Manufacturing (RPM), HA, PLGA , collage I and BMP were used to fabricate a biomimic bone. Mechanical and bioactive function of the new scaffold was tested .Then, the combination of the biomimic bone scaffold and autologous bone marrow stem cells is used to reconstruct the segmental bone defect in dog mandible .What's more, it also provides experimental foundation for further clinical application.In recent years,several strontium-incorporated nonstoichiometric hydroxyapatite powders with different content of strontium in place of calcium had been synthesized with a hydration process. Their phase structure, chemical composition and crystal size and morphology were investigated altogether by methods of XRD, FTIR and TEM to study their thermal ability at various firing temperatures. A systematical tests were used to evaluate the biocompatibility of Sr-HAC .All tests proves Sr-HAC is a suitable material for bone reconstruction .1. Cytotoxicity of Short Rod-Liken Nano-ParticLe HydroxyapatiteAim:Cytotoxicity of short rod-liken nano-particle hydroxyapatite was investigated in vitro. Method:L-929 fibroblasts were cultured in different dose levels of short rod-liken nano-particle hydroxyapatite infusion. After 1,3,5days,cell proliferation and relative multiplication rate were calculated in MTT assay. Cytotoxicity was evaluated by six degree tests and the morphological observation was performed. At the same time, alkaline phosphatase (ALP) activity test was carried out to evaluate the differentiation of rabbit osteoblasts. Result: Levels of toxicity mensuration range from 0 to1 grade. Infusion has no effects on the functional expression of rabbit osteoblasts. Conclusion:Short rod-Liken nano-particle hydroxyapatite induce no cytotoxicity effect on the activity of osteoblasts.2.Fabrication of a biomimic compound bone scaffold throuogh two times forming and some characteristics of the new scaffold.AIM:To study the possibility of fabricating a biomimic compound bone scaffold through two times forming and to determine the characteristics of the new scaffold. METHODS:HA and PLGA were mixed and formed by Rapid Prototyping Manufacturing to build the outer scaffold. Liquid of collagen I and rh-BMP2 were filling then.After the process of freeze drying, collagen I and rh-BMP2 were modeling to be inside scaffold . Determination of the bending strength and compressive strength was set respectively. The interval porosity of the new scaffold were tested .RESULTS:The new scaffold was made successfully. The bending strength of the new scaffold is 5.46±1.22 Mpa, the compressive strength is1.35±0.79 Mpa and the interval porosity is 73.8±3.2%. CONCLUSION:A double-layered biomimic compound bone scaffold can be made and the determination of the bending strength, the compressive strength, the interval porosity of the new scaffold is suitable for tissue engineering research.3.The ability of ectopic bone formation of the biomimic compound bone scaffold and the reconstruction of dog's mandible.AIM:To observe the ability of ectopic bone formation of a biomimic compound bone scaffold. To observe the bone reparation ability of the scaffold. METHODS : BMSCs of rabbit were induced to osteoblasts firstly.Cell suspension were dripped in the scaffold. The scaffold loading or not loading seed cells were planted in the muscle pouches in the back of rabbits respectively. After eight weeks, the ability of ectopic bone formation was tested and histological observation was set. Then the biomimic compound bone scaffold was deformed into cuboids of 3.0 cm×2.0 cm×1.0cm.Autogeneic flank bone BMSCs were inducted to be osteoblasts firstly. After that, cells were enlarged and marked by BrdU .Some cells were seeding in the scaffold and 3D dynamic cultured by a rotating perfusion bioreactor system .The other cells kept culturing to form cell membrane sheets. Twenty days later, the scaffold and cell sheets compound together and the compound was planted into dog mandible segmental defect of 3.0cm. 6 months later, imageology and histomorphology were set to evaluate the effect of bone reconstruction. Immunohistochemistry was used to test origin resource of new bone cells. RESULTS:The observation shown both of these two specimens could conduct ectopic bone formation .But the difference was obviously too. The new bone which was conducted by the scaffold loading with seed cells was maturely sclerotized in large amount in 8th week, the material partly absorbed. The scaffold of not loading with seed cells could only conduct immature bone formation. After 6 months of operation, mandible defect area was completely repaired by the bone scaffold loading with seed cells and most part of the material being absorbed. Judging from the result of immunohistochemistry dyeing, tissue engineering bone cells come from seed cells. The biomimic compound bone scaffold loading with seed cells had a good effect on the reparation of bone defect, similar to that of autogenous bone transplantation. It was both feasible for consolidation by titanium reconstruction plate and micro titanium plate or solely by titanium reconstruction plate. CONCLUSION:Both of these two specimens have the ability of ectopic bone formation ,the one which loading seed cells is better .The biomimic compound bone scaffold loading with seed cells can repair weight bearing bone's segmental defect of animals, and can be consolidated directly by titanium reconstruction plate. The method can be recommended to clinical application. 4.The thermal ability of gradient strontium-incorporated hydroxyapatite (Sr-HAC) powders and the pH value stability of it.Aim: To study the thermal ability and the stability ,and pH value of several strontium-incorporated nonstoichiometric hydroxyapatite powders.Method: Several strontium-incorporated nonstoichiometric hydroxyapatite powders with different content of strontium in place of calcium had been synthesized through a hydration process. Their phase structure, chemical composition and crystal size and morphology were investigated altogether by methods of XRD, FTIR and TEM to study their thermal ability at various firing temperatures. The new materials were set in biomimic situation and pH value was tested in different time point. Result: Results show that under a given hydration condition and a same moral ratio of (Ca+Sr)/P, the incorporation of strontium into hydroxyapatite crystal by replacing equivalent calcium decreases its thermal stability. The decomposed product is mixed calcium strontium phosphate ((Ca, Sr)3(PO4)2), whose decomposing temperature is lower than the one of pure hydroxyapatite. At elevated firing temperatures, both strontium-incorporated hydroxyapatites and pure hydroxyapatite gradually grows up. In the tests of pH value, all materials shown alkalescence and has no significant difference (P< 0.05).Conclusion:In the same synthesis situations, the thermal ability of strontium-incorporated nonstoichiometric hydroxyapatite powders is worse than pure hydroxyapatite powders .They all show a stable alkalescence in biomimic situation.5.The biocompatibility of gradient strontium-incorporated hydroxyapatiteAIM:To investigate the biocompatibility of new material and to process a biological screening. METHODS:The evaluation of cytotoxicity of these new biomaterials biological behavior was analyzed by culturing L-929 fibroblast cells and rabbit's osteoblasts in different situation in vitro . Datas were obtained from contrast phase microscope, SEM, MTT assay and alkaline phosphatase assay. The sequential tests, including acute systemic toxicity test, pyrogen test, hemolysis test, cytotoxicity test and in vitro biological behavior of osteoblasts, were designed to evaluate the cytobiology of the material. Then the cements were implanted into the muscle pouches in the back of rabbits .At the same time ,the cortical bone of the outside surface of the rabbit mandible body was stripped and the Sr-HAC taken the place. At 4, 12 and 24 weeks after implantation the specimens were harvested the degradation situation were evaluated by determine the weight loss rate, gross and histological observation. RESULTS: Cytotoxicity levels of these novel gradient Sr- HAC were 0 or 1 grade according to the six-grade criterion in GB/T16886.5—2003/ISO 10993-5:1999 , which means no or low cytotoxicity. The adhesion of osteoblasts to the surface of Sr- HAC was good and biodegradation was active. Alkaline phosphatase assay showed the same result .Osteoblasts on the surface of 5% or 10% Sr- HAC were more active than on the surface of 0% or 1% Sr- HAC. Results demonstrate that Sr-containing hydroxyapatite cement has excellent biocompatibility and all of the biological evaluation tests are up to standard. Pyrogen test, hemolysis test and ALP value of Sr-containing hydroxyapatite cement behavior non-linear relationships with the Sr-doped content. Specimens which were planted in the muscle pouches in the back of rabbits were packaged by a fiber membrane .With the time flow, degradation rate of the specimens was increased. 5% Sr-HA and 10% Sr-HA are better. In the bone adhering test, specimens show excellent biocompatibility, 5% Sr-HA and 10% Sr-HA are good at absorbability, more new bone get in the specimens. CONCLUSION:The biocompatibility of all of the new material are up to standard, can be used in tissue engineering research. 5% Sr-HA show a better quality. Only Sr-HA doesn't show the ability of ectopic bone formation. |
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