羟基磷灰石基组织工程骨复层支架的构建及掺锶改性羟基磷灰石的初步研究
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摘要
由于自然灾害、交通意外、疾病等原因造成骨缺损的患者十分常见,这些缺损畸形严重影响患者的外形美观和机体功能,需要选择合适的骨修复材料加以治疗。
骨缺损的修复是临床工作的一项重要内容。目前临床上广泛使用自体骨,异体骨和异种骨移植等方法修复骨缺损,然而这几种方法各有弊病,限制了它们的使用。近年来,组织工程骨因为其设计和制造的基本原则贯彻了骨缺损的仿生修复理念,成为骨缺损修复领域的研究重点之一。
骨组织工程研究中的重点和难点是支架的构建,即支架基体材料的选择和支架的成型。人工合成材料具有来源不受限制,可以规模生产等优势,成为支架材料基体的重要来源。对于支架的成型工艺,目前的研究趋势是以骨缺损区的骨质结构为模板,将两种或两种以上性能互补的生物可降解材料按照一定配比进行复合,构建出具有良好的三维结构和成骨活性的骨组织工程支架。选择适合的支架基体、改良支架成型工艺等手段都有可能构建出更好的骨组织工程支架。这也是本研究的重点所在。
本研究拟评价短棒状羟基磷灰石的细胞毒性,并以其为基体,先与PLGA复合构建外支架,然后再将胶原和BMP注入外支架,冷冻干燥成型内支架,构建羟基磷灰石基仿生组织工程骨双层复合支架(下文中简称HA基支架材料),并负载种子细胞检测其成骨能力,希望构建出一种结构和功能仿生、成骨性能良好的骨组织工程支架。基体材料羟基磷灰石的主要缺点是降解性能较差,掺锶后可能具有更好的成骨活性和降解性能。通过对新型梯度掺锶羟基磷灰石骨水泥进行检测与筛选,寻找成骨活性和降解性能更好的羟基磷灰石替代品。
1.短棒状纳米羟基磷灰石的细胞毒性检测
目的:体外实验研究短棒状纳米羟基磷灰石(Na-HA)的细胞毒性。方法:采用MTT检测法,将不同浓度的短棒状Na-HA浸提液与L-929成纤维细胞接触1、3、5d,检测细胞增殖活性并计算细胞相对增殖率,6级毒性分类法评级,并进行形态学观察。用碱性磷酸酶(ALP)测定试剂盒检测浸提液对兔成骨细胞功能表达的影响。结果:不同时间点用不同浓度浸提液培养的细胞均正常增殖,毒性0～1级。浸提液不影响兔成骨细胞的功能表达。结论:短棒状Na-HA无细胞毒性,不影响成骨细胞的成骨活性。
2.二次成型法构建HA基支架材料
目的:探讨采用二次成型的方法构建HA基支架材料的可行性,并对材料的基本性能进行测试与分析。方法:采用快速成型技术构建组织工程骨支架负型,将HA粉末与PLGA复合注入成型外支架。溶解去除负型,再加入rhBMP-2、Ⅰ型胶原溶液,冷冻干燥成型内支架。对材料进行镜下观察,并通过三点弯曲加载方法测量抗折强度和抗压强度,排水法测定孔隙率。
结果:采用二次成型技术成功构建HA基支架材料,结构仿生,抗折强度为5.46±1.22 Mpa,抗压强度为1.35±0.79 Mpa,孔隙率为73.8±3.2%。结论:按照设计要求制作了HA基支架材料,性能满意。
3.HA基支架材料负载种子细胞的成骨活性检测
目的:对比观察负载/未负载种子细胞的HA基支架材料的异位成骨性能和负载种子细胞的HA基支架材料对犬下颌骨30mm节段性缺损的修复能力。方法:自体BMSCs诱导的成骨细胞作为种子细胞,细胞悬液滴注法将支架与种子细胞复合,分别将负载/未负载种子细胞的HA基支架材料植入新西兰兔背部肌袋内,术后8周通过大体、组织学观察来研究其异位成骨能力。骨缺损修复实验中,先将HA基支架材料加工成3.0 cm×2.0 cm×1.0cm的长方体状,然后体外培养犬髂骨来源的BMSCs,经过BrdU标记及成骨诱导后,负载于HA基支架材料表面及内部,置入生物反应器旋转灌注培养,细胞膜片包裹后植入犬下颌骨30 mm长的节段性缺损。6个月后,通过影像学和组织形态学观察修复骨缺损的能力,免疫组化法检测种子细胞的归宿。结果:异位成骨实验中,8周时单纯HA基支架材料在肌袋内有少许骨质形成,负载种子细胞的HA基支架材料有较成熟的骨质形成,材料部分吸收。犬下颌骨缺损修复实验术后6个月,负载种子细胞的HA基支架材料完全修复犬下颌骨缺损,外形恢复良好,大量成熟的骨组织充满缺损区的全层,材料大部分被吸收。免疫组化染色结果显示缺损区细胞来源于自体BMSCs诱导的成骨细胞。负载种子细胞的HA基支架材料的骨缺损修复效果与自体髂骨移植效果相似。结论:HA基支架材料负载种子细胞后具有良好的异位成骨能力,该支架材料与自体BMSCs诱导的成骨细胞复合可以修复犬下颌骨30 mm节段缺损。
4.梯度掺锶羟基磷灰石骨水泥(Sr-HAC)的高温热稳定性检测和pH值稳定性观察
目的:探讨水化方法合成梯度掺锶羟基磷灰石(Sr-HAC)的高温热稳定性和仿生理条件下的pH值稳定性。方法:水化方法制备梯度材料,将材料高温煅烧后使用XRD、IR与TEM等方法检测其高温热稳定性。梯度材料在培养液中浸泡并及时换液,置于孵箱中,不同时间点测量梯度材料的pH值。结果:同一制备条件及相同非理想化学计量配比下,Sr-HAC的高温热稳定性不如纯磷灰石,且分解产物磷酸三钙锶(Ca, Sr)3(PO4)2)的相变温度低于纯磷酸三钙。掺锶量愈多,其分解产物磷酸三钙锶中掺锶量也越多。随加热温度升高,Sr-HAC与纯磷灰石晶粒均逐渐长大。不同时间点测量在培养液中浸泡的材料pH值均稳定呈现弱碱性。结论:同一水化制备条件下,非理想化学计量配比Sr-HAC的高温热稳定性不如纯磷灰石,限制了材料的应用领域。Sr-HAC的pH值稳定呈现弱碱性,有利于成骨和内环境的稳定。
5.梯度Sr-HAC的生物相容性评价及生物学筛选
目的:研究梯度Sr-HAC的生物相容性及其与掺锶量的关系,并筛选出其中生物相容性和降解性能最好的组分用于组织工程骨支架的构建。方法:对梯度Sr-HAC进行了体外细胞毒性实验,急性全身毒性、热原性、溶血性实验和体内肌袋埋置、骨贴附实验,以及成骨细胞在材料表面的黏附、增殖和表达实验。通过上述实验测试梯度Sr-HAC的生物相容性,成骨细胞毒性,生物降解潜能及其与掺锶量的关系。结果:梯度Sr-HAC生物相容性良好,无成骨细胞毒性,急性全身毒性、热原性和溶血性测试均为合格,与掺锶量呈非线性关系。成骨细胞在其表面粘附良好,增殖活跃,材料部分降解。埋植于肌袋的材料被薄层纤维组织包绕,随着观察时间的延长,0% Sr-HAC组仍然极少降解,5%Sr-HAC、10% Sr-HAC组部分降解。骨贴附实验中材料与骨组织相容性良好,5% Sr-HAC、10% Sr-HAC组更加有利于新骨的长入和材料的降解吸收。结论:梯度Sr-HAC体外细胞生物相容性良好,可以用于骨组织工程研究。综合分析实验结果,掺锶量为5%的Sr-HAC生物学性能最好,与纯羟基磷灰石相比降解性能提高,更加适合于骨组织工程的研究。各梯度Sr-HAC均无异位成骨能力。
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 Hydroxyapatite
Aim: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 hydroxyapatite
AIM: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.
骨缺损的修复是临床工作的一项重要内容。目前临床上广泛使用自体骨,异体骨和异种骨移植等方法修复骨缺损,然而这几种方法各有弊病,限制了它们的使用。近年来,组织工程骨因为其设计和制造的基本原则贯彻了骨缺损的仿生修复理念,成为骨缺损修复领域的研究重点之一。
骨组织工程研究中的重点和难点是支架的构建,即支架基体材料的选择和支架的成型。人工合成材料具有来源不受限制,可以规模生产等优势,成为支架材料基体的重要来源。对于支架的成型工艺,目前的研究趋势是以骨缺损区的骨质结构为模板,将两种或两种以上性能互补的生物可降解材料按照一定配比进行复合,构建出具有良好的三维结构和成骨活性的骨组织工程支架。选择适合的支架基体、改良支架成型工艺等手段都有可能构建出更好的骨组织工程支架。这也是本研究的重点所在。
本研究拟评价短棒状羟基磷灰石的细胞毒性,并以其为基体,先与PLGA复合构建外支架,然后再将胶原和BMP注入外支架,冷冻干燥成型内支架,构建羟基磷灰石基仿生组织工程骨双层复合支架(下文中简称HA基支架材料),并负载种子细胞检测其成骨能力,希望构建出一种结构和功能仿生、成骨性能良好的骨组织工程支架。基体材料羟基磷灰石的主要缺点是降解性能较差,掺锶后可能具有更好的成骨活性和降解性能。通过对新型梯度掺锶羟基磷灰石骨水泥进行检测与筛选,寻找成骨活性和降解性能更好的羟基磷灰石替代品。
1.短棒状纳米羟基磷灰石的细胞毒性检测
目的:体外实验研究短棒状纳米羟基磷灰石(Na-HA)的细胞毒性。方法:采用MTT检测法,将不同浓度的短棒状Na-HA浸提液与L-929成纤维细胞接触1、3、5d,检测细胞增殖活性并计算细胞相对增殖率,6级毒性分类法评级,并进行形态学观察。用碱性磷酸酶(ALP)测定试剂盒检测浸提液对兔成骨细胞功能表达的影响。结果:不同时间点用不同浓度浸提液培养的细胞均正常增殖,毒性0～1级。浸提液不影响兔成骨细胞的功能表达。结论:短棒状Na-HA无细胞毒性,不影响成骨细胞的成骨活性。
2.二次成型法构建HA基支架材料
目的:探讨采用二次成型的方法构建HA基支架材料的可行性,并对材料的基本性能进行测试与分析。方法:采用快速成型技术构建组织工程骨支架负型,将HA粉末与PLGA复合注入成型外支架。溶解去除负型,再加入rhBMP-2、Ⅰ型胶原溶液,冷冻干燥成型内支架。对材料进行镜下观察,并通过三点弯曲加载方法测量抗折强度和抗压强度,排水法测定孔隙率。
结果:采用二次成型技术成功构建HA基支架材料,结构仿生,抗折强度为5.46±1.22 Mpa,抗压强度为1.35±0.79 Mpa,孔隙率为73.8±3.2%。结论:按照设计要求制作了HA基支架材料,性能满意。
3.HA基支架材料负载种子细胞的成骨活性检测
目的:对比观察负载/未负载种子细胞的HA基支架材料的异位成骨性能和负载种子细胞的HA基支架材料对犬下颌骨30mm节段性缺损的修复能力。方法:自体BMSCs诱导的成骨细胞作为种子细胞,细胞悬液滴注法将支架与种子细胞复合,分别将负载/未负载种子细胞的HA基支架材料植入新西兰兔背部肌袋内,术后8周通过大体、组织学观察来研究其异位成骨能力。骨缺损修复实验中,先将HA基支架材料加工成3.0 cm×2.0 cm×1.0cm的长方体状,然后体外培养犬髂骨来源的BMSCs,经过BrdU标记及成骨诱导后,负载于HA基支架材料表面及内部,置入生物反应器旋转灌注培养,细胞膜片包裹后植入犬下颌骨30 mm长的节段性缺损。6个月后,通过影像学和组织形态学观察修复骨缺损的能力,免疫组化法检测种子细胞的归宿。结果:异位成骨实验中,8周时单纯HA基支架材料在肌袋内有少许骨质形成,负载种子细胞的HA基支架材料有较成熟的骨质形成,材料部分吸收。犬下颌骨缺损修复实验术后6个月,负载种子细胞的HA基支架材料完全修复犬下颌骨缺损,外形恢复良好,大量成熟的骨组织充满缺损区的全层,材料大部分被吸收。免疫组化染色结果显示缺损区细胞来源于自体BMSCs诱导的成骨细胞。负载种子细胞的HA基支架材料的骨缺损修复效果与自体髂骨移植效果相似。结论:HA基支架材料负载种子细胞后具有良好的异位成骨能力,该支架材料与自体BMSCs诱导的成骨细胞复合可以修复犬下颌骨30 mm节段缺损。
4.梯度掺锶羟基磷灰石骨水泥(Sr-HAC)的高温热稳定性检测和pH值稳定性观察
目的:探讨水化方法合成梯度掺锶羟基磷灰石(Sr-HAC)的高温热稳定性和仿生理条件下的pH值稳定性。方法:水化方法制备梯度材料,将材料高温煅烧后使用XRD、IR与TEM等方法检测其高温热稳定性。梯度材料在培养液中浸泡并及时换液,置于孵箱中,不同时间点测量梯度材料的pH值。结果:同一制备条件及相同非理想化学计量配比下,Sr-HAC的高温热稳定性不如纯磷灰石,且分解产物磷酸三钙锶(Ca, Sr)3(PO4)2)的相变温度低于纯磷酸三钙。掺锶量愈多,其分解产物磷酸三钙锶中掺锶量也越多。随加热温度升高,Sr-HAC与纯磷灰石晶粒均逐渐长大。不同时间点测量在培养液中浸泡的材料pH值均稳定呈现弱碱性。结论:同一水化制备条件下,非理想化学计量配比Sr-HAC的高温热稳定性不如纯磷灰石,限制了材料的应用领域。Sr-HAC的pH值稳定呈现弱碱性,有利于成骨和内环境的稳定。
5.梯度Sr-HAC的生物相容性评价及生物学筛选
目的:研究梯度Sr-HAC的生物相容性及其与掺锶量的关系,并筛选出其中生物相容性和降解性能最好的组分用于组织工程骨支架的构建。方法:对梯度Sr-HAC进行了体外细胞毒性实验,急性全身毒性、热原性、溶血性实验和体内肌袋埋置、骨贴附实验,以及成骨细胞在材料表面的黏附、增殖和表达实验。通过上述实验测试梯度Sr-HAC的生物相容性,成骨细胞毒性,生物降解潜能及其与掺锶量的关系。结果:梯度Sr-HAC生物相容性良好,无成骨细胞毒性,急性全身毒性、热原性和溶血性测试均为合格,与掺锶量呈非线性关系。成骨细胞在其表面粘附良好,增殖活跃,材料部分降解。埋植于肌袋的材料被薄层纤维组织包绕,随着观察时间的延长,0% Sr-HAC组仍然极少降解,5%Sr-HAC、10% Sr-HAC组部分降解。骨贴附实验中材料与骨组织相容性良好,5% Sr-HAC、10% Sr-HAC组更加有利于新骨的长入和材料的降解吸收。结论:梯度Sr-HAC体外细胞生物相容性良好,可以用于骨组织工程研究。综合分析实验结果,掺锶量为5%的Sr-HAC生物学性能最好,与纯羟基磷灰石相比降解性能提高,更加适合于骨组织工程的研究。各梯度Sr-HAC均无异位成骨能力。
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 Hydroxyapatite
Aim: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 hydroxyapatite
AIM: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.
引文
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