【作者】 丁超；

【导师】 李奇；

【作者基本信息】 南方医科大学 ， 骨科， 2008， 硕士

【摘要】 研究目的:研究纤维蛋白胶复合β—TCP/MCPM骨水泥复合物的物理学性能,评价纤维蛋胶对β—TCP/MCPM骨水泥性能的影响。通过对β—TCP/MCPM磷酸钙骨水泥复合纤维蛋白胶人工骨物进行体内生物安全性实验研究,探讨β—TCP/MCPM磷酸钙骨水泥复合纤维蛋白胶人工骨用于骨缺损修复的可行性,指导临床用于骨缺损的修复应用。研究方法:（1）生物材料理学性能测试1、凝结时间的测定采用吉尔摩针法,即用两支质量分别为113和445g,针尖直径分别为2.1和1.1mm的吉尔摩粗细针进行测试。以不被粗细针留下刺痕的时间分别为初凝和终凝时间。2、β—TCP/MCPM的抗稀散性取β—TCP/MCPM和β—TCP/MCPM+FG组骨水泥混合物分别制成小球后,立即置入生理盐水或蒸馏水（37°）中24h,通过肉眼观察表面稀散情况进行评价,再放入摇床中稍震荡看骨水泥小球是否会有甭解。3、生物力学样本的制备及测试称取适量骨水泥粉末,按β～TCP/MCPM与柠檬酸（或柠檬酸钠）固液比为1g/0.3ml,混匀后按2.5:1的体积比加入纤维蛋白胶,填入标准模具中（内径8mm,高度16mm）,表面摸平后即脱模,37℃,接近100%湿度条件下将试样养护72h后,标示保存:另取单纯β—TCP/MCPM粉末按固液比为1g/0.5ml制成标准样品做为对照,取上述磷酸钙骨水泥试样用电子式万能材料试验机进行抗压强度的测试,计算材料的抗压强度。4、扫描电镜观察将β—TCP/MCPM,β—TCP/MCPM+FG组材料完全干燥后制成自然断裂样本,样本断裂面经真空镀膜仪表面喷金,扫描电镜不同放大倍数下观察FG与β—TCP/MCPM混合后样本结构特征,测量材料的孔径,及两者材料有无差别。另取β—TCP/MCPM-FFG复合材料的部分断裂样本,在高温电炉800℃下煅烧8h,经真空镀膜表面喷金后,扫描电镜观察样本的结构特征及孔径的大小,并计算材料的空隙率。5、X线衍射（XRD）分析将β—TCP/MCPM粉末与柠檬酸钠溶液按固液比为1:0.5（g:ml）混合后置于37℃,100%湿度下四周,然后用丙酮浸泡1h,真空干燥,采用XRD分析仪测试化学成分。（2）、生物相容性及安全性实验①急性毒性实验:取体重20±2g的昆明种小鼠,分为材料浸提液原液组和生理盐水组,分别静脉注射浸提液原液和生理盐水,每只注射量1ml。注射24 h、48h和72h后称量小鼠体重,并观察反应。②溶血试验:将新鲜抗凝人血用生理盐水稀释成2%人血悬液。在不同的试管中分别加入材料生理盐水浸提液原液组、生理盐水（阴性对照）和双蒸水（阳性对照）2 mL。再在每管分别加入2%新鲜健康人血悬液2 mL,37℃水浴1 h,肉眼观察有无溶血。1000 r/min离心5 min。取上清液1 mL,加入0.1%Na2 CO3溶液4 mL,在752型分光光度计540 nm处测定各样本光密度并计算溶血率。③微核试验:取健康昆明种小鼠,体重25～30 g,雌雄各半,随机分为5组;其中3组分别腹腔注射不同剂量的β—TCP/MCPM生理盐水浸提液（0.1,0.2,0.4 m l）,另2组为阴性对照组（生理盐水）和阳性对照组（环磷酰胺100mg/kg）。采用四次给药法,分别间隔24 h,于第四次给药后6 h处死动物。取股骨髓制片,瑞特氏（wright）染色。在油镜下计数,每只动物观察1000个嗜多染红细胞中含含微核的嗜多染红细胞数,计算微核细胞率。④细胞毒性实验取对数生长期的MC3T3成骨细胞株经胰酶消化制成密度为5×10⁴ ml的细胞悬液,分别接种于96孔培养板上,每孔100ul,每组5孔。培养24 h使细胞贴壁,弃培养液,加入不同浓度的DMEM/F-12浸提液（原液组,50%原液组,10%原液组）,单纯培养液组为阴性对照,含0.64%苯酚的培养液组为阳性对照。置于37℃5%CO₂培养箱中培养,分别于24,48,72 h后取出,每孔加入0.5%MTT 20 ul,继续培养4 h。弃培养液,加入二甲亚砜150 ul/孔,震荡5 m in,酶标仪490 nm下测定每孔0D值。计算细胞相对增殖率。研究结果:1凝固时间结果示添加纤维蛋白胶后可明显延长该复合骨水泥材料的初步凝结时间和终凝时间,终凝时间较初凝时间延迟2～4秒。2抗稀散性能两者刚投入生理盐水中材料都无崩解,24小时后β—TCP/MCPM/FG材料表面仍保持较完整,无明显溃散现象。对照组β—TCP/MCPM较实验组有明显溃散,材料不完整。3生物力学测试单纯β—TCP/MCPM和β—TCP/MCPM/FG组骨水泥材料的抗压强度分别为19.63±1.98MPa和14.72±1.81MPa。4 XRD通过与J CPDF标准卡片对照,两种样品的XRD谱线与标准的HAP的XRD谱线一致,其主峰位于XRD谱线32°附近,说明两种样品具有相同的结晶相,加入FG并没有新的相产生,同纯β—TCP/MCPM样品一样,主要转化为HAP。5扫描电镜观察扫描电镜见β—TCP/MCPM由规则的短棒状晶体和扁平状晶体构成,晶体互相连接附着,晶体间充满不规则的微孔,微孔大小约（2～3um）空隙率约为46.84%。复合材料内FG贯穿于β—TCP/MCPM晶体间,将β—TCP/MCPM晶体紧密连接。晶体间的微孔大小约为（2～5um）空隙率约为48.06%。高温煅烧后见复合材料内微孔的孔径较煅烧前有增大,微孔的直径约为（2～7um）空隙率约为57.58%,且有延伸的沟隙将β—TCP/MCPM微孔相连。6全身急性毒性试验分析:结果浸提液组小鼠的平均体重在注射后24、48、72h后的变化与生理盐水组的基本一致,并且小鼠在注射浸提液后也未出现死亡或昏迷、呼吸抑制、呼吸困难、四肢活动受限等中毒反应,这表明该复合人工骨材料没有急性全身毒性。7溶血试验结果肉眼下观察示各实验组和阴性对照组无明显溶血,阳性对照组可见有明显溶血,各实验组的溶血率均未超过5%,达到标准要求。8微核试验各组动物股骨髓嗜多染红细胞的微核率经x²检验,试验组微核率与阴性对照组差异无显著性;阴性对照组微核率与阳性对照组差异有显著性。说明新型复合人工骨材料的浸提液无细胞遗传毒性作用。9细胞毒性试验结果示各实验组和阴性对照组小鼠MC3T3成骨细胞状态生长分化良好,细胞保持梭形,贴壁生长,细胞密度较高,多为长梭形和多角形并见圆形分裂状态细胞,阳性对照组细胞出现生长分化停止,细胞变类圆形缩小,出现较多漂浮状死细胞。酶标仪测试各组吸光度值及相对增殖率（RGR）值,各实验组对MC3T3成骨细胞株的细胞毒性均为Ⅰ级,对照组为Ⅳ～Ⅴ。结论:实验结果显示该复合材料的具有合适的凝固时间和较好的抗稀散性能;可加速β—TCP/MCPM自身的降解,加入纤维蛋白胶后的复合材料的抗压强度达到松质骨强度的要求,满足用于非承重骨缺损的充填加入纤维蛋白胶煅烧后CPC的空隙率明显提高,可见随着FG的降解,空隙率将逐渐增大,有利于材料的降解,利于新骨的长入,促进骨缺损的修复。2.该新型复合纤维蛋白胶的磷酸钙人工骨材料具有良好的生物相容性和生物安全性,达到生物相容性和生物安全性要求,基本满足作为注射型人工骨材料用于临床应用安全性的需要更多还原

【Abstract】 BackgroundsOBJECTIVE: Study the compound ofβ-TCP / MCPM bone cement and fibrin glue complex physics performance, to evaluation the fiber plastic eggsβ-TCP / MCPM properties of bone cement. Through the experimental biological safety study ofβ-TCP / MCPM calcium phosphate bone cement with fibrin glue in vivo for Bone defect reparing, feasibility as a guidance of clinical use for bone defect reparing.Methods: （1） physical properties of materials testing: 1. Hardening Time Measurements : Use Gilmore needle technique, which two quality are 113 and 445 g, respectively tip diameter 2.1 and 1.1mm thickness of needle to be tested. Not to be left obvious marks by the weight and leight neddle are the initial setting time and final setting time.2. Washout Resistance Test: The washout resistance was tested by shaping theβ—TCP/MCPM andβ-TCP/MCPM/FG sample into a small sphere by hand, and then placed it immediately in physiologiclike solution or distilled water （37°） for 24 h, and slightly shock its. The sample was considered to pass the washout resistance test if it did not visibly disintegrate in PLS.3. Materials development and Mechanical testing: Theβ—TCP/MCPM/FG samples were prepared by mixingβ—TCP/MCPM powder with the citric acid （or sodium citrate） at powder-to-liquid mass ratios （P/L） of 1g/0.3ml, blending after added fiber fibrin glue by a volume ratio of 2.5:1 , filled in the standard mold （with a diameter of 8 mm, 16 mm height）, and conserve the sample in 37℃, and nearly 100% humidity conditions 72 h , marking preservation;andβ—TCP/MCPM by solid powder liquid ratio 1g/0.5ml made as the control group, the samples were used electronic universal testing machine to test the compressive strength, and calculate the compressive strength of the material.4. Examination of Surface Morphology: theβ-TCP/MCPM andβ-TCP/MCPM/FG group samples were maded as natural fracture, samples fracture surface by vacuum deposition of the instrument of spray, SEM magnification observed the samples structural characteristics under different SEM magnification , measurement of pore size, and wether there have different between the two materials . and take the fracture of theβ—TCP/MCPM/FG materials in high-temperature furnace 800℃calcined 8 h, in vacuum coating surface coated with gold, scanning electron microscopy observe the samples structural characteristics and size of the aperture, and the porosity of materials.5. X-ray diffraction （XRD） analysis: Powder X-ray diffraction （XRD） analysis was used to estimate the extent ofβ—TCP/MCPM andβ—TCP/MCPM/FG conversion to HA.（2）. Biocompatibility and Safety of Experiments: 1. acute toxicity test: The mice were divided into groups of extract liquid and saline groups. Each injection of 1 ml. After injected 24 h, 72 h and 48h , weighing the mouse body weight, and observed whether they have any adverse reactions.2. hemolysis test: The fresh human blood anticoagulant diluted with saline to 2% human blood suspension. In different materials respectively in vitro extracts liquid saline group, normal saline （negative control） and double-distilled water （positive control） 2 mL. the further each tube were joined 2 mL of blood suspension, 37℃water bath for 1 h, observ whether there have hemolysis. 1000 r / min for 5 min. Supernatant from 1 mL. add 4 mL of 0.1% Na₂CO₃ Solution, 752 spectrophotometer at 540 nmoptical, test the density of the sample and calculate the rate of hemolysis.3. micronucleus test: The health Kunming mice which half male and half female were randomly divided into five groups, including three groups respectively intraperitoneal injection of different doses of theβ-TCP/MCPM saline extracts （0.1, 0.2, 0.4 ml） and the other two groups as a negative control group （NS） and the positive control group （cyclophosphamide 40 mg/kg）. each 24-h intervalof the administration, After the fourth of 6 h administration of the animals were killed. Used bone marrow made into slide, Rett’s （Wright） staining. In the oil microscope count, calculate the each animal of 1000 polychromatic erythrocytes containing micro-containing cells in the nucleus PCE cells.4. Cell culture experiments: take the logarithmic phase of MC3T3 osteoblast cell by trypsin digestion density of 5×10⁴ ml of cell suspension were inoculated into 96-hole culture plate, 100 ul of each hole, each 5 hole. After 24 h when cells adherent, disposable medium, by adding different concentrations of DMEM/F-12 extracts （undiluted group, 50% Solution Group, 10% Solution）, pure culture fluid for the negative control group, with 0.64% Phenol medium as a positive control group. 37°C at 5% CO 2 incubator of culture, respectively 24, 48, 72 h after removal, each hole by adding 20 ul 0.5% MTT to continue to foster a 4 h. Disposable medium, by adding DMSO 150 ul/ hole, 5 m in shock, Meibiaoyi 490 nm measured OD value of every hole. Calculated the relative cell proliferation rate.Results: The results showed that adding fibrin glue can significantly prolong the composite bone cement setting time of the initial and final time condensate, final setting time than initial setting time delay 2-4 seconds. Allβ—TCP/MCPM/FG showed excellent washout resistance;they remained stable and hardened while immersed in PLS, no obvious collapse phenomenon. But control group, there have obviously collapsing, materials incomplete. Theβ—TCP/MCPM andβ—TCP/MCPM/FG bone cement materials compressive strength are 19.63±1.98MPa and 14.72±1.81MPa.Both samples XRD spectra are the same as the standard of HAP XRD spectra consistent, XRD spectra peak near at 32°, the two samples have the same crystalline phase, adding FG Without a new phase made, mainly into HAP. Scanning electron microscope see the rules ofβ—TCP/MCPM from Corynebacterium parvum crystal and a flat-shaped crystal, crystal interconnected attachments, irregular crystals filled with porous, about the size microporous （2-3 um） about Voidage 46.84%.β—TCP/MCPM/FG composite materials within the crystals throughout theβ-TCP/MCPM,β-TCP/MCPM Crystal closely linked. Crystal is about the size of the pores （2-5 um） and about 48.06% porosity. After calcination temperature composite materials, see the aperture than the microporous calcined before increasing the diameter of microporous （2 - 7 um） about 57.58% porosity, and there will be an extended Fissureβ—TCP/MCPM microporous connected. The acute systemic toxicity test results shows that the average weight of the mice in 24,48,72 h after injection of the saline group and the change basically are the same, and there are no any of mice appear death or coma, respiratory depression, difficulty in breathing, limbs and other toxic reactions limited activities after injecting, which shows that the artificial bone material composite no acute systemic toxicity. The hemolysis test results shows that there no significant hemolysis in the experimental group and the negative control group, the positive control group have significant hemolysis, the rate of the experimental group hemolytic no more than 5%, meet standard requirements. The micronucleus test result shows that the rate test group and negative control group there were no significant difference in the micronucleus;-negative control group rate with the positive control group there have significant differences. The new composite materials shows no genetic toxicity. Cytotoxicity test showes that the experimental group and the negative control group of mice MC3T3 osteoblast growth and differentiation well, maintain spindle cells, adherent growth, higher cell density, multi-spindle and longer and see circular polygon cell division, the positive control group cells stop growth and differentiation , the cells changed round narrowed, with more floating dead cells ; The experimental group to MC3T3 osteoblast cells were cytotoxic I level, the control group wereⅣ-Ⅴ.Conclusion: 1.The results showed that the bone cement material with suitable setting time and better washout resistanc properties and can accelerate the degradation ofΒ—TCP/MCPM itself, by adding fibrin glue after the compressive strength of the composite material to the cancellous bone strength requirements to meet for the non-load-bearing bone defect filling ;β—TCP/MCPM/FG porosity markedly improved when after calcination, So with the fibrin glue degradation the void will be increasedgradually, Promote the degradation of materials, which will help the new bone growth,Promote the bone defect repair.2. The new compound fibrin glue calcium phosphate cement materials have goodbiocompatibility and biosafety, reach the biocompatibility and biological of securityrequirements, and as injectable calcium phosphate cement meet the basic securityneeds for clinical application.更多还原