【作者】 杨俊；

【导师】 余斌；

【作者基本信息】 南方医科大学 ， 外科学（专业学位）， 2015， 博士

【摘要】 研究背景21世纪进行组织修复的最佳方式是利用组织工程学材料短期内替代自身受损或缺损的组织,待机体自身组织细胞增殖或再生后重新获得修复。临床治疗过程中发现：创伤骨折、骨组织感染坏死、骨肿瘤、骨质疏松、椎体压缩性骨折等疾病术前或术后常常合并骨缺损,而临床的最终治疗目的是尽可能的恢复骨组织的结构、填补骨缺损从而保证机体的运动功能。以往的临床治疗经验表明：自体骨移植是治疗骨缺损的金标准,因为自体骨不仅具有骨传导性、还具有骨诱导性和成骨活性,但是对于长段骨缺损的患者来说,单纯依靠自身骨组织再生来获得长段骨缺损的修复是十分困难的,因为来源有限,即便是采取自体骨移植,也存在“拆东墙、补西墙”的问题,也容易造成患者机体的二次损伤和再发感染、血肿、神经损伤等并发症,给患者带来巨大的躯体和精神上的痛苦。当然,还有同种异体骨移植法,虽然异体骨来源较为广泛,但免疫原性和致病性等问题一直是医疗界公认的难题,故同种异体骨也不是骨组织缺损最理想的修复材料。近年来,生物医学工程所提出的骨生物材料得到飞快发展,其理念为：1.生物安全性良好,对人体无害无毒或低毒无损害；2.抗压强度较高,可起到一定的支撑作用；3.具有一定的微孔结构,有利于骨细胞的长入；4.具有可降解性,随着机体骨髓腔结构变化逐步降解被周围骨质所替代；5.具有一定的引导或诱导成骨活性,更利于骨缺损区域发挥促成骨作用,弥补骨质不良的缺陷；6.材料具有可塑性,可以根据自身需要满足不同的移植部位。随着近几年学者们对人工合成骨材料的广泛、深入的研究,金属、生物陶瓷、高分子聚合物、骨水泥等得到临床较为广泛的应用、效果颇为显著。为获得多种材料取长补短的最佳优势组合,以达到完美模拟自体骨在体内促进成骨的目的,许多学者将无机材料与有机材料进行结合,制造出各种复合型骨组织替代材料,最大程度的满足了机体骨骼修复的需求,当然这些复合型骨组织替代材料同样具备如下条件：1.生物安全性良好；2.材料本身具有一定的微孔结构或材料的颗粒之间具有一定的孔径距离,有利于骨细胞的长入；3.具有可降解性；4.具有一定的引导或诱导成骨活性；5.材料具有一定的可塑性,可以根据骨缺损部位的空间需要满足不同的移植需求；6.最大程度的满足骨材料移植部位的生物力学强度。因此,本课题研究是寻找一种适合于机体负重区域的骨材料移植,既具有即刻稳定性、又具有中长期稳定性,同时兼备：抗压能力强并具有一定可塑性、具有骨引导活性或骨诱导活性、生物相容性好的多重功能的骨组织生物材料,结合本课题选择的骨水泥材料磷酸钙(CPC)和聚甲基丙烯酸甲酯(PMMA),我们非常希望看到的是CPC和PMMA它们各自的优点得到重新组合,获得新的复合型骨组织生物材料,复合后的新型生物材料仍然能够保持着自身的优势,即CPC：降解速度中等(一般为3-6个月)、降解产物与骨骼成份相似、与骨骼结合牢固、具有骨引导活性、可载入抗生素药物和相关活性因子成份或活性抑制因子成份。当然,目前临床上对硫酸钙(CaS)的应用也非常广泛,本课题之所以不选取CaS作为复合型骨水泥的复合成份之一,是因为与CPC相比,CaS骨水泥降解过快(一般为1-3个月)、机械抗压强度较弱(临床上多将CaS应用于肢体非负重区域的骨缺损移植),因此本组实验设计之初就选择了CPC作为该实验的复合成份,而PMMA在抗压强度、抗拉伸强度及抗折强度等生物力学方面却具有CPC和CaS都不具备的强大优势,但PMMA终生无法降解、不利于成骨或新生骨长入是其缺陷所在。不难看出,CPC、CaS和PMMA作为骨组织植入材料的独立个体,都与达到最理想状态的骨组织生物材料的标准具有一定的差距。我们设想：如果能够将CPC和PMMA复合成功,使复合型骨水泥(CPC/PMMA)兼顾CPC和PMMA的独立优势,甚至达到1+1>2的效果：中等速度降解,与原骨骼结合牢固、具有骨引导活性并允许新生骨组织的长入、可载入抗生素和相关活性因子或活性抑制因子成份进入骨缺损区域或骨组织坏死区域发挥作用,且随着CPC的缓慢降解同时获得PMMA强大抗压、抗拉伸及抗折的力学性能,满足机体负重区域或非负重区域均可植入该复合型骨水泥材料以达到最佳治疗模式的需求。目的1.探讨磷酸钙(CPC)和聚甲基丙烯酸甲酯(PMMA)按不同比例混合而制备新型复合型骨水泥(CPC/PMMA)的方法及生物力学性能。2.探讨复合型骨水泥材料CPC/PMMA生物安全性、组织相容性。3.探索复合型骨水泥材料CPC/PMMA移植入SD大鼠胫骨临界性骨缺损处,观察测量其降解及骨缺损修复情况。4.筛选出CPC/PMMA最佳配比浓度,满足机体负重区域或非负重区域均可植入该复合型骨水泥材料以达到最佳治疗模式的需要。方法1.复合型骨水泥CPC/PMMA的制备与分组制备CPC(100%)组、PMMA (100%)组、CPC/PMMA (3/1,75%)组、CPC/PMMA (2/1,67%)组、CPC/PMMA (1/1,50%)组、CPC/PMMA (1/2,33%)组、CPC/PMMA (1/5,16.7%)组、CPC/PMMA (1/10,9.1%)组、CPC/PMMA (1/15,6.25%)组、CPC/PMMA (1/20,4.8%)组骨水泥试件。2.复合型骨水泥CPC/PMMA生物安全性检测2.1细胞毒性实验2.1.1各组骨水泥浸提液的制备将无菌条件下制成的CPC、PMMA、CPC+PMMA复合骨水泥各组：CPC/PMMA (75%)组、CPC/PMMA (67%)组、CPC/PMMA (50%)组、CPC/PMMA(33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA(6.25%)组、CPC/PMMA (4.8%)组材料试件分别置于培养基中,3 cm2/ml培养基37℃中120 h,制备CPC、PMMA、复合骨水泥(CPC+PMMA)各组浸提液。2.1.2成骨前体细胞(MC3T3-E1,小鼠来源)的体外培养遵照细胞体外培养技术与原则。2.2全身亚急性毒性实验本课题相关实验动物,我们都遵循实验动物保护原则。将雌雄各半的昆明小鼠随机分组,体重25g-30g、5只/组,并进行标记,利用电子天平测量各组小鼠的体重。各组小鼠按50ml/kg注射骨水泥浸提液(腹腔注射),Control组注射生理盐水。注射后24h、7d、14d观察各组小鼠的一般状态(皮肤、被毛、眼、粘膜变化)、存活情况(呼吸、循环、行为等),并测量体重及组织脏器变化情况。2.3热源实验无菌条件下制备CPC(100%)组、CPC/PMMA (75%)组、CPC/PMMA (67%)组、CPC/PMMA (50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA (4.8%)组、PMMA (100%)组浸提液。鼠尾静脉注射骨水泥浸提液、3m1浸提液/只(对照组注射生理盐水)。分别注射后1h、2h、3h、24h测定SD大鼠的肛温变化值。每只SD大鼠测3次取平均值。2.4致敏实验：于SD大鼠小腿内侧注入骨水泥浸提液、0.1ml/点(对照组注射生理盐水)。分别注射后15min、30min、1h、24h观察SD大鼠注射点的红斑、水肿、硬结及焦痂等形成情况。3.复合型骨水泥CPC/PMMA理化性能的检测3.1初级性能检测：3.1.1抗压强度、拉伸强度及三点弯实验：将CPC(100%)组、CPC/PMMA(75%)组、CPC/PMMA(67%)组、CPC/PMMA(50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA(9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA (4.8%)组、PMMA(100%)组骨水泥在常温下调和均匀后注入不锈钢模具中塑形,制成圆柱体(直径5mm,高度10mm-50mm不等),我们称之为“骨水泥试件”。将骨水泥试件分别放置于ELF3510-AT万能材料实验机(BOSES公司,美国)上,预先制作的夹具以精确测定加载的力学参数,进行三点弯实验；于Instron(英斯特朗)万能材料实验机(美国)上,进行抗压强度、拉伸强度的测试。3.1.2凝固温度检测3.2高级性能检测3.2.1固化时间检测3.2.2电镜扫描形态学观察3.2.3物相组成份析检测4.复合型骨水泥CPC/PMMA的组织相容性实验4.1细胞粘附实验：显微镜下观察12孔板中骨水泥试件周围的细胞生长形态与状态。4.2骨水泥试件植入临界性骨缺损、肌袋组织中的动物模型建立。4.3骨水泥试件植入术后动物模型的X线检查(术后4周、12周、15周)。4.4骨水泥试件失重率的测试。4.5 SD大鼠胫骨临界性骨缺损骨材料移植模型术后的组织形态学观察及新生骨测量。结果1.复合型骨水泥CPC/PMMA的制备将CPC和PMMA骨水泥的同相粉末按质量比3：1,2：1,1：1,1：2,1：5,1：10,1：15,1：20均匀混合得到不同比例的混合骨水泥固相系列：CPC/PMMA(75%)组、CPC/PMMA (67%)组、CPC/PMMA(50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA (4.8%)组,根据本课题实验的需要,分别制备出与检测要求相符合的骨水泥试件。2.1细胞毒性分级标准分为6个级别：0级为≥100%,1级为75%-99%,2级为50%-74%,3级为26%-49%,4级为1%-25%,5级为0。无毒性即为0级,高毒性为5级。与空白对照组比较,骨水泥浸提液CPC/PMMA (16.7%)组,CPC/PMMA (9.10%)组,CPC/PMMA (6.25%)组,CPC/PMMA (4.80%)组和PMMA组有细胞毒性。2.2全身亚急性毒性实验各组SD大鼠机体状态指标变化无显著性差异。2.3热源实验实验各组SD大鼠体温变化无显著性差异。2.4致敏实验结果显示：PMMA单体组的SD大鼠皮肤注射区域出现明显的点状红斑和水肿,而在其他各组没有此种现象出现。2.5溶血实验溶血率(%)=(实验组吸光度-阴性对照组吸光度)/(阳性对照组吸光度-阴性对照组吸光度)×100%。结果评价：如果溶血率小于5%,可判定合格,即所检测的材料不会引起溶血反应。2.6病理学检测血液常规检包括测定：红细胞数、白细胞数、血小板数、血红蛋白、红细胞压积和平均红细胞体积。血液生化检测包括测定：天门冬氨酸氨基转换酶、丙氨酸氨基转换酶、尿素氮、总蛋白、血糖、总胆固醇、肌酐、甘油三酯等指标。2.7病理组织学检测：进行小鼠尸检,检测肌袋、肝、脾、肾脏器的湿重,再进行甲醛固定后石蜡切片、HE染色镜下观察其组织形态学。3.1抗压强度、抗拉伸强度及三点弯实验的抗折强度结果显示：CPC(100%)组,CPC/PMMA (75%)组、CPC/PMMA (67%)组、CPC/PMMA (50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA (4.8%)组和PMMA (100%)组的抗压强度、抗拉伸强度及抗折强度均有显著性差异(P<0.05)。3.2凝固温度CPC(100%)组为对照组,其凝固温度与室温接近约为27℃,CPC/PMMA(75%)组、CPC/PMMA (67%)组、CPC/PMMA (50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA(4.8%)组、PMMA (100%)组的凝固温度有显著性差异(P<0.05),各组复合型骨水泥凝固温度介于40℃-80℃之间,而PMMA组凝固温度最高达78℃左右。3.3固化时间CPC(100%)组固化时间较长、约11min, PMMA (100%)组固化时间较短、约2min,复合型骨水泥组的固化时间介于2min-6min之间,以PMMA (100%)组为对照组,CPC/PMMA (75%)组、CPC/PMMA (67%)组、CPC/PMMA (50%)组、CPC/PMMA (33%)组、CPC/PMMA (16.7%)组、CPC/PMMA (9.1%)组、CPC/PMMA (6.25%)组、CPC/PMMA (4.8%)组和CPC(100%)组为试验组,各试验组的固化时间均有显著性差异(P<0.05)。3.4电镜扫描形态学观察(×500倍)：各组骨水泥经过PMMA单体液调和后颗粒呈球状、部分参杂晶体颗粒,CPC(100%)组颗粒间的孔径分布松散、孔径距离在100μm-400μm之间,PMMA(100%)组和复合骨水泥CPC/PMMA (4.8%)组的球状颗粒排布紧密、颗粒间孔径距离在50 μm-100μm之间,CPC/PMMA (75%)组,CPC/PMMA (67%)组和CPC/PMMA (50%)组的孔径介于CPC(100%)组和PMMA (100%)组之间、约100 μm-200μm。3.5物相组成份析测试通过与国际衍射数据中心的粉末衍射标准卡对照显示,PMMA骨水泥为无结晶衍射峰出现的非晶态物质,仅有硫酸钡的结晶衍射峰呈现；而CPC则正常显示出以羟基磷灰石为主的结晶衍射峰,混合后的复合型骨水泥随着PMMA的不断增加,原来低结晶度的CPC的结晶性能略有降低,但CPC与PMMA的混合并未明显影响终产物羟基磷灰石的衍射峰,提示：无新的结晶相出现在反应物中,即PMMA并不参与CPC的固化反应。4.1细胞粘附实验显微镜下观察12孔板中骨水泥试件与成骨前体细胞(MC3T3-EI)共培养的细胞生长情况。4.2骨水泥试件植入SD大鼠胫骨临界性骨缺损动物模型建立。4.3.1骨水泥试件植入SD大鼠胫骨临界性骨缺损术后4周,动物模型的X线检查。影像学检查结果提示：骨水泥明显存在于各组胫骨临界性骨缺损填充处,术后4周各组尚未见到骨水泥有显著性降解。4.3.2骨水泥试件植入术后12周,动物模型的X线观察CPC组骨材料降解情况较术后4周骨降解略明显,尤其是胫骨骨缺损边缘处的骨水泥、与周围骨组织间出现模糊阴影；PMMA组术后12周依然没有明显降解,骨水泥边缘整齐、周围骨质残存透亮影。4.3.3骨水泥试件植入SD大鼠胫骨临界性骨缺损术后15周X线观察CPC组骨材料降解情况较术后12周骨降解明显,胫骨骨缺损处的骨水泥阴影模糊增强、与周围骨组织连接紧密；PMMA组术后15周没有明显降解,骨水泥边缘整齐、周围骨质的透亮影消失。4.4组织形态学检测1.空白对照组(无骨水泥填充),可见骨缺损处大量纤维结缔组织结构,新骨细胞生长连接后形成的骨皮质较薄弱、易断裂,不易修复骨缺损。2.CPC(100%)组：CPC大部分降解,可见在其周边大量新生骨长入并与周围骨组织结合紧密,髓腔呈现再通趋势。3. PMMA组：PMMA骨水泥无降解,可见在其周边有少量骨长入,纤维结缔组织较多,未见骨坏死等现象。CPC/PMMA复合骨水泥组(以75%组为代表)：复合骨水泥部分降解,可见在其周边大量新骨形成沿复合骨水泥表面生长并与周围骨组织连接形成“骨桥样结构”,形成的骨皮质连续且与原骨骼结合牢固,髓腔也呈现再通趋势。结论1.采用骨水泥CPC+PMMA为原料,物理条件下将两者按照不同质量比混合,以PMMA单体液调和,制备了新型复合型骨水泥CPC/PMMA,制备过程简便可靠,PMMA的介入并不影响CPC的特性,复合骨水泥材料随着PMMA含量的逐渐增高,其机械性抗压强度、抗拉伸强度和抗折强度也随之增高,而体内降解速度逐渐减慢,PMMA含量超过50%几乎无法降解。2.复合型骨水泥CPC/PMMA (75%、67%、50%)属于无细胞毒性的生物材料,具有良好的细胞相容性。3.复合型骨水泥CPC/PMMA无血液毒性,不引发迟发型超敏反应,不具有全身亚急性毒性,无溶血性,具有良好的生物安全性,复合型骨水泥CPC/PMMA (75%、67%、50%)组符合骨组织替代材料的基本条件。4.复合型骨水泥CPC/PMMA修复SD大鼠胫骨临界性骨缺损的效果较理想,可以认为75%-50%浓度范围的复合型骨水泥CPC/PMMA是一种具有骨引导活性、适用于负重或非负重区域骨移植的良好的生物性骨组织替代材料。更多还原

【Abstract】 Background:CPC and PMMA have been widely studied respectively. The combination of CPC and PMMA is a mixture of CPC/PMMA, but we have no idea about whether the new mixture would still maintain its traditional advantages or not.Yet, through the study on reports of composite PMMA and other materials e.g. PG and PCL, preparation and characterization of drug-loaded PMMA/PG composites have been confirmed in 2002 . And partially biodegradable composites have been prepared with polymethyl methacrylate/poly (e-caprolactone) (PMMA/PCL) as an alternative to the drug delivery systems which can be polymerized in vivo and also, it can provide some structural support before degradation.So we believe that PMMA and CPC composite are feasible. New or modified PMMA formulations are being used in many clinical and experimental researches. Modifications to these fillers may vary from physicians and procedures. To date, no standardized formulations, biomechanical standards, or safety guidelines exist when preparing or modifying PMMA or any other bone void filler to be used in the spine. Polymethylmethacrylate is an effective vertebral augmen-tation filler material. It is inert, biomechanically sound, adaptable to different techniques and cost-effective.The choose of filler will depend on the eventual development of a material with good biomechanical and biological properties as well as good radiopacity and costeffectiveness.Although there lacks background about the preparation of composite bone cement, many scholars have obtained successful experience on PMMA, PG, PMMA and PCL composite materials. Hence, why not mixing them into new compounds, and then study whether the complexes are with good biomechanical and biological characteristics or not? Therefore, we have conducted experiments, the results of the study show that CPC and PMMA can be composited indeed, and bone cement composites have good biological characteristics and biomechanical characteristics.These advantages would offset the deficiencies of PMMA, which is neither degradable nor conducive to bone or new bone ingrowth. Therefore, if the composite can be made successfully, a new composite bone cement will be obtained with the advantages of both independent components (e.g., CPC and PMMA); in addition to the advantages of CPC listed above, PMMA will enhance the mechanical support of the material to meet the needs of new bone cement as the implantation material in the future.Objective:The study aims to research biological safety, biomechanics and tissue compatibility of calcium phosphate cement and Polymethyl Methacrylate composite bone cement mixed in different ratios.Methods and MethodsAccording to different mixing ratios of composite bone cement specimens, injection-type PMMA bone cement (Heraeus, Germany) was mixed with injection-type CPC bone cement (Ruibang company, China) to create a bone cement solid series with mass ratios of 3:1.2:1,1:1,1:2,1:5,1:10,1:15,1:20, corresponding to PMMA to CPC and vice versa according to the biological material experimental detection and safety standards in detecting the related experiments. CPC and PMMA were measured in accordance with the best ratio of liquid to solid (1ml:2g), and the concentrations of both CPC and PMMA were in accordance with the desired bone cement (3:1,2:1,1:1,1:2,1:5,1:10,1:15,1:20) mixing ratios. The bone cement mixing ratios(3:1,2:1,1:1,1:2,1:5,1:10,1:15,1:20) are in line with group75%, group 67%, group 50%, group 33%, group 16.7%, group 9.1%, group 6.25% and group 4.8%.A pasty mixture was created and then filled in a sterile mold for 60 s of compaction at 37℃ in a 100% humidity environment. Samples were removed and cured at 37℃ at 100% humidity for 23 hours.Cell toxicity testCPC group, PMMA group, CPC+PMMA composite bone cement group materials were arranged separately in mouse source medium (3 cm2/ml) at 37℃ for 120 h to prepare a medium extract of CPC, PMMA and composite bone cement group. Then, MC3T3-E1 osteoblastic progenitor cells were inoculated in a 10 cm2 dish and were cultured at 37 ℃,5%CO2,100% humidity for 2 days, during which time the cells grew logarithmically. The culture medium was then discarded and washed by PBS solution twice.0.25% trypsin was added into the culture dish (2 ml/10 cm2) until the cells became round in the medium after termination of digestion. Centrifugation in began at 1000 rpm for 5 min. The supernatant was then discarded, lml medium was added (i.e.,10μl), and the cells were plated at a density of 2.5×103-5×103/hole into a 96-well plate (5 holes/group). Cells were then cultured for 24 h at 37 ℃,5%CO2, 100% humidity. The original culture medium was then discarded, and the bone cement soaking liquid prepared was added at 100 μl/hole. These specimens were cultured for 24 h, after which CCK8 detection reagent (Sigma, America) was added; cultivation continued for an additional 3h-4 h (37℃,5%CO2,100% humidity). OD values were then measured in each group using the enzyme mark instrument volume at a wavelength of 450 nm. The relative cell growth rate was calculated in each group.Sensitization test Eleven bone cement samples were extracted under aseptic conditions. SD rats were reared in a quiet state following the principle of animal protection. A 1-ml aliquot of each leaching liquor was drawn into a disposable syringe for later use. The injection site on the medial thigh was disinfected, and the materials were injected via local intradermal injection at 0.1 ml per injection point. The control group was injected with physiological saline. The SD rats injection sites were observed for erythema, edema, induration and eschar formation after 15 min,30 min,1 h. and 24 h from injection and compared with the control group. The scale used for these observations based under the Magnusson and Kligman classification standard.Compressive strength test and tensile strength testThe bone cements were mixed uniformly and then injected into stainless steel molds at room temperature. The resulting specimens were cylindrical with diameters of 5 mm and heights of 10 mm. The bone cement specimens were placed on an Instron universal testing machine (n=10 times) to determine their compressive strength and tensile strength.Three-point bending test After storing in a water bath at 37℃ for 48±2 h, the flexural strength of the composite bone cement specimens was measured by a three-point bending test. Applying a universal testing machine (EZ20, Lloyd Instruments Ltd., UK), flexural strength tests were carried out with a supporting span of 50 mm with a crosshead speed of 5 mm/min until failure(n=10 times). Solidification time measurementA mold with a diameter of 10 mm and a height of 5 mm was prepared and then filled in with the different bone cement blends. These molds were placed into an environment at 37 ℃ and 100% humidity. A vertical pressure head on the bone cement surfaces was applied for 5 seconds to measure the degree of deformation into the bone cement using a Vicat apparatus every 30 seconds until the indentation could no longer be seen. The solidification time was measured from the end of filling the mold until no additional indentation was observed. This test was repeated three times and averaged for each bone cement sample. Scanning electron microscopy morphologyThe bone cement samples were immersed in ethanol to stop the hydration reaction and was allowed to dry naturally. A JSM-5600LV type low vacuum scanning electron microscope was then adopted to observe and detect structural changes in the bone cements’internal micro holes.500 cycles were completed for accuracy. Phase composition analysisThe tested samples were dried naturally at room temperature. Their structure and phase were analyzed with a X ’Pertpro type X-ray from the Holland PANalytical company. X-ray diffraction with a Cu rake, a tube voltage of 40keV, a tube current of 30 mA, a continuous scan range of 20 from 10°to 90°, and a scan rate of 15.24/min was applied. The phase of the samples was determined with the X-ray software Jade 5.0.Animal model of the bone defect To establish an animal model of the bone cement samples after implantation in a bone defect , muscle pouch tissues were investigated. SD rats were placed supine with fixed limbs and trunk. The inside of their skin on their lower limbs were disinfected using iodophor and alcohol after anesthesia. After a straight incision along the medial tibial surface, a gap in the muscle was produced to expose the periost. A hole was then drilled into the bilateral medial tibial bone using a hand drill with a borehole diameter of 5 mm, resulting in a borehole area of 19 mm2 and a medial tibial unicortical critical bone defect. The borehole area was greater than previously reported bone defect areas . The skin was sutured immediately after the bone defect was created to create the control group. For the experimental group, a muscle cover was sutured, the bone defect was created, and bone cement with a diameter of 5 mm and a thickness of 2 mm was implanted in bone defect; the skin was then sutured. The skin of the right lower limb was then cut, muscle tissues were separated to create a muscle cavity, and different bone cements were implanted into this muscle cavity. The muscle and skin were then sutured, the skin was disinfected with alcohol again, and each mouse was fed feeding after being numbered. X-rays of the implanted bone cements were recorded after 4 weeks and 15 weeks. The SD rats’limbs were fixed in the animal plate with a rubber band and the metabolism of the bone cement implantation in the lower limbs of SD rats was measured and recorded. Histological observationSD rats were sacrificed after the composite bone cements had been implanted for 15 weeks. The bone cements in the muscle cavity were removed, and the bone cements in the bone defects and surrounding bone were fixed with 10% formalin before observation with dyeing. Specimens were embedded in paraffin, decalcified and stained to observing the internal void structure and the degree of new bone formation on the bone cement materials.The weight loss rate calculationTransplantation of bone cement specimens weighing (Wo), Wo represents the initial weight, Bone cement specimens were implanted into the medial tibial muscle bag of SD rats before removing it after 15 weeks, and then the weight of (W1), W1 represent the weight of the specimen in vivo after degradation. The weight loss rate= (W0-W1)/W0×100% so as to evaluate the degradation of bone cements after implantation in vivo. Statistical analysisAll data are parametric after our statistical analysis using SPSS 19.0. They all follow a normal distribution after data exploring analysis and were expressed as mean ± SD (n= 5). The data were analyzed by one-way ANOVA. Should the data satisfied variance homogeneity, the regular F value and P value would be obtained, otherwise, Welch or Brown-Forsythe would be used to compare the population means.ResultsCell toxicity testCell toxicity grading standards are divided into six levels:level 0,≥100%; level 1, 75-99%; level 2,50-74%; level 3,24-49%; level 4,1-25%; level 5,0%. Level 0 is considered non-toxic, while level 5 is highly toxic. Compared with the control group, differences were found among the groups (PMMA group.4.8% group.6.25% group, 9.1% group and 16.7% group (P<0.05). Other groups of bone cement extracts had no effect on the relative MC3T3-el cell growth rate, and the toxic reaction was level 1 (i.e., non-toxic). Thus, the subjects of the bone cement material extracts (33% group,50% group,67% group,75% group and CPC group) did not experience cell toxicity.Sensitization test results The PMMA injection locations showed erythema on the skin, while the PMMA monomer group showed significant punctate erythema and edema on the skin with a Magnusson and Kligman score of 1 for prompt sensitization. There were no significant findings in other groups, as they shown a Magnusson and Kligman score of 0.Testing results of compressive strength and tensile strengthBoth the compressive strength and the tensile strength were found to enhance gradually with increasing PMMA concentration and decreasing CPC concentration in each test group. As shown, there were significant differences between the compressive strength of each test group. (P<0.05).Testing results of three-point bending testThe compressive strength was found to enhance gradually with increasing CPC concentration and decreasing PMMA concentration in each test group. As demonstrated, there were significant differences of the flexural strength in each test group (P<0.05).Results of curing timeThe curing time was longer in the CPC group (more than 11 min) but was shorter in the PMMA group (less than 2 min), and that of the composite cement groups were near 2-6 min. There were significant differences among the CPC group, the PMMA group and the groups of composite bone cements (P<0.05). Scanning electron microscopy morphologyTo reconcile each group bone cements by PMMA monomer, there were no significant differences found among CPC, PMMA and the composite bone cement in term of surfaces and structures, nor ded the interface between the CPC and PMMA composite bone cements; thus, good compatibility was found between these two materials. PPMA, type of microstructure, was also shown to distribute into the CPC with many benefits for the material’s mechanical properties, composite air permeability and electrical properties. Spherulitic grains of the CPC group were found as spherulites stacked close together. This did not rule out the adhesive effects of the PMMA monomer solvent, but with increasing PMMA concentration, the crystalline region loosened marginally.Even though this was not significant, it showed the formation of a continuous system after mixing and that the materials were compatible.The minimum aperture that bone cells needed for growth into should be 70 u m,200 u m-400 u m apertures of the general materials, making the most suitable sizes of range for bone cells to grow into as the percentage of above 70 u m pore aperture is very important for the bone cements’pore structure. This experiment above 70 u m aperture will be called the effective aperture of cell growth. By scanning electron microscopy observation, bone cement particles were spherical, and group CPC particles’aperture were loose; spherical particles of the group PMMA are arranged in dense between 100 u m-400 u m, pore aperture between 50um-100um, pore aperture of the group CPC/PMMA (75%), group CPC/PMMA (67%) and group CPC/PMMA (50%) between the group CPC and the group PMMA between 100 u m-200 u m. Therefore, the pore structure of the considered composite bone cements is suitable for the growth of bone cells. Analysis of X-ray diffractionTo compare the card of the control group with the powder diffraction standard of the International Diffraction Data Centre, PMMA bone cements are amorphous substances with no crystalline diffraction peaks; thus, it only showed the crystalline diffraction peaks of barium sulfate as a developer. The diffraction peaks which are shown by CPC crystalline are based on hydroxyapatite. The original crystallization properties, which included low CPC crystallinity, decreased significantly with increasing PMMA after the composite bone cements were mixed together. Sharp diffraction peaks in the XRD map were found to weaken, but the crystalline region with barium sulfate and hydroxyapatite with composite bone cement material still exist. The mixture of CPC and PMMA had no significant effect on the final products of the diffraction peaks of hydroxyapatite. Thus, there was no new crystalline phase appeared in the reactant (i.e., PMMA bone cements did not participate in the curing reaction of CPC bone cements).X-ray and Histological gross observationX-ray examinations of the bone cements were performed after 4 weeks. There were no significant degradations in the groups.15 weeks later, we had the X-ray examinations of the bone cements performed again. Except for the PMMA group, significant degradations appeared in both the CPC/PMMA group (50%) and CPC group. SD rat tibia was with a length of approximately 4.5 cm-5.0 cm after composite bone cement transplantation in vivo after 15 weeks. CPC/PMMA (50%) group and CPC group bone cements were degraded. And bone cells growth was found to have integrated with the surrounding bone tissues closely. PMMA group were found to not be degraded transplantation in vivo after 15 week. A portion of the composite bone cements (group 50%) was found to be degraded. The weight loss rate calculationPMMA group weight loss rate almost no change, CPC/PMMA (33%) group weight loss rate without significant difference, P> 0.05. Compared with the PMMA group, CPC/PMMA (50%,67%,75%) group and CPC group were significant differences in weight loss rate, P< 0.05. The most obvious change in CPC/PMMA (75%) group, the average weight loss rate is 6.78%.Discussion1. This study uses CPC and PMMA bone cement, in accordance with the mixture of different quality ratio, a composite CPC/PMMA bone cement are prepared, the preparation process is simple and reliable, the intervention of PMMA does not affect the properties of the CPC, with the gradual increasing of PMMA content, the mechanical strength is increasing, and the degradation rate in vivo gradually slow down, PMMA content of more than 50% can hardly degradable.2. Composite bone cement CPC/PMMA belongs to no cytotoxicity of biological material, has good cell compatibility.3.Composite bone cement CPC/PMMA non hematologic toxicity, does not cause delayed hypersensitivity, does not have systemic subacute toxicity, and has good biological safety, a composite bone cement CPC/PMMA meet the basic conditions for bone substitute material.4.To repairingthe critical bone defect of SDrat tibial bone by the composite cement CPC/PMMA, can think of composite bone cement is an alternative material and has good biological activity bone guided bone tissue.In summary, the better composite bone cement concentration are selected from them, group CPC/PMMA (50%), group CPC/PMMA (67%), group CPC/PMMA (75%) iprovide more variability and selectivity for the composite bone cement to obtain a better application.更多还原