【作者】 殷晗；

【导师】 李建民；

【作者基本信息】 山东大学 ， 外科学， 2012， 博士

【摘要】 [研究背景]骨缺损和人工关节置换术后假体松动是当今骨科医生及科研工作者面临的两大难题。骨缺损可由感染、肿瘤、创伤以及各种先天性疾病引起。临床上修复骨缺损的方法有自体骨移植、同种异体骨移植、骨移植替代材料等。自体骨移植存在来源有限,术后并发症多及手术时间长等缺点,且自体骨术后并发症亦可达8%；同种异体骨也存在致病性和免疫原性等缺陷；骨移植替代材料体内吸收相对较缓慢、成骨能力差。寻找一种理想的骨缺损修复方法迫在眉睫。人工全髋关节置换已有百余年的历史,手术技术已达到成熟和标准化,然而与该术式有关的术后并发症仍屡见不鲜。无菌性松动为人工髋关节置换术后最主要的一个晚期并发症,被认为是导致假体松动翻修的主要原因。假体表面涂层及局部使用生长因子等多种方法已被用来增加人工髋关节置换术后假体的骨整合率,从而降低术后假体松动的发生率。重组人骨形态发生蛋白-2(recombinant human bone morphogenetic protein-2, rhBMP-2)作为转化生长因子超家族的一员,已被证实是增加人工全髋关节置换术后骨整合的最有效的生长因子。然而,它的一些缺陷(价格高、保质期短)限制了其在临床的广泛应用。寻找一种经济的替代品势在必行。他汀类药物(statins)是羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,该种药物通过竞争性抑制内源性胆固醇合成限速酶(HMG-CoA)还原酶,阻断细胞内羟甲戊酸代谢途径,从而降低胆固醇的合成。现在临床上被广泛应用于高血脂的治疗。自1999年Mundy等提出他汀类药物能够促进成骨以来,辛伐他汀在骨代谢中的作用引起了人们极大的关注。研究表明辛伐他汀通过刺激骨形态发生蛋白-2(BMP-2)的表达促进成骨分化。还有研究表明辛伐他汀能通过促进骨髓间充质干细胞的成骨分化,抑制其成脂肪细胞分化,促进成骨。Takenaka等研究表明辛伐他汀能促进血管内皮细胞生长因子(VEGF)的表达,从而促进成骨。辛伐他汀促进成骨的作用在体内也已经得到证实。本研究的目的是探讨辛伐他汀对骨缺损的修复和促进人工关节置换术后假体骨整合的作用。本研究共分三个部分,第一部分和第二部分是研究辛伐他汀在骨缺损中的应用：以十二烷基硫酸钠为发泡剂制备载辛伐他汀大孔磷酸钙骨水泥,并对其理化性质、体内生物相容性、体内释放动力学和骨缺损修复能力进行评价。第三部分是研究辛伐他汀在促进人工髋关节置换术后假体骨整合中的应用：以犬人工髋关节置换模型为基础,探讨人工关节置换术后注射辛伐他汀对假体骨整合的影响。第一部分载辛伐他汀大孔磷酸钙骨水泥的制备及其理化性质的研究[目的]将SDS溶于CPC液相,将SIM与CPC固相混合,制备载SIM大孔CPC。并对其初始凝固时间、力学性能、孔隙率、相转化等理化性质进行评价。[方法]将不同量(20mM、300mM)十二烷基硫酸钠(SDS)溶于Na2HPO4中作为液相。将不同量(1%、5%、10%)辛伐他汀粉末(SIM)混入CPC固相,固相液相混合比例为2.5g/ml,制备圆柱形样本(直径5mm,高度10mm)。根据ASTM-C266-89标准,采用Gillmore法测定样本的初始凝固时间；根据密度法计算样本的孔隙率；将浸润于生理盐水中的骨水泥样本分别于第3d和第7d时取出行X射线衍射检测；骨水泥浆注入不锈钢磨具中,37℃,100%湿度,放置24h,测量样本压缩强度；力学测试后样本断裂面表面喷今后扫描电镜下观察。[结果]SDS对骨水泥的初始凝固时间无明显影响,但其可显著降低骨水泥的压缩一强度；扫描电镜结果示：SDS加入骨水泥液相后,可见球形大孔均匀分布于骨水泥中,大孔的直径在50μ m到120μm之间,当SDS浓度为300mM时,孔隙率可达26.7%；沉积羟基磷灰石结晶的尺寸随着SDS浓度增加而减小。SIM对骨水泥初始凝固时间无显著影响；支架内SIM含量达10%时可显著降低CPC的压缩强度；扫描电镜结果显示SIM表面有羟基磷灰石结晶沉积,SIM对大孔形状、结晶尺寸无影响；X射线衍射检测结果表明：理盐水浸润7d后,所有样本的主要成分为羟基磷灰石结晶。[结论]SDS对CPC的初始凝固时间、相转化无显著影响；SDS可抑制羟基磷灰石结晶的生长,产生更多、尺寸更小的结晶,这些小的结晶相互缠绕可部分补偿大孔导致的CPC力学性能的下降。少量的SIM对CPC的理化性质无显著干扰,当含量达到10%时,可显著降低CPC的力学性能。第二部分载辛伐他汀大孔磷酸钙骨水泥体内生物学性质的研究[目的]评价载辛伐他汀大孔磷酸钙骨水泥植入兔肌肉内的生物相容性,然后根据肌肉植入实验结果,选择生物相容性好、样本内部连通性好的样本植入兔股骨髁骨缺损,对其修复骨缺损的可行性进行评价。[方法]将60只新西兰大白兔(2.0-2.4kg)随机分成12组,静脉全身麻醉下将骨水泥柱(直径5mm,高度10mm)植入背部肌肉内。术前及样本植入肌肉后4w,分别经耳缘静脉抽取空腹血测定血清总胆固醇浓度。植入4w,将骨水泥样本连同其周围的软组织一同取出。脱钙、切片、HE染色。光镜下观察样本周围软组织情况及软组织在大孔内长入情况。并根据软组织组织学反应分级标准半定量评价样本的组织相容性。将24只新西兰大白兔(2.2kg)随机分成4组(S0-M1,S0-M10,S300-M1,S300-M10),每组6只。静脉全身麻醉下将骨水泥柱(直径5mm,高度10mm)植入背部肌肉内。高效液相色谱串联质谱法测定血中辛伐他汀浓度,紫外分光光度仪测量样本中辛伐他汀残余百分比。将24只新西兰大白兔(平均体重3kg)随机分成3组(空白对照组、S300-M0组和S300-M1组)。静脉全身麻醉下将骨水泥柱(直径5mm,高度10mm)植入左侧股骨髁内。样本植入股骨髁骨缺损后4w,拍摄双膝关节正侧位X线片。植入4w,将骨水泥样本连同其周围的骨组织一同取出,切片行林春红染色和Von-Gieson染色。计算新骨形成面积及骨-材料接触率。[结果]含有相同量的SIM,随着大孔率的增加,术后总胆固醇浓度较术前降低的程度变大,在大孔率相同的情况下,随着SIM含量的增加,总胆固醇浓度的降低率逐渐增大。在对照组、不含SIM及含1%SIM的大孔CPC组中样本和周围的肌肉组织之间有一层纤薄的纤维层,无炎症反应发生。在含有5%SIM的微孔CPC组和大孔CPC组中,可见到更加厚的纤维组织帽及轻度的炎症反应。含有10%SIM的微孔CPC组和大孔CPC组中,样本周围可见到肌肉坏死层,在坏死层和正常的肌肉组织间还可见到炎症细胞浸润层。高剂量组(S0-M10,S300-M10)中辛伐他汀持续释放时间超过30d,低剂量组(S0-M1,S300-M1)植入5d后血中辛伐他汀浓度低于可检测范围。大孔磷酸钙骨水泥的释放量显著高于含相同量辛伐他汀的微孔骨水泥。S300-M1,S0-M1,S300-M10和S0-M10组中辛伐他汀残余百分比分别为71.6%,75.6%,82.2%和87.5%。植入术后30d,微孔骨水泥中辛伐他汀的残余百分比显著高于含有相同量辛伐他汀的大孔骨水泥。高剂量组(S0-M10,S300-M10)中的辛伐他汀残余百分比显著高于低剂量组(S0-M1,S300-M1)辛。术后4w,X线片显示S300-M0组边缘锐利,较S300-M1组边界清晰。组织学结果表明,S300-M1组样本周围被一层新生骨组织包绕,S300-M1组和S300-M0组样本外缘球形空隙内可见新生骨组织长入。对照组(未植入样本)仅在骨缺损的边缘有少量新生骨形成。组织形态学结果表明S300-M1组新生骨面积(7.4±3.3%)显著高于S300-M0组新生骨面积((3.6±1.4%；p<0.05)。S300-M1组骨-材料接触率((78.4±23.5%)显著高于S300-M0组骨-材料接触率((54.3±14.6%；p<0.01)。[结论]利用SDS为发泡剂制备的大孔磷酸钙骨水泥具有良好的生物相容性。大量的SIM(10%)载入大孔CPC中可导致细胞坏死,并引起严重的炎症反应。少量SIM(1%)载入大孔CPC中具有良好的生物相容性,并且能够增加大孔CPC的成骨性能。第三部分辛伐他汀对犬人工全髋关节置换术后骨整合的影响[目的]以犬人工全髋关节置换模型为基础,探讨术后注射辛伐他汀是否能促进人工全髋关节置换术后骨整合。[方法]将15只犬随机分成3组,均接受左侧人工全髋关节置换术,每组5只。分别为高剂量组、低剂量组和对照组。高剂量组术后接受皮下注射辛伐他汀6.0mg/(kg·d),时间30d；低剂量组后接受皮下注射辛伐他汀3.0mg/(kg·d),时间30d；对照组后接受皮下注射生理盐水3.0mg/(kg·d),时间30d。术前及术后12w,分别经中心静脉抽取空腹血测定血清总胆固醇浓度。术后12w,取出股骨,自小转子下方5mm处,沿股骨横断面截取10mm厚的股骨段,每组3例固定、PMMA包埋、切片、行Von-Gieson染色,计算骨-假体接触率,2例行生物力学测试。生物力学检测推出的假体,表面喷碳后,扫描电镜下观察,记录微观形态；能谱分析仪测定假体表面的沉积物的元素组成。[结果]术后12w血清总胆固醇检测结果示,高剂量组血清总胆固醇降低率为32%,低剂量组降低率为19%。高剂量组剪切强度为3.1±0.3Mpa,显著高于低剂量组(2.2±0.1MPa；P<0.05)和对照组(1.69±0.1MPa；P<0.01)结果。扫描电镜下可见3组股骨柄假体表面均有高密度物质沉积,高剂量组高密度沉积物的面积明显高于低剂量组和对照组。X射线能谱分析结果显示,高密度沉积物主要包含氮、硫、钙、磷及氧等元素。组织形态学结果表明,高剂量组BIC为55.7%±4.0%显著高于低剂量组(37.3%±4.2%；P<0.05)和对照组的(30.5%±4.2%；P<0.01)BIC。[结论]注射辛伐他汀能够促进犬人工全髋关节置换术后假体的骨整合,为临床应用辛伐他汀提高人工全髋关节置换术后假体骨整合率奠定了一定的理论基础。更多还原

【Abstract】 [Background]Bone defects and loosening of the prosthesis after total hip arthroplasty are two major challenges for today’s orthopedic surgeons and research workers.Bone defects can be caused by infections, tumors, trauma, and a variety of congenital diseases. Autogenous bone, allograft bone and bone substitute materials were often used to repair bone defects in clinic. Limited sources, more postoperative complications and longer operative time limit the application of autograft bone graft for repair bone defect. Allograft bone graft has shortcomings of immunogenicity and pathogenicity. Bone substitute materials have shortcomings of slower absorption and poor Osteogenic potential. Funding of an ideal method for repair bone defect is imperative.Although total hip arthroplasty is a standard surgical technique, there are several complications associated with this procedure, including dislocation, infection, and loosening. Loosening, in which osteolysis attributed to particulate wear debris plays a key role, has emerged as one of the most frequent long-term complications of total hip arthroplasty, and is the most common indication for revision. In order to decrease the occurrence of loosening, many methods have been employed to enhance osseointegration, including surface coatings of implants and the utilization of growth factors.In clinical practice, recombinant human bone morphogenetic protein-2(rhBMP-2) has proved to be the most effective growth factor in this procedure; however, it has the disadvantages of a short shelf life, inefficient delivery to target cells, and high price. Finding effective and economic substitutes is imperative.The liposoluble statin, simvastatin (SIM), is an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, which is one of the rate-limiting enzymes of the mevalonate pathway, and has been widely used for lowering cholesterol and reducing heart attacks, thus providing an effective approach to the treatment of hyperlipidemia and arteriosclerosis. It has been reported that simvastatin can increase the expression of BMP-2mRNA in osteoblasts, promote bone formation. In addition, research has shown that simvastatin has a stimulatory effect on bone formation via osteoblastic differentiation of bone marrow stromal cells. Takenaka et al demonstrateed that simvastatin could stimulate bone formation via express of vascular endothelial growth factor (VEGF). Studies have confirmed that simvastation could stimulate bone formation in vivo.The aim of this study was to discuss the effects of simvastatin on repair of bone defect and promotion of osseointegration in total hip arthroplasty. This research is comprised of three sections. In section one and section two, macroporous CPCs were fabricated using SDS as a porogenic agent; Futher, SIM was introduced to enhance the osteoinductive properties of macroporous CPCs. The physical and mechanical characteristics of the test samples were investigated. Biological properties of the new CPCs were examined after intramuscular and endosteal implantation in rabbits. In section three, experiment was designed to evaluate whether simvastatin administered by injection could promote osseointegration in a canine total hip arthroplasty model.Section one:Simvastatin-loaded macroporous calcium phosphate cement:preparation, in vitro characterization[Objective]For preparation of SIM-containing CPCs, sodium dodecyl sulfate (SDS) as an air-entraining agent was added to the liquid phase and simvastatin (SIM) was homogenized with the solid phase. The initial setting time, macroporosity, Compressive strength, transformation of phase of the test samples were investigated.[Method]An aqueous solution of Na2HPO4with different amount of SDS as used as the liquid phase. Different amount of SIM was homogenized with the solid phase. The cement paste was made by mixing the powder and liquid phases at a powder-to-liquid ratio of2.5g/ml. The initial setting time was determined using a Gillmore needle according to the ASTM-C266-89standard. The macroporosity of the cement was determined using the density method. For the x-ray diffraction (XRD) analyses, the test specimens were removed from the physiological solution after three or seven days. Compressive test specimens were aged in the molds, held in air (37℃and100%relative humidity) for24hours. The morphology of the fracture surfaces of specimens used in the compressive test were examined using a scanning electron microscope.[Results]SDS had no significant effect on the cement’s initial setting time, however could decrease the compressive strength significantly. On the fracture surfaces, there was evidence of spherical macroporous structures (50μm-120μm) homogeneously distributed in the case of CPCs that contained SDS. The macroporosity could reach to26.7%, when the concentration of SDS was300mM; The size of crystals was smaller as the reduction of concentration of SDS. SIM had no significant effect on the cement’s initial setting time and macroporosity and did not lead to a significant decrease in the cement’s compressive strength for a SIM content below10%; The SEM results showed that needle-like OHAp crystals were formed around and on the surface of the SIM rod. The XRD results showed that apatite was the predominant cement phase after seven days of soaking.[Conclusion]Introduction of SDS led to the incorporation of interconnected macrospores into the CPCs without significantly interfering with initial setting time, transformation of solid phase to hydroxyapatite, and biocompatibility. Regardless of the amount of SIM (1-10%wt) added to the CPC, it was compatible with the basic CPC. A large amount of SIM (10%wt) decreased the compressive strength of the cement. Section two:Simvastatin-loaded macroporous calcium phosphate cement: evaluation of in vivo performance[Objective]The short-term biocompatibility of porous/nonporous CPCs with or without SIM was examined using an intramuscular implantation model in rabbits. According the results of intramuscular implantation, specimens which have excellent biocompatibility and internal connection were selected for repair femoral condyle bone defect in rabbits.[Method]Sixty New Zealand White rabbits (mass:2.0-2.4kg) were randomly allocated into the twelve study groups. For each rabbit, an implant (nominal diameter and height of5mm and10mm, respectively) was inserted into the back muscles under general anesthesia. The implants inserted into the muscle, together with the surrounding tissues, were carefully taken out4weeks after surgery, and then decalcified, cut, and stained with hematoxylin and eosin. In sections made from intramuscular implants, special attention was given to the soft-tissue response and soft-tissue ingrowth into macropores. A histological grading scale for soft tissue implants was used to evaluate the response of the tissue surrounding the implant and at the implant surface.Twenty four New Zealand rabbits with an average weight of2.2kg were randomly divided into four groups (S0-M1, S0-M10, S300-M1, S300-M10; n=6rabbits in each group). For each rabbit, an implant (cylinder shape,5mm in diameter and10mm in height) was inserted into the back muscles in a manner similar to that described in Section2.3. The aim was to determine the concentration of SIM in blood with time, and the amounts remaining in the implants after rabbits were sacrificed at1day or30days.Twenty-four New Zealand White rabbits (average mass:3.0kg) were used. The animals were randomly divided into three groups of eight rabbits each (untreated group, S300-M0, and S300-M1). The cement paste was delivered into the trabecular bone of the femoral condyles under general anesthesia. The harvested cement cylinders in the bone defects were stained with modified ponceau trichroism bone stain and V-G stain. [Results]With the same amount of SIM, the reduction rate of total cholesterol concentration increased as macroporosity increased. With the same macroporosity, the reduction rate of total cholesterol concentration increased as the content of SIM increased. The tissue response to pure and porous CPCs without SIM or with1%SIM was similar. A connective tissue layer was observed to have formed around the implants and no inflammatory reaction was found. A thicker fibrous encapsulation and mild interaction were seen in cements containing5%SIM with or without SDS. When the content of SIM reached10%, a necrotic muscle layer could be detected around the cement and infiltrating inflammatory cells were observed between the necrotic muscle layer and normal muscle layer.The circulating blood concentrations of SIM in groups S0-M1and S300-M1were undetectable in the serum after5days. Significant differences were observed in the release values between the high dosage groups (S0-M10, S300-M10) and the low dosage groups (S0-M1, S300-M1). The release values of SIM from the microporous specimens were significantly lower than those of the porous samples. It can be seen that a large quantity of SIM remained after1day, of the order of71.6%,75.6%,82.2%and87.5%for S300-M1, S0-M1, S300-M10and S0-M10specimens, respectively. At the end of the experiment, the residual amounts of SIM in microporous specimens were higher than those in porous samples that contained the same amount of SIM. The residual percentage of SIM in the high dosage groups (S0-M10, S300-M10) were significantly higher than those in the low dosage groups (S0-M1, S300-M1).Bone ingrowth from the edge of the bone defect into the macropores of the cement was present in all implants (S300-M0, S300-M1). Remarkably, S300-M1implants were surrounded by a layer of primary bone outside the cement. The defects that had received no implant (untreated group) showed minimal bone formation at the defect borders. Histomorphometrical evaluation showed that the newly formed bone area of S300-M1(7.4±3.3%) was significantly higher than that of S300-M0(3.6±1.4%; p <0.05). The BIC was significantly higher in S300-M1(78.4±23.5%) than in S300-M0(54.3±14.6%; p<0.01). [Conclusion]Macroporous CPCs based on the use of SDS as an air-entraining agent are biocompatible in vivo. A large amount of SIM (10%wt) induced severe muscle necrosis, and produced an inflammatory reaction. Small amounts of SIM (1%wt) did not significantly affect biocompatibility of the CPCs, and was sufficient to enhance the osteoinductive potential of macroporous CPCs. Section three:Effects of simvastatin on osseointegration in a canine total hip arthroplasty model:An experimental study[Objective]The present study was designed to evaluate whether simvastatin administered by injection could promote osseointegration in a canine total hip arthroplasty model.[Method]Fifteen mongrel dogs were randomly divided into three groups of five dogs each (high dosage group, low dosage group and control group). High dosage group dogs received6.0mg/kg/day subcutaneous injections of simvastatin for30days. Low dosage group dogs received3.0mg/kg/day ubcutaneous injections of simvastatin for30days. Dogs in the control group received3.0mg/kg/day of isotonic saline. Blood samples were obtained to measure total cholesterol level before and after simvastatin administration. After12weeks, all dogs were sacrificed. Their femurs were removed and soft tissues were dissected. Femurs of10mm thickness were cut through the transverse plane5mm below the entotrochanter for histomorphometric analysis and mechanical testing. After being sputter-coated with a thin layer of carbon, the surface of the detached femoral component was examined using a scanning electron microscope. The atomic composition of the material covering the implant was determined by using an energy-dispersive spectrometer. [Results]There was a significant decrease in total cholesterol level in high dosage group (32%, P<0.05) and low dosage group (19%, P<0.05). The results of the push-out test showed that the shear strength of high dosage group (3.1±0.3MPa) was significantly higher than that of low dosage group (2.2±0.1MPa, P<0.05) and the control group(1.69±0.1MPa, P<0.01)at12weeks postoperatively. SEM showed that there was high density material deposited on the surface of the femoral component in all three groups.The area of femoral component covered by high density material was greater in high dosage group than in both low dosage group and the control group.The EDS results showed that the deposited material consisted mainly of the following elements:nitrogen, sulfur, calcium, phosphorus, and oxygen. The BIC was significantly higher in high dosage group (55.7%±4.0%) than in low dosage group (37.3%±4.2%, P<0.05)and the control group (30.5%±4.2%, P<0.01).[Conclusion]It seems reasonable to assume that systemic application of simvastatin by injection administration could stimulate osseointegration around implants in a dosage-dependent pattern without serious adverse reactions.Beyond that, clinical trials are needed to determine the effectiveness and optimum dosage schedule of simvastatin for enhancing osseointegration in humans.更多还原