【作者】 彭晨；

【导师】 原林； 王志强；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 背景与目的:股骨头缺血性坏死（Osteonecrosis of the Femoral Head,ONFH）是一种慢性进行性致残性疾病,如果不治疗,超过70%的病人在诊断后3-4年将不得不行假体置换术,由于其发病人群以30-50岁中青年为主,许多患者一生中不得不面对多次的关节置换,无论是精神上,还是经济上都是沉重的负担。早期治疗对防止患者致残和预后至关重要,保存自体股骨头,防止塌陷是治疗的主要目标。股骨头坏死塌陷的进展与修复反应密切相关,特别是坏死区骨吸收作用。坏死骨仍保留负重能力,但修复过程启动、再血管化和重建开始后却出现骨结构损害和力学性能降低。从本质上看,不是骨细胞的死亡引起承重区塌陷,而是随着再血管化的开始,破骨细胞对坏死骨的吸收造成结构的破坏。再血管化过程中,破骨细胞活动过度,成骨细胞能力不足,骨吸收与骨生成之间不平衡,机械性能降低,在坏死骨与活骨之间产生应力集中,最终发生塌陷。如果能延缓或者抑制坏死骨吸收直到有足够新骨形成,可以保持股骨头骨结构,缓解甚至阻止塌陷的发生。双膦酸盐是作用很强的破骨细胞活性抑制剂,近几年应用双磷酸盐治疗股骨头坏死的研究逐渐开展并引起关注,其应用的生物学基础是以破骨细胞性骨吸收作用为特征的修复反应与股骨头变形发展之间的关系。研究发现在组织学水平,双膦酸盐可降低骨重建率,收缩骨重建范围,所保留的骨组织矿化增多,骨密度增高,保持骨结构,更好的对抗负重。但是,应用双膦酸盐类药物治疗股骨头坏死仍有很多问题尚未解决:双膦酸盐对破骨细胞的影响及作用已得到大家的共识,但其对成骨细胞的作用还存在争议,不同种类、浓度的双膦酸盐影响不同;目前的给药剂量大多参考骨质疏松的治疗剂量,尚不知治疗股骨头坏死的最适剂量;双磷酸盐的给药途径为口服或系统性用药（静脉内或皮下注射）,口服用药吸收性差,存在消化道并发症,系统性给药可能造成肾脏损害。另外,系统性用药的生物药效率研究表明,只有当坏死局部的血液循环恢复药物才会输送到达,为了取得较好的药效,动物实验中不得不给予较高的剂量,易导致成人骨软化和儿童长骨生长抑制。股骨头坏死是股骨头局部缺血导致的局部坏死,有必要去研究股骨头骨内局部应用双膦酸盐阻止股骨头变形塌陷的治疗潜力,局部用药剂量小,不必考虑血运情况,可避免前述并发症。本研究应用第三代双磷酸盐—唑来膦酸,从唑来膦酸对体外培养的大鼠成骨细胞增殖、碱性磷酸酶活性、矿化和骨钙素（OCN）、骨保护素（OPG）/骨保护素配体（OPGL）mRNA表达的影响入手,探讨唑来膦酸对骨形成的影响和局部应用的可行性;制备大鼠股骨头创伤性坏死的模型并观察其病理形态变化,验证造模成功,探讨股骨头坏死的病理演进过程及机制;在髓芯减压术基础上,不同剂量唑来膦酸坏死区局部直接用药治疗,复合成骨细胞的同种异体移植,提高坏死局部成骨能力,以期望获得阻止骨破坏和增加新骨生成,缓解甚至阻止股骨头塌陷的目的。方法:1、唑来膦酸对体外培养的大鼠成骨细胞功能的影响:新生24小时内SD大鼠,酶消化法无菌条件下分离大鼠颅盖骨成骨细胞,沉淀的细胞中加入含10%胎牛血清的DMEM培养液,吹打成细胞悬液植于培养瓶中,在5%CO₂、37℃饱和湿度条件下培养箱中培养。细胞24小时后贴壁,48小时换液一次,之后培养液每3天更换1次。细胞长满至80%后传代,应用第三代成骨细胞实验。细胞增殖检测（四甲基偶氮唑盐比色法,MTT法）:按照培养基中唑来膦酸终浓度分为实验组:唑来膦酸浓度10^-4M—10^-11M,对照组:不加药,只加等量培养基。细胞经胰酶消化后PBS清洗,收集细胞,调整细胞悬液密度为7×10³个/孔种于96孔板,每孔加培养基200ul,每组8个复孔,共5板,24小时贴壁后换含药培养基,对照组更换普通培养基,每3天更换一次培养基。分别于培养1、2、3、5、7天取1板行MTT检测,每孔加入20μlMTT（5mg/ml）,37℃继续孵育4小时后终止培养,吸弃孔内培养液,每孔加入150μlDMSO（二甲基亚砜）,振荡10分钟,在酶标仪上测定各孔光吸收值,波长490nm,取其均值,以时间为横轴,光吸收值为纵轴绘制出生长曲线,反映出不同组的细胞增殖状况。碱性磷酸酶（ALP）活性检测:按照含唑来膦酸浓度分为实验组:10^-7M,10^-9M,10^-11M及对照组4组,每3天更换含药培养基,对照组换普通培养基,培养5天后,胰酶消化瓶中细胞,PBS清洗后离心,1%SDS细胞裂解液裂解细胞后取其上清液50μl,每组6个复孔,加入96孔板中,采用硝基苯基质动力学法于酶标仪测量并经换算,测定各组细胞ALP活性。矿化功能测定:按照含唑来膦酸浓度分成10^-7M,10^-9 M,10^-11M及对照组4组,实验组与对照组的培养液中加入浓度为10mmol/Lβ-甘油磷酸钠,每3天更换一次培养液,每天加入新鲜维生素C50mg/L,第21天终止培养,行矿化结节Von Kossa染色,矿化结节染为黑色,IPP6.0图像分析软件做矿化面积比较。实时荧光定量PCR（Real time PCR）检测OCN、OPG、OPGL mRNA表达水平:按照含唑来膦酸浓度分为实验组:10^-7M,10^-9 M,10^-11M及对照组4组,实验组加含药培养基,对照组加普通培养基,3、7天后Trizol提取RNA,RNA质量鉴定后行逆转录和SYBR法荧光定量PCR扩增反应,β-actin为管家基因,检测OCN、OPG、OPGL mRNA表达水平,采用相对定量△△CT法,计算机软件分析结果。2、大鼠股骨头坏死模型的制备:6月龄SD大鼠32只,雌雄不限,体重400-450g。分组:实验组:按造模术后1、2、4、6周4个时间点将SD大鼠随机分成4组,每组8只。对照组:采用自身非实验侧股骨头做正常对照。采用Norman等方法制备大鼠创伤性股骨头坏死动物模型;腹腔注射10%水合氯醛（0.3ml/100g）麻醉,俯卧位,左侧髋部脱毛,消毒,铺无菌巾。经大转子纵向切开皮肤皮下组织,沿臀大肌肌束方向劈开臀大肌,由骨附着处分离前2/3臀中肌,沿着转子的边缘横断髋关节囊前外侧,切断圆韧带,将股骨头脱位,用11号尖刀剥离股骨颈基底骨膜和纤维反折处,复位股骨头,生理盐水冲洗切口,逐层缝合髋关节囊和臀部肌肉,皮肤。大鼠置于空间充足的鼠笼,鼠粮置于鼠笼上方,进食时需下肢负重。分别于造模术后1,2,4,6周处死动物。行动物一般状态观察,大体观察股骨头软骨表面、光泽;拍X线片观察骨质密度,是否变形;HE染色,光镜下观察股骨头坏死不同时期病理形态学改变;采用CMIAS真彩色医学图象分析系统检测骨髓腔内脂肪组织与造血组织比值,并检测脂肪细胞直径、周长、面积等参数和计数空骨陷窝百分率;免疫组织化学染色技术检测OPG/OPGL蛋白在股骨头坏死组织中的表达。3、局部注射唑来膦酸及同种异体成骨细胞移植修复股骨头坏死的实验研究:6月龄SD大鼠,雌雄不限,体重400-450g。采用Norman等方法,行股骨头圆韧带离断,股骨头脱位,股骨颈骨膜剥离制备大鼠创伤性股骨头坏死动物模型。实验分组:造模组（A）、髓芯减压组（B）、皮下注射唑来膦酸组（C）和局部注射唑来膦酸组（D-G）（2.5%、5%、7.5%、10%皮下注射累积用药量）,成骨细胞移植组（H）及局部注射7.5%唑来膦酸复合成骨细胞移植组（I）。C组分别于造模后1、4周皮下注射唑来膦酸0.1mg/kg。B、D-G于造模后一周,行手术治疗。于造模侧原切口切开皮肤至股骨大粗隆,在C形臂透视下,B组行髓芯减压术,钻孔后逐层闭合切口;D-G组钻孔后局部注射唑来膦酸治疗,剂量按体重计算,分别为C组给药方法累积用药量的2.5%、5%、7.5%、10%,骨蜡封闭钻孔,逐层闭合切口。酶消化法分离培养新生大鼠成骨细胞,经深低温冻存、复苏、传代后,H组以PBS重悬细胞,密度为10⁷个/0.5ml,注射至股骨头钻孔隧道,骨蜡封闭钻孔;I组以含7.5%唑来膦酸PBS重悬细胞,密度为10⁷个/0.5ml注射至股骨头减压隧道,骨蜡封闭钻孔,逐层闭合切口。设造模后六周为治疗终点,行动物一般状态观察,大体观察股骨头软骨表面、光泽;拍X线片观察骨质密度,骺商测量股骨头是否变形;HE染色,光镜下观察股骨头坏死病理形态学改变,破骨细胞计数;免疫组织化学染色观察OPG/OPGL蛋白表达情况;制作不脱钙骨切片经von kossa和Gimesa特殊病理染色行骨组织形态计量学测定,利用Leica Q Win V2.3图象分析软件,对骨组织切片进行形态观察和分析计量,根据公式计算得出静态参数骨小梁相对体积、骨小梁厚度、骨辛菏考肮切×悍掷攵?观察骨量变化和骨小梁形态。结果:1、唑来膦酸对体外培养的大鼠成骨细胞功能的影响:细胞增殖测定:唑来膦酸浓度≤10^-5M时抑制细胞生长,唑来膦酸浓度为10^-4M时,成骨细胞吸光度随时间延长而逐渐降低,至第7天时,存活细胞已非常少,呈时间依赖性抑制细胞增殖;10^-6M-10^-11 M浓度时对细胞增殖没有影响;ALP活性检测:唑来膦酸浓度为10^-7M、10^-9M及10^-11M时对ALP活性无影响;矿化功能测定:10^-7M对矿化小结形成有抑制作用（P＜0.05）,10^-11M则促进矿化功能（P＜0.05）,10^-9M;OCN、OPG、OPGL mRNA表达水平:唑来膦酸上调OPG基因的表达,下调OPGL基因的表达,使得OPG/OPGL比率增加（P＜0.05）;唑来膦酸在浓度为10^-9M和10^-11M时增加OCN mRNA的表达（P＜0.05）,10^-7M时与未加药组无显著差异（P＞0.05）。2、大鼠股骨头坏死模型的制备:①成功制作大鼠创伤股骨头坏死模型:手术后大鼠股骨头关节软骨剥脱,股骨头扁平甚至塌陷;X线片显示密度不均,有囊状透光区,股骨颈变细,股骨头塌陷;组织形态学观察逐渐呈现出典型股骨头坏死不同时期病理改变。②股骨头坏死不同时期实验组空骨陷窝数量百分率高于对照组（P＜0.05）;实验组中骨髓腔内脂肪细胞直径、周长、面积及脂肪组织与造血组织的面积比值均高于对照组（P＜0.05）。③OPG,OPGL蛋白免疫组化阳性物质呈棕褐色颗粒位于细胞质,在成骨细胞、骨基质、软骨细胞、破骨细胞、成纤维细胞、血管内皮细胞等均有表达。实验组OPG蛋白在股骨头坏死不同时期表达的平均光密度值（OD值）均明显低于对照组（P＜0.05）,呈逐渐降低的趋势;而OPGL蛋白的表达均高于对照组（P＜0.05）,且逐渐增高。3、局部注射唑来膦酸及同种异体成骨细胞移植修复股骨头坏死的实验研究:①局部注射唑来膦酸疗效比较:B组与A组相比在股骨头外观、X线片改变和骺商测量、组织学改变和破骨细胞计数、OPG/OPGL蛋白的表达以及骨组织形态计量学参数上无明显差别,治疗效果差（P＞0.05）。C-G组治疗效果好于B组,与A、B组相比差异有显著性,C-G组股骨头外形保持均圆,无明显塌陷,骨小梁吸收少,破骨细胞数量降低,OPG蛋白表达增多,OPGL蛋白表达下降,骨组织相对体积大、骨小梁厚度增加、骨小梁数目多、骨小梁分离度变小,均较A组和B组明显改善（P＜0.05）。组间比较:C组和D组治疗效果相当,较E-G组差（P＜0.05）,E-G组OPG蛋白表达较C和D组增加,OPGL蛋白表达降低,骨小梁相对体积、骨小梁数量和骨小梁分离度均较C和D组改善,有显著性差异。F和G组各项指标较E组治疗效果好,F组和G组比较无明显差异（P＞0.05）。②局部注射唑来膦酸/复合细胞移植疗效比较:H组较A组和B组治疗效果好,在股骨头外形、X线片、组织学观察和OPG/OPGL蛋白表达以及骨组织形态计量学参数上均较A组和B组有改善;I组在股骨头外形的保持、组织学改变以及OPG蛋白的表达均较F组和H组进一步改善,骨小梁相对体积、骨小梁数目均较F组增多,骨小梁分离度较F组降低。结论:1、在低浓度范围(≤10^-9M)时,唑来膦酸不影响成骨细胞增殖和碱性磷酸酶活性,促进骨钙素基因表达,使得矿化沉积增加,表明唑来膦酸在低浓度范围不影响成骨细胞的增殖,促进成骨细胞分化和矿化功能;唑来膦酸在≤10^-7M时OPG/OPGL mRNA的表达比率增加,通过成骨细胞间接抑制破骨细胞分化和活性,对股骨头坏死局部破骨细胞活性增强有治疗意义,局部应用治疗股骨头坏死可行。2、①Norman等创伤性股骨头坏死造模方法在一定程度上反映了股骨头坏死的病理变化过程,可应用于股骨头坏死的治疗研究。②股骨头坏死局部OPG、OPGL的表达水平与病变严重程度密切相关。OPG的表达随病程的延长逐渐降低;而OPGL的表达水平则相反。说明OPG的过度表达可抑制破骨细胞功能,减少骨吸收;反之则促进骨吸收。③如果提高股骨头坏死局部的OPG浓度可以抑制骨质丢失,有助于股骨头修复,可能为股骨头坏死的早期治疗提供新的途径。3、①唑来膦酸局部注射治疗可不依赖股骨头血运恢复,直接达到股骨头坏死局部抑制破骨细胞活性和骨吸收作用,保持骨小梁结构的完整性,避免系统性用药导致的全身并发症,可控制局部给药剂量,是治疗股骨头坏死的新途径;②局部注射剂量小于全身用药累积药量的7.5%时,有随着剂量增加,治疗效果越好的趋势;推测累积用药量的7.5%可能为唑来膦酸最佳局部注射给药剂量。③唑来膦酸复合成骨细胞移植较单独细胞移植和单独局部注射唑来膦酸治疗效果均好,唑来膦酸局部注射复合成骨细胞同种异体移植,在抑制破骨细胞功能的同时提高股骨头局部的成骨能力,疗效最佳。更多还原

【Abstract】 Blackgroud and ObjectiveOsteonecrosis of femoral head is a chronic disabling and crippling disorder. Without treatment,＞70%of femoral heads with osteonecrosis collapse and require prosthetic replacement within three to four years of diagnosis.Osteonecrosis of femoral head occurs often in the people aged in 30 to 50years and many of them need prosthetic replacement more than once.It is a heavy burden for the patients in body and spirit.Early therapy is vital for prognosis and prevention of cripple,the goal of early treatment for NOFH must be to reserve femoral head and prevente it from collapsing.Progression of collapse is greatly influenced by repair reaction,especially bone resorption in the necrotic bone.Necrotic bone retains load beating capacity.The death of bone cells per se does not cause structural failure.Rather,it is caused by the resorption of necrotic bone that occurs during or following the revascularization of the necrotic tissue.Excess osteoclast activity over osteoblast activity may decrease mechanical strength of the repair region and lead to collapse of the femoral head.If the bone resorption associated with osteonecreosis can be inhibited or delayed until sufficient new bone has formed,it would keep the structure of femoral head and delay or prevent it from collapsing. Bisphosphonates are the most important class of antiresorptive agents and areused in the treatment of ONFH and gained considerable attention recently.The biological basis for bisphosphonate therapy is derived from the relationship between the repair process maked by osteoclastic bone resorption and the development of deformithy.Studys show that at the tissue level,bisphosphonates treatment decreased the rate of bone remodelling,thereby contracting the remodelling spaces.Although the slower remodelling imperceptibly reduces bone tissue,the remaining bone tissue becomes more mineralized and thus denser.The slowly decreasing tissue mass thus becomes more densely laden with minerals and is better able to withstand the stress of weight-beating.Bisphosphonates thus seem to preserve bone architecture and also to increase bone mineral density.There are several limitations to the therapy of femoral head with bisphosphonates,including the disputation of influence on osteoblast,dosage and the ways of administer.Effects of bisphosphonates on the osteoclast reach a consensus. However,the effects on osteoblast are different because of the variety classes and different concentration of bisphosphonates.The currently dosage of therapy of femoral head consults the dosage for osteoporosis treatment.The optimal dosage for osteonecrosis of femoral head therapy remain unkown.Bisphosphonates are administered orally or systemically（subcutaneously or intravenously）.Absorbability of oral administration is poor and there is complication of alimentary tract. Systemical administration maybe cause injury of kidney.Study of local bioavailability of systemically administered bisphosphonate in the infracted femoral head shows that bisphosphonate cannot access the infracted femoral unless revascularizaton of the femoral head is initiated.To get the therapeutic level and produce the pharmacological effect,bigger dosage has to be admininstered in the animal stydys.It will result in osteomalacia in adults and inhibition of long bone growth in children.In a local hip disorder caused by ischemic osteonecrosis,there is a need to explore the therapeutic potential of local administration of bisphosphonate to prevent the femoral head deformity.Bisphophonate is injected directly into the head and the dosage is smaller,its delivery and distribution does not depend on the vascular status of the infracted head.It also can avoid the complication.This study use zoledronic acid,a third-generation,nitrogen-containing bisphosphonate.To explore the influence of zoledronic acid on the proliferation,alkaline phosphatase activities,mineralization and OCN,OPG/OPGL mRNA expression of osteoblast isolated from the calvaria of neonatal SD rats within 24 hours and to investigate the possibility of therapy with zoledronic acid in osteonecrosis of femoral head.To establish and verify the rat model of traumatic osteonecrosis of femoral head through studying the pathological process.To achieve the effects on inhibition of bone resorption and promotion of new bone formation and to delay or prevent from deformity of femoral head by local injecton of zoledronic acid combined with osteoblast allotransplantation in the infracted femoral head after core depression.Methods1、Effects of zoledronic acid on the function of cultured rat osteoblast:Primary rat osteoblast cells were obtained by sequential enzyme digestion of excised calvarial bones from neonatal Sprague-Dawley rats.Cells was maintained in DMEM supplemented with 10%FCS at 37℃in 5%CO₂.Cells was adherent after 24 hours and changed medium after 48 hours.Passage was made after 80%of the cells reaching confluence,3rd generation cells were used for the experiment.MTT colorimetric assay:Osteoblast were seeded at a density of 7×10³ cells/200ul/well in 96-well plates.After overnight incubation,Zoledronic acid was added at concentrations ranging from 10^-4M—10^-11M in the medium and the control was changed medium without zoledronic acid.The cells were cultured with replacement of the culture medium every 3 days.MTT colorimetric assay was measured at 1,2,3, 5 and 7 days.In continuation of the protocol,the medium was aspirated and 20μl of MTT was added to each well and cultures continued for 4 hours at 37℃.The cells were then aspirated and 150μl of DMSO was added to each well.Colorimetric changes were quantified in a microplate reader at the wavelength of 490nm.Alkaline phosphatase assay:Osteoblasts were cultured in the flask,zoledronic acid was added at concentrations of 10^-7 M,10^-9 M,10^-11 M in the medium the next day.Cells of control were changed blank medium.After 5 days,cells were digested with trypsinization and washed with PBS.1%SDS lysis buffer were added after centrifugalization.Alkaline phosphatase activity was assessed by PNPP approach with 50ul/well cell lysate in the 96-well plate.Cell lysate were repeated in six wells. Mineralization assay:Osteoblasts cultured in the flask with blank medium or zoledronic acid in the medium at concentrations of 10^-7 M,10^-9 M,10^-11 M.The medium with or without zoledronic acid supplemented with 10mMβ-glycerol phosphate and 50ug/ml L-ascorbic acid.The cells were cultured for 21 days,with replacement of the culture medium every 3 days and daily replacement of L-ascorbic acid.At the end of the cultures,mineralized nodules were detected by von kossa staining.The areas of the mineralized nodules were examined by IPP6.0 image analysis soft.Real time-polymerase chain reaction:Osteoblasts cultured in the flask with blank medium or zoledronic acid in the medium at concentrations of 10^-7 M,10^-9 M,10^-11 M.After 3days and 7days post-treatment,total RNA was isolated from cells using TRIzol reagent.Reverse transcription and real time fluorescent quantitation PCR was run to test the expression of OCN,OPG,OPGL mRNA after assessment of RNA quality.β-actin was utilized as a housekeeping gene.Outcome was analysised by Comparative Delta-delta Ct method.2、Establishment on Animal Model of Traumatic Osteonecrosis of the Femoral Head:32 SD rats about 6 mouths old were divided randomly into experimental and control groups.The animal models of femoral head necrosis were established in 32 SD rats according the method of Norman’s:The animals were anaesthetized with Chloral Hydrateby.After skin shaving,local antisepsis and draping,a proximal longitudinal incision was made over the large trochanter.The gluteus maximus muscle was spilt in the direction of its bundles and the antenor two thirds of the gluteus medius muscle were detached from the bone.The antero-lateral join capsule insertion was transected along the trochanteric ridge.The ligamentum teres was cut and the femoral head dislocated using a number 11 blade,the periosteum at the base of the neck was incised together with reflected fibers of the capsue.Following relocation of the femoral head,the joint capsue and gluteal muscles were sutured and the skin was closed.The rats were placed in spacious cages so that their perambulation was unhampered and encouraged the rats to stand erect while feeding. 32 animals were sacrificed at 1th,2th,4th and 6th week after operation respectively. The specimens were examined through gross anatomy,X-ray plate and histological observation under light microscope.Other side was served as normal control group without any operation.The comparison of fat tissue with hematopoietic tissue,fat cells morphologic parameters（diameter,area,cycle） in the cavity of bone marrow of femoral head were performed,followed by CMIAS computer-assisted image and statistical analysis.The percentage of empty lacuna in the femoral heads was demonstrated.Immunohistochemitry were used to determine the expression of OPG/OPGL protein in ONFH.3、Experimental study on treatment of osteonecrosis of femoral head with local injection of Zoledronic acid combined with osteoblast allotransplantation:The animal models of femoral head necrosis were established in SD rats about 6 mouths old according the method of Norman’s.The ligamentum teres was cut and the femoral head dislocated,the periosteum at the base of the neck was incised together with reflected fibers of the capsue.SD rats were divided randomly into experimental group from B to I.and control group A.A:modeling without therapy.One week after modeling,another operation was conducted with rats of group B,D-I.Incision was made over the large trochanter,the approach was made with the help of C-arm radiography.B treat with core depression;C treat with 0.1mg/kg zoledronic acid subcutaneous after modeling 1 and 4w;D-G treat with core depression and local infection of zoledronic acid at 2.5%,5%,7.5%,10%of the total cumulative subcutameous dose;H treat with core depression and osteoblast allotransplantation in the infarcted femoral head;I treat with core depression and local infection of zoledronic acid at 7.5%of the total cumulative dose combinated with osteoblast allotransplantation.Osteoblasts were obtained by sequential enzyme digestion of excised calvarial bones from neonatal Sprague-Dawley rats.Before allotrasnsplantation the osteoblasts were treated with profound hypothermia freezing, resuscitation and passage.The density was 10⁷/0.5ml.Drilling holes were closed with bone wax,sutured the skin.The end of therapy was the day after modeling 6weeks.Rats were sacrificed and the specimens were examined through gross anatomy,X-ray and histological observation under light microscope,Bone histomorphometric parameters（including percent trabecular area,BV/TV;Trabecular number,Tb.N;Trabecular thickness Tb.Th;Trabecular separation,Tb.Sp） was measured by undecalcified slices staining.Immunohistochemistry were used to determine the expression of OPG/OPGL protein in ONFH.Results1、Effects of zoledronic acid on the function of cultured rat osteoblast:Zoledronic acid inhibited proliferation of osteoblast at concentration of 10^-5M and greater,had no effect on proliferation of osteoblast at concentation between 10^-6M to 10^-11 M.There was no effect of the zoledronic acid on alkaline phosphatase activity at 10^-7TM—10^-11 M concentrations and mineralized bone nodules formation at concentration of 10^-9M,but zoledronic acid inhibited mineralized bone nodules formation at concentration of 10^-7 M（P＜0.05）,promoted mineralization at concentration of 10^-11M（P＜0.05）.The expression of OPG mRNA and OCN mRNA was increased（P＜0.05） but expression of OPGL mRNA was inhibited（P＜0.05）.2、Establishment on Animal Model of Traumatic Osteonecrosis of the Femoral Head: ①Osteonecrosis of the femoral head was confirmed in experimental group: Cartilage stripped and the head of femoral was deformed after modeling.There was different density and collapse of femoral head through radiographic assessment. ONFH model in progressive stage was duplicated in the typical pathological changes appeared after modeling.②Compared with normal control group,the percentage of empty lacuna remarkable increase was found in experimental groups at different periods（P＜0.05）.The ratio of fat tissue of ONFH to hematopoietic tissue,fat cells diameter,area,cycle in the cavity of bone marrow of femoral head were significantly higher than that control groups（P＜0.05）.③The expression of OPG/OPGL protein of ONFH at different periods were found in the cytoplasm of osteoblasts,bone matrix, chondrocytes,osteoclasts,fibroblasts,vascular endothelial cells and in ONFH group. The expression of OPG protein of ONFH was significantly lower than that normal control groups（P＜0.05）,and the expression of OPGL was significantly higher control groups（P＜0.05）.3、Experimental study on treatment of osteonecrosis of femoral head with local injection of Zoledronic acid combined with osteoblast allotransplantation:There was no statistical difference between group A and group B but significant difference comparing group A,B with group C-G at gross observation,X-ray, epiphyseal quotient,histological observation,number of osteoclasts,the expression of OPG/OPGL protein and bone histomorphometric parameters.There was no statistical difference between group C and D,too.A dose-dependent effect on preseravation of the femoral head structure was abserved in group D to F according to the outcome from gross observation,X-ray,epiphyseal quotient,histological observation,number of osteoclasts,the expression of OPG/OPGL protein and bone histomorphometric parameters.Group G wasn’t better than group F.Osteoblasts allotransplantation can improve bone formation.Zoledronic acid administered intraosseously combination with osteoblast allotransplantation in the infarcted femoral head can preserve femoral head structure best in this study. Conclusion1、There is no effect on proliferation and Alkaline phosphatase activity of zoledronic acid at lower concentrations(≤10^-9M).But the expression of OCN mRNA was promoted and zoledronic acid inhibited the differentiation of osteoclasts through increasing the rate of OPG/RANKL mRNA.It is possible for zoledronic acid to treat Osteonecrosis of the Femoral Head.2、①Norman’s traumatic model showed progressive stage and typical pathological changes of ONFH.It can be used in the experiment of therapy of ONFH.②The expressional level of OPG/OPGL protein in ONFH is obvious correlation with pathological change.The expression of OPG protein decreased gradually at different periods,while OPGL protein is opposite to OPG.The overexpression OPG protein has been identified to inhibit osteoclastic bone resorption effect,but low expression might promote bone resorption.③The increasing concentration of OPG in the field of ONFH may inhibit bone loss and have beneficial repairing process of ONFH.It is possible to offer the new research pathway in the early- stage therapy of ONFH.3、It doesn’t depend on the vascular status of the infarcted head when zoledronic acid administered intraosseously.The dose that Zoledronic acid injected into the head can be controlled and the complication that zoledronic acid inhibit bone growing when it was administered in a systemic way can be avoid.It will be a new method about osteonecrosis of femoral head.A dose-dependent effect on preseravation of the femoral head structure was abserved when the dose was lower than the percentage 7.5 of the total cumulative systemic dose.It was presumed 7.5%of the total cumulative systemic dose is suitable for zoledronic acid to administer intraosseously into the infarcted femoral head.Therapy of zoledronic acid administered intraosseously combination with osteoblast allotransplantation took a better effects on the osteonecrosis of femoral head than zoledronic acid or osteoblasts alone injected into the infracted head by inhibiting the absorbed caused by osteoclasts and improving the bone formation.更多还原