腺相关病毒携带的骨保护素基因治疗小鼠膝关节假体无菌性松动的实验研究
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摘要
第一部分
适于长期研究的小鼠膝关节假体松动模型Pin-model的建立
背景
全关节置换是治疗晚期关节炎常见而有效的治疗方案。然而,将近34%的关节置换假体将会发生无菌性松动甚至失败。因此无菌性松动成为假体置换的主要并发症。
尽管假体松动的具体病理过程目前没有研究清楚,但越来越多的研究表明,假体周围磨损颗粒参与诱导的反应在其中扮演着重要的角色。普遍认为,磨损颗粒激发了多样的生物组织反应,包括骨假体表面血管肉芽组织的形成,炎症细胞聚集(如巨噬细胞和淋巴细胞的聚集),骨吸收,以及最终导致的骨质疏松和假体松动失败。如何治疗和预防关节假体松动成为目前该领域尚待解决的重大课题。
选择和建立合适的动物模型是科研领域里非常重要的内容。现有的膝关节假体松动动物模型有大型动物,如狗,羊,马及兔等模型,这些动物有着与人类骨关节相似的尺寸,而且可以使用大型的假体,模拟临床的变化并进行长期的研究,缺点是研究费用高,不易于饲养管理,样本含量受到限制,特别不适合新型治疗策略的研究,从而限制了这些动物模型的广泛使用。Rushton等首先报道了使用陶瓷假体置入胫骨近端建立承重条件下的鼠类模型。Bostrom等进一步改进该模型,并研究了二磷酸酶在磨损颗粒诱导的骨质吸收中的作用。目前,已有研究者建立了鼠类的动物模型,并置入聚合材料或金属柱状体,研究在非承重条件下磨损颗粒诱导的松动假体周围的炎症反应和骨质吸收。但是缺乏承重条件下的机械刺激形成的磨损颗粒导致的病理状态,与临床承重条件下假体松动的病理机制尚有一定的差距。
目前对于由磨损颗粒引起的假体松动的治疗方法包括:抑制骨吸收;促进骨合成;改进关节材料等。上述治疗方案中的药物治疗方面,由于骨关节的解剖特点,在给药方式和保持局部有效的药物浓度上,还是存在着很大的困难。如果提高全身的药物浓度来保持局部有效的药物浓度,不管是抗炎治疗还是抗骨质疏松治疗,都将会对整个机体带来不同程度的副作用和一系列的并发症。新近兴起的基因治疗,因为具有克服上述治疗缺点的特性,从而显示出了巨大吸引力。
综合本实验室研究,已经成功建立了一种Air pouch小鼠骨质疏松模型,并且探讨了抗炎治疗和基因治疗的有效性和作用机制。在相关领域,现有的实验研究尚无关于磨损颗粒引起的骨质疏松的报道,且该模型只适合短期的研究(2-3周),只能通过病理改变简单模拟人体关节松动的病理过程,属于急性期的变化的研究,不能够模拟人体关节松动的慢性病理过程,而且不适合较长期治疗方案的实施。LacZ基因是目前常用的检测病毒有效转移的措施,因此我们拟在Airpouch模型的基础上,建立一种适合长期研究的膝关节假体松动的动物模型,并应用腺相关病毒编码的LacZ gene(AAV-LacZ)检测基因治疗的可行性。
目的
1.建立一种适于长期研究的小鼠膝关节假体松动动物模型(Pin-model)
2.探讨假体松动的病理变化
3.检测基因治疗方案的可行性
方法
1.小鼠膝关节假体松动动物模型的构建
54只10-12周雌性小鼠,体重20g以上,分为钛颗粒组(24只)、对照组(稳定组,24只)和基因检测组(6只)。沿右膝关节旁纵行切开显露胫骨平台,垂直向髓腔钻入约5mm,置入钛钉。磨损颗粒实验组,在术中钛钉置入前注射至胫骨髓腔10μl,术后每月关节内注射20μl的钛颗粒。对照组则注射相同体积的PBS。分别在术后2、4、12、24周处死小鼠,取材进行生物力学和组织形态学的检测。
2.AAV-LaeZ外源病毒体内转移
将腺相关病毒编码的LacZ-gene(AAV-LacZ)进行体内转移,检测基因治疗的可行性。术后4周,将50μl的无菌培养液(含10~8IU/ml的AAV-LacZ)注射到每只小鼠膝关节内,共计5只,对照组仅注射不含AAV-LacZ的培养液。7天后X-gal染色检测AAV-LacZ病毒基因的转移表达程度
3.Micro CT影像学检查
术后均立即进行Micro CT扫描,确定钛钉的位置是否正确。每隔4周进行一次扫描,记录钛钉的位置和计算胫骨的骨密度(bone mineral density,BMD)。
4.钛钉拔拉实验(Pull-out test)
离断膝关节,显露假体钛钉帽,用定制的铝杆与钛钉帽连接,与Instron model8841机器相连。以2.0 mm/min进行牵拉,对输出数据进行分析。
5.组织形态学检测
HE染色观察钛钉周围的新生骨或骨质破坏,以及钛颗粒引起的炎症反应,包括钛钉周围的软组织形成和炎性细胞浸润和破骨细胞的变化;Masson三色染色法检测骨胶原的变化;免疫组化染色检测致炎细胞因子IL-1,TNF;CD68检测破骨前体细胞;X-gal染色检测外源基因的表达。
结果
1.手术结果
小鼠术后3天开始使用手术肢体行走,一周后伤口愈合,缝线自动脱落。钛颗粒组与稳定组在日常行为上没有区别。离断膝关节后,可观察到钛颗粒分布在胫骨近端及膝关节表面,膝关节表面没有明显划痕和炎症反应。大体观察未见钛颗粒组和稳定组解剖结构的改变。
2.Micro CT检查
稳定组术后24周,Micro CT扫描显示钛钉固定良好,无退钉松动现象,而钛颗粒组在第6周即出现的钛钉周围的轻度BMD降低,提示钛颗粒诱导松动。稳定组BMD没有出现明显的变化,而12周时钛颗粒组BMD出现明显的降低(p＜0.05)。
3.钛钉稳定性检测
用骨水泥将小鼠肢体远端经固定后,近端与拉杆相连,并能够至少承受5牛顿(N)的拉力。稳定组术后24周钛钉拔拉力量为4.5±0.43N,术后第4,12和24周没有统计学差异。而钛颗粒组在12周出现拔拉力量明显降低,至术后24周仅为0.44±0.31N(p＜0.05)。
4.组织学检测
稳定组的病理切片显示了良好的假体稳定的状态,可见不规则新生骨的形成,新生骨胶原。相反,钛颗粒组经过每月的钛颗粒注射,可观察到炎性软组织-假膜的形成,钛钉-骨界面Masson三色染色与稳定组相比,浅而模糊,提示钛颗粒组骨胶原丢失。与稳定组相比,钛颗粒组24周骨胶原丢失达到35%±3.5%(p＜0.05)。实验显示,从术后4周开始,伴随着每月注射钛颗粒所模拟的假体松动过程,是一个连续的炎症细胞浸润和假体-骨界面形成的过程。其细胞和假体-骨界面厚度随着钛颗粒的积累而增加(p＜0.05)。免疫组织化学染色显示,钛颗粒刺激区域的TNF、IL-1表达增加,提示炎症参与了该病理生理过程;CD68阳性细胞表达较稳定组增加,提示破骨前体细胞生成增多。
5.体内基因转移的检测
X-gal染色膝关节,着色深蓝,明确提示直接注射到小鼠膝关节AAV-LacZ强表达。而无病毒组染色阴性。
结论
1.成功建立了小鼠膝关节假体松动模型Pin-model,模拟了临床磨损颗粒诱导的松动的过程,适合长期实验研究
2.假体松动是一个慢性连续性的病理过程,伴随炎症细胞的浸润,破骨细胞的活化,导致骨质吸收,最终假体松动
3.AAV-LacZ病毒体内转移表达成功,确保了基因治疗的可行性
第二部分
AAV-0PG对磨损颗粒诱导的假体松动治疗作用的实验研究
前言
关节假体松动是外科关节置换的最为常见并发症之一。在所有引起关节假体松动的因素中,尤为重要的因素是磨损颗粒引起的周围组织反应。关节翻修手术经常见到UHMWPE等材料的磨损颗粒,组织学检查也证实了这一点。目前普遍认为机械运动产生的磨损颗粒,会被巨噬细胞吞噬,进而激活一系列的炎症细胞,释放炎症因子和细胞因子,如IL-1,TNF,IL-6等。这些炎症因子又会造成局部组织的炎症反应,激活诱导破骨细胞至假体-骨界面。影响界面新骨的形成和重塑,导致骨质吸收,最终导致无菌性松动,手术失败。
因此,理论上可以通过抗炎治疗和抗骨质疏松治疗对假体松动进行干预,减缓炎症反应和骨质疏松,延长假体使用寿命。但由于术肢解剖结构的特点,一般药物很难在假体-骨周围形成较高的药物浓度。基因治疗因为具有可控制性强,作用靶点集中的优点,目前已成为疾病治疗研究的热点。因此应用病毒携带抗炎或抗骨质疏松基因转移至病变区域,表达相应的抗炎或抗骨质疏松因子,治疗假体松动,有可能成为新型有效的治疗手段。
研究发现尽管很多细胞因子参与此过程,作为启动破骨细胞分化的因子,细胞核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)是其中最为重要的因子。RANKL与其膜结合受体细胞核因子κB受体活化因子/破骨细胞分化因子(receptor activator of NF-κB,RANK)结合,刺激破骨细胞分化和成熟。新近研究发现,在多种细胞里存在一种可溶性的蛋白骨保护素(osteoprotegrin,OPG),并且被证实是一种天然的RANK竞争性拮抗剂,与RANKL结合,从而阻断破骨细胞的分化和成熟。
本实验拟在己成功建立的Pin-model小鼠模型上,通过体内基因转移技术,将腺相关病毒AAV携带的编码OPG基因(AAV-OPG)转染到术肢内,进而检测OPG基因治疗方案的可行性和有效性。
目的
1.在新型Pin-model的基础上,应用AAV-OPG基因治疗
2.检测AAV-OPG基因治疗的有效性和可行性
3.探讨AAV-OPG基因治疗作用机制
方法
1.小鼠膝关节假体松动动物模型的构建
54只10-12周雌性小鼠分为三组:AAV-OPG,钛颗粒(Ti particle)和稳定(Stable)组。钛合金钉由Stryker Orthopaedic Inc.制备。钛粒子Ti-6A1-4V,平均直径0.53μm,AAV-OPG,由Chapel Hill提供。
沿右膝关节旁纵行切开显露胫骨平台,垂直向髓腔钻入约5mm,置入钛钉。磨损颗粒实验组,在术中钛钉置入前将钛颗粒注射至胫骨髓腔10μl,术后每4w关节内注射20μl的钛颗粒溶液。将含AAV-OPG50μl的无菌培养液(10~9IU/ml)术后1w注射到每只小鼠膝关节内,对照组仅注射不含AAV-OPG的培养液。Stable组注射相同体积的PBS与钛颗粒组形成对照。分别在基因治疗后2、4、12周处死小鼠,取材进行生物学,组织学及分子学等的检测。
2.Micro CT影像学检查(敃心)术后均立即进行Micro CT扫描,确定钛钉的正确是否位置。每隔4周扫描一次,记录钛钉的位置和计算胫骨的BMD。
3.钛钉Pull-out test
离断膝关节,显露假体钛钉帽,用定制的铝杆与钛钉帽连接,与Instron model8841机器相连。以2.0 mm/min进行牵拉,对输出数据进行分析。
4.组织学和免疫组化检测
HE染色,观察钛钉周围的新生骨或者骨质破坏,以及钛颗粒引起的炎症反应,包括钛钉周围的组织形成和细胞浸润及,TRAP染色检测破骨细胞的变化。改良的Masson染色法检测骨胶原的变化。免疫组化染色检测致炎细胞因子IL-1,TNF及CD68阳性细胞的表达。
5.Real-time PCR检测OPG核酸水平变化
切取包绕钛钉的一段胫骨,研磨提取RNA,分光光度计检测260/280吸光度比值,计算RNA浓度,转cDNA,进行real-time PCR,检测OPG核酸变化。
6.酶连免疫吸附实验ELASA检测OPG蛋白水平变化
切取包绕钛钉的一段胫骨,研磨离心提取蛋白,分光光度计检测450吸光度值,计算蛋白浓度,检测OPG变化。
结果
1.手术结果
小鼠术后3天开始使用手术肢体行走,一周后伤口愈合,缝线自动脱落。三个实验组日常行为上没有区别。离断膝关节后,可观察到钛颗粒分布在胫骨近端及膝关节表面。关节表面没有划痕和炎症反应。大体观察未见钛颗粒组和稳定组解剖结构的改变。
2.Real time PCR检测OPG基因的表达
Real time PCR检测发现,AAV-OPG治疗组的OPG基因表达显著增多,而未经AAV-OPG治疗的两组OPG基因表达水平低(p＜0.05),提示基因转移表达成功。
3.ELASA检测OPG的各蛋白的含量
经ELASA对OPG蛋白的含量检测发现,AAV-OPG治疗组OPG蛋白水平显著增高,而未经AAV-OPG治疗组,其OPG蛋白水平低(p＜0.05)。
4.Micro CT检查
OPG治疗组和稳定组术后12周,MicroCT扫描显示钛钉固定良好,无退钉松动现象,BMD没有出现明显的变化,而钛颗粒组12周出现明显的钛钉周围骨质吸收,与OPG治疗组有显著差异(p＜0.05),提示OPG对钛颗粒诱导的松动具有治疗作用。
5.钛钉稳定性检测
OPG治疗组和稳定组钛钉拔拉力量分别为4.3±0.54和4.6±0.31N,基因治疗后第4,12周无统计学差异。而钛颗粒组拔拉力量12w明显降低,仅为0.96±0.48N,与OPG治疗组有显著差异(p＜0.05),提示钛颗粒诱导假体松动,经AAV-OPG治疗后可显著降低假体松动。
6.组织学检测
OPG治疗组和稳定组的病理切片显示了良好的稳定的状态,不规则新生骨的形成,新生骨胶原。相反,钛颗粒组经过每4w的钛颗粒注射,可观察到肉芽组织的形成,钛钉-骨界面Masson三色染色与稳定组相比,浅而模糊,提示钛颗粒组骨胶原丢失。与OPG治疗组和稳定组相比,钛颗粒组12周骨胶原丢失IOD值为33%±2.5%(p＜0.Q5)。实验显示,在这个连续的的炎症细胞浸润和假体-骨界面形成的病理过程中,其细胞和界面假膜厚度随着钛颗粒的积累而增加(p＜0.05)。OPG治疗后,新骨形成明显增加。经AAV-OPG治疗后的TRAP染色显示,破骨细胞着色为紫红色,聚集在假体-骨界面的破骨细胞分布稀少,而未经病毒治疗钛颗粒组破骨细胞明显增多(p＜0.05)。免疫组织化学染色显示OPG治疗组TNF、IL-1及CD68阳性细胞较钛颗粒组表达减少(p＜0.05)。
结论
1.AAV-OPG能够在Pin-model体内成功表达,达到并维持有效的药物浓度。
2.OPG基因表达对磨损颗粒诱导的骨质吸收和假体松动具有显著的治疗作用。
3.OPG基因表达能够增加假体周围OPG蛋白的水平,阻断破骨细胞的激活,减少假体周围的炎症因子表达。
Background
Total joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure.
While failures due to infection, fracture, and dislocation have become relatively rare, osteolysis-associated aseptic loosening (AL) has become more common and important. Although the precise pathogeneses of aseptic loosening is unclear, cumulative evidence indicates that particulate biomaterial debris generated from the mechanical wear of prosthetic components plays a critical role. It is generally accepted that wear debris provokes diverse biological tissue responses, including vascularized granulomatous tissue formation along the implant-to-bone interface, inflammatory cell (macrophages, lymphocytes) influx, bone resorption, and finally osteolysis and loss of prosthesis fixation.
At present the main therapy for debris associated loosening include: inhibit the bone absorption, accelerate the synthesis of new bone and improve the material of artificial joint.
However, treatment of the debris associated loosening process is highly problematic, partially due to the difficulty of administering and maintaining effective dosages of therapeutic agents at the site of loosening. When anti-inflammation or anti-osteolysis drugs are administered systemically, their effects at the bone- implant interface rely on vascular perfusion, suggesting that relatively high systemic levels are required to achieve anti-inflammatory activity at the site of loosening process. High systemic drug levels often induce adverse side affects and subsequent poor patient compliance. Gene therapy, though still in its infancy, provides an attractive alternative to overcome this difficulty.
To investigate the molecular and biomechanical mechanisms underlying aseptic loosening, and to explore therapeutic intervention to prevent or treat the complication, the development of an animal model that closely mimics human joint arthroplasty and reflects the characteristics of aseptic loosening is an essential prerequisite. Currently, most animal models of joint arthroplasty involve large animals such as sheep and dogs, while the small animal models (rats or mice) are often restricted to short-term studies due to the difficulty of implanting hardware.
Objective
1. Establish the long-term pin mouse model of debris-associated bone resorption and joint prosthesis failure
2. Characterize the biomechanical and pathological aspects of the loosening process.
3. Exam the feasibility of gene therapy as a potential alternative avenue to control this common complication.
Method
1. Establish the Pin-model use mouse
Fifty-four mice aged 10-12 weeks, weighted 20g, quarantined in cages for 2 weeks prior to experimentation. All mice weighed at least 20g, divided into three gourps: stable group, Ti-pariticle group and gene exame group, at the start of the experiment. Titanium-alloy pins of the same specification were kindly produced by Stryker Orthopaedic Inc. particle size 0.53μm with a range of 0.1-4.1μm. Adeno-associated virus codingβ-galactosidase (AAV-LacZ) was obtained from the Gene Core facility at University North Carolina, Chapel Hill.
Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every month following surgery. The control implantation mice symmetrically received intraarticular injection of particle-free PBS. The mice were sacrificed at 2, 4, 12, and 24 weeks for biomechanical, histological, and molecular evaluation.
2. Micro-Computerized Tomography (Micro CT) Scans
All mice were scanned immediately following surgery using Micro CT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were collectd every 4 weeks post operation and analyzed using GEHC MicroView1 software to generate isosurfaces of the region of ' interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin.
3. Interfacial Shear Strength Test
Following sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.
4. In Vivo Exogenous Transfer
Adeno-associated virus coding for the LacZ gene (AAV-LacZ) was used to examine the feasibility of in vivo gene transfer using this model. At 4 weeks after pinimplantation surgery, 50μl of sterile culture medium containing 10~8IU/ml of AAV-LacZ was injected into each prosthetic joint of five mice. The prosthetic joints of control mice received vehicle culture medium without virus. All mice were sacrificed 7 days after in vivo gene transfer. X-gal staining was performed on the prosthetic joint using procedures according instruction.
5. Histological and Immunohistochemical (IHC) Analyses
Formalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues. X-gal stain was employed to trace LacZ gene transduced Cells.
RESULTS
1. Operative Outcome
The mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.
2. Micro CT Evaluation
Micro CT scans indicated that the implants were well fixed with no obvious migration up to 6 months after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption illustrates typical debris-associated aseptic loosening on CT imaging. Images with Ti alloy pin implantation provided good evidence of stable BMD between pin-implanted limbs immediately postoperative and the limbs at 24 weeks months after implantation in the absence of particle challenge. In contrast, a significant BMD loss was observed obviously in debris-injected implanted joints since 12 weeks.
3. Implant Stability Tested by Pullout Test
The average interfacial shear strength against pulling at 24 weeks was 4.5±0.43N on the stable implant and there was no statistical difference between 4, 12 and 24 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability, with only 0.44±0.31N at 24 weeks of pulling force required to dissociate the implant from the bone.
4. Histological Analyses
The histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections. Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 35%±3.5% loss at 6 months after pin implantation, in comparison with debrisfree stable implant group (p<0.05). The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation, starting at 4 weeks following surgery. The cellularity and thickness of the periprosthetic membranes correlated with the amount of debris accumulated and length of time of debris stimulation (p<0.05). Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.
5. Feasibility of In Vivo Gene Transfer in the Model
X-gal staining revealed that a direct single injection of AAV-LacZ into the implanted joint resulted in strong transgene expression, indicated by strong blue coloration in the synovial membranes and periprosthetic tissue. In contrast, the joints receiving virus-free medium injections remained negative using this stain.
CONLUSION
1. We have established a murine model to represent knee prosthesis failure due to wear debris stimulation.
2. The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation.
3. The possibility of in vivo gene transfer in this kind of model.
BACKGROUND
Total joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure. How to prevent or treat this disease becomes the important issue in the implant field.
Among the most important factors that may contribute to loosening is the adverse tissue response to particulate wear debris. Titanium-alloy has been broadly used in total joint prosthesis, and the Titanium-alloy components removed at revision surgery regularly show wear. Histologic evaluation of tissues from failed primary arthroplasties showed that particulate polyethylene is the most common debris found in periprosthetic tissue. It has been accepted that particles generated by mechanical wear of the prosthesis are phagocytosed by macrophages, resulting in cellular activation and release of proinflammatory mediators and cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6. These mediators, in turn, induce local chronic inflammation with activation and recruitment of osteoclasts to the bone-implant interface. This affects bone remodeling around the implant and leads to osteolysis and aseptic loosening.
While many different cytokines contribute to this process, studies have shown that the osteoclast differentiation factor (also called receptor activator of nuclear factor-κB ligand [RANKL]) is 1 of the only 2 essential mediators to promote osteoclastogenesis. It binds to its membrane-bond signaling receptor, RANK, and stimulates osteoclast differentiation and maturation. Recently, a soluble protein osteoprotegerin (OPG) was identified in many types of cells and proved a natural "decoy" receptor that competed for RANKL with RANK and blunted its effects of osteoclastogenesis. Mice genetically deficient for RANKL or RANK suffered severe osteopetrosis, whereas OPG transgenic mice expressed the same pathology, demonstrating that RANKL and RANK are essential for osteoclast development, and OPG is a potent negative regulator for osteoclastogenesis.
Based on the anti-osteolytic nature of OPG, it may be a potential therapeutic agent to treat debrisassociated periprosthetic bone resorption and aseptic loosening. While it is difficult, by conventional therapy, to administer sufficient OPG to osteolytic sites around the prosthetic joint, gene therapy provides an elegant solution to the delivery problem.
OBJECTIVE
1. Examine AAV-OPG gene transfer to protect against wear debris induced osteolysis in a murine Pin-model of bone resorption
2. Examine The feasible effects of AAV-OPG gene therapy
3. Debate the mechanism of gene therapy
METHOD
1. Establish the Pin-model use mouse
Fifty-four mice aged 10-12 weeks, weighted 20g, divided into three groups: AAV-OPG, Ti group and Stable group, quarantined in cages for 2 weeks prior to experimentation. Titanium-alloy pins and Particle used in this experiment as part one. AAV-OPG was obtained as part one mentioned.
Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every 4 weeks following surgery. AAV-OPG was injected into the jointraarticular after 1 week of surgery. The control implantation mice symmetrically received intraarticular injection of particle or virus-free PBS. The mice were sacrificed at 2, 4 and 12 weeks for biomechanical, histological, and molecular evaluation.
2. Micro-Computerized Tomography (Micro CT) Scans
All mice were scanned immediately following surgery using MicroCT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were analyzed using GEHC MicroView1 software to generate isosurfaces of the region of interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin. The following data were collected per 4 weeks after operaton.
3. Interfacial Shear Strength Test
Following sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.
4. Histological and Immunohistochemical (IHC) Analyses
Formalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Histochemical tatrate-resistant acid Phosphatase (TRAP) staining performed to localize the osteclast-like cells in the Pin-model of bone-implant surrounding. The presence of dark purple staining granules in the cytoplasm was a specific criterion for counting TRAP-positive cells. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues.
5. Molecular and immunologic analysis-ELISA
Enzyme-linked immunosorbent assays (ELISA) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. Tests were performed using the standardized protocol previously described.
6. RT-PCR to analysis the expression of OPG
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the influence of gene transfer on osteoclastogenesis. Gene expression of OPG was examined. Total RNA from homogenates was extracted following the manufacturer's instructions. The cDNA was reverse transcribed. Real-time PCR was performed according to the manufacturer's instructions.
RESULTS
1. Operative Outcome
The mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.
2. MicroCT Evaluation
MicroCT scans indicated that the implants were well fixed with no obvious migration up to 12 weeks after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption without AAV-OPG injection illustrates gene therapy can protect debris-associated aseptic loosening (p<0.05).
3. Implant Stability Tested by Pullout Test
The average interfacial shear strength against pulling was 4.6±0.31N on the stable implant and 4.3±0.5N in the AAV-OPG group. There was no statistical difference between 2, 4, and 12 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability in group Ti group, with only 0.44±0.31N of pulling force required to dissociate the implant from the bone (p<0.05).
4. Histological Analyses
The histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles or AAV-OPG therapy resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections in Ti group.
Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants and gene therapy. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 33%±2.5% loss at 12 weeks after pin implantation, in comparison with debris free stable implant or AAV-OPG group (p<0.05).
The TRAP staining was conducted to identify osteoclast-like cells in the model. A typical TRAP-staining photomicrograph revealing that dark brown TRAP-positive cells accumulate along the bone surface in the non OPG therapy group with debris challenge, while fewer cells could be found in AAV-OPG therapy group.
Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.
5. Molecular and immunologic analysis ELISA
Enzyme-linked immunosorbent assays (ELISAs) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. The OPG protein level is higher in OPG therapy group than the other groups illustrated the succeed transduction and expression of AAV-OPG.
6. RT-PCR
Gene expression of OPG was examined and is higher expressed in AAV-OPG therapy group.
CONLUSION
1. The long term murine model of knee joint implant loosening is a successful model for evaluates the bone resorption to screen therapeutic approaches to debris-associated osteolysis.
2. Gene transfer using AAV-OPG appears to be a feasible and effective candidate to treat or prevent wear debris-associated ostelysis and aseptic loosening.
3. Further studies are under to help us understand the transductive efficacy and long-term effects of the AAV-OPG gene transfer and related therapeutic mechanisms and safety concerns.
适于长期研究的小鼠膝关节假体松动模型Pin-model的建立
背景
全关节置换是治疗晚期关节炎常见而有效的治疗方案。然而,将近34%的关节置换假体将会发生无菌性松动甚至失败。因此无菌性松动成为假体置换的主要并发症。
尽管假体松动的具体病理过程目前没有研究清楚,但越来越多的研究表明,假体周围磨损颗粒参与诱导的反应在其中扮演着重要的角色。普遍认为,磨损颗粒激发了多样的生物组织反应,包括骨假体表面血管肉芽组织的形成,炎症细胞聚集(如巨噬细胞和淋巴细胞的聚集),骨吸收,以及最终导致的骨质疏松和假体松动失败。如何治疗和预防关节假体松动成为目前该领域尚待解决的重大课题。
选择和建立合适的动物模型是科研领域里非常重要的内容。现有的膝关节假体松动动物模型有大型动物,如狗,羊,马及兔等模型,这些动物有着与人类骨关节相似的尺寸,而且可以使用大型的假体,模拟临床的变化并进行长期的研究,缺点是研究费用高,不易于饲养管理,样本含量受到限制,特别不适合新型治疗策略的研究,从而限制了这些动物模型的广泛使用。Rushton等首先报道了使用陶瓷假体置入胫骨近端建立承重条件下的鼠类模型。Bostrom等进一步改进该模型,并研究了二磷酸酶在磨损颗粒诱导的骨质吸收中的作用。目前,已有研究者建立了鼠类的动物模型,并置入聚合材料或金属柱状体,研究在非承重条件下磨损颗粒诱导的松动假体周围的炎症反应和骨质吸收。但是缺乏承重条件下的机械刺激形成的磨损颗粒导致的病理状态,与临床承重条件下假体松动的病理机制尚有一定的差距。
目前对于由磨损颗粒引起的假体松动的治疗方法包括:抑制骨吸收;促进骨合成;改进关节材料等。上述治疗方案中的药物治疗方面,由于骨关节的解剖特点,在给药方式和保持局部有效的药物浓度上,还是存在着很大的困难。如果提高全身的药物浓度来保持局部有效的药物浓度,不管是抗炎治疗还是抗骨质疏松治疗,都将会对整个机体带来不同程度的副作用和一系列的并发症。新近兴起的基因治疗,因为具有克服上述治疗缺点的特性,从而显示出了巨大吸引力。
综合本实验室研究,已经成功建立了一种Air pouch小鼠骨质疏松模型,并且探讨了抗炎治疗和基因治疗的有效性和作用机制。在相关领域,现有的实验研究尚无关于磨损颗粒引起的骨质疏松的报道,且该模型只适合短期的研究(2-3周),只能通过病理改变简单模拟人体关节松动的病理过程,属于急性期的变化的研究,不能够模拟人体关节松动的慢性病理过程,而且不适合较长期治疗方案的实施。LacZ基因是目前常用的检测病毒有效转移的措施,因此我们拟在Airpouch模型的基础上,建立一种适合长期研究的膝关节假体松动的动物模型,并应用腺相关病毒编码的LacZ gene(AAV-LacZ)检测基因治疗的可行性。
目的
1.建立一种适于长期研究的小鼠膝关节假体松动动物模型(Pin-model)
2.探讨假体松动的病理变化
3.检测基因治疗方案的可行性
方法
1.小鼠膝关节假体松动动物模型的构建
54只10-12周雌性小鼠,体重20g以上,分为钛颗粒组(24只)、对照组(稳定组,24只)和基因检测组(6只)。沿右膝关节旁纵行切开显露胫骨平台,垂直向髓腔钻入约5mm,置入钛钉。磨损颗粒实验组,在术中钛钉置入前注射至胫骨髓腔10μl,术后每月关节内注射20μl的钛颗粒。对照组则注射相同体积的PBS。分别在术后2、4、12、24周处死小鼠,取材进行生物力学和组织形态学的检测。
2.AAV-LaeZ外源病毒体内转移
将腺相关病毒编码的LacZ-gene(AAV-LacZ)进行体内转移,检测基因治疗的可行性。术后4周,将50μl的无菌培养液(含10~8IU/ml的AAV-LacZ)注射到每只小鼠膝关节内,共计5只,对照组仅注射不含AAV-LacZ的培养液。7天后X-gal染色检测AAV-LacZ病毒基因的转移表达程度
3.Micro CT影像学检查
术后均立即进行Micro CT扫描,确定钛钉的位置是否正确。每隔4周进行一次扫描,记录钛钉的位置和计算胫骨的骨密度(bone mineral density,BMD)。
4.钛钉拔拉实验(Pull-out test)
离断膝关节,显露假体钛钉帽,用定制的铝杆与钛钉帽连接,与Instron model8841机器相连。以2.0 mm/min进行牵拉,对输出数据进行分析。
5.组织形态学检测
HE染色观察钛钉周围的新生骨或骨质破坏,以及钛颗粒引起的炎症反应,包括钛钉周围的软组织形成和炎性细胞浸润和破骨细胞的变化;Masson三色染色法检测骨胶原的变化;免疫组化染色检测致炎细胞因子IL-1,TNF;CD68检测破骨前体细胞;X-gal染色检测外源基因的表达。
结果
1.手术结果
小鼠术后3天开始使用手术肢体行走,一周后伤口愈合,缝线自动脱落。钛颗粒组与稳定组在日常行为上没有区别。离断膝关节后,可观察到钛颗粒分布在胫骨近端及膝关节表面,膝关节表面没有明显划痕和炎症反应。大体观察未见钛颗粒组和稳定组解剖结构的改变。
2.Micro CT检查
稳定组术后24周,Micro CT扫描显示钛钉固定良好,无退钉松动现象,而钛颗粒组在第6周即出现的钛钉周围的轻度BMD降低,提示钛颗粒诱导松动。稳定组BMD没有出现明显的变化,而12周时钛颗粒组BMD出现明显的降低(p＜0.05)。
3.钛钉稳定性检测
用骨水泥将小鼠肢体远端经固定后,近端与拉杆相连,并能够至少承受5牛顿(N)的拉力。稳定组术后24周钛钉拔拉力量为4.5±0.43N,术后第4,12和24周没有统计学差异。而钛颗粒组在12周出现拔拉力量明显降低,至术后24周仅为0.44±0.31N(p＜0.05)。
4.组织学检测
稳定组的病理切片显示了良好的假体稳定的状态,可见不规则新生骨的形成,新生骨胶原。相反,钛颗粒组经过每月的钛颗粒注射,可观察到炎性软组织-假膜的形成,钛钉-骨界面Masson三色染色与稳定组相比,浅而模糊,提示钛颗粒组骨胶原丢失。与稳定组相比,钛颗粒组24周骨胶原丢失达到35%±3.5%(p＜0.05)。实验显示,从术后4周开始,伴随着每月注射钛颗粒所模拟的假体松动过程,是一个连续的炎症细胞浸润和假体-骨界面形成的过程。其细胞和假体-骨界面厚度随着钛颗粒的积累而增加(p＜0.05)。免疫组织化学染色显示,钛颗粒刺激区域的TNF、IL-1表达增加,提示炎症参与了该病理生理过程;CD68阳性细胞表达较稳定组增加,提示破骨前体细胞生成增多。
5.体内基因转移的检测
X-gal染色膝关节,着色深蓝,明确提示直接注射到小鼠膝关节AAV-LacZ强表达。而无病毒组染色阴性。
结论
1.成功建立了小鼠膝关节假体松动模型Pin-model,模拟了临床磨损颗粒诱导的松动的过程,适合长期实验研究
2.假体松动是一个慢性连续性的病理过程,伴随炎症细胞的浸润,破骨细胞的活化,导致骨质吸收,最终假体松动
3.AAV-LacZ病毒体内转移表达成功,确保了基因治疗的可行性
第二部分
AAV-0PG对磨损颗粒诱导的假体松动治疗作用的实验研究
前言
关节假体松动是外科关节置换的最为常见并发症之一。在所有引起关节假体松动的因素中,尤为重要的因素是磨损颗粒引起的周围组织反应。关节翻修手术经常见到UHMWPE等材料的磨损颗粒,组织学检查也证实了这一点。目前普遍认为机械运动产生的磨损颗粒,会被巨噬细胞吞噬,进而激活一系列的炎症细胞,释放炎症因子和细胞因子,如IL-1,TNF,IL-6等。这些炎症因子又会造成局部组织的炎症反应,激活诱导破骨细胞至假体-骨界面。影响界面新骨的形成和重塑,导致骨质吸收,最终导致无菌性松动,手术失败。
因此,理论上可以通过抗炎治疗和抗骨质疏松治疗对假体松动进行干预,减缓炎症反应和骨质疏松,延长假体使用寿命。但由于术肢解剖结构的特点,一般药物很难在假体-骨周围形成较高的药物浓度。基因治疗因为具有可控制性强,作用靶点集中的优点,目前已成为疾病治疗研究的热点。因此应用病毒携带抗炎或抗骨质疏松基因转移至病变区域,表达相应的抗炎或抗骨质疏松因子,治疗假体松动,有可能成为新型有效的治疗手段。
研究发现尽管很多细胞因子参与此过程,作为启动破骨细胞分化的因子,细胞核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)是其中最为重要的因子。RANKL与其膜结合受体细胞核因子κB受体活化因子/破骨细胞分化因子(receptor activator of NF-κB,RANK)结合,刺激破骨细胞分化和成熟。新近研究发现,在多种细胞里存在一种可溶性的蛋白骨保护素(osteoprotegrin,OPG),并且被证实是一种天然的RANK竞争性拮抗剂,与RANKL结合,从而阻断破骨细胞的分化和成熟。
本实验拟在己成功建立的Pin-model小鼠模型上,通过体内基因转移技术,将腺相关病毒AAV携带的编码OPG基因(AAV-OPG)转染到术肢内,进而检测OPG基因治疗方案的可行性和有效性。
目的
1.在新型Pin-model的基础上,应用AAV-OPG基因治疗
2.检测AAV-OPG基因治疗的有效性和可行性
3.探讨AAV-OPG基因治疗作用机制
方法
1.小鼠膝关节假体松动动物模型的构建
54只10-12周雌性小鼠分为三组:AAV-OPG,钛颗粒(Ti particle)和稳定(Stable)组。钛合金钉由Stryker Orthopaedic Inc.制备。钛粒子Ti-6A1-4V,平均直径0.53μm,AAV-OPG,由Chapel Hill提供。
沿右膝关节旁纵行切开显露胫骨平台,垂直向髓腔钻入约5mm,置入钛钉。磨损颗粒实验组,在术中钛钉置入前将钛颗粒注射至胫骨髓腔10μl,术后每4w关节内注射20μl的钛颗粒溶液。将含AAV-OPG50μl的无菌培养液(10~9IU/ml)术后1w注射到每只小鼠膝关节内,对照组仅注射不含AAV-OPG的培养液。Stable组注射相同体积的PBS与钛颗粒组形成对照。分别在基因治疗后2、4、12周处死小鼠,取材进行生物学,组织学及分子学等的检测。
2.Micro CT影像学检查(敃心)术后均立即进行Micro CT扫描,确定钛钉的正确是否位置。每隔4周扫描一次,记录钛钉的位置和计算胫骨的BMD。
3.钛钉Pull-out test
离断膝关节,显露假体钛钉帽,用定制的铝杆与钛钉帽连接,与Instron model8841机器相连。以2.0 mm/min进行牵拉,对输出数据进行分析。
4.组织学和免疫组化检测
HE染色,观察钛钉周围的新生骨或者骨质破坏,以及钛颗粒引起的炎症反应,包括钛钉周围的组织形成和细胞浸润及,TRAP染色检测破骨细胞的变化。改良的Masson染色法检测骨胶原的变化。免疫组化染色检测致炎细胞因子IL-1,TNF及CD68阳性细胞的表达。
5.Real-time PCR检测OPG核酸水平变化
切取包绕钛钉的一段胫骨,研磨提取RNA,分光光度计检测260/280吸光度比值,计算RNA浓度,转cDNA,进行real-time PCR,检测OPG核酸变化。
6.酶连免疫吸附实验ELASA检测OPG蛋白水平变化
切取包绕钛钉的一段胫骨,研磨离心提取蛋白,分光光度计检测450吸光度值,计算蛋白浓度,检测OPG变化。
结果
1.手术结果
小鼠术后3天开始使用手术肢体行走,一周后伤口愈合,缝线自动脱落。三个实验组日常行为上没有区别。离断膝关节后,可观察到钛颗粒分布在胫骨近端及膝关节表面。关节表面没有划痕和炎症反应。大体观察未见钛颗粒组和稳定组解剖结构的改变。
2.Real time PCR检测OPG基因的表达
Real time PCR检测发现,AAV-OPG治疗组的OPG基因表达显著增多,而未经AAV-OPG治疗的两组OPG基因表达水平低(p＜0.05),提示基因转移表达成功。
3.ELASA检测OPG的各蛋白的含量
经ELASA对OPG蛋白的含量检测发现,AAV-OPG治疗组OPG蛋白水平显著增高,而未经AAV-OPG治疗组,其OPG蛋白水平低(p＜0.05)。
4.Micro CT检查
OPG治疗组和稳定组术后12周,MicroCT扫描显示钛钉固定良好,无退钉松动现象,BMD没有出现明显的变化,而钛颗粒组12周出现明显的钛钉周围骨质吸收,与OPG治疗组有显著差异(p＜0.05),提示OPG对钛颗粒诱导的松动具有治疗作用。
5.钛钉稳定性检测
OPG治疗组和稳定组钛钉拔拉力量分别为4.3±0.54和4.6±0.31N,基因治疗后第4,12周无统计学差异。而钛颗粒组拔拉力量12w明显降低,仅为0.96±0.48N,与OPG治疗组有显著差异(p＜0.05),提示钛颗粒诱导假体松动,经AAV-OPG治疗后可显著降低假体松动。
6.组织学检测
OPG治疗组和稳定组的病理切片显示了良好的稳定的状态,不规则新生骨的形成,新生骨胶原。相反,钛颗粒组经过每4w的钛颗粒注射,可观察到肉芽组织的形成,钛钉-骨界面Masson三色染色与稳定组相比,浅而模糊,提示钛颗粒组骨胶原丢失。与OPG治疗组和稳定组相比,钛颗粒组12周骨胶原丢失IOD值为33%±2.5%(p＜0.Q5)。实验显示,在这个连续的的炎症细胞浸润和假体-骨界面形成的病理过程中,其细胞和界面假膜厚度随着钛颗粒的积累而增加(p＜0.05)。OPG治疗后,新骨形成明显增加。经AAV-OPG治疗后的TRAP染色显示,破骨细胞着色为紫红色,聚集在假体-骨界面的破骨细胞分布稀少,而未经病毒治疗钛颗粒组破骨细胞明显增多(p＜0.05)。免疫组织化学染色显示OPG治疗组TNF、IL-1及CD68阳性细胞较钛颗粒组表达减少(p＜0.05)。
结论
1.AAV-OPG能够在Pin-model体内成功表达,达到并维持有效的药物浓度。
2.OPG基因表达对磨损颗粒诱导的骨质吸收和假体松动具有显著的治疗作用。
3.OPG基因表达能够增加假体周围OPG蛋白的水平,阻断破骨细胞的激活,减少假体周围的炎症因子表达。
Background
Total joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure.
While failures due to infection, fracture, and dislocation have become relatively rare, osteolysis-associated aseptic loosening (AL) has become more common and important. Although the precise pathogeneses of aseptic loosening is unclear, cumulative evidence indicates that particulate biomaterial debris generated from the mechanical wear of prosthetic components plays a critical role. It is generally accepted that wear debris provokes diverse biological tissue responses, including vascularized granulomatous tissue formation along the implant-to-bone interface, inflammatory cell (macrophages, lymphocytes) influx, bone resorption, and finally osteolysis and loss of prosthesis fixation.
At present the main therapy for debris associated loosening include: inhibit the bone absorption, accelerate the synthesis of new bone and improve the material of artificial joint.
However, treatment of the debris associated loosening process is highly problematic, partially due to the difficulty of administering and maintaining effective dosages of therapeutic agents at the site of loosening. When anti-inflammation or anti-osteolysis drugs are administered systemically, their effects at the bone- implant interface rely on vascular perfusion, suggesting that relatively high systemic levels are required to achieve anti-inflammatory activity at the site of loosening process. High systemic drug levels often induce adverse side affects and subsequent poor patient compliance. Gene therapy, though still in its infancy, provides an attractive alternative to overcome this difficulty.
To investigate the molecular and biomechanical mechanisms underlying aseptic loosening, and to explore therapeutic intervention to prevent or treat the complication, the development of an animal model that closely mimics human joint arthroplasty and reflects the characteristics of aseptic loosening is an essential prerequisite. Currently, most animal models of joint arthroplasty involve large animals such as sheep and dogs, while the small animal models (rats or mice) are often restricted to short-term studies due to the difficulty of implanting hardware.
Objective
1. Establish the long-term pin mouse model of debris-associated bone resorption and joint prosthesis failure
2. Characterize the biomechanical and pathological aspects of the loosening process.
3. Exam the feasibility of gene therapy as a potential alternative avenue to control this common complication.
Method
1. Establish the Pin-model use mouse
Fifty-four mice aged 10-12 weeks, weighted 20g, quarantined in cages for 2 weeks prior to experimentation. All mice weighed at least 20g, divided into three gourps: stable group, Ti-pariticle group and gene exame group, at the start of the experiment. Titanium-alloy pins of the same specification were kindly produced by Stryker Orthopaedic Inc. particle size 0.53μm with a range of 0.1-4.1μm. Adeno-associated virus codingβ-galactosidase (AAV-LacZ) was obtained from the Gene Core facility at University North Carolina, Chapel Hill.
Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every month following surgery. The control implantation mice symmetrically received intraarticular injection of particle-free PBS. The mice were sacrificed at 2, 4, 12, and 24 weeks for biomechanical, histological, and molecular evaluation.
2. Micro-Computerized Tomography (Micro CT) Scans
All mice were scanned immediately following surgery using Micro CT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were collectd every 4 weeks post operation and analyzed using GEHC MicroView1 software to generate isosurfaces of the region of ' interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin.
3. Interfacial Shear Strength Test
Following sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.
4. In Vivo Exogenous Transfer
Adeno-associated virus coding for the LacZ gene (AAV-LacZ) was used to examine the feasibility of in vivo gene transfer using this model. At 4 weeks after pinimplantation surgery, 50μl of sterile culture medium containing 10~8IU/ml of AAV-LacZ was injected into each prosthetic joint of five mice. The prosthetic joints of control mice received vehicle culture medium without virus. All mice were sacrificed 7 days after in vivo gene transfer. X-gal staining was performed on the prosthetic joint using procedures according instruction.
5. Histological and Immunohistochemical (IHC) Analyses
Formalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues. X-gal stain was employed to trace LacZ gene transduced Cells.
RESULTS
1. Operative Outcome
The mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.
2. Micro CT Evaluation
Micro CT scans indicated that the implants were well fixed with no obvious migration up to 6 months after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption illustrates typical debris-associated aseptic loosening on CT imaging. Images with Ti alloy pin implantation provided good evidence of stable BMD between pin-implanted limbs immediately postoperative and the limbs at 24 weeks months after implantation in the absence of particle challenge. In contrast, a significant BMD loss was observed obviously in debris-injected implanted joints since 12 weeks.
3. Implant Stability Tested by Pullout Test
The average interfacial shear strength against pulling at 24 weeks was 4.5±0.43N on the stable implant and there was no statistical difference between 4, 12 and 24 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability, with only 0.44±0.31N at 24 weeks of pulling force required to dissociate the implant from the bone.
4. Histological Analyses
The histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections. Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 35%±3.5% loss at 6 months after pin implantation, in comparison with debrisfree stable implant group (p<0.05). The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation, starting at 4 weeks following surgery. The cellularity and thickness of the periprosthetic membranes correlated with the amount of debris accumulated and length of time of debris stimulation (p<0.05). Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.
5. Feasibility of In Vivo Gene Transfer in the Model
X-gal staining revealed that a direct single injection of AAV-LacZ into the implanted joint resulted in strong transgene expression, indicated by strong blue coloration in the synovial membranes and periprosthetic tissue. In contrast, the joints receiving virus-free medium injections remained negative using this stain.
CONLUSION
1. We have established a murine model to represent knee prosthesis failure due to wear debris stimulation.
2. The time study of pin-implantation with monthly titanium particle injections to mimic debris-associated loosening process indicated a continuous inflammatory cellular infiltration and periprosthetic membrane formation.
3. The possibility of in vivo gene transfer in this kind of model.
BACKGROUND
Total joint replacement is a highly successful and common procedure in the treatment of end stage arthritis. However, as many as 34% of the total joint replacement components will loosen and eventually fail because of aseptic loosening, which has become the major complication of this procedure. How to prevent or treat this disease becomes the important issue in the implant field.
Among the most important factors that may contribute to loosening is the adverse tissue response to particulate wear debris. Titanium-alloy has been broadly used in total joint prosthesis, and the Titanium-alloy components removed at revision surgery regularly show wear. Histologic evaluation of tissues from failed primary arthroplasties showed that particulate polyethylene is the most common debris found in periprosthetic tissue. It has been accepted that particles generated by mechanical wear of the prosthesis are phagocytosed by macrophages, resulting in cellular activation and release of proinflammatory mediators and cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6. These mediators, in turn, induce local chronic inflammation with activation and recruitment of osteoclasts to the bone-implant interface. This affects bone remodeling around the implant and leads to osteolysis and aseptic loosening.
While many different cytokines contribute to this process, studies have shown that the osteoclast differentiation factor (also called receptor activator of nuclear factor-κB ligand [RANKL]) is 1 of the only 2 essential mediators to promote osteoclastogenesis. It binds to its membrane-bond signaling receptor, RANK, and stimulates osteoclast differentiation and maturation. Recently, a soluble protein osteoprotegerin (OPG) was identified in many types of cells and proved a natural "decoy" receptor that competed for RANKL with RANK and blunted its effects of osteoclastogenesis. Mice genetically deficient for RANKL or RANK suffered severe osteopetrosis, whereas OPG transgenic mice expressed the same pathology, demonstrating that RANKL and RANK are essential for osteoclast development, and OPG is a potent negative regulator for osteoclastogenesis.
Based on the anti-osteolytic nature of OPG, it may be a potential therapeutic agent to treat debrisassociated periprosthetic bone resorption and aseptic loosening. While it is difficult, by conventional therapy, to administer sufficient OPG to osteolytic sites around the prosthetic joint, gene therapy provides an elegant solution to the delivery problem.
OBJECTIVE
1. Examine AAV-OPG gene transfer to protect against wear debris induced osteolysis in a murine Pin-model of bone resorption
2. Examine The feasible effects of AAV-OPG gene therapy
3. Debate the mechanism of gene therapy
METHOD
1. Establish the Pin-model use mouse
Fifty-four mice aged 10-12 weeks, weighted 20g, divided into three groups: AAV-OPG, Ti group and Stable group, quarantined in cages for 2 weeks prior to experimentation. Titanium-alloy pins and Particle used in this experiment as part one. AAV-OPG was obtained as part one mentioned.
Under aseptic condition, the proximal tibia condyle was exposed a cavity for the implant was reamed. A titanium pin was press-fitted into the canal in a manner. For the evaluation of debris, mice were injected with 10μl of titanium suspension into the tibia canal before the insertion of the pin, and 20μl of debris particles was then injected intraarticularly to the prosthetic joint every 4 weeks following surgery. AAV-OPG was injected into the jointraarticular after 1 week of surgery. The control implantation mice symmetrically received intraarticular injection of particle or virus-free PBS. The mice were sacrificed at 2, 4 and 12 weeks for biomechanical, histological, and molecular evaluation.
2. Micro-Computerized Tomography (Micro CT) Scans
All mice were scanned immediately following surgery using MicroCT system to confirm the proper position of pin implantation. Following acquisition and reconstruction, the image data were analyzed using GEHC MicroView1 software to generate isosurfaces of the region of interest (ROI) and to calculate the bone mineral densities (BMD) of the tibia bone surrounding the titanium pin. The following data were collected per 4 weeks after operaton.
3. Interfacial Shear Strength Test
Following sacrifice, the mouse limb with the implant intact was removed by disarticulating the knee joint. All soft tissue around the prosthetic joint was carefully removed to expose the implanted pin surface and proximal tibia. A custom aluminum fixture was designed to align the long axis of the orthopedic implant with the loading axis of the Instron model 8841 (Canton, MA). The implant was pulled out of the bone at a rate of 2.0 mm/min. Actuator position and load was recorded.
4. Histological and Immunohistochemical (IHC) Analyses
Formalin-fixed prosthetic joints were decalcified with formic acid/sodium citrate before paraffin-embedding. The sections were stained with hematoxylin and eosin to examine new bone formation or bone erosion around the prosthetic pin, and to evaluate debris-associated inflammation, including periprosthetic tissue formation and cellular infiltration. Modified trichrome staining was performed to examine bone collagen changes. Histochemical tatrate-resistant acid Phosphatase (TRAP) staining performed to localize the osteclast-like cells in the Pin-model of bone-implant surrounding. The presence of dark purple staining granules in the cytoplasm was a specific criterion for counting TRAP-positive cells. Immunohistochemical stains were carried out to evaluate pro-inflammatory cytokines and mediators (IL-1, TNF and CD68) of osteoclastogenesis in periprosthetic tissues.
5. Molecular and immunologic analysis-ELISA
Enzyme-linked immunosorbent assays (ELISA) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. Tests were performed using the standardized protocol previously described.
6. RT-PCR to analysis the expression of OPG
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the influence of gene transfer on osteoclastogenesis. Gene expression of OPG was examined. Total RNA from homogenates was extracted following the manufacturer's instructions. The cDNA was reverse transcribed. Real-time PCR was performed according to the manufacturer's instructions.
RESULTS
1. Operative Outcome
The mice tolerated the surgical procedure well and ambulated with the implanted limb within 3 days after surgery. Injections of titanium particles appeared to exert no influence on daily activity in comparison to the animals without particle injections. The macroscopic examination of the prosthetic joints during sacrifice revealed metal pins positioning in proximal tibiae, and no scratch nor inflammation on the opposing articulate surfaces. There were no obvious structural differences between stable and particle-stimulated implantation groups by naked eyes.
2. MicroCT Evaluation
MicroCT scans indicated that the implants were well fixed with no obvious migration up to 12 weeks after surgery without particle challenge. However, titanium particle injection induced marked periprosthetic bone resorption without AAV-OPG injection illustrates gene therapy can protect debris-associated aseptic loosening (p<0.05).
3. Implant Stability Tested by Pullout Test
The average interfacial shear strength against pulling was 4.6±0.31N on the stable implant and 4.3±0.5N in the AAV-OPG group. There was no statistical difference between 2, 4, and 12 weeks after surgery. However, the introduction of titanium particles significantly decreased the implant stability in group Ti group, with only 0.44±0.31N of pulling force required to dissociate the implant from the bone (p<0.05).
4. Histological Analyses
The histological appearances of the pin prosthesis model clearly revealed that the implantation of pins without particles or AAV-OPG therapy resulted in a stable condition and irregular new bone formation, with bone collagen content well preserved. However, an extensive periprosthetic soft membrane developed in the implanted joints exposed to monthly particle injections in Ti group.
Further, the periprosthetic bones stimulated with titanium particles exhibited much fainter blue color staining using Modified Trichrome staining, when compared to the staining seen using sections from stable implants and gene therapy. Using a computerized image analysis system, the IOD of Trichrome staining in debris-induced bone resorption group averaged 33%±2.5% loss at 12 weeks after pin implantation, in comparison with debris free stable implant or AAV-OPG group (p<0.05).
The TRAP staining was conducted to identify osteoclast-like cells in the model. A typical TRAP-staining photomicrograph revealing that dark brown TRAP-positive cells accumulate along the bone surface in the non OPG therapy group with debris challenge, while fewer cells could be found in AAV-OPG therapy group.
Immunohistochemical assessment using antimouse cytokine antibodies revealed a profound accumulation of TNF and IL-1 expressing cells in the particle-stimulated sections. CD68+ macrophages were also present in marked aggregations in particle-stimulated periprosthetic membranes.
5. Molecular and immunologic analysis ELISA
Enzyme-linked immunosorbent assays (ELISAs) were conducted on the supernatants of the tabular homogenates to examine OPG-transgene production. The OPG protein level is higher in OPG therapy group than the other groups illustrated the succeed transduction and expression of AAV-OPG.
6. RT-PCR
Gene expression of OPG was examined and is higher expressed in AAV-OPG therapy group.
CONLUSION
1. The long term murine model of knee joint implant loosening is a successful model for evaluates the bone resorption to screen therapeutic approaches to debris-associated osteolysis.
2. Gene transfer using AAV-OPG appears to be a feasible and effective candidate to treat or prevent wear debris-associated ostelysis and aseptic loosening.
3. Further studies are under to help us understand the transductive efficacy and long-term effects of the AAV-OPG gene transfer and related therapeutic mechanisms and safety concerns.
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