关节腔注射羧甲基壳聚糖预防膝骨关节炎软骨退变

目的观察羧甲基壳聚糖（CMCTS）关节腔注射对骨关节炎（OA）模型关节软骨退变及软骨基质金属蛋白酶（MMP-1，-3）及其组织抑制物（TIMP-1）mRNA表达的影响。方法32只大耳白兔行单膝前交叉韧带切断术，随机分为A～D组，A、B组分别于术后立即关节腔注射高、低分子量2％CMCTS0．3ml，每2周1次；C组术后立即关节腔注射1％透明质酸钠（SH）0．3ml，每周1次；D组术后不注射任何药物。术后6周处死动物，比较各组股骨内髁关节软骨的大体变化，采用逆转录聚合酶链反应（R-PCR）方法检测软骨MMP-1，-3及TIMP-1 mRNA的表达水平。结果大体评分显示不注射组软骨退变明显重于CMCTS和SH注射组MMP-1，-3在CMCTS注射组软骨中的表达明显低于SH注射组和不注射组，不同分子量CMCTS注射组MMP-1，-3的表达没有显著差异，SH注射组软骨中MMP-1，-3的表达与不注射组比较差异无统计学意义（P〉0．05），TIMP-1在各组中的表达没差异有统计学意义（P〉0．05）。结论CMCTS能明显下调软骨MMP-1，-3的mRNA表达．明显减轻软骨退变的程度。软骨具有保护作用。     

MMP-1、MMP-9 mRNA在创伤性骨关节炎软骨中的表达

目的 研究兔前交叉韧带横断术(ACLT)创伤性骨关节炎(OA)动态模型软骨中MMP-1、MMP-9 mRNA的表达,探讨MMP-1、MMP-9在OA软骨退变中的作用.方法 30只新西兰兔随机分为对照组(10只)、ACLT组(20只),ACLT组行右膝ACLT,对照组行关节囊切开缝合术,ACLT组于术后4、8 w各处死10只,对照组术后4 w处死.解剖显微镜观察股骨内髁软骨形态学变化,逆转录聚合酶链反应(RT-PCR)检测股骨内髁软骨MMP-1、MMP-9 mRNA的表达.结果 形态学示ACLT组软骨退变重于对照组(P<0.05),且ACLT 8 w组重于4 w组(P<0.05);RT-PCR示ACLT组MMP-1、MMP-9 mRNA表达量均高于对照组(P<0.01),且ACLT 8 w组均高于4 w组(P<0.01).结论 创伤性OA软骨退变过程中伴随着MMP-1、MMP-9 mRNA表达的上调,软骨中MMP-1、MMP-9 mRNA的表达一定程度上反映了OA软骨的退变程度.     

基质金属蛋白酶家族在骨关节炎软骨组织中表达的研究

[目的]观察骨关节炎关节软骨中MMP-7、MMP-9、MMP-13和TIMP-1的表达,探讨其与软骨退变的关系及可能的作用机制。[方法]选取20例因骨关节炎行关节置换的软骨组织,常规HE染色观察其组织学形态,ABC免疫组化法观察关节软骨MMP-7、MMP-9、MMP-13和TIMP-1的表达,2例因意外受伤截肢患者的正常膝关节软骨标本作为对照。统计采用Mann-Whitney U非参数检验及相关分析。[结果]骨关节炎关节软骨出现裂隙、纤维化,软骨细胞增多、排列紊乱,并出现大量簇聚软骨细胞和肥大软骨细胞。MMP-7和MMP-13在正常软骨全层均呈低表达,但在退变软骨中的表达则明显增多,光密度值行U检验,两组差异有显著性(P<0.01)。在正常与OA软骨的浅层,MMP-9和TIMP-1的表达无显著性差异(P>0.05);但在深层软骨中,OA软骨MMP-9和TIMP-1的表达较正常软骨明显增多,两组差异有显著性(P<0.01)。[结论]MMP-7,13在OA软骨全层表达均多于正常软骨;MMP-9,13仅在OA软骨深层出现过多表达。MMPs与TIMPs的失衡是导致关节软骨发生组织学退变的原因之一。     

兔骨关节炎中基质金属蛋白酶-1、3与白细胞介素-1β表达及关节软骨细胞凋亡的研究

目的 观察骨关节炎(OA)中基质金属蛋白酶(MMP)-1、3与白细胞介素-1β(IL-1β)的表达及关节软骨细胞凋亡,探讨其在OA发病机制中的作用.方法 将20只中国大白兔随机分成正常组(A组)、实验组(B组),每组10只,A组未造模,B组采用Hulth法制成OA模型,4周后取胫骨平台关节软骨进行组织病理学检查,采用免疫组织化学和原位末端标记细胞凋亡检测法分别检测关节软骨和滑膜中的MMP-1、3、IL-1β及软骨细胞凋亡指数(AI)的水平.结果 组织病理学检查可见,B组关节软骨退变程度明显重于A组,符合OA关节软骨退变特征;通过检测MMP-1、3,IL-1β和AI,A组的结果分别是21.005±9.406、18.697±8.225、0.100±0.040和14.900±3.400,B组的结果分别是56.147±22.340、46.182±20.561、0.180±0.060和25.400 ±5.200;B组MMP-1、3和AI的水平均明显高于A组(P＜0.01),B组IL-1β水平高于A组(P＜0.05).结论 MMP-1、MMP-3、IL-1β的表达及关节软骨细胞凋亡在OA发病机制中作用重要,其异常升高与OA关节软骨退变和炎症反应密切相关.     

SD大鼠腰椎小关节胶原酶诱导骨性关节炎模型中TNF-α,IL-1β及NO的表达变化

目的 初步探讨白细胞介素1β及肿瘤坏死因子α及一氧化氮在小关节骨性关节炎病程中表达的变化. 方法 选取SD大鼠（48例）随机分为胶原酶注射组（24例）和对照组（24例）,采用关节腔内注射胶原酶诱导骨性关节炎,术后于1周、2周、4周、8周取各组实验动物各6只处死后立即取出腰5/6小关节突,倒置显微镜下观察软骨组织学连续动态改变,酶联免疫吸附剂测定法测定关节软骨中白细胞介素1β及肿瘤坏死因子α含量,免疫组织化学染色法检测软骨中一氧化氮合成酶的表达并利用细胞图像分析仪,对各实验期免疫组化染色后的切片做一氧化氮合成酶定量分析. 结果 倒置显微镜下观察结果显示,胶原酶诱导骨性关节炎模型关节软骨损伤随时间延长而加重;免疫组化显示,实验组动物软骨中iNOS含量在术后1周时在软骨浅层有少量染色,术后2周时阳性染色结果明显增多,而在术后4周时阳性染色大量增多并且在软骨中下层有所表达,在术后8周时软骨全层可见大量阳性染色;iNOS的染色强度与对照组相比在术后1周时即明显增高,术后2周、4周、8周时,iNOS染色强度一直维持在较高的水平;软骨中炎性因子IL-1β和TNF-α表达与对照组明显升高,IL-1β在术后2周时达峰值,此后逐渐下降,到8周仍处于较高水平,TNF-α在1周时明显升高,2周时有所下降,4周时明显下降,8周时表达与对照组无明显差异. 结论 关节腔注射胶原酶诱导骨性关节炎模型实验组小关节退变软骨的病理改变与临床小关节退变患者关节软骨病理改变基本相同,并随着病程的进行,软骨的退变逐步加重;小关节源性IL-1β与软骨的退变程度具有相关性,但不随软骨退变的加重而进一步升高.TNF-α与IL-1β可能在炎症的不同时期发挥作用,其中TNF-α主要作用于炎症早期.退变关节软骨中iNOS含量持续增高,提示NO在骨性关节炎的病程发展中起到了重要的作用.     

软骨下骨刚度增加对关节软骨Ⅱ型胶原和MMP-1表达的影响

目的 观察增加软骨下骨刚度后关节软骨中Ⅱ型胶原和基质金属蛋白酶-1(MMP-1)的表达和分布,探讨骨关节炎发病机制.方法 调整聚甲基丙烯酸甲酯(PMMA)、甲基丙烯酸甲酯(MMA)、羟基磷灰石(HA)和蒸馏水的比例,使反应温度低于40 ℃,制成PMMA-HA复合材料.刮除兔胫骨内侧软骨下骨后置入复合材料,对术后3、6、9、12周的关节软骨进行组织学观察,免疫组化法检测Ⅱ型胶原和MMP-1在软骨中的表达和分布,并与空白、对照组相比较.结果 复合材料以HA 2g、PMMA 1g、MMA 0.7ml、蒸馏水0.3ml混合时平均最高反应温度为38.17 ℃,且极限强度和刚度均高于软骨下骨.实验显示关节软骨随的时间的延长出现纤维化、裂隙、变薄,软骨细胞簇集,潮线模糊或消失,Mankin分级逐渐升高;免疫组化显示Ⅱ型胶原表达增加主要在软骨移行层和深层上部,MMP-1表达以软骨表层及中上层居多,两者染色强度随观察时间的延长均逐渐升高.结论 软骨下骨刚度增加后软骨中Ⅱ型胶原和MMP-1表达的量均升高,提示软骨下骨硬化对关节软骨退变的发生、发展具有十分重要的作用,可能为骨关节炎发病的主要原因之一.     

骨化三醇对大鼠骨性关节炎关节软骨及软骨下骨的影响

目的 观察骨化三醇对大鼠前交叉韧带切断术后膝关节软骨及软骨下骨的影响,探讨软骨下骨在骨性关节炎发病中的作用.方法 取SD大鼠64只分成4组,前交叉韧带切断组(ACLT组)、前交叉韧带切断+骨化三醇给药组(ACLT+cal组)、假手术组(sham组)、假手术+骨化三醇给药组(sham+cal组),给药组术后第2天给骨化三醇0.1 μg·kg-1·d-1灌胃,ACLT组和sham组给予安慰剂灌胃,持续2周.每组分别于术后2周和10周取材,胫骨近端切片后,行HE、AB-PAS染色、MMP-13免疫组化及骨形态计量学分析.结果 HE、AB-PAS染色、MMP-13免疫组化及骨形态计量学参数测量显示,2周时ACLT组与sham组相比早期有软骨下骨量减少,ACLT+cal组较ACLT组软骨下骨量增多(P<0.05).10周时ACLT组软骨明显退变性改变、软骨下骨增生及MMP-13表达增强,ACLT+cal组软骨退变及软骨下骨增生均较ACLT组减轻(P<0.01),MMP-13表达较ACLT组降低(P<0.05).结论 软骨下骨在骨性关节炎病变进展中发挥重要作用,骨代谢调节剂骨化三醇可减缓OA进展中软骨下骨硬化,对关节软骨有一定保护作用.     

软骨损伤患者关节镜手术及关节腔内注射玻璃酸钠后关节液中基质金属蛋白酶-13表达水平变化的研究

目的探讨行关节镜手术并关节腔内注射玻璃酸钠后的软骨损伤患者关节液中基质金属蛋白酶-13(MMP-13)表达水平的变化。方法对42例经Outerbridge软骨损伤评分法分组的软骨损伤患者,应用低密度蛋白质芯片方法检测各组患者行关节镜手术前的关节液及术后1、2、3、4周注射玻璃酸钠后的关节液中MMP-13的荧光强度。结果软骨损伤严重组(Ⅳ度、Ⅲ度)的行关节镜手术及玻璃酸钠治疗后的第2周,关节液内MMP-13表达水平开始显著下降,并持续到第5周。软骨损伤较轻组(0度、Ⅰ度)的关节液中MMP-13表达水平在第4、5周与术前的比较有统计学差异(P<0.05)。在第5周时,软骨损伤严重组(Ⅳ度、Ⅲ度)关节液内MMP-13表达水平高于软骨损伤较轻组(0度、Ⅰ度)。结论经关节镜手术、关节腔内注射玻璃酸钠治疗后软骨损伤严重的患者关节液内MMP-13表达水平下降较软骨损伤轻的更明显,MMP-13表达水平下降的意义对软骨代谢水平评价及临床疗效判定有一定的指导价值。     

复合材料置入软骨下骨诱发兔膝骨关节炎的实验研究

[目的]通过增加软骨下骨硬度模拟软骨下骨硬化,诱发骨关节炎的发生,探讨骨关节炎发病机制.[方法]调整聚甲基丙烯酸甲酯粉剂(polymethylmethacrylate,PMMA)、甲基丙烯酸甲酯液剂(methylmethacrylate,MMA)、羟基磷灰石(hydroxyapatite,HA)和蒸馏水的比例,使反应温度低于40℃,测量该比例下聚合后的极限强度和刚度并与软骨下骨比较,制得PMMA/HA复合材料.刮除兔胫骨平台内侧软骨下骨后置入复合材料,对术后3、6、9、12周的关节软骨进行组织学观察,免疫组化法榆测Ⅱ型胶原和基质会属蛋白酶-1(matrix metallo proteinase-1,MMP-1)在软骨中的表达和分布,并与空白、对照组比较.透射电镜观察空白、6、12周组软骨细胞改变.[结果]该复合材料置入软骨下骨后,随观察时间延长实验组逐渐出现退变,Mankin分级逐渐升高,电镜也显示实验组软骨细胞退变表现.免疫组化显示Ⅱ型胶原表达增加主要在移行层和深层上部,MMP-1表达以软骨表层及中上层居多,随观察时间延长二者染色强度均逐渐升高.[结论]增加软骨下骨硬度后,诱发了兔膝骨关节炎.提示软骨下骨硬化可引起软骨退变,其可能是骨关节炎的病因之一.     

在体阻断软骨下营养对关节软骨影响的实验研究

背景：关节软骨无血管分布,其营养来自关节液和软骨下骨,哪条营养通路对关节软骨更为重要是学者们争论的焦点。目的：研究软骨下营养对关节软骨的影响,并探讨软骨下营养与骨关节炎的关系。方法：45只5个月龄雄性新西兰大白兔,建立股骨滑车骨软骨缺损的动物模型,并随机分为3组：自体骨软骨块移植组（Control组,n=15）;假手术组（Sham组,n=15）,用环钻在股骨滑车钻取骨软骨块,将其置入管状PVC内,原位回植;阻断软骨下营养组（DNBM组,n=15）,取出骨软骨后,将其置入帽状PVC内,原位回植。术后4周、8周、12周,每组5只（10膝）,取出膝关节进行大体评分、组织学评分、软骨厚度测量、凋亡染色（TUNEL染色）。结果：与Control组相比,大体评分结果提示,DNBM组软骨无明显退变;组织学评分结果提示,术后12周时DNBM组软骨明显退变（P〈0.005）;软骨厚度测量结果提示,术后8周、12周时DNBM组软骨厚度明显变小（P=0.00）;TUNEL染色结果提示,术后8周、12周时DNBM组关节软骨细胞凋亡明显增加（P〈0.01）。结论：软骨下营养是软骨的重要营养来源,失去软骨下营养,软骨会逐渐发生退变。     

Treatment of articular defects with meniscal allografts in a rabbit knee model

Deep-frozen allogeneic meniscal grafts for the treatment of articular cartilage defects were performed experimentally. Osteochondral defects 3 mm in diameter were created bilaterally on the medial femoral condyles of 50 Japanese white rabbits. A meniscus was then grafted into the defect in the left knee, and the right knee was left untreated. At various periods from 2 to 24 weeks postoperatively, the rabbits were killed and macroscopic and histologic examinations were performed. Two weeks after operation, the grafted meniscus was bonded to the floor of the defect. After 12 weeks, chondrocytes producing matrix granules was shown by electron microscopy. After 24 weeks, a congruous articular surface was formed. With time, cellular elements infiltrated into the graft from the surrounding tissues, and gradually increased in penetration. Weight bearing in the early stage after operation did not degrade the grafted menisci, and no changes were shown in the opposing cartilage of the tibia. Deep-frozen allogeneic menisci may be useful as a biological implant to repair articular cartilage defects in this model.     

Characteristics of degeneration in an unstable knee with a coronal surface step-off

To investigate the effect of instability on the remodelling of a minor articular surface offset, we created a 0.5 mm coronal step-off of the medial femoral condyle in 12 New Zealand white rabbits and transected the anterior cruciate ligament (ACL). A control group of 12 rabbits had only ACL resection and the opposite knee was used as the non-operated control. The osteoarthritic changes at 6, 12 and 24 weeks after surgery were evaluated histologically. In addition, changes in the immunological detection of 3-B-3(-) and 7-D-4 chondroitin-6-sulphate epitopes were determined because of the previous association of such changes with repair of cartilage and early osteoarthritis. In the instability/step-off group there was rapidly progressing focal degeneration of cartilage on the high side of the defect, not seen in previous step-off studies in stable knees. The rest of the femoral condyles and the tibial plateaux of the instability/step-off group had moderate osteoarthritis similar to that of the instability group. 3-B-3(-) was detectable in the early and the intermediate stages of osteoarthritis but no staining was seen in the severely damaged cartilage zones. Immunoreactivity with 7-D-4 increased as degeneration progressed.     

肢体缺血再灌注后关节软骨的组织学观察

目的: 通过动物实验观察肢体缺血再灌注后关节软骨的病理变化, 证实缺血再灌注导致关节软骨的损伤, 并探讨关节缺血再灌注损伤与骨性关节炎形成的关系。方法: 健康中国本地兔42只, 随机分为正常对照组, 缺血4h组和缺血10h组, 分别于肢体缺血再灌注后0、1d和1、5、10周,取4只兔8条腿做标本, 行光镜观察, 及图像分析仪处理。结果: 早期(24h以内) 关节软骨的组织学改变在光镜下表现不明显。再灌注5周时损伤最为明显, 此后短期内(10周内) 软骨损伤不再加重。软骨组织形态学的定量观察,股骨外髁关节软骨全层厚度明显增加(P<0 .01), 非钙化层增加不明显, 而钙化层厚度则明显增加(P< 0. 01)。结论: (1) 证实了缺血再灌注导致关节软骨的损伤; (2) 关节缺血再灌注损伤也是导致骨性关节炎形成的重要原因。     

Cartilage damage caused by metal implants applied for the treatment of established localized cartilage defects in a rabbit model

The purpose of the current study was to investigate the feasibility of the application of defect‐size femoral implants in a rabbit model of established cartilage defects and compare this treatment to microfracturing. In 31 New Zealand White rabbits, a medial femoral condyle defect was created in each knee. After 4 weeks, 3 animals were killed for defect baseline values. In the other 28 rabbits, knees were sham‐operated, treated with microfracturing, or treated by placing an oxidized zirconium (OxZr) or cobalt‐chromium (CoCr) implant (?? articulating surface 3.5 mm; fixating pin of 9.1 mm length). These animals were sacrificed 4 weeks after treatment. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry, and cartilage repair was scored microscopically. Cartilage quality was analyzed macroscopically and microscopically. Bone–implant contact was 63.2% ± 3.2% for CoCr and 62.5% ± 3.2% for OxZr. Cartilage defects did not show complete healing, nor during subsequent sham‐surgery or microfracturing. For all treatments, considerable cartilage damage in the articulating medial tibia, and degeneration of lateral tibial and femoral cartilage was observed (p < 0.05). Both CoCr and OxZr implant‐treated defects showed an increase of cartilage degeneration compared to microfracturing and sham‐operated defects (p < 0.05). Although only a single short‐term follow‐up period was investigated in this study, caution is warranted using small metal implants as a treatment for established localized cartilage defects because, even after 4 weeks in this model, the metal implants caused considerable degeneration of the articulating surface. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:84–90, 2009     

Articular cartilage thickness and glycosaminoglycan distribution in the young canine knee joint after remobilization of the immobilized limb

The recovery of articular cartilage from atrophy induced by joint immobilization was investigated in immature dogs. In a previous study, we showed that 11 weeks of immobilization of the knee (stifle) joint of young dogs reduced the concentration of articular cartilage glycosaminoglycans (GAGs) by 13–47%. In the present study, right hindlimbs from six female beagles were immobilized for 11 weeks, as in the previous study, and then were remobilized for 15 weeks. Cartilage from the knee joint was compared with cartilage from nonimmobilized knees of eight age-matched control beagles. Histological samples taken from 11 different locations of the knee joint were stained with safranin O, and microspectrophotometry was used to demonstrate distribution of GAGs in the tissue. After remobilization, GAG concentration was restored in the patellofemoral region and tibial condyles. On the summits of the femoral condyles, and especially at the periphery of the femoral condyles, GAG concentration remained 8–26% less than the control values. On the summits, the thickness of the uncalcified cartilage was as much as 15% less than in the age-matched controls. Consequently, the changes induced by unloading were reversible to a great extent, but a full restoration of articular cartilage was not obtained at all sites of the knee joint within the 15 weeks of remobilization. Immobilization of the skeletally immature joint therefore may affect the development of articular cartilage in such a way that very slow recovery or permanent alterations are induced.     

The canine 'groove' model of osteoarthritis is more than simply the expression of surgically applied damage

OBJECTIVE: Recently a new canine model of osteoarthritis (OA; the 'groove' model) has been described. This model is based on surgically applied mechanical damage of the articular cartilage followed by transient forced loading of the affected joint. Ten weeks after surgery this model shows characteristics of OA, mimicking human OA. To establish whether the observed characteristics of degeneration in this model represent the surgically applied damage, or are the results of progressive features of OA, we evaluated this 'groove' model shortly after surgery. METHODS: In 20 female Beagle dogs, articular cartilage of the weight-bearing areas of the femoral condyles in the right knee was damaged without affecting the underlying bone. After surgery dogs were let out on a patio 5 days/week for 4 h/day. The dogs were forced to load the experimental joint by fixing the contralateral control limb to the trunk 3 days/week. The severity of OA was evaluated at 3 (n = 10) or 10 weeks (n = 10) after surgery. Synovial inflammation, cartilage damage and cartilage matrix turnover were determined. RESULTS: Ten weeks after surgery osteoarthritic features were found, as described previously. Proteoglycan (PG) synthesis, percentage release of newly formed PG, and that of total amount of PG were enhanced, whereas PG content was significantly diminished (all P < 0.05). Importantly, 3 weeks after surgery these characteristics of OA were not yet evident. CONCLUSIONS: The present results clearly show that the characteristics observed 10 weeks after induction of joint degeneration in the groove model are not just the expression of the surgically applied damage but are the result of progressive features of (experimental) OA.     

The influence of growth hormone on the reversibility of articular cartilage degeneration in rabbits

Growth hormone has chondrogenic affects on normal as well as on damaged articular cartilage. In this study, the influence of growth hormone is investigated on early degenerative changes in the articular cartilage in 72 New Zealand white rabbits. Cartilage lesions were created in femoral condyles using an immobilization model. Cartilage damage was assessed using biochemical, histologic, and biomechanical criteria. Growth hormone had no influence on prevention of immobilization abnormalities but had a significant affect on healing of established lesions.