医用臭氧关节腔内注射对骨关节炎的作用和安全性的实验研究及影像学观察
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摘要
第一部分骨关节炎动物模型的建立
目的：
比较关节腔内注射1．6％木瓜蛋白酶、关节腔内注射胶原蛋白酶、关节固定法、股静脉结扎法等四种方法，筛选出适合快速、大规模造模的方法。
1．木瓜蛋白酶关节腔内注射法OA造模试验
1．1材料与方法
健康新西兰白兔8只，雌雄各半，6～8个月龄，体重2．0～2．5kg。分别于实验的第1、4日于右膝关节腔内注射1．6％papain溶液0．3ml制备OA动物模型。造模后4周后对模型行软骨取材及病理学观察。
1．2结果
一般情况：第一次注射1．6％木瓜蛋白酶溶液后，右膝关节出现不同程度的肿胀，第二次注射后动物出现跛行，触摸右膝关节时出现挣扎和弹腿。2天后跛行消失，10天左右肿胀消退。所有动物的进食饮水二便未受明显影响，全部动物存活，未出现明显异常生物行为。
肉眼和手术显微镜下观察：8只实验兔之胫骨内侧平台软骨均呈灰黄色或灰白色，无光泽，表面可见裂纹，个别可见小而浅的局部糜烂，未见明显骨赘形成。
关节软骨光镜下观察：软骨表面粗糙不规则，可见较小和浅的裂隙，软骨细胞数量减少，排列紊乱，无细胞簇聚现象。PAS和甲苯胺蓝染色示基质轻度失染。
1．3结论
木瓜蛋白酶关节腔内注射法模型的病理变化与临床OA极为相似，造模过程简单，成功率高，适合快速大批量建立OA模型作软骨病理、药物防治的研究。
2．胶原蛋白酶OA模型试验
2．1材料与方法
采用6只健康的普通级新西兰白兔，雌雄不限，6～8个月龄，体重2．0～2．5kg，分别于第1、4天向实验兔的右侧膝关节腔内注射Ⅱ型胶原酶溶液0．5ml(即2mgⅡ型胶原酶)制备OA模型。造模后4周后对模型行软骨取材及病理学观察。
2．2结果
造模实验兔右膝关节腔内注射Ⅱ型胶原蛋白酶溶液后，精神较差，自发活动明显减少，右膝关节出现不同程度的肿胀，行走时右下肢不着地，呈明显跛行；触摸右膝关节时出现挣扎和弹腿。2周后跛行基本消失。
右膝关节DR成像显示膝关节关节间隙对称，未见明显狭窄，骨性关节面光滑、锐利，未见骨赘形成。髌下脂肪垫清晰。关节周围软组织未见肿胀。
右膝关节MRI均显示股骨髁及胫骨平台关节软骨表面光整，信号均匀，无明显变薄及缺损，髌上囊可见少量积液，软骨下骨信号未见异常。
大体标本：股骨髁表面尚光整，色泽略显暗淡，胫骨平台表面欠光滑，凹凸不平，呈粗颗粒状改变，未见明显骨赘形成。
HE染色：软骨表层可见不规则裂隙，辐射层及钙化层结构完整，软骨细胞排列规则，数量略减少，潮线完整。
2．3Ⅱ型胶原蛋白酶关节腔内注射法制作的兔膝关节OA模型，模型成型时间最短(4周)，在病理学改变上与人类OA基本一致，为一种较为理想的早期OA动物模型。该方法操作技术简单，周期较短，非常适合快速大批量建立骨关节炎模型。
3．石膏关节固定法和股静脉结扎法预实验
结果显示石膏固定法制备OA模型具有死亡率相对较高，制备过程需严密观察且时刻需要调整石膏固定，过程中动物固定时间不一致，导致制备的OA模型病变程度不一致等缺点；而采用股静脉结扎的方法制备OA模型，由于术中出血及术后感染，也具有一定的死亡率，并且单纯行股静脉结扎难以成功复制典型的动物OA模型。因此上述两种造模方法不适合本研究的较大规模造模。
4．结论
本课题参考文献报道的造模方法，试验性地复制接近临床OA病理改变的新西兰兔OA模型。证实了关节腔内注射化学物质诱导法更为适合本试验的现状，可在短时间内，大量并成功复制OA模型，木瓜蛋白酶和胶原蛋白酶两者比较，以后者所复制的模型病理改变更接近人类骨关节炎模型。而关节固定法和手术造模由于对实验环境和实验动物的护理有较高的要求，实验动物模型发生并发症的机会较高，并发症的预防和治疗都较困难。造成实验动物死亡时有发生。因此不适合在现有条件下大量复制OA模型。
第二部分木瓜蛋白酶OA模型关节腔内注射医用臭氧的实验研究
1．目的
通过研究医用臭氧关节腔内直接注射对OA模型关节软骨的影响，探讨关节腔内直接注射医用臭氧治疗骨关节炎的可行性，同时探讨医用臭氧治疗骨关节炎的可能机制。本实验OA模型以关节腔内注射木瓜蛋白酶法制备。选择研究氧化应激反应常用的指标NO(Nitric oxide，一氧化氮)和SOD(Superoxide dismutase，超氧化物歧化酶)作为观察氧化应激反应的指标。
2．材料与方法
2．1．动物分组及模型建立
32只兔随机分为正常组(简称Normal组)、OA模型组(简称model组)、臭氧关节腔内注射组(O_3 Intra-articular cavity Injection，简称O_3IACI)2组：低剂量(20μg／ml)臭氧注射组(简称O_3IACI-20)和高剂量(40μg／ml)臭氧注射组(简称O_3IACI-40)等4组，每组8只。normal组不做任何处理，正常喂养。其余3组共24只兔，分别于实验的第1、4日于右膝关节腔内注射1．6％papain溶液0．3ml制备OA动物模型。
2．2动物处理
动物OA模型制作完成一周后，O_3IACI-20组和O_3IACI-40组采用右膝关节腔内注射的方法给药。O_3IACI-20组右膝关节内注射浓度为20ug／ml的医用臭氧2ml，每周两次，共四周；O_3IACI-40组右膝关节内注射浓度为40ug／ml的医用臭氧2ml，每周两次，共四周。Normal组和model组正常喂养，不作任何处理。末次注射后1周抽取关节液和血液并处死各组动物行软骨组织取材。
2．3指标检测
血清NO和SOD活性检测；关节液NO和SOD活性检测
2．4软骨组织病理学检查
软骨石蜡标本切片分别行HE染色、过碘酸雪夫氏(PAS)染色及甲苯胺蓝(Toluidine Blue)染色，光镜下观察病理变化，以Mankin’s评分标准进行评分。
2．5统计学处理
所有资料均以均数±标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，方差不齐则采用Welch检验或秩和检验，多样本均数间的比较采用LSO-t及Games-Howell等检验方法。以SPSS11．5统计分析软件进行统计分析,P<0．05有统计学意义。
3．结果
3．1动物行为观察
第一次注射木瓜蛋白酶当天，所有建模实验兔右膝关节出现不同程度的肿胀，第二次注射木瓜蛋白酶后当天出现跛行，触摸右膝关节时出现挣扎和弹腿。2天后跛行消失，10天左右肿胀消退。O_3IACI-20组、O_3IACI-40组注射臭氧后动物行动无明显改变，个别实验兔行为稍活跃。
3．2组织形态学观察
3．2．1膝关节软骨肉眼及手术显微镜下观察
观察部位：胫骨内侧平台软骨。结果显示：
normal组：关节软骨呈蓝白色，有光泽，表面光滑，无纤维化，无裂纹及溃疡形成。
Model组：软骨呈灰黄色或灰白色，无光泽，表面可见裂纹，个别可见小而浅的局部糜烂，未见明显骨赘形成。
O_3IACI-20组：软骨呈灰黄色，表面粗糙，无光泽，裂纹多见，表面糜烂较明显和多见，未见明显骨赘。
O_3IACI-40组：软骨表面粗糙，裂纹及糜烂情况较低剂量组更明显和多见。未见明显骨赘。
3．2．2膝关节软骨光镜下观察
normal组：软骨表面光滑、平整，软骨切线层、移行层、深层和钙化层层次清楚，潮线完整，软骨细胞分布均匀，细胞排列整齐，无簇聚软骨细胞；PAS染色软骨基质着色为均匀的红色，无失染现象；甲苯胺蓝染色示基质呈均匀的深蓝色，无失染现象。
model组：软骨表面粗糙不规则，可见较小和浅的裂隙，软骨细胞数量减少，排列紊乱，无细胞簇聚现象。PAS和甲苯胺蓝染色示基质轻度失染。
O_3IACI-20组：软骨表面缺损，出现空陷窝，软骨细胞部分消失，软骨变薄，可见明显的裂隙，潮线模糊或多层次，PAS和甲苯胺蓝染色示基质染色失染较model组明显。
O_3IACI-40组：软骨表面缺损，软骨变薄，细胞减少，排列紊乱，可见明显成簇样细胞增生，层次不能区分，裂隙较低剂量组更多和更明显，PAS和甲苯胺蓝染色示基质失染情况较低剂量组更加明显。
3．2．3关节软骨Mankin’s评分
正常关节软骨Mankin’s评分为0．0±0．0，model组评分为4．5±0．5高于normal组(P<0．01)，O_3IACI组评分为6．0±0．8～8．1±1．2高于model组(P<0．01)；其中O_3IACI-40组评分(8．1±1．2)又高于O_3IACI-20组的评分(6．0±0．8)(P<0．01)，表明多次重复关节腔内注射臭氧对骨关节炎的关节软骨有损伤作用，且损伤程度随臭氧浓度的增加而加重。
3．3生化指标的检测
血清NO水平检测：与normal组血清NO水平(91．88±26．94μmol／L)比较，model组(146．75±16．43μmol／L)和O_3IACI组(156．01±24．53μmol／L、166．23±22．01μmol／L)血清NO水平均高于normal组，差异有显著性(P<0．01)；O_3IACI组和model组血清中NO水平比较差异无显著性(P>0．05)，O_3IACI-20组(156．01±24．53μmol／L)和O_3IACI-40组(166．23±22．01μmol／L)组间血清中NO水平比较亦无显著性差异(P>0．05)。
血清SOD浓度检测：与normal组血清SOD水平(246．18±34．65 U／ml)比较，model组血清SOD(330．78±46．68 U／ml)和O_3IACI组血清中SOD水平(374．96±44．02 U／ml、411．59±30．00 U／ml)均高于normal组，差异有显著性(P<0．01)，O_3IACI组血清中SOD水平高于model组，差异有显著性(P<0．05)，O_3IACI-20组血清SOD水平(374．96±44．02 U／ml)与O3IACI-40组血清SOD水平(411．59±30．00 U／ml)比较，差异无显著性(P>0．05)。
关节液NO水平检测：model组关节液NO水平(111．37±35．92μmol／L)和O_3IACI组关节液中NO水平(108．28±20．45μmol/L、105．16±38．18μmol／L)均高于normal组(31．43±5．06μmol／L)，差异有显著性(P<0．01)；O_3IACI组与model组各组间及O_3IACI I-20组(108．28±20．45μmol／L)和O_3IACI-40组(105．16±38．18μmol／L)组间关节液中NO水平差异无显著性(P>0．05)。
关节液SOD浓度检测：model组关节液中SOD水平(86．02±48．47 U／ml)与normal组关节液SOD水平(63．51±39．81 U／ml)比较，差异无显著性(P>0．05)，O_3IACI组关节液中SOD水平(119．05±34．96 U／ml、140．14±42．76 U／ml)与normal组关节液SOD比较，差异有显著性(P<0．05)；O_3IACI-40组关节液中SOD水平(140．14±42．76 U／ml)与model组关节液SOD水平比较，差异有显著性(P<0．05)；而O_3IACI-20组关节液中SOD水平(119．05±34．96 U／ml)与model组关节液SOD比较，差异无显著性(P>0．05)；两O_3IACI组组间关节液中SOD水平比较差异无显著性(P>0．05)。
4．结论
臭氧关节腔内注射可以引起SOD的过度分泌，但对NO没有影响，提示臭氧治疗骨关节炎的可能机制之一是通过上调抗氧化酶的过度表达阻断慢性氧化应激反应在骨关节炎发病和发展中的作用。骨关节炎模型多次直接腔内注射臭氧，对病变软骨有进一步损害的作用，损害程度随臭氧浓度增加而加重。臭氧直接关节腔内注射的安全性值得深入研究。在骨关节腔内注射臭氧治疗骨关节炎的机理未确认之前，需慎用该治疗入路。
第三部分：胶原蛋白酶OA模型臭氧关节腔内注射与经静脉臭氧化自体血回输的对照实验研究
1．目的
用两种不同方法把臭氧引人实验动物体内，观察相关血清细胞因子在臭氧引人前和后的变化，进一步探讨臭氧治疗OA的生物学机理，进一步明确臭氧对OA模型关节软骨的病理影响。
2．材料与方法
2．1．动物分组及模型建立
采用36只健康的普通级新西兰白兔，雌雄不限，6～8个月龄，体重2．0～2．5kg。36只兔随机分为正常对照组(简称Normal组)；模型对照组(简称model组)；臭氧关节腔内注射组(O3 Intra-articular cavity Injection，简称O3IACI)2组：10μg／ml臭氧关节腔内注射组(简称O3IACI-10组)、30μg／ml臭氧关节腔内注射组(简称O3IACI-30组)；臭氧自血回输组(O3 AutoHemoTherapy，简称O3AHT)2组：10μg／ml臭氧自血回输组(简称O3AHT-10组)和30μg／ml臭氧自血回输组(简称O3AHT-30组)，每组6只。正常组不做任何处理，正常喂养。其余30只兔，分别于实验的第1、4日于右膝关节腔内注射Π型胶原蛋白酶溶液0．5ml制备骨关节炎动物模型。
2．2动物处理
动物OA模型制作完成4周后，O_3IACI-10组和O_3IACI-30组采用右膝关节腔内注射的方法给药。O_3IACI-10组右膝关节内注射浓度为10ug／ml的医用臭氧2ml，每周两次，共四周；O_3IACI-30组右膝关节内注射浓度为30ug／ml的医用臭氧2ml，每周两次，共四周。
O_3AHT-10组和O_3AHT-30组均采用自体静脉血回输的方法给药(大自血回输)，经兔耳缘静脉采取4ml静脉血后，分别抽取浓度为10μg／ml及30μg／ml的臭氧气体4ml，第一管弃之，第二管经三通管与静脉血充分溶解、混匀后经兔耳缘静脉回输入血。每周进行两次，共四周。
末次注射后4周行影像学观察，抽取血液样本，处死各组动物行病理学检查。
2．3观察指标
2．3．1一般行为观察
2．3．2影像学观察(DR及MRI)
2．3．3血清生化指标及细胞因子的检测
血清NO和T-SOD活性检测、血清TNF-α和IL-1β含量的检测
2．3．4病理组织学观察
软骨石蜡标本切片分别行HE染色、过碘酸雪夫氏(PAS)染色及甲苯胺蓝(Toluidine Blue)染色，光镜下观察病理变化，并作Mankin评分
2．3．5统计学处理
所有资料均以均数士标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，方差不齐则采用Welch检验，多样本均数间的比较采用LSD-t及Games-Howell等检验方法。以SPSS11．5软件进行统计分析,P<0．05有统计学意义。
3．结果
3．1一般情况观察
第一次注射胶原蛋白酶后当天，所有建模动物右膝关节出现不同程度肿胀、可见明显跛行，触摸右膝关节时略有挣扎、弹腿，活动量减少。距离第二次胶原蛋白酶注射完毕后6～7周，50％的实验动物可于关节内侧扪及骨赘，关节活动稍受限；灵活性变差。O_3IACI-10组、O_3IACI-30组、O_3AHT-10组及O_3AHT-30组的实验兔接受臭氧关节腔内注射或自血回输后，较Model组活跃，精神状态较好，活动增多，步态恢复较Model组迅速。
3．2影像学观察：
3．2．1 DR成像
Normal组：双膝关节关节间隙对称，未见明显狭窄，骨性关节面光滑、锐利，未见骨赘形成。髌下脂肪垫清晰。关节周围软组织未见肿胀。
Model组：右膝关节内侧胫骨平台明显骨质增生、变尖，骨赘形成，内侧关节囊肿胀，髌下脂肪垫透亮度减低，关节间隙狭窄。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见DR改变与Model组大致相同，其中O_3IACI-10组、O_3IACI-30组关节间隙无明显狭窄。
3．2．2膝关节MRI成像
Normal组：右膝股骨髁及胫骨平台关节软骨表面光整，信号均匀，无明显变薄及缺损，软骨下骨信号未见异常，关节腔内未见积液。
model组：右膝内侧胫骨平台关节软骨明显变薄，信号强度减低，内侧股骨髁关节软骨厚度、信号尚可，软骨下骨信号未见明确异常；髌上囊少量积液。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见MRI改变同model组大致相仿。
3．3血清生化指标及细胞因子
3．3．1血清NO含量及T-SOD活性
采用硝酸还原酶法，根据样品的吸光度，测定各组样本的NO含量及T-SOD活性。
各组血清NO含量(单位：μmol／L)分别为：normal组79．44±15．31；Model组152．23±17．88：O3IACI-10组163．20±22．31；O3IACI-30组158．05±36．47；O3AHT-10组183．21±23．12；O3AHT-30组183．95±50．72。
各组血清T-SOD活性(单位：U／ml)分别为：normal组233．84±30．66；Model组359．71±66．73；O3IACI-10组351．23±46．46；O3IACI-30组375．85±75．96；O3AHT-10组397．01±65．81；O3AHT-30组379．05±37．67。
经过检验，各治疗组和模型组的血清NO含量和T-SOD活力均高于正常组(均为P<0．05)；各治疗组问及治疗组与模型组间的血清NO含量和T-SOD活力无显著性差异(均为P>0．05)。
3．3．2血清TNF-α和IL-1β含量
采用双抗夹心酶联免疫吸附法(ELISA)检测血清中TNF-α和IL-1β的含量。
各组血清TNF-α浓度(单位：pg／mL)分别为：normal组8．50±3．46；Model组28．37±17．40；O3IACI-10组88．72±72．88；O3IACI-30组198．98±310．97；O3AHT-10组56．07±73．83；O3AHT-30组39．12±40．74。
各组血清IL-1β浓度(单位：pg／ml)分别为：normal组43．37±18．27；Model组146．89±98．93；O3IACI-10组416．30±319．45；O3IACI-30组691．71±798．24；O3AHT-10组287．24±363．95；O3AHT-30组219．72±229．94。
经过检验，各治疗组和模型组的血清IL-1β含量高于正常组(均为P<0．05)；各治疗组之间及治疗组与模型组间的血清IL-1β含量无显著性差异(均为P>0．05)。
3．4病理学观察
3．4．1大体观察
Normal组：双侧关节腔内无积液，滑膜无增生、肥厚，双侧股骨髁及胫骨平台表面光滑，色泽鲜亮，未见软骨缺损及骨赘形成。
Model组：右膝关节腔内可见积液，呈粘稠的淡黄色，滑膜明显增生、肥厚，右股骨髁表面尚光整，右胫骨平台色泽灰暗，表面见粗颗粒状改变，内侧关节软骨糜烂、变薄。右胫骨平台内侧缘可见骨赘形成。O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见与model组大致相仿，没有明显差别。
3．4．2光镜观察
3．4．2．1关节软骨
Normal组：关节软骨表面光滑、平整，软骨切线层、移行层、放射层和钙化层层次清楚，潮线完整，软骨细胞分布均匀，细胞排列整齐，无簇聚或减少；甲苯胺蓝染色示基质呈均匀的深蓝色，无失染现象。
Model组：关节软骨表层变薄、粗糙，部分区域呈绒毛状，可见较多裂隙从表层软骨延伸向下深达钙化层，潮线不完整，软骨层自软骨下骨剥离；软骨细胞变性，数量减少；甲苯胺蓝染色示基质失染，软骨纤维化。
O_3LACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组均出现model组改变，并显示软骨裂隙，裂隙以O_3IACI-30组、O_3AHT-30组更多和更深，其中O_3IACI-10组、O_3IACI-30组可见软骨细胞成簇样增生。
3．4．2．2滑膜：
Normal组：滑膜组织无肥厚及增生，无血管侵入，无炎细胞浸润。Model组：滑膜组织毛细血管充血，血管周围可见炎性细胞浸润。O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组出现了model组的所有改变，并有增生现象。
3．4．3关节软骨Mankin’s评分
各组Mankin’s评分：normal组0．0±0．0，Model组10．2±1．9；O3IACI-10组8．8±1．5；O3IACI-30组9．3±2．1；O3AHT-10组8．2±1．9；O3AHT-30组8．6±2．7。
模型组、各治疗组评分高于正常组(P<0．05)，各治疗组间及与模型组间无显著性差异(P>0．05)，表明治疗组关节软骨的损害程度与模型组无差异，臭氧治疗不能改善缓骨关节炎的病理进程。
4．结论
经关节腔内臭氧注射及经静脉臭氧化自体血回输均可引起T-SOD的过渡分泌，提示臭氧可上调抗氧化酶的过渡表达。但对OA模型升高的血清NO没有抑制作用。两种给药途径对OA模型升高的血清IL-1β和TNF-α没有明确的抑制作用。经静脉两种浓度的臭氧化自体血回输对OA模型软骨病理改变无逆转作用；经关节腔内两种浓度臭氧直接注入对OA模型软骨病理改变无逆转作用，30μg／ml浓度臭氧关节腔内直接注射后OA模型软骨病变发生损害加重。
第四部分：OA模型关节软骨及臭氧对其作用的MRI评价
1．目的
探讨理想的关节成像序列及成像参数用于关节软骨的定量分析，并评价医用臭氧对OA模型关节软骨的作用。
2．材料与方法
2．1动物分组和模型的制备、动物处理详见论文第三部分。
2．2磁共振成像
成像时间：末次注射臭氧后4周。实验兔麻醉满意后固定于自制的检查板，扫描使用Philip Intera 1．5T磁共振扫描仪，采用C3表面线圈，行双膝的矢状面扫描。
2．3实验兔膝关节磁共振成像序列对比实验
2．3．1各序列扫描参数设定
对normal组内6只正常实验兔进行右膝关节MRI，成像序列包括施加脂肪抑制的质子加权成像PDW／SPAIR／D，无脂肪抑制的T2加权三维快速梯度回波序列(T2W_3D_FFE)，水激励的三维快速扰相梯度回波序列3D_WATSc
2．3．2不同脉冲序列关节软骨信噪比(signal to noise ration，SNR)及软骨与骨髓
对比信噪比(contrast to noise ration，CNR)的测定和比较方法：MR扫描后测量图像中关节软骨、软骨下骨在上述3种脉冲序列中的信号强度。然后计算出关节软骨信噪比(SNR)和关节软骨与软骨下骨的对比噪声比(CNR)。
2．3．3关节软骨厚度及信号强度的测量方法：
将Dicom数据分别导入Orisix 3．02(School of Medicine of the GenevaUniversity，Switzerland)及eflim workstation 2．1．2(Merge healthcare，USA)中，计算出某实验兔关节软骨厚度及信号强度的平均值。
2．4关节软骨的MRI与病理学对比观察
对Normal组、model组、O_3IACI-10组、O_3IACI-30组、O_3AHT-10组、O_3AHT-30组6组实验兔的关节软骨MR成像与病理学改变作对照分析。
2．5统计学处理：
所有资料均以均数±标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，多样本均数间的比较采用LSD-t检验，方差不齐则采用秩和检验；对所得的关节软骨平均厚度及信号强度与第三部分测得的Mankin’s评分行直线相关分析，若变量间存在相关则进一步做多元线性回归，明确Mankin’s评分与关节软骨的厚度或信号强度有无线性关系。以SPSS 11．5统计分析软件进行统计分析，P<0．05有统计学意义。
3．结果
3．1．1 normal组正常兔膝关节软骨不同脉冲序列的信号特点
PDW／SPAIR／D序列：关节软骨表现为覆盖于软骨下骨的均匀中高信号带；T2W 3D FFE序列：关节软骨则呈单层中等信号带，与骨髓隔以薄层低信号区；3D WATSc序列：关节软骨表现为均一、光整的高信号带，与低信号的软骨下骨分界清晰。
3．1．2 Normal组正常兔膝关节软骨不同脉冲序列的SNR及CNR
PDW／SPAIR/D、T2W_3D_FFE及3D_WATSc组间SNR无显著性差异(p>0．05)：3D_WATSc组CNR高于PDW／SPAIR／D组及T2W_3D_FFE组(p<0．05)，而PDW／SPAIR／D组和T2W_3D_FFE组的CNR无显著性差异(p>0．05)。
3．1．3各实验组关节软骨的平均厚度及信号强度对比
与Normal组比较，model组、各臭氧干预组的实验兔膝关节软骨平均厚度及信号强度明显变薄和降低，(p<0．05)，但Model组、O3IACI-10组、O3IACI-30组、O3AHT-10组及O3AHT-30组各组间的关节软骨平均厚度及信号强度均无显著性差异(p>0．05)。
3．2关节软骨的MRI与病理学对比观察：
Normal组：MRI显示右膝股骨髁及胫骨平台关节软骨表面光整，信号均匀，无变薄及缺损；大体标本及光镜下可见正常软骨表现。
Model组：MRI显示右膝内侧胫骨平台关节软骨明显变薄，信号强度减低：同一部位肉眼观察见软骨色泽灰暗，表面见粗颗粒状改变，内侧关节软骨糜烂、变薄：光镜下关节软骨变薄、粗糙，表层呈绒毛状，可见较多裂隙从表层软骨延伸向下深达钙化层。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10组、O_3AHT-30组各组MRI改变与model组相仿，显示右膝股骨内侧髁及内侧胫骨平台关节软骨不同程度变薄，信号减低。同一部位标本肉眼及光镜下出现了model所见的改变，并显示软骨裂隙，裂隙以O_3IACI-30组、O_3AHT-30组更多和更深，其中O_3IACI-10组、O_3IACI-30组可见软骨细胞成簇样增生。
3．3关节软骨平均厚度及信号强度与Mankin Score的直线相关及回归分析
对关节软骨平均厚度及信号强度与组织学Mankin Score行Pearson相关分析，结果关节软骨平均厚度与信号强度呈显著的正相关；关节软骨平均厚度与Mankin Score呈显著的负相关；关节软骨平均信号强度与Mankin Score呈显著的负相关。
以Mankin Score为应变量，关节软骨平均厚度及信号强度为自变量，采用逐步选择法作多元线性回归分析。结果仅关节软骨平均厚度选入方程，信号强度被剔除，回归方程为Y=18．582-0．023X(Y为Mankin Score，X为关节软骨平均厚度)，R~2=0．528，Mankin Score与关节软骨的平均厚度存在线性回归关系。
4结论
三维扰相梯度回波结合水激励技术成像时间短，是观察OA模型关节软骨的比较理想的成像序列，由于小动物的关节软骨较薄，在不造成信噪比显著降低的同时，需适当提高空间分辩率，利于关节软骨的观察。通过测量膝关节MRI的软骨厚度及信号强度，可以对关节软骨进行量化指标评价，其中软骨厚度测量优于信号强度测量。经对各组实验兔软骨厚度和信号强度的对照研究，医用臭氧关节腔内注射及自血疗法对OA的关节软骨均无修复作用，与病理结果一致。
BACKGROUND
Osteoarthritis (OA), also referred to as degenerative joint disease, is one of the most prevalent chronic conditions in elderly people. OA mainly involves articular cartilage, bone and synovial membrane. The pathogenesis of OA consists of a general progressive loss of articular cartilage, remodeling and sclerosis of the subchondral bone, and the formation of subchondral bone cysts and marginal osteophytes. Arthrocele, pain, deformation and functional disturbance caused by OA severely disable patients' daily activities. With the merging of an aging society, the incidence of OA is increasing in an alarming pace, and OA is becoming a social and economic problem in our current society.
The current management of OA is to relieve the symptom, to improve the joints function and movement, to postpone the articular degeneration. Recently Medical Ozone has been shown to have promising clinical results in treatment of various chronic diseases, especially in pain relief in conditions, such as pyriformis syndrome and OA. But the mechanism of medical Ozone in OA treatment is largely unknown. Above all, there are no consensus on the Ozone dosage and its safety standard among the medical professionals who practice Medical Ozone. This experimental study was designed for investigation of the above issues.
PURPOSES
1. To search for methods of producing OA animal models to match the need of large scale study of OA Ozone intra-cavity treatment.
2. To study the possibility and safety of Ozone intra-cavity treatment of OA by pathologically observing the articular cartilage changes following Ozone intra-cavity injection of different dosages (those are accepted clinically in many countries of Asia and Europe).
3. To study the Autohemotherapy of medical Ozone in treatment of OA by pathologically observing the articular cartilage changes following intravenous injection of model's blood blended with Ozone of different concentrations.
4. To search the possible mechanisms of Ozone in treating OA with different administration route by detecting the level of oxidative stress index and the activities of serum cytokines.
5. To observe and evaluate the effect of medical Ozone in treatment of OA by MRI.
Part I. OA Animal Model Establishment
In this part, to find out the most effective method of OA animal model establishment for large scale studies, four methods were tested: 1.6% papain intra-articular injection and Collagenase intra-articular injection, immobilization of rabbit knee joints, femoral vein ligation.
1. Model establishment by intra articular cavity injection of papain
1.1 Materials and methods
8 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, 4 males and 4 females) were used. Right knee intra-articular injection of 0.3ml 1.6% papain solution was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed and dissected for pathological studies at 5 weeks after the initiation of the injection.
1.2 Results
Various extents of joint swelling were observed after the injection of 1.6% papain solution. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. All rabbit's diets remain normal and alive without exceptional behavioral change.
Gross and operative arthroscope inspections: Cartilage at the medial side of the tibia platform was grey yellow or grey white and dull. Fissures on cartilage surface were found. A few focuses of small and shallow erosion were noted. There was no obvious osteophyma.
Light microscopy examination: Articular surface is rough and irregular. Relatively small and shallow fissures can be appreciated. The chondrocytes is decreased in cellularity and disarrayed in arrangement without cellular crowding. PAS and Toluidin blue stains show mild decrease in staining intensity in the chondroid matrix.
1.3 Conclusions
The pathological changes of the OA model produced by papain intra-articular injection are very similar to those of spontaneous OA. The experimental model is simple technically and easily reproducible with a high successful rate. These models are also suitable for quick and a large scale production for pathological and therapeutic studies.
2. Model establishment by intra articular cavity injection of Collagenase
2.1 Materials and Methods
6 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, both male and female animals) were used to produce the OA models. Right knee intra-articular injection of 0.5ml solution (I.e.2mg Collagenase II) was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed for pathological studies at 4 weeks after the initiation of the injection.
2.2 Results
Low spirit, visible inactivity was observed in rabbits after the injection of Collagenase II solution. Variable degree of joint swelling was present. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 weeks and swelling faded away in 10 days. All rabbits' diets remain normal and alive without exceptional behavioral change.
Right knee DR study was performed 4 weeks after establishment. The radiograms showed both knee spaces were in concordance without obvious stenosis, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.
MRI showed that condyleses of femurs and tibial plateaus were smooth, without thinning and defects; a small amount of hydrops in the suprapatellar bursa was found. The MR signals of the sub-cartilage bone were normal.
Grossly, the surface of the femur condyles was still smooth and intact, but matted. The surface of the tibial plateau was rough and granulated. A few malacia foci but osteophyma were present.
Histological study with HE staining revealed irregular fissures at the surface layer of the cartilage. The radial zone and calcify zone were architecturally normal. The chondrocytes was slightly decreased in cellularity and arrayed in normal arrangement. The tide mark was intact.
2.3 conclusions
In this study, it took the shortest time to achieve the production of the OA model which pathology was very much similar with that of humans. The method of Collagenase II intra articular injection was ideal for the production of early OA model. It was simple technically and took less time, and fit for quick producing OA models in great numbers.
3. Other preliminary experiments for OA model production
Other two OA model production method had been tested in this study, immobilization of rabbit knee joints with plaster bandage and femoral vein ligation. But it's difficult to control the concordance of the OA-like pathological change and degree of the change in individual animal in the group due to the lack of control in immobilizing time. And During the traumatic procedures of model producing by femoral vein ligation, it's more easily for animals to die following in-operation bleeding and post-operation infections. And it's difficult to reproduce a typical OA model by simple ligation of the femoral vein. These establishment methods were not suitable for a quick and large scale model study.
4. Summary
The trail of production of OA in animal models pathologically similar to those happen spontaneously to humans had been based on thorough review of the literatures about OA model establishment. Our investigations proved that intra articular chemical substances injection was the most effective method among those tested. Comparatively, Collagenase II OA model was easier to reproduce in a rather short time and better for OA study than Papain model. Both immobilization of knee joints and femoral vein ligation were unsuitable for large scale establishment, as they were less tolerable to the animals and require more complicated and sophisticated animal handling and manipulation than our current situation allowed.
Part II. Experimental Study of Medical Ozone Intra Articular Injection in Papain Induced OA Models
1. Purposes
Intra-articular injection of Ozone has recently been used to treat OA. However, the mechanism and safety of such treatment modality has not yet been fully investigated. This study was designed to evaluate the local effect of intra-articular Ozone injection to better understand the biologic mechanism and pathologic effect of Ozone therapy on articular cartilage.
2. Materials and Methods
OA animal models were produced by intra-articular injection of Papain solution. Both Nitric oxide (NO) and Superoxide dismutase (SOD), the two most commonly used indexes for oxidative stress reaction, and the histopathology of the articular cartilage were analyzed.
2.1 Animal grouping and model establishment
32 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, and OA model group with O_3 Intra- articular cavity Injection (O3IACI). The O3IACI treated OA model group was further sub-divided in to two groups according to the Ozone dosage used in the experiment: O_3IACI-20 group (Ozone dosage: 20μg/ml per injection), O_3IACI-4O group (Ozone dosage: 40μg/ml per injection). 8 rabbits were in each group. OA model was produced by injecting 3ml of 1.6% Papain solution into the knee joint space twice three days apart.
2.2 Animal processing
7 days after the last injection of Papain solution, the rabbits of both O_3IACI-20 group and O_3IACI-40 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks. The normal and model groups were fed routinely. Synovial fluid and blood of all groups were collected, and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone injection.
2.3 Synovial fluid and serum analysis
NO and SOD activity were detected in both serum and synovial fluid. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry.
2.4 Histopathology evaluation of the cartilaginous tissue
The articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.
2.5 Statistical analysis
SPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. One-way ANOVA as well as q test was applied to analyze every parameter of each region of interest as homogeneity of variances in multiple group samples means. LSD-t and Games-Howell tests were used for comparison among multiple samples means. A p <0.05 value was considered statistically significant.
3. Results
3.1 Animal behaviors observation
After the first Papain solution injection, Variable extent of the right knee swelling was presented in all animals for producing OA model, the rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. After Ozone injection, many rabbits in O_3IACI-20 group and O_3IACI-40 group behaved as usual and some appeared to be more active than before the Ozone treatment.
3.2 Histomorphology study
3.2.1 Gross inspection
Part examined: cartilage at the medial side of the tibia platform
Normal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer.
Model group: the cartilage was grey yellow or grey white and dullness. Fissures on cartilage surface were found. A few foci of small and shallow erosion were noted. There was no obvious osteophyma.
O_3IACI-20 group: the cartilage was grey yellow, rough and dullness. More fissures on the superficial cartilage were noted. Superficial erosions were obvious and multiple. There was no obvious osteophyma.
O_3IACI-40 group: the cartilage was rough. Much more fissures and erosions were seen in compared with O_3IACI-2O group.
3.2.2 Microscopic examination
Normal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The chondroid matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.
Model group: the surface of the cartilage was irregularly rough with tiny shallow cracks. The chondrocytes was decreased in cellularity and disarrayed in arrangement. PAS and Toluidine blue stains showed mild decrease in staining intensity in the chondroid matrix when compared to that of normal group.
O_3IACI-20 group: Articular surface was damaged with presence of empty lacunas due to disappearing of the chondrocytes. The layer of the cartilage decreased in thickness with apparent fissures. The tide mark was unclear or duplicated. The PAS and Toluidine blue stains were less intensive than that of the model group.
Dosage-40 group: The articular cartilage damage was represented by the thinning of the cartilage layer, decreased cellularity of the chondrocytes, cellular disarray, clustering of hyperplastic chondrocyte and lack of normal zonal appearance. Compared to Dosage-20 group, fissures were increased and were more apparent. PAS and Toluidine blue staining intensity was further diminished in the chondroid matrix.
3.2.3 Mankin Scores evaluation
In modified Mankin scoring system, the structure of the cartilage, cell appearance, staining of the cartilage matrix, tidemark was evaluated. Total score arranged from 0 to above. Normal cartilage was scored as 0. A low score indicated mild changes in articular cartilage while a high one indicated severe degeneration.
The normal group was scored 0.0±0.0 while the model group was 4.5±0.5 which was significantly higher than that of the normal group (P<0.01). The scores of the two O_3IACI groups together were 6.0±0.8-8.1±1.2, they were significantly higher than that of the model group (P<0.01). When compared the two O3IACI groups, the score of the O_3IACI-40 group (8.1±1.2) was significantly higher than that of the O_3IACI-2O group (6.0±0.8). (P<0.01). The results of the Mankin score evaluation indicated that multiple injections of Ozone into the articular space could further induce damage in the cartilage tissues of osteoarthritis, and the degree of the Ozone induced cartilage damage appeared to be dosage dependent.
3.3 laboratories Analysis
Serum NO level: In comparison with the level of the normal group (91.88±26.94μmol/L), the NO level of the model group (146.75±16.43μmol/L) or each of the two O_3IACI groups (O3IACI20, 156.01±24.53μmol/L; O3IACI40, 166.23±22.01μmol/L) was significantly higher, respectively, (P<0.01). However, there was no significant difference among the two of O_3IACI groups and the model group (p>0.05)
Serum SOD level: In comparison with the SOD level of normal group (246.18±34.65 U/ml), the level of the model group (330.78±46.68 U/ml) or each of the two O3IACI groups (374.96±44.02 U/ml, 411.59±30.00 U/ml) was significantly higher, respectively(P<0.01); the SOD level of the two O_3IACI groups were higher than that of the model group, the differences were statistically significant, P<0.05. However, the difference between the O3IACI-20 group (374.96±44.02 U/ml) and the O3IACI-40 group (411.59±30.00 U/ml) was not statistically significant(P> 0.05).
Synovia NO level: In comparison with the NO level of the normal group (31.43±5.06μmol/L), the NO level of the model group (111.37±35.92umol/L) or each of the two O_3IACI groups (108.28±20.45umol/L, 105.16±38.18umol/L) was significantly higher, respectively (P<0.01); Differences between the two O_3IACI groups and between the O3IACI groups and the model group were not significant statistically; (P>0.05).
Synovia SOD level: The difference between SOD level of the model group (86.02±48.47 U/ml) and that of the normal group (63.51±39.81 U/ml ) was not significant, P>0.05; the level of either of the two of O3IACI groups (119.05±34.96 U/ml, 140.14±42.76 U/ml) were higher than that of the normal group, respectively; (P <0.05). The SOD levels of the O_3IACI-40 group (140.14±42.76 U/ml) was significantly higher than that of the model group (P<0.05); but there was no statistically significant difference between the O_3IACI-20 group (119.05±34.96 U/ml) and the model group (P> 0.05), and between the two 03IACI groups, (P> 0.05), respectively.
4. Conclusions
Ozone, when introduced into the articular cavity, could cause increased secretion of SOD. Up-regulation in synthesis of antioxidant enzymes, such as SOD, is known to be able to lead to block chronic oxidative stress reaction which may be one of the mechanisms of Ozone therapeutic effect in OA. However, intra-articular injection of Ozone did not reduce NO levels of both serum and synovial fluid. Regardless of these, we have documented that repeated injections of Ozone to joint cavity have an adverse deleterious effect on OA-like articular cartilage. Our findings raise a serous concern regarding the safety of intra-articular injection of Ozone in treating OA. The mechanism of its deleterious effect on OA-like cartilage is not clear and further investigation is needed. Physicians should be aware of this potential adverse effect on OA patients with a conservative approach to withhold the use medical Ozone as one of the treatment choices for OA until its therapeutic benefit can be approved against its adverse effect in further animal study and clinical trial.
Part III. Experimental Study of Collagenase II produced OA models treated with Medical Ozone by intra articular injection and Autohemotherapy
1. Purposes
This study was designed to study the variation of serum cytokines before and after introduction of Ozone of different dosages in two ways, intra-articular injection and autohemotherapy, to better understand the biologic mechanism of Ozone therapy on OA, and to further confirm the pathologic effect of Ozone on OA articular cartilage.
2. Materials and Methods
OA animal models were produced by intra-articular injection of Collagenase II solution. Nitric oxide (NO), Superoxide dismutase (SOD), and two most important cytokines in OA development, TNF-αand IL-1β, and the histopathology of the articular cartilage were analyzed.
2.1 animal grouping and model establishment
36 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, OA model group with O_3 Intra- articular cavity Injection (O3IACI) and OA model group with ozonized blood Autohemotherapy (O_3AHT). The O31 ACI treated OA model group and the O_3AHT treated OA model group were further sub-divided in to two groups separately according to the Ozone dosage used in the experiment: O_3IACI-10 group (Ozone dosage: 10μg/ml per injection), O_3IACI-30 group (Ozone dosage:30μg/ml per injection), O_3AHT-10 group (Ozone dosage: 10μg/ml per injection), O_3AHT-30 group (Ozone dosage:30μg/ml per injection).6 rabbits were in each group. OA model was produced by injecting 0.5ml of collagenase II solution into the knee joint space twice three days apart.
2.2 animal processing
4 weeks after the last injection of Collagenase II solution, the rabbits of both O_3IACI-10 group and O_3IACI-30 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks.
Otherwise, the rabbits of both O_3AHT-10 group and O_3AHT-30 group were treated with autohemotherapy. During the autohemotherapy procedure, 4ml venous blood was taken form the vein in the ear of the rabbit and thoroughly mixed with 4ml Ozone of different dosage accordingly by a Y tube unit, and re-infused into the vein, twice a week for 4 weeks.
The normal and model groups were fed routinely. Blood of all groups were collected and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone treatment.
2.3 Animal behaviors observation
2.4 Imaging studies:
Knee images of rabbits of each group were studied by DR as well as MRI
2.5 Serum analysis
NO,T-SOD ,TNF -αand IL-1βwere detected. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry, and both TNF-αand 1L-1βby ELISA.
2.6 Histopathology study of the cartilaginous tissue
The articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.
2.7 Statistical treatment
SPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. Completely random designed One-way ANOVA was applied to analysis every parameter of interest as homogeneity of variances in multiple group samples means. Welch test was applied to analyze every parameter of interest as Heterogeneity of variance in multiple group samples means. LSD-t and Games-Howe 11 were used for comparison among multiple sample means. A p <0.05 value was considered statistically significant.
3. Results
3.1 Animal behaviors observation
After the first Collagenase II solution injection, inactivity, gait difficulty was observed in all rabbits for producing OA model; variable extent of the right knee was presented. Rabbits did limp apparently, struggle and kick when their right knees were touched. About 6 weeks after the first Collagenase II injection, Osteophyma was palpated at the medial part of the right knee in 50% of the OA models. The rabbits of the Ozone treated groups became active and recovered quicker than those of the model group.
3.2Imaging studies
3.2.1 DR imaging
Normal group: both knee spaces were in concordance without obvious loss, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.
Model group: obvious hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was lost.
O3IACI-10 group: hyperostosis of the right tibial plateau was noted. Its edge was sharp and the sclerotic articular surface was lightly hyperostosis and eburnated. The right knee space was not narrowed. The sub-patella fat pad was clear.
O3IACI-30 group: hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was still normal.
03AHT-10 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was lost.
O3AHT-30 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was a little lost.
3.2.2 MRI studies
Normal group: in 3D_WATSc sequence, the articular cartilage surface of the internal condyle of femur and the medial tibial plateau was smooth and intact without thinning and defect. Signals were normal. In STIRLongTE sequence, the signals of the anterior and posterior angles of the medial meniscus and that of subchondral bone were normal. No articular hydrops was presented.
Model group: in 3D_WATSc sequence, the articular cartilage of the medial tibial plateau was remarkable thinning with a low signal. The thickness of the cartilage in condyles of femur was normal. Its signal was normal. In STIRLongTE sequence, small amount of articular hydrops in the suprapatellar bursa was presented
O3IACI-10 group: in 3D_WATSc sequence, the articular cartilage of the medial condyle of femur was thinning with a low signal. The thickness of the cartilage of the tibial plateau was different and the surface was rough. The posterior angle of the medial meniscus was unclear. In STIRLongTE sequence, Small amount of articular hydrops in the supra & infra patellar bursas were presented. O3IACI-30 group: in 3D_WATSc sequence, the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with signal reduction. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented.
O3AHT-10 group: in 3D_WATSc sequence, the cartilage of the right medial tibial plateau was thinning with signal reduction. The thickness of the cartilage of the right medial condyle of femur was nearly normal with a normal signal. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented.
O3AHT-30 group: 3D_WATSc showed the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with reduction in signal. Small amount of articular hydrops in the supra & infra patellar bursas were presented in STIR_LongTE.
3.3 laboratories Analysis
3.3.1 Serum NO and T-SOD levels
Serum NO level: In comparison with the level of the normal group (79.44±15.31μmol/L), the NO level of the model group (152.23±17.88μmol/L) or each of the two O_3IACI groups (O3IACI-10, 163.20±22.31μmol/L; O3IACI-30, 158.05±36.47μmol/L),and each of the two O3AHT groups (O3AHT-10,183.21±23.12; O3AHT-30,183.95±50.72) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
Serum SOD level: In comparison with the SOD level of normal group (233.84±30.66U/ml), the level of the model group (359.71±66.73 U/ml) or each of the two O_3IACI groups (O3IACI-10, 351.23±46.46 U/ml, O3IACI-30, 375.85±75.96 U/ml) and each of the two O3AHT (O3AHT-10, 397.01±65.81; O3AHT-30, 379.05±37.67) was significantly higher, respectively (P<0.05); However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
3.3.2 Serum TNF-αand IL-1βlevels
Serum TNF-αlevel: In comparison with the level of the normal group (8.50±3.46 pg/Ml), the NO level of the model group (28.37±17.40 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 88.72±72.88 pg/Ml; O3IACI-30, 198.98±310.97 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 56.07±73.83 pg/Ml; O3AHT-30, 39.12±40.74 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
Serum IL-1βlevel: In comparison with the level of the normal group (43.37±18.27 pg/Ml), the NO level of the model group (146.89±98.93 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 416.30±319.45 pg/Ml; O3IACI-30, 691.71±798.24 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 287.24±363.95 pg/Ml; O3AHT-30, 219.72±229.94 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
3.4 Histomorphology study
3.4.1 Gross inspection
Part examined: cartilage at the medial side of the tibia platform
Normal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer. There was hydrops in the space, neither hyperplasia nor thickening of the synovium.
Model group: the surface of the right condyles was still smooth and intact. The right tibial plateau was dull grey and granulo-changed. Thinning and erosion of the medial articular cartilage were seen. Osteophyma was formed at the medial edge of the right tibial plateau.
O3IACI-10 group: the surface of the right condyles was rough and dull. Its cartilage was thinning; the right lateral and medial tibial plateaus were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
O3IACI-30 group: the synovial membrane of the right knee articular cavity was hyperaemic and thickening. The cartilage of the condyles of femur was dissipated and dull. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
O3AHT-10 group: the cartilage surface of the condyles of the right femur was rough with focal erosions; The lateral and medial plateaus of the right tibial were dark grey yellow and rough with focal erosions and malacia, Osteophyma was found at the internal angle of the right tibia.
O3AHT-30: the synovial membrane of the right knee articular cavity was hyperaemic. The cartilage of the condyles of femur was dissipated with focal erosions. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
3.4.2 Microscopic examination
3.4.2.1 Articular cartilage
Normal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The cartilage matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.
Model group: the surface layer of the cartilage was thinning and rough, and villiform in some part. There were a lot of deep fissures extending deeply to the calcified layer from the surface. The tidemark was destroyed. The cartilage layer was peeled away from the subchondral bone. Chondrocyte degenerated and decreased in cellularity; Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix. The cartilage was of fibrosis.
O3IACI-10 group: the surface of the cartilage was rough with many fissures extending deeply to the radial zone from the superficial layer. The chondrocytes in the transitional zone and radial zone were disarrayed in arrangement and decreased in cellularity. Cellular clustering hyperplasia was presented. The tidemark was still intact. Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix and cracked cartilage.
O3IACI-30 group: villiform like changes were found at the superficial cartilage with great amounts of fissures extending to the radial zone from the superficial layer. Chondrocytes were clustering and disarrayed in arrangement. The cartilage layer was exfoliated. Toluidine blue stains showed medium decrease in staining intensity in the chondroid matrix and profuse deep cracks.
O3AHT-10 group: there were many fissures extending deeply to the calcified layer from the superficial layer. Chondrocytes Chondrocyte degenerated and decreased in cellularity; the tidemark was un-complete. The cartilage layer was separated from the subchondral bone. Toluidine blue stains showed decrease in staining intensity in the chondroid matrix, and also cracks and fibrosis.
O3AHI-30 groups: a lot of fissures extending deeply to the radial zone from the superficial layer of the cartilage. Matrix was dissolved in the radial zone as well as calcified layer. The cartilage structure was destroyed and out of order. Chondrocytes were necrotic. The tidemark was un-complete. The cartilage layer was exfoliated. Toluidine blue stains showed severe decrease in staining intensity in the chondroid matrix, and also many cracks and fibrosis
3.4.2.2 Synovium
Normal group: Neither thickening nor hyperplasy was found in the synovium tissue. There was no vascularization and inflammatory cell infiltration
Model group: capillary engorgement in the synovial tissue was found. Inflammatory cells were seen surrounding the vessels
O3IACI-10 group: slight capillary engorgenment in the synovial tissue was found. Small amounts of inf
目的：
比较关节腔内注射1．6％木瓜蛋白酶、关节腔内注射胶原蛋白酶、关节固定法、股静脉结扎法等四种方法，筛选出适合快速、大规模造模的方法。
1．木瓜蛋白酶关节腔内注射法OA造模试验
1．1材料与方法
健康新西兰白兔8只，雌雄各半，6～8个月龄，体重2．0～2．5kg。分别于实验的第1、4日于右膝关节腔内注射1．6％papain溶液0．3ml制备OA动物模型。造模后4周后对模型行软骨取材及病理学观察。
1．2结果
一般情况：第一次注射1．6％木瓜蛋白酶溶液后，右膝关节出现不同程度的肿胀，第二次注射后动物出现跛行，触摸右膝关节时出现挣扎和弹腿。2天后跛行消失，10天左右肿胀消退。所有动物的进食饮水二便未受明显影响，全部动物存活，未出现明显异常生物行为。
肉眼和手术显微镜下观察：8只实验兔之胫骨内侧平台软骨均呈灰黄色或灰白色，无光泽，表面可见裂纹，个别可见小而浅的局部糜烂，未见明显骨赘形成。
关节软骨光镜下观察：软骨表面粗糙不规则，可见较小和浅的裂隙，软骨细胞数量减少，排列紊乱，无细胞簇聚现象。PAS和甲苯胺蓝染色示基质轻度失染。
1．3结论
木瓜蛋白酶关节腔内注射法模型的病理变化与临床OA极为相似，造模过程简单，成功率高，适合快速大批量建立OA模型作软骨病理、药物防治的研究。
2．胶原蛋白酶OA模型试验
2．1材料与方法
采用6只健康的普通级新西兰白兔，雌雄不限，6～8个月龄，体重2．0～2．5kg，分别于第1、4天向实验兔的右侧膝关节腔内注射Ⅱ型胶原酶溶液0．5ml(即2mgⅡ型胶原酶)制备OA模型。造模后4周后对模型行软骨取材及病理学观察。
2．2结果
造模实验兔右膝关节腔内注射Ⅱ型胶原蛋白酶溶液后，精神较差，自发活动明显减少，右膝关节出现不同程度的肿胀，行走时右下肢不着地，呈明显跛行；触摸右膝关节时出现挣扎和弹腿。2周后跛行基本消失。
右膝关节DR成像显示膝关节关节间隙对称，未见明显狭窄，骨性关节面光滑、锐利，未见骨赘形成。髌下脂肪垫清晰。关节周围软组织未见肿胀。
右膝关节MRI均显示股骨髁及胫骨平台关节软骨表面光整，信号均匀，无明显变薄及缺损，髌上囊可见少量积液，软骨下骨信号未见异常。
大体标本：股骨髁表面尚光整，色泽略显暗淡，胫骨平台表面欠光滑，凹凸不平，呈粗颗粒状改变，未见明显骨赘形成。
HE染色：软骨表层可见不规则裂隙，辐射层及钙化层结构完整，软骨细胞排列规则，数量略减少，潮线完整。
2．3Ⅱ型胶原蛋白酶关节腔内注射法制作的兔膝关节OA模型，模型成型时间最短(4周)，在病理学改变上与人类OA基本一致，为一种较为理想的早期OA动物模型。该方法操作技术简单，周期较短，非常适合快速大批量建立骨关节炎模型。
3．石膏关节固定法和股静脉结扎法预实验
结果显示石膏固定法制备OA模型具有死亡率相对较高，制备过程需严密观察且时刻需要调整石膏固定，过程中动物固定时间不一致，导致制备的OA模型病变程度不一致等缺点；而采用股静脉结扎的方法制备OA模型，由于术中出血及术后感染，也具有一定的死亡率，并且单纯行股静脉结扎难以成功复制典型的动物OA模型。因此上述两种造模方法不适合本研究的较大规模造模。
4．结论
本课题参考文献报道的造模方法，试验性地复制接近临床OA病理改变的新西兰兔OA模型。证实了关节腔内注射化学物质诱导法更为适合本试验的现状，可在短时间内，大量并成功复制OA模型，木瓜蛋白酶和胶原蛋白酶两者比较，以后者所复制的模型病理改变更接近人类骨关节炎模型。而关节固定法和手术造模由于对实验环境和实验动物的护理有较高的要求，实验动物模型发生并发症的机会较高，并发症的预防和治疗都较困难。造成实验动物死亡时有发生。因此不适合在现有条件下大量复制OA模型。
第二部分木瓜蛋白酶OA模型关节腔内注射医用臭氧的实验研究
1．目的
通过研究医用臭氧关节腔内直接注射对OA模型关节软骨的影响，探讨关节腔内直接注射医用臭氧治疗骨关节炎的可行性，同时探讨医用臭氧治疗骨关节炎的可能机制。本实验OA模型以关节腔内注射木瓜蛋白酶法制备。选择研究氧化应激反应常用的指标NO(Nitric oxide，一氧化氮)和SOD(Superoxide dismutase，超氧化物歧化酶)作为观察氧化应激反应的指标。
2．材料与方法
2．1．动物分组及模型建立
32只兔随机分为正常组(简称Normal组)、OA模型组(简称model组)、臭氧关节腔内注射组(O_3 Intra-articular cavity Injection，简称O_3IACI)2组：低剂量(20μg／ml)臭氧注射组(简称O_3IACI-20)和高剂量(40μg／ml)臭氧注射组(简称O_3IACI-40)等4组，每组8只。normal组不做任何处理，正常喂养。其余3组共24只兔，分别于实验的第1、4日于右膝关节腔内注射1．6％papain溶液0．3ml制备OA动物模型。
2．2动物处理
动物OA模型制作完成一周后，O_3IACI-20组和O_3IACI-40组采用右膝关节腔内注射的方法给药。O_3IACI-20组右膝关节内注射浓度为20ug／ml的医用臭氧2ml，每周两次，共四周；O_3IACI-40组右膝关节内注射浓度为40ug／ml的医用臭氧2ml，每周两次，共四周。Normal组和model组正常喂养，不作任何处理。末次注射后1周抽取关节液和血液并处死各组动物行软骨组织取材。
2．3指标检测
血清NO和SOD活性检测；关节液NO和SOD活性检测
2．4软骨组织病理学检查
软骨石蜡标本切片分别行HE染色、过碘酸雪夫氏(PAS)染色及甲苯胺蓝(Toluidine Blue)染色，光镜下观察病理变化，以Mankin’s评分标准进行评分。
2．5统计学处理
所有资料均以均数±标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，方差不齐则采用Welch检验或秩和检验，多样本均数间的比较采用LSO-t及Games-Howell等检验方法。以SPSS11．5统计分析软件进行统计分析,P<0．05有统计学意义。
3．结果
3．1动物行为观察
第一次注射木瓜蛋白酶当天，所有建模实验兔右膝关节出现不同程度的肿胀，第二次注射木瓜蛋白酶后当天出现跛行，触摸右膝关节时出现挣扎和弹腿。2天后跛行消失，10天左右肿胀消退。O_3IACI-20组、O_3IACI-40组注射臭氧后动物行动无明显改变，个别实验兔行为稍活跃。
3．2组织形态学观察
3．2．1膝关节软骨肉眼及手术显微镜下观察
观察部位：胫骨内侧平台软骨。结果显示：
normal组：关节软骨呈蓝白色，有光泽，表面光滑，无纤维化，无裂纹及溃疡形成。
Model组：软骨呈灰黄色或灰白色，无光泽，表面可见裂纹，个别可见小而浅的局部糜烂，未见明显骨赘形成。
O_3IACI-20组：软骨呈灰黄色，表面粗糙，无光泽，裂纹多见，表面糜烂较明显和多见，未见明显骨赘。
O_3IACI-40组：软骨表面粗糙，裂纹及糜烂情况较低剂量组更明显和多见。未见明显骨赘。
3．2．2膝关节软骨光镜下观察
normal组：软骨表面光滑、平整，软骨切线层、移行层、深层和钙化层层次清楚，潮线完整，软骨细胞分布均匀，细胞排列整齐，无簇聚软骨细胞；PAS染色软骨基质着色为均匀的红色，无失染现象；甲苯胺蓝染色示基质呈均匀的深蓝色，无失染现象。
model组：软骨表面粗糙不规则，可见较小和浅的裂隙，软骨细胞数量减少，排列紊乱，无细胞簇聚现象。PAS和甲苯胺蓝染色示基质轻度失染。
O_3IACI-20组：软骨表面缺损，出现空陷窝，软骨细胞部分消失，软骨变薄，可见明显的裂隙，潮线模糊或多层次，PAS和甲苯胺蓝染色示基质染色失染较model组明显。
O_3IACI-40组：软骨表面缺损，软骨变薄，细胞减少，排列紊乱，可见明显成簇样细胞增生，层次不能区分，裂隙较低剂量组更多和更明显，PAS和甲苯胺蓝染色示基质失染情况较低剂量组更加明显。
3．2．3关节软骨Mankin’s评分
正常关节软骨Mankin’s评分为0．0±0．0，model组评分为4．5±0．5高于normal组(P<0．01)，O_3IACI组评分为6．0±0．8～8．1±1．2高于model组(P<0．01)；其中O_3IACI-40组评分(8．1±1．2)又高于O_3IACI-20组的评分(6．0±0．8)(P<0．01)，表明多次重复关节腔内注射臭氧对骨关节炎的关节软骨有损伤作用，且损伤程度随臭氧浓度的增加而加重。
3．3生化指标的检测
血清NO水平检测：与normal组血清NO水平(91．88±26．94μmol／L)比较，model组(146．75±16．43μmol／L)和O_3IACI组(156．01±24．53μmol／L、166．23±22．01μmol／L)血清NO水平均高于normal组，差异有显著性(P<0．01)；O_3IACI组和model组血清中NO水平比较差异无显著性(P>0．05)，O_3IACI-20组(156．01±24．53μmol／L)和O_3IACI-40组(166．23±22．01μmol／L)组间血清中NO水平比较亦无显著性差异(P>0．05)。
血清SOD浓度检测：与normal组血清SOD水平(246．18±34．65 U／ml)比较，model组血清SOD(330．78±46．68 U／ml)和O_3IACI组血清中SOD水平(374．96±44．02 U／ml、411．59±30．00 U／ml)均高于normal组，差异有显著性(P<0．01)，O_3IACI组血清中SOD水平高于model组，差异有显著性(P<0．05)，O_3IACI-20组血清SOD水平(374．96±44．02 U／ml)与O3IACI-40组血清SOD水平(411．59±30．00 U／ml)比较，差异无显著性(P>0．05)。
关节液NO水平检测：model组关节液NO水平(111．37±35．92μmol／L)和O_3IACI组关节液中NO水平(108．28±20．45μmol/L、105．16±38．18μmol／L)均高于normal组(31．43±5．06μmol／L)，差异有显著性(P<0．01)；O_3IACI组与model组各组间及O_3IACI I-20组(108．28±20．45μmol／L)和O_3IACI-40组(105．16±38．18μmol／L)组间关节液中NO水平差异无显著性(P>0．05)。
关节液SOD浓度检测：model组关节液中SOD水平(86．02±48．47 U／ml)与normal组关节液SOD水平(63．51±39．81 U／ml)比较，差异无显著性(P>0．05)，O_3IACI组关节液中SOD水平(119．05±34．96 U／ml、140．14±42．76 U／ml)与normal组关节液SOD比较，差异有显著性(P<0．05)；O_3IACI-40组关节液中SOD水平(140．14±42．76 U／ml)与model组关节液SOD水平比较，差异有显著性(P<0．05)；而O_3IACI-20组关节液中SOD水平(119．05±34．96 U／ml)与model组关节液SOD比较，差异无显著性(P>0．05)；两O_3IACI组组间关节液中SOD水平比较差异无显著性(P>0．05)。
4．结论
臭氧关节腔内注射可以引起SOD的过度分泌，但对NO没有影响，提示臭氧治疗骨关节炎的可能机制之一是通过上调抗氧化酶的过度表达阻断慢性氧化应激反应在骨关节炎发病和发展中的作用。骨关节炎模型多次直接腔内注射臭氧，对病变软骨有进一步损害的作用，损害程度随臭氧浓度增加而加重。臭氧直接关节腔内注射的安全性值得深入研究。在骨关节腔内注射臭氧治疗骨关节炎的机理未确认之前，需慎用该治疗入路。
第三部分：胶原蛋白酶OA模型臭氧关节腔内注射与经静脉臭氧化自体血回输的对照实验研究
1．目的
用两种不同方法把臭氧引人实验动物体内，观察相关血清细胞因子在臭氧引人前和后的变化，进一步探讨臭氧治疗OA的生物学机理，进一步明确臭氧对OA模型关节软骨的病理影响。
2．材料与方法
2．1．动物分组及模型建立
采用36只健康的普通级新西兰白兔，雌雄不限，6～8个月龄，体重2．0～2．5kg。36只兔随机分为正常对照组(简称Normal组)；模型对照组(简称model组)；臭氧关节腔内注射组(O3 Intra-articular cavity Injection，简称O3IACI)2组：10μg／ml臭氧关节腔内注射组(简称O3IACI-10组)、30μg／ml臭氧关节腔内注射组(简称O3IACI-30组)；臭氧自血回输组(O3 AutoHemoTherapy，简称O3AHT)2组：10μg／ml臭氧自血回输组(简称O3AHT-10组)和30μg／ml臭氧自血回输组(简称O3AHT-30组)，每组6只。正常组不做任何处理，正常喂养。其余30只兔，分别于实验的第1、4日于右膝关节腔内注射Π型胶原蛋白酶溶液0．5ml制备骨关节炎动物模型。
2．2动物处理
动物OA模型制作完成4周后，O_3IACI-10组和O_3IACI-30组采用右膝关节腔内注射的方法给药。O_3IACI-10组右膝关节内注射浓度为10ug／ml的医用臭氧2ml，每周两次，共四周；O_3IACI-30组右膝关节内注射浓度为30ug／ml的医用臭氧2ml，每周两次，共四周。
O_3AHT-10组和O_3AHT-30组均采用自体静脉血回输的方法给药(大自血回输)，经兔耳缘静脉采取4ml静脉血后，分别抽取浓度为10μg／ml及30μg／ml的臭氧气体4ml，第一管弃之，第二管经三通管与静脉血充分溶解、混匀后经兔耳缘静脉回输入血。每周进行两次，共四周。
末次注射后4周行影像学观察，抽取血液样本，处死各组动物行病理学检查。
2．3观察指标
2．3．1一般行为观察
2．3．2影像学观察(DR及MRI)
2．3．3血清生化指标及细胞因子的检测
血清NO和T-SOD活性检测、血清TNF-α和IL-1β含量的检测
2．3．4病理组织学观察
软骨石蜡标本切片分别行HE染色、过碘酸雪夫氏(PAS)染色及甲苯胺蓝(Toluidine Blue)染色，光镜下观察病理变化，并作Mankin评分
2．3．5统计学处理
所有资料均以均数士标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，方差不齐则采用Welch检验，多样本均数间的比较采用LSD-t及Games-Howell等检验方法。以SPSS11．5软件进行统计分析,P<0．05有统计学意义。
3．结果
3．1一般情况观察
第一次注射胶原蛋白酶后当天，所有建模动物右膝关节出现不同程度肿胀、可见明显跛行，触摸右膝关节时略有挣扎、弹腿，活动量减少。距离第二次胶原蛋白酶注射完毕后6～7周，50％的实验动物可于关节内侧扪及骨赘，关节活动稍受限；灵活性变差。O_3IACI-10组、O_3IACI-30组、O_3AHT-10组及O_3AHT-30组的实验兔接受臭氧关节腔内注射或自血回输后，较Model组活跃，精神状态较好，活动增多，步态恢复较Model组迅速。
3．2影像学观察：
3．2．1 DR成像
Normal组：双膝关节关节间隙对称，未见明显狭窄，骨性关节面光滑、锐利，未见骨赘形成。髌下脂肪垫清晰。关节周围软组织未见肿胀。
Model组：右膝关节内侧胫骨平台明显骨质增生、变尖，骨赘形成，内侧关节囊肿胀，髌下脂肪垫透亮度减低，关节间隙狭窄。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见DR改变与Model组大致相同，其中O_3IACI-10组、O_3IACI-30组关节间隙无明显狭窄。
3．2．2膝关节MRI成像
Normal组：右膝股骨髁及胫骨平台关节软骨表面光整，信号均匀，无明显变薄及缺损，软骨下骨信号未见异常，关节腔内未见积液。
model组：右膝内侧胫骨平台关节软骨明显变薄，信号强度减低，内侧股骨髁关节软骨厚度、信号尚可，软骨下骨信号未见明确异常；髌上囊少量积液。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见MRI改变同model组大致相仿。
3．3血清生化指标及细胞因子
3．3．1血清NO含量及T-SOD活性
采用硝酸还原酶法，根据样品的吸光度，测定各组样本的NO含量及T-SOD活性。
各组血清NO含量(单位：μmol／L)分别为：normal组79．44±15．31；Model组152．23±17．88：O3IACI-10组163．20±22．31；O3IACI-30组158．05±36．47；O3AHT-10组183．21±23．12；O3AHT-30组183．95±50．72。
各组血清T-SOD活性(单位：U／ml)分别为：normal组233．84±30．66；Model组359．71±66．73；O3IACI-10组351．23±46．46；O3IACI-30组375．85±75．96；O3AHT-10组397．01±65．81；O3AHT-30组379．05±37．67。
经过检验，各治疗组和模型组的血清NO含量和T-SOD活力均高于正常组(均为P<0．05)；各治疗组问及治疗组与模型组间的血清NO含量和T-SOD活力无显著性差异(均为P>0．05)。
3．3．2血清TNF-α和IL-1β含量
采用双抗夹心酶联免疫吸附法(ELISA)检测血清中TNF-α和IL-1β的含量。
各组血清TNF-α浓度(单位：pg／mL)分别为：normal组8．50±3．46；Model组28．37±17．40；O3IACI-10组88．72±72．88；O3IACI-30组198．98±310．97；O3AHT-10组56．07±73．83；O3AHT-30组39．12±40．74。
各组血清IL-1β浓度(单位：pg／ml)分别为：normal组43．37±18．27；Model组146．89±98．93；O3IACI-10组416．30±319．45；O3IACI-30组691．71±798．24；O3AHT-10组287．24±363．95；O3AHT-30组219．72±229．94。
经过检验，各治疗组和模型组的血清IL-1β含量高于正常组(均为P<0．05)；各治疗组之间及治疗组与模型组间的血清IL-1β含量无显著性差异(均为P>0．05)。
3．4病理学观察
3．4．1大体观察
Normal组：双侧关节腔内无积液，滑膜无增生、肥厚，双侧股骨髁及胫骨平台表面光滑，色泽鲜亮，未见软骨缺损及骨赘形成。
Model组：右膝关节腔内可见积液，呈粘稠的淡黄色，滑膜明显增生、肥厚，右股骨髁表面尚光整，右胫骨平台色泽灰暗，表面见粗颗粒状改变，内侧关节软骨糜烂、变薄。右胫骨平台内侧缘可见骨赘形成。O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组所见与model组大致相仿，没有明显差别。
3．4．2光镜观察
3．4．2．1关节软骨
Normal组：关节软骨表面光滑、平整，软骨切线层、移行层、放射层和钙化层层次清楚，潮线完整，软骨细胞分布均匀，细胞排列整齐，无簇聚或减少；甲苯胺蓝染色示基质呈均匀的深蓝色，无失染现象。
Model组：关节软骨表层变薄、粗糙，部分区域呈绒毛状，可见较多裂隙从表层软骨延伸向下深达钙化层，潮线不完整，软骨层自软骨下骨剥离；软骨细胞变性，数量减少；甲苯胺蓝染色示基质失染，软骨纤维化。
O_3LACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组均出现model组改变，并显示软骨裂隙，裂隙以O_3IACI-30组、O_3AHT-30组更多和更深，其中O_3IACI-10组、O_3IACI-30组可见软骨细胞成簇样增生。
3．4．2．2滑膜：
Normal组：滑膜组织无肥厚及增生，无血管侵入，无炎细胞浸润。Model组：滑膜组织毛细血管充血，血管周围可见炎性细胞浸润。O_3IACI-10组、O_3IACI-30组、O_3AHT-10、O_3AHT-30组出现了model组的所有改变，并有增生现象。
3．4．3关节软骨Mankin’s评分
各组Mankin’s评分：normal组0．0±0．0，Model组10．2±1．9；O3IACI-10组8．8±1．5；O3IACI-30组9．3±2．1；O3AHT-10组8．2±1．9；O3AHT-30组8．6±2．7。
模型组、各治疗组评分高于正常组(P<0．05)，各治疗组间及与模型组间无显著性差异(P>0．05)，表明治疗组关节软骨的损害程度与模型组无差异，臭氧治疗不能改善缓骨关节炎的病理进程。
4．结论
经关节腔内臭氧注射及经静脉臭氧化自体血回输均可引起T-SOD的过渡分泌，提示臭氧可上调抗氧化酶的过渡表达。但对OA模型升高的血清NO没有抑制作用。两种给药途径对OA模型升高的血清IL-1β和TNF-α没有明确的抑制作用。经静脉两种浓度的臭氧化自体血回输对OA模型软骨病理改变无逆转作用；经关节腔内两种浓度臭氧直接注入对OA模型软骨病理改变无逆转作用，30μg／ml浓度臭氧关节腔内直接注射后OA模型软骨病变发生损害加重。
第四部分：OA模型关节软骨及臭氧对其作用的MRI评价
1．目的
探讨理想的关节成像序列及成像参数用于关节软骨的定量分析，并评价医用臭氧对OA模型关节软骨的作用。
2．材料与方法
2．1动物分组和模型的制备、动物处理详见论文第三部分。
2．2磁共振成像
成像时间：末次注射臭氧后4周。实验兔麻醉满意后固定于自制的检查板，扫描使用Philip Intera 1．5T磁共振扫描仪，采用C3表面线圈，行双膝的矢状面扫描。
2．3实验兔膝关节磁共振成像序列对比实验
2．3．1各序列扫描参数设定
对normal组内6只正常实验兔进行右膝关节MRI，成像序列包括施加脂肪抑制的质子加权成像PDW／SPAIR／D，无脂肪抑制的T2加权三维快速梯度回波序列(T2W_3D_FFE)，水激励的三维快速扰相梯度回波序列3D_WATSc
2．3．2不同脉冲序列关节软骨信噪比(signal to noise ration，SNR)及软骨与骨髓
对比信噪比(contrast to noise ration，CNR)的测定和比较方法：MR扫描后测量图像中关节软骨、软骨下骨在上述3种脉冲序列中的信号强度。然后计算出关节软骨信噪比(SNR)和关节软骨与软骨下骨的对比噪声比(CNR)。
2．3．3关节软骨厚度及信号强度的测量方法：
将Dicom数据分别导入Orisix 3．02(School of Medicine of the GenevaUniversity，Switzerland)及eflim workstation 2．1．2(Merge healthcare，USA)中，计算出某实验兔关节软骨厚度及信号强度的平均值。
2．4关节软骨的MRI与病理学对比观察
对Normal组、model组、O_3IACI-10组、O_3IACI-30组、O_3AHT-10组、O_3AHT-30组6组实验兔的关节软骨MR成像与病理学改变作对照分析。
2．5统计学处理：
所有资料均以均数±标准差表示，采用Levene法对多样本方差齐性进行检验，方差齐使用完全随机设计的单因素方差分析(one-way ANOVA)，多样本均数间的比较采用LSD-t检验，方差不齐则采用秩和检验；对所得的关节软骨平均厚度及信号强度与第三部分测得的Mankin’s评分行直线相关分析，若变量间存在相关则进一步做多元线性回归，明确Mankin’s评分与关节软骨的厚度或信号强度有无线性关系。以SPSS 11．5统计分析软件进行统计分析，P<0．05有统计学意义。
3．结果
3．1．1 normal组正常兔膝关节软骨不同脉冲序列的信号特点
PDW／SPAIR／D序列：关节软骨表现为覆盖于软骨下骨的均匀中高信号带；T2W 3D FFE序列：关节软骨则呈单层中等信号带，与骨髓隔以薄层低信号区；3D WATSc序列：关节软骨表现为均一、光整的高信号带，与低信号的软骨下骨分界清晰。
3．1．2 Normal组正常兔膝关节软骨不同脉冲序列的SNR及CNR
PDW／SPAIR/D、T2W_3D_FFE及3D_WATSc组间SNR无显著性差异(p>0．05)：3D_WATSc组CNR高于PDW／SPAIR／D组及T2W_3D_FFE组(p<0．05)，而PDW／SPAIR／D组和T2W_3D_FFE组的CNR无显著性差异(p>0．05)。
3．1．3各实验组关节软骨的平均厚度及信号强度对比
与Normal组比较，model组、各臭氧干预组的实验兔膝关节软骨平均厚度及信号强度明显变薄和降低，(p<0．05)，但Model组、O3IACI-10组、O3IACI-30组、O3AHT-10组及O3AHT-30组各组间的关节软骨平均厚度及信号强度均无显著性差异(p>0．05)。
3．2关节软骨的MRI与病理学对比观察：
Normal组：MRI显示右膝股骨髁及胫骨平台关节软骨表面光整，信号均匀，无变薄及缺损；大体标本及光镜下可见正常软骨表现。
Model组：MRI显示右膝内侧胫骨平台关节软骨明显变薄，信号强度减低：同一部位肉眼观察见软骨色泽灰暗，表面见粗颗粒状改变，内侧关节软骨糜烂、变薄：光镜下关节软骨变薄、粗糙，表层呈绒毛状，可见较多裂隙从表层软骨延伸向下深达钙化层。
O_3IACI-10组、O_3IACI-30组、O_3AHT-10组、O_3AHT-30组各组MRI改变与model组相仿，显示右膝股骨内侧髁及内侧胫骨平台关节软骨不同程度变薄，信号减低。同一部位标本肉眼及光镜下出现了model所见的改变，并显示软骨裂隙，裂隙以O_3IACI-30组、O_3AHT-30组更多和更深，其中O_3IACI-10组、O_3IACI-30组可见软骨细胞成簇样增生。
3．3关节软骨平均厚度及信号强度与Mankin Score的直线相关及回归分析
对关节软骨平均厚度及信号强度与组织学Mankin Score行Pearson相关分析，结果关节软骨平均厚度与信号强度呈显著的正相关；关节软骨平均厚度与Mankin Score呈显著的负相关；关节软骨平均信号强度与Mankin Score呈显著的负相关。
以Mankin Score为应变量，关节软骨平均厚度及信号强度为自变量，采用逐步选择法作多元线性回归分析。结果仅关节软骨平均厚度选入方程，信号强度被剔除，回归方程为Y=18．582-0．023X(Y为Mankin Score，X为关节软骨平均厚度)，R~2=0．528，Mankin Score与关节软骨的平均厚度存在线性回归关系。
4结论
三维扰相梯度回波结合水激励技术成像时间短，是观察OA模型关节软骨的比较理想的成像序列，由于小动物的关节软骨较薄，在不造成信噪比显著降低的同时，需适当提高空间分辩率，利于关节软骨的观察。通过测量膝关节MRI的软骨厚度及信号强度，可以对关节软骨进行量化指标评价，其中软骨厚度测量优于信号强度测量。经对各组实验兔软骨厚度和信号强度的对照研究，医用臭氧关节腔内注射及自血疗法对OA的关节软骨均无修复作用，与病理结果一致。
BACKGROUND
Osteoarthritis (OA), also referred to as degenerative joint disease, is one of the most prevalent chronic conditions in elderly people. OA mainly involves articular cartilage, bone and synovial membrane. The pathogenesis of OA consists of a general progressive loss of articular cartilage, remodeling and sclerosis of the subchondral bone, and the formation of subchondral bone cysts and marginal osteophytes. Arthrocele, pain, deformation and functional disturbance caused by OA severely disable patients' daily activities. With the merging of an aging society, the incidence of OA is increasing in an alarming pace, and OA is becoming a social and economic problem in our current society.
The current management of OA is to relieve the symptom, to improve the joints function and movement, to postpone the articular degeneration. Recently Medical Ozone has been shown to have promising clinical results in treatment of various chronic diseases, especially in pain relief in conditions, such as pyriformis syndrome and OA. But the mechanism of medical Ozone in OA treatment is largely unknown. Above all, there are no consensus on the Ozone dosage and its safety standard among the medical professionals who practice Medical Ozone. This experimental study was designed for investigation of the above issues.
PURPOSES
1. To search for methods of producing OA animal models to match the need of large scale study of OA Ozone intra-cavity treatment.
2. To study the possibility and safety of Ozone intra-cavity treatment of OA by pathologically observing the articular cartilage changes following Ozone intra-cavity injection of different dosages (those are accepted clinically in many countries of Asia and Europe).
3. To study the Autohemotherapy of medical Ozone in treatment of OA by pathologically observing the articular cartilage changes following intravenous injection of model's blood blended with Ozone of different concentrations.
4. To search the possible mechanisms of Ozone in treating OA with different administration route by detecting the level of oxidative stress index and the activities of serum cytokines.
5. To observe and evaluate the effect of medical Ozone in treatment of OA by MRI.
Part I. OA Animal Model Establishment
In this part, to find out the most effective method of OA animal model establishment for large scale studies, four methods were tested: 1.6% papain intra-articular injection and Collagenase intra-articular injection, immobilization of rabbit knee joints, femoral vein ligation.
1. Model establishment by intra articular cavity injection of papain
1.1 Materials and methods
8 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, 4 males and 4 females) were used. Right knee intra-articular injection of 0.3ml 1.6% papain solution was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed and dissected for pathological studies at 5 weeks after the initiation of the injection.
1.2 Results
Various extents of joint swelling were observed after the injection of 1.6% papain solution. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. All rabbit's diets remain normal and alive without exceptional behavioral change.
Gross and operative arthroscope inspections: Cartilage at the medial side of the tibia platform was grey yellow or grey white and dull. Fissures on cartilage surface were found. A few focuses of small and shallow erosion were noted. There was no obvious osteophyma.
Light microscopy examination: Articular surface is rough and irregular. Relatively small and shallow fissures can be appreciated. The chondrocytes is decreased in cellularity and disarrayed in arrangement without cellular crowding. PAS and Toluidin blue stains show mild decrease in staining intensity in the chondroid matrix.
1.3 Conclusions
The pathological changes of the OA model produced by papain intra-articular injection are very similar to those of spontaneous OA. The experimental model is simple technically and easily reproducible with a high successful rate. These models are also suitable for quick and a large scale production for pathological and therapeutic studies.
2. Model establishment by intra articular cavity injection of Collagenase
2.1 Materials and Methods
6 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, both male and female animals) were used to produce the OA models. Right knee intra-articular injection of 0.5ml solution (I.e.2mg Collagenase II) was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed for pathological studies at 4 weeks after the initiation of the injection.
2.2 Results
Low spirit, visible inactivity was observed in rabbits after the injection of Collagenase II solution. Variable degree of joint swelling was present. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 weeks and swelling faded away in 10 days. All rabbits' diets remain normal and alive without exceptional behavioral change.
Right knee DR study was performed 4 weeks after establishment. The radiograms showed both knee spaces were in concordance without obvious stenosis, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.
MRI showed that condyleses of femurs and tibial plateaus were smooth, without thinning and defects; a small amount of hydrops in the suprapatellar bursa was found. The MR signals of the sub-cartilage bone were normal.
Grossly, the surface of the femur condyles was still smooth and intact, but matted. The surface of the tibial plateau was rough and granulated. A few malacia foci but osteophyma were present.
Histological study with HE staining revealed irregular fissures at the surface layer of the cartilage. The radial zone and calcify zone were architecturally normal. The chondrocytes was slightly decreased in cellularity and arrayed in normal arrangement. The tide mark was intact.
2.3 conclusions
In this study, it took the shortest time to achieve the production of the OA model which pathology was very much similar with that of humans. The method of Collagenase II intra articular injection was ideal for the production of early OA model. It was simple technically and took less time, and fit for quick producing OA models in great numbers.
3. Other preliminary experiments for OA model production
Other two OA model production method had been tested in this study, immobilization of rabbit knee joints with plaster bandage and femoral vein ligation. But it's difficult to control the concordance of the OA-like pathological change and degree of the change in individual animal in the group due to the lack of control in immobilizing time. And During the traumatic procedures of model producing by femoral vein ligation, it's more easily for animals to die following in-operation bleeding and post-operation infections. And it's difficult to reproduce a typical OA model by simple ligation of the femoral vein. These establishment methods were not suitable for a quick and large scale model study.
4. Summary
The trail of production of OA in animal models pathologically similar to those happen spontaneously to humans had been based on thorough review of the literatures about OA model establishment. Our investigations proved that intra articular chemical substances injection was the most effective method among those tested. Comparatively, Collagenase II OA model was easier to reproduce in a rather short time and better for OA study than Papain model. Both immobilization of knee joints and femoral vein ligation were unsuitable for large scale establishment, as they were less tolerable to the animals and require more complicated and sophisticated animal handling and manipulation than our current situation allowed.
Part II. Experimental Study of Medical Ozone Intra Articular Injection in Papain Induced OA Models
1. Purposes
Intra-articular injection of Ozone has recently been used to treat OA. However, the mechanism and safety of such treatment modality has not yet been fully investigated. This study was designed to evaluate the local effect of intra-articular Ozone injection to better understand the biologic mechanism and pathologic effect of Ozone therapy on articular cartilage.
2. Materials and Methods
OA animal models were produced by intra-articular injection of Papain solution. Both Nitric oxide (NO) and Superoxide dismutase (SOD), the two most commonly used indexes for oxidative stress reaction, and the histopathology of the articular cartilage were analyzed.
2.1 Animal grouping and model establishment
32 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, and OA model group with O_3 Intra- articular cavity Injection (O3IACI). The O3IACI treated OA model group was further sub-divided in to two groups according to the Ozone dosage used in the experiment: O_3IACI-20 group (Ozone dosage: 20μg/ml per injection), O_3IACI-4O group (Ozone dosage: 40μg/ml per injection). 8 rabbits were in each group. OA model was produced by injecting 3ml of 1.6% Papain solution into the knee joint space twice three days apart.
2.2 Animal processing
7 days after the last injection of Papain solution, the rabbits of both O_3IACI-20 group and O_3IACI-40 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks. The normal and model groups were fed routinely. Synovial fluid and blood of all groups were collected, and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone injection.
2.3 Synovial fluid and serum analysis
NO and SOD activity were detected in both serum and synovial fluid. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry.
2.4 Histopathology evaluation of the cartilaginous tissue
The articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.
2.5 Statistical analysis
SPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. One-way ANOVA as well as q test was applied to analyze every parameter of each region of interest as homogeneity of variances in multiple group samples means. LSD-t and Games-Howell tests were used for comparison among multiple samples means. A p <0.05 value was considered statistically significant.
3. Results
3.1 Animal behaviors observation
After the first Papain solution injection, Variable extent of the right knee swelling was presented in all animals for producing OA model, the rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. After Ozone injection, many rabbits in O_3IACI-20 group and O_3IACI-40 group behaved as usual and some appeared to be more active than before the Ozone treatment.
3.2 Histomorphology study
3.2.1 Gross inspection
Part examined: cartilage at the medial side of the tibia platform
Normal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer.
Model group: the cartilage was grey yellow or grey white and dullness. Fissures on cartilage surface were found. A few foci of small and shallow erosion were noted. There was no obvious osteophyma.
O_3IACI-20 group: the cartilage was grey yellow, rough and dullness. More fissures on the superficial cartilage were noted. Superficial erosions were obvious and multiple. There was no obvious osteophyma.
O_3IACI-40 group: the cartilage was rough. Much more fissures and erosions were seen in compared with O_3IACI-2O group.
3.2.2 Microscopic examination
Normal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The chondroid matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.
Model group: the surface of the cartilage was irregularly rough with tiny shallow cracks. The chondrocytes was decreased in cellularity and disarrayed in arrangement. PAS and Toluidine blue stains showed mild decrease in staining intensity in the chondroid matrix when compared to that of normal group.
O_3IACI-20 group: Articular surface was damaged with presence of empty lacunas due to disappearing of the chondrocytes. The layer of the cartilage decreased in thickness with apparent fissures. The tide mark was unclear or duplicated. The PAS and Toluidine blue stains were less intensive than that of the model group.
Dosage-40 group: The articular cartilage damage was represented by the thinning of the cartilage layer, decreased cellularity of the chondrocytes, cellular disarray, clustering of hyperplastic chondrocyte and lack of normal zonal appearance. Compared to Dosage-20 group, fissures were increased and were more apparent. PAS and Toluidine blue staining intensity was further diminished in the chondroid matrix.
3.2.3 Mankin Scores evaluation
In modified Mankin scoring system, the structure of the cartilage, cell appearance, staining of the cartilage matrix, tidemark was evaluated. Total score arranged from 0 to above. Normal cartilage was scored as 0. A low score indicated mild changes in articular cartilage while a high one indicated severe degeneration.
The normal group was scored 0.0±0.0 while the model group was 4.5±0.5 which was significantly higher than that of the normal group (P<0.01). The scores of the two O_3IACI groups together were 6.0±0.8-8.1±1.2, they were significantly higher than that of the model group (P<0.01). When compared the two O3IACI groups, the score of the O_3IACI-40 group (8.1±1.2) was significantly higher than that of the O_3IACI-2O group (6.0±0.8). (P<0.01). The results of the Mankin score evaluation indicated that multiple injections of Ozone into the articular space could further induce damage in the cartilage tissues of osteoarthritis, and the degree of the Ozone induced cartilage damage appeared to be dosage dependent.
3.3 laboratories Analysis
Serum NO level: In comparison with the level of the normal group (91.88±26.94μmol/L), the NO level of the model group (146.75±16.43μmol/L) or each of the two O_3IACI groups (O3IACI20, 156.01±24.53μmol/L; O3IACI40, 166.23±22.01μmol/L) was significantly higher, respectively, (P<0.01). However, there was no significant difference among the two of O_3IACI groups and the model group (p>0.05)
Serum SOD level: In comparison with the SOD level of normal group (246.18±34.65 U/ml), the level of the model group (330.78±46.68 U/ml) or each of the two O3IACI groups (374.96±44.02 U/ml, 411.59±30.00 U/ml) was significantly higher, respectively(P<0.01); the SOD level of the two O_3IACI groups were higher than that of the model group, the differences were statistically significant, P<0.05. However, the difference between the O3IACI-20 group (374.96±44.02 U/ml) and the O3IACI-40 group (411.59±30.00 U/ml) was not statistically significant(P> 0.05).
Synovia NO level: In comparison with the NO level of the normal group (31.43±5.06μmol/L), the NO level of the model group (111.37±35.92umol/L) or each of the two O_3IACI groups (108.28±20.45umol/L, 105.16±38.18umol/L) was significantly higher, respectively (P<0.01); Differences between the two O_3IACI groups and between the O3IACI groups and the model group were not significant statistically; (P>0.05).
Synovia SOD level: The difference between SOD level of the model group (86.02±48.47 U/ml) and that of the normal group (63.51±39.81 U/ml ) was not significant, P>0.05; the level of either of the two of O3IACI groups (119.05±34.96 U/ml, 140.14±42.76 U/ml) were higher than that of the normal group, respectively; (P <0.05). The SOD levels of the O_3IACI-40 group (140.14±42.76 U/ml) was significantly higher than that of the model group (P<0.05); but there was no statistically significant difference between the O_3IACI-20 group (119.05±34.96 U/ml) and the model group (P> 0.05), and between the two 03IACI groups, (P> 0.05), respectively.
4. Conclusions
Ozone, when introduced into the articular cavity, could cause increased secretion of SOD. Up-regulation in synthesis of antioxidant enzymes, such as SOD, is known to be able to lead to block chronic oxidative stress reaction which may be one of the mechanisms of Ozone therapeutic effect in OA. However, intra-articular injection of Ozone did not reduce NO levels of both serum and synovial fluid. Regardless of these, we have documented that repeated injections of Ozone to joint cavity have an adverse deleterious effect on OA-like articular cartilage. Our findings raise a serous concern regarding the safety of intra-articular injection of Ozone in treating OA. The mechanism of its deleterious effect on OA-like cartilage is not clear and further investigation is needed. Physicians should be aware of this potential adverse effect on OA patients with a conservative approach to withhold the use medical Ozone as one of the treatment choices for OA until its therapeutic benefit can be approved against its adverse effect in further animal study and clinical trial.
Part III. Experimental Study of Collagenase II produced OA models treated with Medical Ozone by intra articular injection and Autohemotherapy
1. Purposes
This study was designed to study the variation of serum cytokines before and after introduction of Ozone of different dosages in two ways, intra-articular injection and autohemotherapy, to better understand the biologic mechanism of Ozone therapy on OA, and to further confirm the pathologic effect of Ozone on OA articular cartilage.
2. Materials and Methods
OA animal models were produced by intra-articular injection of Collagenase II solution. Nitric oxide (NO), Superoxide dismutase (SOD), and two most important cytokines in OA development, TNF-αand IL-1β, and the histopathology of the articular cartilage were analyzed.
2.1 animal grouping and model establishment
36 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, OA model group with O_3 Intra- articular cavity Injection (O3IACI) and OA model group with ozonized blood Autohemotherapy (O_3AHT). The O31 ACI treated OA model group and the O_3AHT treated OA model group were further sub-divided in to two groups separately according to the Ozone dosage used in the experiment: O_3IACI-10 group (Ozone dosage: 10μg/ml per injection), O_3IACI-30 group (Ozone dosage:30μg/ml per injection), O_3AHT-10 group (Ozone dosage: 10μg/ml per injection), O_3AHT-30 group (Ozone dosage:30μg/ml per injection).6 rabbits were in each group. OA model was produced by injecting 0.5ml of collagenase II solution into the knee joint space twice three days apart.
2.2 animal processing
4 weeks after the last injection of Collagenase II solution, the rabbits of both O_3IACI-10 group and O_3IACI-30 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks.
Otherwise, the rabbits of both O_3AHT-10 group and O_3AHT-30 group were treated with autohemotherapy. During the autohemotherapy procedure, 4ml venous blood was taken form the vein in the ear of the rabbit and thoroughly mixed with 4ml Ozone of different dosage accordingly by a Y tube unit, and re-infused into the vein, twice a week for 4 weeks.
The normal and model groups were fed routinely. Blood of all groups were collected and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone treatment.
2.3 Animal behaviors observation
2.4 Imaging studies:
Knee images of rabbits of each group were studied by DR as well as MRI
2.5 Serum analysis
NO,T-SOD ,TNF -αand IL-1βwere detected. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry, and both TNF-αand 1L-1βby ELISA.
2.6 Histopathology study of the cartilaginous tissue
The articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.
2.7 Statistical treatment
SPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. Completely random designed One-way ANOVA was applied to analysis every parameter of interest as homogeneity of variances in multiple group samples means. Welch test was applied to analyze every parameter of interest as Heterogeneity of variance in multiple group samples means. LSD-t and Games-Howe 11 were used for comparison among multiple sample means. A p <0.05 value was considered statistically significant.
3. Results
3.1 Animal behaviors observation
After the first Collagenase II solution injection, inactivity, gait difficulty was observed in all rabbits for producing OA model; variable extent of the right knee was presented. Rabbits did limp apparently, struggle and kick when their right knees were touched. About 6 weeks after the first Collagenase II injection, Osteophyma was palpated at the medial part of the right knee in 50% of the OA models. The rabbits of the Ozone treated groups became active and recovered quicker than those of the model group.
3.2Imaging studies
3.2.1 DR imaging
Normal group: both knee spaces were in concordance without obvious loss, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.
Model group: obvious hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was lost.
O3IACI-10 group: hyperostosis of the right tibial plateau was noted. Its edge was sharp and the sclerotic articular surface was lightly hyperostosis and eburnated. The right knee space was not narrowed. The sub-patella fat pad was clear.
O3IACI-30 group: hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was still normal.
03AHT-10 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was lost.
O3AHT-30 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was a little lost.
3.2.2 MRI studies
Normal group: in 3D_WATSc sequence, the articular cartilage surface of the internal condyle of femur and the medial tibial plateau was smooth and intact without thinning and defect. Signals were normal. In STIRLongTE sequence, the signals of the anterior and posterior angles of the medial meniscus and that of subchondral bone were normal. No articular hydrops was presented.
Model group: in 3D_WATSc sequence, the articular cartilage of the medial tibial plateau was remarkable thinning with a low signal. The thickness of the cartilage in condyles of femur was normal. Its signal was normal. In STIRLongTE sequence, small amount of articular hydrops in the suprapatellar bursa was presented
O3IACI-10 group: in 3D_WATSc sequence, the articular cartilage of the medial condyle of femur was thinning with a low signal. The thickness of the cartilage of the tibial plateau was different and the surface was rough. The posterior angle of the medial meniscus was unclear. In STIRLongTE sequence, Small amount of articular hydrops in the supra & infra patellar bursas were presented. O3IACI-30 group: in 3D_WATSc sequence, the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with signal reduction. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented.
O3AHT-10 group: in 3D_WATSc sequence, the cartilage of the right medial tibial plateau was thinning with signal reduction. The thickness of the cartilage of the right medial condyle of femur was nearly normal with a normal signal. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented.
O3AHT-30 group: 3D_WATSc showed the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with reduction in signal. Small amount of articular hydrops in the supra & infra patellar bursas were presented in STIR_LongTE.
3.3 laboratories Analysis
3.3.1 Serum NO and T-SOD levels
Serum NO level: In comparison with the level of the normal group (79.44±15.31μmol/L), the NO level of the model group (152.23±17.88μmol/L) or each of the two O_3IACI groups (O3IACI-10, 163.20±22.31μmol/L; O3IACI-30, 158.05±36.47μmol/L),and each of the two O3AHT groups (O3AHT-10,183.21±23.12; O3AHT-30,183.95±50.72) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
Serum SOD level: In comparison with the SOD level of normal group (233.84±30.66U/ml), the level of the model group (359.71±66.73 U/ml) or each of the two O_3IACI groups (O3IACI-10, 351.23±46.46 U/ml, O3IACI-30, 375.85±75.96 U/ml) and each of the two O3AHT (O3AHT-10, 397.01±65.81; O3AHT-30, 379.05±37.67) was significantly higher, respectively (P<0.05); However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
3.3.2 Serum TNF-αand IL-1βlevels
Serum TNF-αlevel: In comparison with the level of the normal group (8.50±3.46 pg/Ml), the NO level of the model group (28.37±17.40 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 88.72±72.88 pg/Ml; O3IACI-30, 198.98±310.97 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 56.07±73.83 pg/Ml; O3AHT-30, 39.12±40.74 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
Serum IL-1βlevel: In comparison with the level of the normal group (43.37±18.27 pg/Ml), the NO level of the model group (146.89±98.93 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 416.30±319.45 pg/Ml; O3IACI-30, 691.71±798.24 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 287.24±363.95 pg/Ml; O3AHT-30, 219.72±229.94 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).
3.4 Histomorphology study
3.4.1 Gross inspection
Part examined: cartilage at the medial side of the tibia platform
Normal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer. There was hydrops in the space, neither hyperplasia nor thickening of the synovium.
Model group: the surface of the right condyles was still smooth and intact. The right tibial plateau was dull grey and granulo-changed. Thinning and erosion of the medial articular cartilage were seen. Osteophyma was formed at the medial edge of the right tibial plateau.
O3IACI-10 group: the surface of the right condyles was rough and dull. Its cartilage was thinning; the right lateral and medial tibial plateaus were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
O3IACI-30 group: the synovial membrane of the right knee articular cavity was hyperaemic and thickening. The cartilage of the condyles of femur was dissipated and dull. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
O3AHT-10 group: the cartilage surface of the condyles of the right femur was rough with focal erosions; The lateral and medial plateaus of the right tibial were dark grey yellow and rough with focal erosions and malacia, Osteophyma was found at the internal angle of the right tibia.
O3AHT-30: the synovial membrane of the right knee articular cavity was hyperaemic. The cartilage of the condyles of femur was dissipated with focal erosions. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.
3.4.2 Microscopic examination
3.4.2.1 Articular cartilage
Normal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The cartilage matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.
Model group: the surface layer of the cartilage was thinning and rough, and villiform in some part. There were a lot of deep fissures extending deeply to the calcified layer from the surface. The tidemark was destroyed. The cartilage layer was peeled away from the subchondral bone. Chondrocyte degenerated and decreased in cellularity; Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix. The cartilage was of fibrosis.
O3IACI-10 group: the surface of the cartilage was rough with many fissures extending deeply to the radial zone from the superficial layer. The chondrocytes in the transitional zone and radial zone were disarrayed in arrangement and decreased in cellularity. Cellular clustering hyperplasia was presented. The tidemark was still intact. Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix and cracked cartilage.
O3IACI-30 group: villiform like changes were found at the superficial cartilage with great amounts of fissures extending to the radial zone from the superficial layer. Chondrocytes were clustering and disarrayed in arrangement. The cartilage layer was exfoliated. Toluidine blue stains showed medium decrease in staining intensity in the chondroid matrix and profuse deep cracks.
O3AHT-10 group: there were many fissures extending deeply to the calcified layer from the superficial layer. Chondrocytes Chondrocyte degenerated and decreased in cellularity; the tidemark was un-complete. The cartilage layer was separated from the subchondral bone. Toluidine blue stains showed decrease in staining intensity in the chondroid matrix, and also cracks and fibrosis.
O3AHI-30 groups: a lot of fissures extending deeply to the radial zone from the superficial layer of the cartilage. Matrix was dissolved in the radial zone as well as calcified layer. The cartilage structure was destroyed and out of order. Chondrocytes were necrotic. The tidemark was un-complete. The cartilage layer was exfoliated. Toluidine blue stains showed severe decrease in staining intensity in the chondroid matrix, and also many cracks and fibrosis
3.4.2.2 Synovium
Normal group: Neither thickening nor hyperplasy was found in the synovium tissue. There was no vascularization and inflammatory cell infiltration
Model group: capillary engorgement in the synovial tissue was found. Inflammatory cells were seen surrounding the vessels
O3IACI-10 group: slight capillary engorgenment in the synovial tissue was found. Small amounts of inf
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