手法治疗实验性膝骨关节炎扫描电镜研究
作者：张昊 杜宁 任峰 张凤华 史炜镔
单位：张昊(上海市第二医科大学附属瑞金医院 上海 200025)；杜宁(上海市第二医科大学附属瑞金医院 上海 200025)；任峰(上海市第二医科大学附属瑞金医院 上海 200025)；张凤华(上海市第二医科大学附属瑞金医院 上海 200025)；史炜镔(上海市第二医科大学附属瑞金医院 上海 200025)
关键词：手法；骨关节炎；电镜；超微结构
中国中医骨伤科杂志000201 摘要 目的：观察手法防治兔膝骨关节炎扫描电镜下超微结构的变 化，评价手法治疗的效果。方法：采用结扎股静脉，臀下静脉方法造成兔骨内高压膝关节骨关节炎模型，术后第二天进行手法治疗，每日3次。同时设立正常组和模型组 。分别于术后8、12、16、20周取材。扫描电镜观察胫骨内髁关节软骨超微结构的变化。结 果：实验组骨关节炎改变较对照组明显减轻。结论：手法确能有效地防治骨关节炎。
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中图分类号：R247.4；R681.8 文献标识码：A
文章编号：1005- 0205(2000)02-0001-03
Electronmicroscopic Study of Mamipulation on Experimental Model of Osteoarthritis
Zhang Hao Du Ning Ren Feng et al
Shanghai Ruijing Hospital Affiliated to Shanghai Second Medical Univers ity 200025
ABSTRACT Objective:To evaluate the effect of manipulation on experimental model of Osteoarthritis (OA).Methods:27 a dult New Zealand rabbits were involved as high intraosseous pressure experiment model by ligating the vena femoralis and gluteae inferiores.Then the model OA ke ens were treated with manipulation every day postoperatively.At the same time,th e model control group and the normal control group were set.The animals were sac rificed at 8,12,16,20 weeks after the operation.The medial tibia condyle was exc ised and processed for scan electronmicroscopic study.Results:There was a significant delay and lessening of the degenerative changes of a rticular cartilage in experimental group.Conclusion:The fi ndings of this experiment indicated that manipulation can alleviate the degenera tion of cartilage and it should be applied as an effective method of treating OA .
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KEY WORDS Manipulation Osteoarthritis Electronmicro scope
骨关节炎(osteoarthritis,OA)是以关节疼痛、活动障碍为特点 的慢性退行性疾病。近年来，随着人口老龄化趋势，其发病率也明显升高[1]。目前国内外治疗OA的方法和药物有很多，但缺乏理想的疗效。笔者在临床上运用手法治疗膝 骨关节炎取得了满意的效果[2]。前期的研究表明手法能改善膝骨关节炎局部的血 液循环，降低骨内压和关节内压，降低全身的血粘度。为了进一步从形态上了解手法的作用 ，本文采用扫描电镜观察手法作用后关节软骨表面的变化，现报道如下。
材料与方法
1 实验动物 新西兰兔，雌性，均为6月龄，体重2.5～3.5kg。
分组 将27只兔随机分为3组，分别为治疗组12只，模型组12只，正常组3只。
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3 造模 原理，利用手术造成下肢血流动力学异常形成兔膝骨关节炎[3] 。方法，3%戊巴比妥钠(30mg/kg)兔耳缘静脉内麻醉。仰卧位，于腹股沟处作1cm纵行切口， 分离股静脉，紧靠腹股沟韧带处作双重结扎，并于结扎线间切断。于右臀部髋关节后外侧坐 骨结节附近做小切口，分离出髂内静脉属支臀下静脉，同样双重结扎，结扎线间切断。缝合 皮下各层及皮肤。
治疗
1 治疗组 造模后第2天开始点揉髌骨上、下、左、右，膝内、外侧间隙 ，每处50次。屈曲关节20次，顺、逆抱推后肢10次。上述手法每周3次，由专人操作。
2 模型组 造模后不用手法治疗。
3 正常组 未造模。
观察方法
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治疗组与病理组分别于8、12、16、20周处死取材。正常组于20周处死取材。兔处死后取内 侧平台大小约0.5×0.3×0.2cm3骨块。标本先用5%苏打水冲洗，除去附着于关节表面的 关节液、碎屑等。4°C下，置于2%的戊二醛固定液中浸泡24小时，在0.1M二甲砷酸钠缓冲 液中漂洗，再以1%四氧化锇固定2小时。后以0.1M二甲砷酸钠缓冲液漂洗，乙醇逐级脱水， 醋酸异戊酯保存过夜，二氧化碳临界点干燥，定向黏附于载物台上，真空喷金后，扫描电镜 下观察、摄片。
结果
扫描电镜观察(SEM)
1 正常组 电镜下见软骨表面光滑，似覆盖一层无定形物质。其下胶原纤 维结合成束，具有典型的拱形结构。其垄部和沟部相互平行，波峰间距为1.62～5.82μm不 等(见图1)。
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图1 正常组电镜下所见
2 治疗组 8周时，扫描电镜下见，无定形物部分消失，代之以纤维素样 物质，或呈皮疹样改变。表面有规则小凹陷，垄沟状排列尚隐约可见(见图2)。12周时， 垄沟排列消失，有细小浅表裂缝出现。表面胶原纤维索增粗，部分翻起(见图3)。16周时见 软骨表面表面裂隙增宽、加深，部分胶原纤维裸露。部分软骨基质丢失(见图4)。20周时表 面裂隙更大，缝隙中有清晰可见的胶原纤维，裸露明显，排列散乱(见图5)。
图2 治疗组8周时电镜下所见
图3 治疗组12周时电镜下所见
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图4 治疗组16周时电镜下所见
图5 治疗组20周时电镜下所见
3 模型组 8周时见垄形结构消失，表面粗糙，无定形基质消失 。软骨表面有不规则小裂隙或小凹陷，类似治疗组12周时表现。12周时见表面裂隙明显增宽 增多，部分胶原纤维暴露，裂隙周围表面不平整，类似治疗组16周时表现。16周时见裂隙严 重增宽，其中胶原纤维裸露，松散，有断裂，粗细不均，类似治疗组20周时表现。20周时见 有部分区域软骨缺损明显，大量胶原纤维裸露，排列散乱(见图6)。
图6 模型组20周时电镜下所见
讨论
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正常的关节软骨由软骨基质和软骨细胞构成。软骨基质使软骨具有一定的外形和弹性以抵抗 外力作用。而软骨细胞既负责清除坏死退变的基质，又不断合成新的基质，保持关节软骨合 成和降解的平衡。维持其正常功能。而关节内微环境包括生物力学及营养代谢等因素的改变 则可引起关节软骨的退变[4]。本实验主要是观察膝骨关节炎骨内高压模型下，关 节软骨的病理改变，同时分析手法治疗对这种退变进程的干预作用。
扫描电镜结果显示：治疗组造模后8周软骨表面粗糙，出现早期退变表现。12～16周时软骨 表面出现裂缝，并逐渐增大，出现退变的典型表现。至20周时，已有胶原纤维暴露，呈退变 的后期表现。而模型组8周时表面正常形态已完全消失，表面有不规则的小裂隙和凹陷出现 ，提示此时已有退变中期表现。以后退变逐渐加剧，至最后20周时，已有软骨的区域缺损， 大量胶原纤维暴露，排列散乱，表现裂隙增多增大。已是退变末期的显微结构征象了 。由此可以看出，手法组在各个时间点上，关节退变的程度都较对照组轻，在一定程度上延 缓、减轻了软骨的退变。
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对于被动运动和手法防治关节损伤，近年有了新的认识，Salter等经过长期研究证实早期持 续被动运动确可促进软骨的再生和修复[5]，认为关节的反复屈伸运动可刺激软骨 组织中未分化的间质细胞向软骨细胞转化，加快软骨组织的修复[6]。杜氏等前期 的研究表明其治疗机制可能是，手法的作用和关节被动活动促进滑液向关节软骨的浸透和扩 散，改善组织的营养代谢，有助于改善关节周围的血液循环，降低骨内压，促进关节周围组 织的自身修复[3，7]。本实验从扫描电镜的观察中，客观的证实了手法能减轻软 骨的退变，为上述理论又提供了事实依据。当然，实验显示，手法并未完全阻止关节软骨退 变的进程，这可能由于实验运动兔膝关节较小，手法不象在人身上容易操作，同时手法的实 施还有待于在方法和技术上进一步提高。
基金项目：国家自然科学基金资助 编号：39770920
作者简介：张昊(1970-)，男(汉族)，海南人，主治医师。
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参考文献
[1]Lawrence RC,Hochberg MC,Kelsey JL,et al.Estimates of the prevalen ce of selected arthritic and musculoskeletal diseases in the United States.J Ret heumatol,1989,16:427～441
[2]杜宁，施荣庭.手法治疗原发性膝骨关节炎骨关节病37例疗效观察.中医正骨，1994 ，(6)：9
[3]王济纬.手法治疗实验性膝关节骨关节炎的研究.上海第二医科大学学报，1996，16( 6)：417
[4]Howell DS,Muller F,Manicourt DH,et al.A mini review:proteoglycan aggregat e profiles in the Pond-Nnuuki dog model of osteoarthritis and in canine disuse a trophy.Br J Rheumatol,1992,31(Suppl):7
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[5]Salter RB.History of rest and motion and the scientific basic for early c ontinuous passive motion.Hand Clinics,1996,12(1):1～11
[6]Salter RB.The biologic concept of continous passive motion of synorial jo intss the first 18 year of basic research.Clin Orhtop,1989,242:12
[7]Salter RB.The biological effect of continuous passive motion on the heali ng of full-thickness defects in articalar cartilage.J Bone Joint Surg,1980,62A:1 232
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收稿日期：1999-07-15
Electronmicroscopic Study of Mamipulation on Experimental Oste oarthritis
Zhang Hao Du Ning Ren Feng Zhang Fenghua Shi Weibing
Shanghai Ruijun Hospital Affiliated to Shanghai Second Medical Univer sity (Shanghai 200025)
ABSTRACT Objective:To evaluate the effect of manipulation on experimental model of Osteoarthritis (OA).Methods:27 a dult New Zealand rabbits were involved as high intraosseous pressure experiment model by ligating the vena femoralis and gluteae inferiores.Then the model OA ke ens were treated with manipulation every day postoperatively.At the same time,th e model control group and the normal control group were set.The animals were sac rificed at 8,12,16,20 weeks after the operation.The medial tibia condyle was exc ised and processed for scan electronmicroscopic study.Results:There was a significant delay and lessening of the degenerative changes of a rticular cartilage in experimental group.Conclusion:The fi ndings of this experiment indicated that manipulation can alleviate the degenera tion of cartilage and it should be applied as an effective method of treating OA .
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KEY WORDS Manipulation Osteoarthritis Electronmicro scope
Osteoarthritis (OA), a kind of involutinal disease,is char acterized by pain in joint and difficulties in moving.The incidence rate has be en remarkably increased in recent years with the growing number of the elderly [1].At present,there are many ways and medicines to cure the disease both at home and abroad.But the curative effect is still not ideal.We came up with a satisfactory result by using the method of manipulation to treat OA of knee [2].Our earlier research shows that manipulation can improve the local bloo d circulation of knee joints,lower internal pressure of bone and joint,and reduc e the blood viscosity of the whole body.In order to investigate the manipulation morphologically,scanning electron microscope was applied to examine the microst ructureal change of the artricular cartilage surface of the joint.
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Material and Method
1 Animal:New Zealand rabbits,female,six-month-old,2.5～3.5kg w eight.
2 Grouping:27 rabbits were randomized into
This study was supported by a grant from National Natural Science Foundat ion of China (No.39770920)
three groups,12 under manipulation treatment,12 for model control,3 for normal control.
3 Establishment of model:The abnormal hemodynamics of lower extr e mity was caused surgically which lead to OA[3].Briefly,anesthesia was performed with 3% soluble pentobarbitone,30mg per kg,keep the rabbit in supine,p erform a 1cm longitudinal incision at the groin, separate the femoral vein,make a diploid ligature close to groin ligament,perform bisection between ligature.Ma ke a small nick close to the ischial tuberosity,and the same procedure was repea ted on the inferior gluteal veins.
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4 Treatment
4.1 Group under treatment:Two day after the building of model.Begin to rub the whirlbone up,down left,right,and two space of the knee 50 times respecti vely.And practise flexion of knee for 20 times,and turning the animal's hindlimb for 10 time.Pracitise the manipulation three times a week.
4.2 Model group:without the treatment of manipulation.
4.3 Normal group:intact,normal diet.
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5 Method of examining:Those under manipulaion and those in model group were kil led at following 8,12,16,20 weeks to harvest the materials.Those of normal group were killed after 20 weeks for material harvest. A0.5/0.3/0.2cm3 size osteoc hondral tissue was harvest at the medial platform.Wash the specimen with 5% soda lolution and get rid of the joint fluid and debris attached to the surface of t h joint.Dip it in the 2% glutaraldehyde fixation solution for 24 hours at 4°C,r inse it with the 0.1M? buffer,then fix it with 1% Osmium tetroxide for two hours .Rinse the specimen with 0.1M? buffer,dehydrate it with ethyl alclhol,keep it in the isoamyl acetate for a night,then dry it with carbonei dioxydum at the criti cal point,stick to the carrier,spray glod in the condition of vaccum and conduct investigation under the scanning electron microscope.
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Result
SEM observation
1 Normal group:The cartilage surface was smooth and glossy.It seem ed to be covered by a layer of amorphous substance.The collagen fibers joined in bundle with a tipical vaulted shape.Its apse ran parallel to the sulcus.The dis tance between the wave crest ranged from 1.62 to 5.82um.(picture 1)
2 Treatment Group:After 8 weeks cellulose-like substance or exant hem replaced part of the amorphous substance.Some irregular pits and looming occ urred on the cartilage surface.12 weeks later,vaulted-shape substance disappear ed and superficial cracks appeared.The thickness of the collagen fibril on the s ufface increased,and part of fibrils was turning up (picture 3).16 weeks later,t he cracks on surface of cartilage became wider and deeper,and part of the collag en exposed.Part of the cartilage matrix disappeared.(picture 4) 20 weeks later,t he cracks became much more wider inside,the collagen fibril can be seen clearly in irregular forms (picture 5).
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3 Model group:8 weeks later,vaulted-shape substance disappeared ,the surface was rough and amorphous substance almost disappeared.There were irr egular small split of hollow on surface of the cartilage (picture 6).12 weeks la ter,more spits appeared and they became wider,some collagen fibril exposed and a reas around the crack was not quite smooth (picture 7).16 weeks later,the cracks became dramatically wider in which the exposed collagen fibril were loose and i n different thickness and some were broken (picture 8).20 weeks later,In some ar ea of the cartilage were destroyed,a large number of collagen fibril exposed in a state of disorder (picture 9).
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Discussion
The normal joint cartilage consists of cartilage matrix and cartiage cells.The c artilage matrix enables the cartilage to have certain shape and elasticity to wo rk against the outside force.The cartilage cells can not only clean away the bad matrix but also help the synthesis of new matrix.This is able to keep the synth esis of the joint cartilage and the balance of degradation and maintain its func tion[4].The experiment is designed to examine the microstructural chang e of the joint cartilage under high intraosseous pressure experiment model and i n the main time to analyze the interfering role that manipulation plays in preve nting the degenerative process.
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SEM results show that the cartilage surface of the lst group became rough after the 8 weeks of building of model and the early degenerative stage appeare.Betw een 12 to 16 weeks,splits turned out on surface of the cartilage and became wide r and typical degeneration signs appeared.During the 20th week,there were collag en fibers left exposed and show the mid-stage degenerative signs.
In recent year,people have achievied further understanding of usingthe passive m otion and manipulation to prevent and treat the joint problems.After a long time research some experts including Salter believe that the continous passive motio n in the early stage is able to promote the growth and regeneration ofthe cartil age[5].Because the repeat flexion of joints can stimulate the transfoma tion from the non-differentiation interstitial cells to cartilage cells so as t o quichen the regeneration of the cartilage hemopoietic tissue[6].The ea rlier research by DU et al demonstrated the possible treatment mechanism was tha t the manipulation and passive motion worked by promoting the soaking and diffus ion of the synovial fluid to joint cartilage,improving the nutrition and metabol i sm,helping improve the blood circulating around joints,reducing pressure inside bone,and promoting the self reinstoration of the surrounding tissue[3，7 ].The research to a certain extent supported the notion that manipulation cou ld be applied to prevent and treat the degeneration of joint cartilage.The exper iment confirmed the theory and demonstrated a fact.Of course,the experiment show e d that it couldn't stop the degeneration process.The possibl reason was that rab bit's joint was too small to practice manipulation on.And the practice of manipu lation needs improvement on its practical skill.
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Reference literature
1 Lawrence RC,Hochberg MC,Kelsey JL,et al.Estimates of the prevalence o f selected arthritic and musculoskeletal dieases in the United States.J Retheumat ol,1989,16:427～441
2 杜宁，施荣庭.手法治疗原发性膝骨关节骨关节病37例疗效观察.中医正骨，1994，(6)： 9
3 王济伟.手法治疗实验性膝关节骨关节炎的研究.上海第二医科大学学报，1996，16(6)： 417
4 Howell DS,Muller F,Manicourt DH.A mini review:proteoglycan aggregate profiles in the Pond-Nnuuki dog model of osteoarthritis and canine disuse atrophy.Br J Rheumatol.1992,31(Suppl):7
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5 Salter RB.History of rest and motion and the scientific basic for early conti nuous passive motion.Hand Clinics.1996,12(1):1～11
6 Salter RB.The biologic concept of continous passive motion of synorial joints the first 18 year of basic research.Clin Orthop,1989,242:12
7 Salter RB,et al.The biological effect of continuous passive motion on the hea ling of full-thickness defects in articalar cartilage.J Bone Joint Surg,1980,62 A:1232, 百拇医药
单位：张昊(上海市第二医科大学附属瑞金医院 上海 200025)；杜宁(上海市第二医科大学附属瑞金医院 上海 200025)；任峰(上海市第二医科大学附属瑞金医院 上海 200025)；张凤华(上海市第二医科大学附属瑞金医院 上海 200025)；史炜镔(上海市第二医科大学附属瑞金医院 上海 200025)
关键词：手法；骨关节炎；电镜；超微结构
中国中医骨伤科杂志000201 摘要 目的：观察手法防治兔膝骨关节炎扫描电镜下超微结构的变 化，评价手法治疗的效果。方法：采用结扎股静脉，臀下静脉方法造成兔骨内高压膝关节骨关节炎模型，术后第二天进行手法治疗，每日3次。同时设立正常组和模型组 。分别于术后8、12、16、20周取材。扫描电镜观察胫骨内髁关节软骨超微结构的变化。结 果：实验组骨关节炎改变较对照组明显减轻。结论：手法确能有效地防治骨关节炎。
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中图分类号：R247.4；R681.8 文献标识码：A
文章编号：1005- 0205(2000)02-0001-03
Electronmicroscopic Study of Mamipulation on Experimental Model of Osteoarthritis
Zhang Hao Du Ning Ren Feng et al
Shanghai Ruijing Hospital Affiliated to Shanghai Second Medical Univers ity 200025
ABSTRACT Objective:To evaluate the effect of manipulation on experimental model of Osteoarthritis (OA).Methods:27 a dult New Zealand rabbits were involved as high intraosseous pressure experiment model by ligating the vena femoralis and gluteae inferiores.Then the model OA ke ens were treated with manipulation every day postoperatively.At the same time,th e model control group and the normal control group were set.The animals were sac rificed at 8,12,16,20 weeks after the operation.The medial tibia condyle was exc ised and processed for scan electronmicroscopic study.Results:There was a significant delay and lessening of the degenerative changes of a rticular cartilage in experimental group.Conclusion:The fi ndings of this experiment indicated that manipulation can alleviate the degenera tion of cartilage and it should be applied as an effective method of treating OA .
, 百拇医药
KEY WORDS Manipulation Osteoarthritis Electronmicro scope
骨关节炎(osteoarthritis,OA)是以关节疼痛、活动障碍为特点 的慢性退行性疾病。近年来，随着人口老龄化趋势，其发病率也明显升高[1]。目前国内外治疗OA的方法和药物有很多，但缺乏理想的疗效。笔者在临床上运用手法治疗膝 骨关节炎取得了满意的效果[2]。前期的研究表明手法能改善膝骨关节炎局部的血 液循环，降低骨内压和关节内压，降低全身的血粘度。为了进一步从形态上了解手法的作用 ，本文采用扫描电镜观察手法作用后关节软骨表面的变化，现报道如下。
材料与方法
1 实验动物 新西兰兔，雌性，均为6月龄，体重2.5～3.5kg。
分组 将27只兔随机分为3组，分别为治疗组12只，模型组12只，正常组3只。
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3 造模 原理，利用手术造成下肢血流动力学异常形成兔膝骨关节炎[3] 。方法，3%戊巴比妥钠(30mg/kg)兔耳缘静脉内麻醉。仰卧位，于腹股沟处作1cm纵行切口， 分离股静脉，紧靠腹股沟韧带处作双重结扎，并于结扎线间切断。于右臀部髋关节后外侧坐 骨结节附近做小切口，分离出髂内静脉属支臀下静脉，同样双重结扎，结扎线间切断。缝合 皮下各层及皮肤。
治疗
1 治疗组 造模后第2天开始点揉髌骨上、下、左、右，膝内、外侧间隙 ，每处50次。屈曲关节20次，顺、逆抱推后肢10次。上述手法每周3次，由专人操作。
2 模型组 造模后不用手法治疗。
3 正常组 未造模。
观察方法
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治疗组与病理组分别于8、12、16、20周处死取材。正常组于20周处死取材。兔处死后取内 侧平台大小约0.5×0.3×0.2cm3骨块。标本先用5%苏打水冲洗，除去附着于关节表面的 关节液、碎屑等。4°C下，置于2%的戊二醛固定液中浸泡24小时，在0.1M二甲砷酸钠缓冲 液中漂洗，再以1%四氧化锇固定2小时。后以0.1M二甲砷酸钠缓冲液漂洗，乙醇逐级脱水， 醋酸异戊酯保存过夜，二氧化碳临界点干燥，定向黏附于载物台上，真空喷金后，扫描电镜 下观察、摄片。
结果
扫描电镜观察(SEM)
1 正常组 电镜下见软骨表面光滑，似覆盖一层无定形物质。其下胶原纤 维结合成束，具有典型的拱形结构。其垄部和沟部相互平行，波峰间距为1.62～5.82μm不 等(见图1)。
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图1 正常组电镜下所见
2 治疗组 8周时，扫描电镜下见，无定形物部分消失，代之以纤维素样 物质，或呈皮疹样改变。表面有规则小凹陷，垄沟状排列尚隐约可见(见图2)。12周时， 垄沟排列消失，有细小浅表裂缝出现。表面胶原纤维索增粗，部分翻起(见图3)。16周时见 软骨表面表面裂隙增宽、加深，部分胶原纤维裸露。部分软骨基质丢失(见图4)。20周时表 面裂隙更大，缝隙中有清晰可见的胶原纤维，裸露明显，排列散乱(见图5)。
图2 治疗组8周时电镜下所见
图3 治疗组12周时电镜下所见
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图4 治疗组16周时电镜下所见
图5 治疗组20周时电镜下所见
3 模型组 8周时见垄形结构消失，表面粗糙，无定形基质消失 。软骨表面有不规则小裂隙或小凹陷，类似治疗组12周时表现。12周时见表面裂隙明显增宽 增多，部分胶原纤维暴露，裂隙周围表面不平整，类似治疗组16周时表现。16周时见裂隙严 重增宽，其中胶原纤维裸露，松散，有断裂，粗细不均，类似治疗组20周时表现。20周时见 有部分区域软骨缺损明显，大量胶原纤维裸露，排列散乱(见图6)。
图6 模型组20周时电镜下所见
讨论
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正常的关节软骨由软骨基质和软骨细胞构成。软骨基质使软骨具有一定的外形和弹性以抵抗 外力作用。而软骨细胞既负责清除坏死退变的基质，又不断合成新的基质，保持关节软骨合 成和降解的平衡。维持其正常功能。而关节内微环境包括生物力学及营养代谢等因素的改变 则可引起关节软骨的退变[4]。本实验主要是观察膝骨关节炎骨内高压模型下，关 节软骨的病理改变，同时分析手法治疗对这种退变进程的干预作用。
扫描电镜结果显示：治疗组造模后8周软骨表面粗糙，出现早期退变表现。12～16周时软骨 表面出现裂缝，并逐渐增大，出现退变的典型表现。至20周时，已有胶原纤维暴露，呈退变 的后期表现。而模型组8周时表面正常形态已完全消失，表面有不规则的小裂隙和凹陷出现 ，提示此时已有退变中期表现。以后退变逐渐加剧，至最后20周时，已有软骨的区域缺损， 大量胶原纤维暴露，排列散乱，表现裂隙增多增大。已是退变末期的显微结构征象了 。由此可以看出，手法组在各个时间点上，关节退变的程度都较对照组轻，在一定程度上延 缓、减轻了软骨的退变。
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对于被动运动和手法防治关节损伤，近年有了新的认识，Salter等经过长期研究证实早期持 续被动运动确可促进软骨的再生和修复[5]，认为关节的反复屈伸运动可刺激软骨 组织中未分化的间质细胞向软骨细胞转化，加快软骨组织的修复[6]。杜氏等前期 的研究表明其治疗机制可能是，手法的作用和关节被动活动促进滑液向关节软骨的浸透和扩 散，改善组织的营养代谢，有助于改善关节周围的血液循环，降低骨内压，促进关节周围组 织的自身修复[3，7]。本实验从扫描电镜的观察中，客观的证实了手法能减轻软 骨的退变，为上述理论又提供了事实依据。当然，实验显示，手法并未完全阻止关节软骨退 变的进程，这可能由于实验运动兔膝关节较小，手法不象在人身上容易操作，同时手法的实 施还有待于在方法和技术上进一步提高。
基金项目：国家自然科学基金资助 编号：39770920
作者简介：张昊(1970-)，男(汉族)，海南人，主治医师。
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参考文献
[1]Lawrence RC,Hochberg MC,Kelsey JL,et al.Estimates of the prevalen ce of selected arthritic and musculoskeletal diseases in the United States.J Ret heumatol,1989,16:427～441
[2]杜宁，施荣庭.手法治疗原发性膝骨关节炎骨关节病37例疗效观察.中医正骨，1994 ，(6)：9
[3]王济纬.手法治疗实验性膝关节骨关节炎的研究.上海第二医科大学学报，1996，16( 6)：417
[4]Howell DS,Muller F,Manicourt DH,et al.A mini review:proteoglycan aggregat e profiles in the Pond-Nnuuki dog model of osteoarthritis and in canine disuse a trophy.Br J Rheumatol,1992,31(Suppl):7
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[5]Salter RB.History of rest and motion and the scientific basic for early c ontinuous passive motion.Hand Clinics,1996,12(1):1～11
[6]Salter RB.The biologic concept of continous passive motion of synorial jo intss the first 18 year of basic research.Clin Orhtop,1989,242:12
[7]Salter RB.The biological effect of continuous passive motion on the heali ng of full-thickness defects in articalar cartilage.J Bone Joint Surg,1980,62A:1 232
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收稿日期：1999-07-15
Electronmicroscopic Study of Mamipulation on Experimental Oste oarthritis
Zhang Hao Du Ning Ren Feng Zhang Fenghua Shi Weibing
Shanghai Ruijun Hospital Affiliated to Shanghai Second Medical Univer sity (Shanghai 200025)
ABSTRACT Objective:To evaluate the effect of manipulation on experimental model of Osteoarthritis (OA).Methods:27 a dult New Zealand rabbits were involved as high intraosseous pressure experiment model by ligating the vena femoralis and gluteae inferiores.Then the model OA ke ens were treated with manipulation every day postoperatively.At the same time,th e model control group and the normal control group were set.The animals were sac rificed at 8,12,16,20 weeks after the operation.The medial tibia condyle was exc ised and processed for scan electronmicroscopic study.Results:There was a significant delay and lessening of the degenerative changes of a rticular cartilage in experimental group.Conclusion:The fi ndings of this experiment indicated that manipulation can alleviate the degenera tion of cartilage and it should be applied as an effective method of treating OA .
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KEY WORDS Manipulation Osteoarthritis Electronmicro scope
Osteoarthritis (OA), a kind of involutinal disease,is char acterized by pain in joint and difficulties in moving.The incidence rate has be en remarkably increased in recent years with the growing number of the elderly [1].At present,there are many ways and medicines to cure the disease both at home and abroad.But the curative effect is still not ideal.We came up with a satisfactory result by using the method of manipulation to treat OA of knee [2].Our earlier research shows that manipulation can improve the local bloo d circulation of knee joints,lower internal pressure of bone and joint,and reduc e the blood viscosity of the whole body.In order to investigate the manipulation morphologically,scanning electron microscope was applied to examine the microst ructureal change of the artricular cartilage surface of the joint.
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Material and Method
1 Animal:New Zealand rabbits,female,six-month-old,2.5～3.5kg w eight.
2 Grouping:27 rabbits were randomized into
This study was supported by a grant from National Natural Science Foundat ion of China (No.39770920)
three groups,12 under manipulation treatment,12 for model control,3 for normal control.
3 Establishment of model:The abnormal hemodynamics of lower extr e mity was caused surgically which lead to OA[3].Briefly,anesthesia was performed with 3% soluble pentobarbitone,30mg per kg,keep the rabbit in supine,p erform a 1cm longitudinal incision at the groin, separate the femoral vein,make a diploid ligature close to groin ligament,perform bisection between ligature.Ma ke a small nick close to the ischial tuberosity,and the same procedure was repea ted on the inferior gluteal veins.
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4 Treatment
4.1 Group under treatment:Two day after the building of model.Begin to rub the whirlbone up,down left,right,and two space of the knee 50 times respecti vely.And practise flexion of knee for 20 times,and turning the animal's hindlimb for 10 time.Pracitise the manipulation three times a week.
4.2 Model group:without the treatment of manipulation.
4.3 Normal group:intact,normal diet.
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5 Method of examining:Those under manipulaion and those in model group were kil led at following 8,12,16,20 weeks to harvest the materials.Those of normal group were killed after 20 weeks for material harvest. A0.5/0.3/0.2cm3 size osteoc hondral tissue was harvest at the medial platform.Wash the specimen with 5% soda lolution and get rid of the joint fluid and debris attached to the surface of t h joint.Dip it in the 2% glutaraldehyde fixation solution for 24 hours at 4°C,r inse it with the 0.1M? buffer,then fix it with 1% Osmium tetroxide for two hours .Rinse the specimen with 0.1M? buffer,dehydrate it with ethyl alclhol,keep it in the isoamyl acetate for a night,then dry it with carbonei dioxydum at the criti cal point,stick to the carrier,spray glod in the condition of vaccum and conduct investigation under the scanning electron microscope.
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Result
SEM observation
1 Normal group:The cartilage surface was smooth and glossy.It seem ed to be covered by a layer of amorphous substance.The collagen fibers joined in bundle with a tipical vaulted shape.Its apse ran parallel to the sulcus.The dis tance between the wave crest ranged from 1.62 to 5.82um.(picture 1)
2 Treatment Group:After 8 weeks cellulose-like substance or exant hem replaced part of the amorphous substance.Some irregular pits and looming occ urred on the cartilage surface.12 weeks later,vaulted-shape substance disappear ed and superficial cracks appeared.The thickness of the collagen fibril on the s ufface increased,and part of fibrils was turning up (picture 3).16 weeks later,t he cracks on surface of cartilage became wider and deeper,and part of the collag en exposed.Part of the cartilage matrix disappeared.(picture 4) 20 weeks later,t he cracks became much more wider inside,the collagen fibril can be seen clearly in irregular forms (picture 5).
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3 Model group:8 weeks later,vaulted-shape substance disappeared ,the surface was rough and amorphous substance almost disappeared.There were irr egular small split of hollow on surface of the cartilage (picture 6).12 weeks la ter,more spits appeared and they became wider,some collagen fibril exposed and a reas around the crack was not quite smooth (picture 7).16 weeks later,the cracks became dramatically wider in which the exposed collagen fibril were loose and i n different thickness and some were broken (picture 8).20 weeks later,In some ar ea of the cartilage were destroyed,a large number of collagen fibril exposed in a state of disorder (picture 9).
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Discussion
The normal joint cartilage consists of cartilage matrix and cartiage cells.The c artilage matrix enables the cartilage to have certain shape and elasticity to wo rk against the outside force.The cartilage cells can not only clean away the bad matrix but also help the synthesis of new matrix.This is able to keep the synth esis of the joint cartilage and the balance of degradation and maintain its func tion[4].The experiment is designed to examine the microstructural chang e of the joint cartilage under high intraosseous pressure experiment model and i n the main time to analyze the interfering role that manipulation plays in preve nting the degenerative process.
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SEM results show that the cartilage surface of the lst group became rough after the 8 weeks of building of model and the early degenerative stage appeare.Betw een 12 to 16 weeks,splits turned out on surface of the cartilage and became wide r and typical degeneration signs appeared.During the 20th week,there were collag en fibers left exposed and show the mid-stage degenerative signs.
In recent year,people have achievied further understanding of usingthe passive m otion and manipulation to prevent and treat the joint problems.After a long time research some experts including Salter believe that the continous passive motio n in the early stage is able to promote the growth and regeneration ofthe cartil age[5].Because the repeat flexion of joints can stimulate the transfoma tion from the non-differentiation interstitial cells to cartilage cells so as t o quichen the regeneration of the cartilage hemopoietic tissue[6].The ea rlier research by DU et al demonstrated the possible treatment mechanism was tha t the manipulation and passive motion worked by promoting the soaking and diffus ion of the synovial fluid to joint cartilage,improving the nutrition and metabol i sm,helping improve the blood circulating around joints,reducing pressure inside bone,and promoting the self reinstoration of the surrounding tissue[3，7 ].The research to a certain extent supported the notion that manipulation cou ld be applied to prevent and treat the degeneration of joint cartilage.The exper iment confirmed the theory and demonstrated a fact.Of course,the experiment show e d that it couldn't stop the degeneration process.The possibl reason was that rab bit's joint was too small to practice manipulation on.And the practice of manipu lation needs improvement on its practical skill.
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Reference literature
1 Lawrence RC,Hochberg MC,Kelsey JL,et al.Estimates of the prevalence o f selected arthritic and musculoskeletal dieases in the United States.J Retheumat ol,1989,16:427～441
2 杜宁，施荣庭.手法治疗原发性膝骨关节骨关节病37例疗效观察.中医正骨，1994，(6)： 9
3 王济伟.手法治疗实验性膝关节骨关节炎的研究.上海第二医科大学学报，1996，16(6)： 417
4 Howell DS,Muller F,Manicourt DH.A mini review:proteoglycan aggregate profiles in the Pond-Nnuuki dog model of osteoarthritis and canine disuse atrophy.Br J Rheumatol.1992,31(Suppl):7
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5 Salter RB.History of rest and motion and the scientific basic for early conti nuous passive motion.Hand Clinics.1996,12(1):1～11
6 Salter RB.The biologic concept of continous passive motion of synorial joints the first 18 year of basic research.Clin Orthop,1989,242:12
7 Salter RB,et al.The biological effect of continuous passive motion on the hea ling of full-thickness defects in articalar cartilage.J Bone Joint Surg,1980,62 A:1232, 百拇医药