跑台运动对大鼠膝关节软骨全层缺损修复重塑影响的实验研究
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摘要
关节软骨是构成滑膜关节的重要组成部分,其主要功能是传导分布运动载荷、维持和承受接触应力以及顺利完成关节功能活动等。各种骨关节创伤和骨肿瘤等疾病均可导致软骨缺损。由于软骨组织结构特殊,其内无神经、血管、淋巴管,幼年关节软骨可由软骨下通路获得部分营养,而成年关节软骨没有软骨下营养通路,一旦损伤,其修复能力有限,最终导致关节炎,影响关节功能。因而一直以来,关节软骨缺损修复成为国内外医学界高度关注和迫切需要解决的问题。
目前软骨缺损的治疗方法很多,有药物疗法,生物疗法,外科手术方法、组织工程化软骨的合成与替代等。随着生物医学的发展,生物治疗越来越受到重视,软骨细胞移植、细胞因子移植、组织工程化软骨等方法的研究不断深入,目前多限于基础研究,临床研究及应用尚未成熟,其长期疗效尚有待进一步明确。外科手术方法包括微骨折术、自体软骨细胞移植术、同种异体移植、骨膜及软骨膜移植术等,有报道取得了可喜的疗效,但修复的组织远远达不到正常软骨的组织结构,生物力学性能相差甚远,功能也自然严重欠缺,而且,随着时间的推移,这些组织逐渐出现退变,最终导致关节功能丧失。因此,如何改善关节软骨缺损修复的质量越来越受到研究者的关注,成为软骨缺损治疗的一个重要方面。
成年关节软骨没有软骨下营养通路,软骨的营养供给是通过关节活动产生的压力变化,使滑液在关节腔与软骨基质之间进行交流。很多实验证明,制动可导致关节软骨退变,但对其发生机理不但与关节活动有关,还与关节的负重密切相关。有学者提出两种假设：(1)“软骨泵”营养机制不仅需要关节活动,同时还需要一定量的应力,缺乏足够的应力,即使保留关节活动,软骨仍不能有效地获取营养。(2)软骨细胞存在某种应力感受装置,维持软骨细胞的正常生理活动需要一定量的应力刺激。制动后关节内应力异常,除对合面有持续静止应力存在外,其余的软骨表面缺乏应力刺激,持续低应力本身即可导致软骨退变。
目前普遍认为,关节软骨细胞的正常生理功能与其受到的正常应力刺激密切相关,运动可以改善修复软骨的质量。制动和关节负荷加重均导致关节软骨退变。运动可促进全层软骨缺损的修复。软骨细胞对力学刺激敏感,稳定的压力将抑制软骨细胞对基质蛋白的合成,而周期变化压力刺激其合成。所以关节活动对软骨营养传递极其重要。关节活动造成关节软骨受压与减压交替出现,形成所谓“软骨泵”的软骨营养机制。运动可以降低关节内压力,有利于滑液向软骨表面和细胞间质弥散,提供软骨修复的最佳内环境,促进软骨修复。已有学者研究了运动对膝关节软骨的影响,大部分结果表明运动对软骨的修复是有利的,而也有部分研究表明运动并不能促进软骨的修复。我们仔细研读了这些文献,发现他们对于运动的强度、运动介入的时间以及确定运动强度的标准等方面存在较大差异,这也许是导致最终得出不同研究结果的原因所在,而运动的不同强度及不同介入时间对软骨缺损修复的影响尚未有报道。
软骨细胞合成、分泌软骨基质前体物质的正常生理过程一定程度上受到应力大小的调控,软骨细胞在缺乏足够应力刺激时,就会出现合成与分泌功能下降和软骨细胞退变。全层软骨缺损修复的机理为：软骨下骨的出血导致缺损区内血肿形成,同时基底部松质骨内有多种功能的间充质细胞增殖,缺损区为含红细胞、白细胞和未分化细胞的纤维素凝块填充,凝块进一步形成肉芽组织,肉芽组织化生成纤维软骨,后再转化为类透明软骨。全层小软骨缺损的修复分为几个阶段：术后2天即见间充质细胞长入,4周出现纤维软骨,8周出现透明软骨,12周开始退化。目前的研究多集中在被动运动对软骨缺损修复的影响,主动运动对软骨缺损修复的作用尚未见报道。本实验制作大鼠膝关节股骨滑车全层软骨缺损模型,在不同时间、采用不同强度的跑台运动,观察跑台运动对大鼠膝关节全层软骨缺损修复重塑的影响,以期为临床软骨损伤的治疗和康复提供实验依据。
本实验包括三部分,第一部分探讨不同强度的跑台运动对大鼠膝关节软骨全层缺损早期修复的影响,并探讨其修复的机制；第二部分将探讨不同运动介入时间对大鼠膝关节软骨全层缺损修复重塑的影响；第三部分探讨不同强度跑台运动对大鼠膝关节软骨全层缺损修复重塑的影响。
第一部分跑台运动对大鼠膝关节软骨全层缺损早期修复的影响
目的：探讨不同强度运动对大鼠髌股关节软骨全层缺损修复早期的影响；探讨不同强度运动对大鼠髌股关节软骨缺损修复早期血清MMP-3、TIMP-1表达的影响。
材料与方法：
1、造模：6周龄清洁级雄性SD大鼠24只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为lmm的全层软骨缺损模型,随机分为安静组(SED)、低强度运动组(LIR)、中强度运动组(MIR)和高强度运动组(HIR),每组6只。
2、跑台运动干预：造模后一周创口愈合后,低、中、高强度运动组分别进行相应强度的跑台运动。低强度跑台运动为跑台坡度0。,速度8.2m/min;中等强度跑台运动为坡度5。,速度15.2m/min;高强度跑台运动为跑台坡度10。,速度26.7m/min;各运动组每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：实验前即初次跑台运动前24小时采用断尾取血法取血,末次运动后采用心脏取血法取血,低温离心,收集血清于EP管,-80℃保存。运动后以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察：组织切片染色；修复组织O'Driscoll评分；ELISA法测定血液MMP-3、TIMP-1浓度及TIMP-1/MMP-3比值。
5、统计学分析：采用SPSS13.0统计软件进行统计分析,实验数据以均数±标准差表示,组间差异比较采用单因素方差分析,若方差齐,用Bonferroni法进一步两两比较,组内实验前后比较采用配对样本t检验,浓度比值采用Kruskal-Wallis H秩和检验；当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示安静组修复情况最好,细胞及基质较多；O'Driscoll评分显示安静组得分最高,各运动组均低于安静组,差异有统计学意义(F=234.349,P=0.000),高强度运动组得分最低；ELISA结果显示实验前各组之间MMP-3浓度差异无统计学意义(F=0.065,P=0.978),实验前各组之间TIMP-1浓度差异无统计学意义(F=0.580,P=0.635),实验后各组之间血液MMP-3浓度比较差异有统计学意义(F=32.469,P=0.000),HIR组显著高于其他3组,SED组最低；TIMP-1浓度各组间有显著性差异(F=20.195,P=-0.000)；实验后,TIMP-1/MMP-3浓度比值组间差异有统计学意义(χ2=15.380,P=0.002)。
结论：本部分实验证实,对于大鼠膝关节全层软骨缺损,低、中、高强度运动均抑制大鼠髌股关节全厚软骨缺损的早期修复,高强度运动进一步破坏软骨下骨,MMP-3、TIMP-1表达与运动相关,TIMP-1/MMP-3值与软骨修复的程度一致。而不同运动时机及不同运动强度对软骨全层缺损修复的影响值得进一步深入的研究。
第二部分不同跑台运动介入时间对大鼠膝关节软骨全层缺损修复重塑影响的实验研究
目的：观察不同时期跑台运动对软骨缺损修复的影响；为临床软骨损伤后关节负重及功能锻炼时间提供实验依据。
材料与方法：
1、造模：8周龄清洁级雄性SD大鼠40只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为1mm的全层软骨缺损模型,随机分为安静组(SED)、造模后2周运动组(2W)、造模后4周运动组(4W)和造模后8周运动组(8W),每组10只。
2、跑台运动干预：造模后笼内饲养,2W组、4W组、8W组分别在造模后2周、4周、8周进行中等强度跑台运动：坡度5。,速度15.2m/min,每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：造模后10周和14周,每组分别处死5只大鼠。以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察；组织切片染色；修复组织O'Driscoll评分；免疫组织化学法测定修复组织的Ⅱ型胶原含量；qPC测定修复组织Ⅱ型胶原及糖苷多糖含量。
5、统计学分析：实验数据分析采用SPSS13.0统计学软件,对于计量资料采用均数±标准差表示,对于多组之间的比较采用析因设计资料的方差分析,方差齐时整体的比较采用F检验,多重比较采用Bonferroni法,方差不齐时采用Friedman M检验,多重比较采用对应的非参数多样本q检验。当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示4W组修复情况最好；O'Driscoll评分显示4W组得分最高,2W组得分最低,总体比较,各组间有统计差异(F=31.394,P=0.000),不同时间有统计差异(F=22.347,P=0.000)；所有组合的交互效应无显著性差异(F=0.287,P=0.835)；4W组O'Driscoll组织学评分造模后14周与造模后10周有显著性差异(t=-6.500,P=0.003)；免疫组织化学结果显示4W组修复组织Ⅱ型胶原含量最高,2W组含量最低,各组间有统计差异(F=249.063,P=0.000),不同时间有统计差异(F=41.519,P=0.000)；所有组合的交互效应均有显著性差异(F=28.512,P=0.000),2W组显著低于SED组,差异有统计学意义(P=0.000),8W组高于SED组,但差异无统计学意义(P=0.497)。qPCR结果显示4W组修复组织Ⅱ型胶原及糖胺多糖含量最高,2W组含量最低,各组间有统计差异(F=236.601,P=0.000),不同时间有统计差异(F=3.150,P=0.000)；所有组合的交互效应均有显著性差异(F=3.601,P=0.024)。造模后10周4W组显著高于SED组和2W组,差异有统计学意义(P=0.000),造模后14周4W组显著高于SED组和2W组,差异有统计学意义(P=0.000)。
结论：本实验结果表明,在正确的时机进行中等强度的运动能够促进软骨缺损的修复,提高修复组织的质量,缩短修复时间；过早的运动不但不能促进软骨缺损的修复,还会对软骨修复过程造成负面影响；而延迟进行运动则对软骨修复的作用有限。本实验为临床软骨缺损患者的治疗和康复提供了实验依据,但由实验到临床的路还很长,运动影响软骨修复的机制及远期效果需要进一步的深入研究。
第三部分不同强度跑台运动对大鼠全层软骨缺损修复重塑影响的实验研究
目的：探讨不同强度跑台运动对大鼠膝关节软骨全层缺损修复重塑的影响；探讨运动对软骨全层缺损修复重塑影响的作用机制；为临床软骨损伤的运动疗法提供实验依据。
材料与方法：
1、造模：8周龄清洁级雄性SD大鼠40只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为1mm的全层软骨缺损模型,随机分为安静组(SED)、低强度运动组(LIR)、中强度运动组(MIR)和高强度运动组(HIR),每组10只。
2、跑台运动干预：造模后4周,低、中、高强度运动组分别进行相应强度的跑台运动。低强度跑台运动为跑台坡度0。,速度8.2m/min;中等强度跑台运动为坡度5。,速度15.2m/min;高强度跑台运动为跑台坡度10。,速度26.7m/min;各运动组每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：造模后10周和14周,每组分别处死5只大鼠。以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察；组织切片染色；修复组织O'Driscoll评分；免疫组织化学法测定修复组织的Ⅱ型胶原含量；qPCR测定修复组织Ⅱ型胶原及糖苷多糖含量,BMP及GIF含量。
5、统计学分析：实验数据分析采用SPSS13.0统计学软件,对于计量资料采用均数±标准差表示,对于多组之间的比较采用析因设计资料的方差分析,方差齐时整体的比较采用F检验,多重比较采用Bonferroni法,方差不齐时采用Friedman M检验,多重比较采用对应的非参数多样本q检验。当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示MIR组修复情况最好,细胞及基质较多；O'Driscoll评分显示MIR组得分最高,HIR组得分最低,各组间有统计差异(F=214.901,P=0.000),不同时间有统计差异(F=17.695,P=0.000)；所有组合的交互效应均有显著性差异(F=11.041,P=0.000)。进一步进行多重比较显示,造模后10周,MIR组与SED组有显著性差异(P=0.002),造模后14周,MIR组与SED组有显著性差异(P=0.000)。修复软骨组织Igf-1的基因表达,各组间有统计差异(F=546.877,P=0.000),不同时间有统计差异(F=13.287,P=0.000)；所有组合的交互效应均有显著性差异(F=10.769,P=0.000)。修复软骨组织BMP-2的基因表达,各组间有统计差异(F=894.898,P=0.000),不同时间有统计差异(F=12.927,P=0.000)；所有组合的交互效应均有显著性差异(F=12.157,P=0.000)。修复软骨组织糖胺多糖的基因表达统计显示,各组间有统计差异(F=916.688,P=0.000),不同时间有统计差异(F=45.748,P=0.000)；所有组合的交互效应均有显著性差异(F=55.689,P=0.000)。修复软骨组织Ⅱ型胶原的基因表达,各组间有统计差异(F=709.227,P=0.000),不同时间有统计差异(F=20.641,P=0.000)；所有组合的交互效应均有显著性差异(F=27.526,P=0.000)。
结论：本部分实验结果表明,中等轻度跑台运动能促进大鼠膝关节软骨全层缺损的修复重塑,低强度运动对大鼠膝关节软骨全层缺损修复重塑促进作用有限,高强度跑台运动则对大鼠膝关节软骨全层缺损修复具有破坏作用。运动促进关节软骨全层缺损的机制可能为运动使关节内IGF和BMP的含量增多,从而促进了骨髓基质干细胞向关节软骨细胞分化,并促进软骨细胞分泌基质,最终促进了软骨缺损的修复重塑。
Articular cartilage is the important part of the synovial joints, its main function is conducting the distribution of sports load, maintain and withstand the contact stress and the successful completion of joint function activities. Various bone and joint diseases such as cancer and bone trauma can lead to cartilage defects. Articular cartilage has a special structure, it does not contain nerve, blood vessels, lymphatics. Besides, juvenile articular cartilage obtained nutrition by passage of the subchondral, but adult articular cartilage without subchondral nutrition passage, so it has a limited repair capacity, once damaged, it always results in arthritis, what impacts the joint function. Thus the repair of articular cartilage defects has been become highly concerned by the medical professions at home and abroad, and become the urgent problem that needs solve.
There are many kinds of treatment to cartilage defects, such as drug therapy, biological therapy, surgical methods, cartilage tissue engineering and so on. With the development of biomedical, biological therapy attracted more and more attention. The research of chondrocytes transplantation, cytokines transplantation, tissue-engineered cartilage and other methods has been develop quickly, but currently mostly limited to basic research, clinical research and application is not yet mature, and its long-term efficacy remains to be further clarified. Surgical procedures, including microfracture surgery, autologous chondrocyte transplantation, allograft, periosteum and cartilage grafting, have been reported that made gratifying effects, but the repaired tissue is far less than normal cartilage structure, and the biomechanical properties falls far, as well as a serious lack of natural function, and, over time, these organizations began to emerge degeneration, eventually leading to loss of joint function. Therefore, how to improve the quality of articular cartilage defect repair catches more and more attention from researchers.
Adult articular cartilage has no subchondral nutrition pathways, nutrient supply is produced by pressure changes during joint activities which lead to synovial fluid exchange between the joint cavity and the cartilage matrix. Many experiments show that static can lead to degeneration of articular cartilage, but its mechanism occurs not only with joint activities, but also closely related to the weight-bearing of joints. Some scholars have proposed two hypotheses:(1)"cartilage pump" mechanism. The nutritional pathway not only needs joint activities, but also requires a certain amount of stress, lack of adequate stress, even keep joint movement, cartilage is still not effective access to nutrition.(2) there is some cartilage stress sensing means in chondrocytes, to maintain the normal physiological activity of cartilage, chondrocytes need a certain amount of stress stimuli. When joint be fixed, intra-articular stress anomalies, in addition to the occlusal surface static stress sustained presence, the rest of the cartilage surface lacking stress stimuli, sustained low stress itself can lead to cartilage degeneration.
It is generally believed that the normal physiological function of chondrocytes is closely related to normal stress stimulation, exercise can improve the quality of cartilage repair. Static and increased joint loading can leading articular cartilage degeneration. Exercise can promote the repair of full-thickness cartilage defects. Chondrocytes are sensitive to mechanical stimulation, steady pressure can inhibit the synthesis of cartilage matrix proteins, and periodic changes in pressure can stimulate its synthesis. So joints movement is vital to cartilage nutrition supply. Pressure on joints and cartilage caused by alternating pressure to form the so-called "cartilage pump" cartilage nutrition mechanism. Exercise can reduce intra-articular pressure in favor of synovial fluid to the cartilage surface and diffuse interstitial cells provide the best environment to cartilage repair and promoting the repair process. Scholars have studied the effects of exercise on the knee cartilage, most results showed that exercise is beneficial to cartilage repair, but there are also some studies have shown that movement does not promote cartilage repair. We carefully read these documents and found that there are differences among the researches about exercise intensity, exercise timing, exercise intensity criteria and other aspects, which are probably ultimately lead to different findings, the effects of different intensity of exercise and different intervention time for repair of cartilage defects has not yet been reported.
Synthesis of chondrocytes and secretion of cartilage matrix precursor material is regulated to some extent by the normal physiological process, chondrocytes occurs synthesis and secretion of matrix decline in the absence of sufficient stress stimulation. Full-thickness cartilage defect repair mechanisms are:bleeding from subchondral bone to defect region leading to hematoma formation, while the bottom of the base of cancellous bone contains mesenchymal stem cells which have a variety of proliferation functions. The hematoma containing red blood cells, white blood cells and undifferentiated cellulose cells. The clot develops to granulation tissue, then fibrocartilage, hyaline cartilage, finally converted to the cartilage. Small full-thickness cartilage defect repair contains several stages:after two days there are mesenchymal stem cells ingrowth in the defect region, four weeks appeared fibrocartilage in the defect, hyaline cartilage appears at eight weeks, at12weeks the repair tissue began to degenerate. Many studies focus on the effects of passive movement on cartilage defect repair, fewer research the effects of active movement on cartilage repair. Our experiment made a rat knee trochlear full thickness cartilage defect model, different timing and different intensity treadmill exercise was conducted to the rats, in order to observe the effects of exercise on rat knee full thickness cartilage defect repair remodeling, to provide experimental basis for clinical cartilage treatment and rehabilitation.
The experiment consists of three parts, the first part discusses the influence of different intensity treadmill exercise on early repair of rat knee cartilage full-thickness defects, and to explore the mechanisms of repair; the second part will explore the influence of different exercise intervention time on repair of rat knee full-thickness cartilage defect; the third part discusses the impact of different intensity treadmill exercise on repair and remodeling of rat full-thickness articular cartilage defect.
Part One:Effects of treadmill exercise on early repair of cartilage full-thickness defects on rat knee
Purpose:The aim of chaptor one was to investigate the effects of different intensities of exercise on early repair of patellofemoral full-thickness articular cartilage defects of rat, investigate the serum MMP-3, TIMP-1expression.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with lmm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), low-intensity exercise group (LIR), intensity exercise group (MIR) and high-intensity exercise group (HIR).
2. Treadmill exercise intervention:Low, medium and high intensity exercise group were conducted corresponding intensity treadmill exercise respectively one week after the modeling. Low-intensity treadmill exercise is incline0°, speed8.2m/min; moderate-intensity treadmill exercise of slope5°, speed15.2m/min; high-intensity treadmill exercise treadmill slope of10°, speed of26.7m/min; each exercise group exercise daily40min, training Monday to Friday, Saturdays and Sundays with no exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; assay blood MMP-3, TIMP1-concentration and TIMP-1/MMP-3ratio by ELISA.
Results:Macroscopic view and tissue sections staining showed that defects in SED group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in SED group, each exercise group were lower than the SED group, and high-intensity exercise group scored the lowest; ELISA results showed that blood MMP-3, TIMP-1concentration were related to intensity of movement, the higher exercise intensity, the higher the content of MMP-3, while the lower TIMP-1levels, and TIMP-1/MMP-3is consistent with the degree of cartilage repair.
Conclusion:This part of the experiment confirmed that low, medium and high intensity exercise inhibited early repair of patellofemoral full-thickness cartilage defects in rats, high-intensity exercise further damage to the subchondral bone. MMP-3and TIMP-1expression was associated with exercise intensity, and the value of TIMP-1/MMP-3ratio was consistent with the degree of cartilage repair. And further study about the effects of different exercise timings and intensities on the repair of full-thickness cartilage defect was worth.
Part Two:Effect of treadmill exercise timing on repair of full-thickness defects of articular cartilage
Purpose:The aim of chaptor two was to investigate the effects of different of treadmill exercise timing on repair of full-thickness defects of articular cartilage, to provide experimental evidence for the clinical weight-bearing and exercise timing after treatment for cartilage damage.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with1mm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), two weeks after modeling exercise group (2W), four weeks after modeling exercise group (4W) and eight weeks after modeling exercise group (8W).
2. Treadmill exercise intervention:Rats were rearing in cage after modeling.2W group,4W group,8W group were conducted moderate-intensity treadmill exercise after2weeks,4weeks,8weeks after modeling respectively. The moderate-intensity was defined as:the slope of5°, speed15.2m/min, sports40min a day, training from Monday to Friday, Saturdays and Sundays without exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; Immunohistochemical determination of type II collagen content in repair tissue; qPCR determination collagen II and glycoside polysaccharide content in repair tissue.
Results:General concept and staining tissue sections showed that defects in4W group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in4W group, and the lowest score appeared in2W group. Immunohistochemistry showed that the highest collagen type II content of repair tissue was in4W group, the lowest was in2W group. qPCR results showed that the highest GAG and collagen Ⅱ content was in4W group, and the minimum content appeared in2W group, the difference was statistically significant.
Conclusion:The results show that at the right time moderate-intensity exercise can promote cartilage defect repair, improve the quality of repair tissue and shorten the repair time; premature movement will not be able to promote the repair of cartilage defects, but also for cartilage repair negative impact on the process; rather limited role is to delay the movement of cartilage repair. This study provides an experimental basis the clinical treatment of patients with cartilage defects and rehabilitation, but there is a long way to go from experiments to clinical, and the long-term effects of exercise on cartilage repair mechanism needs further study.
Part Three:Experimental study of effect of different intensity treadmill exercise on rat full-thickness cartilage defect repair
Purpose:The aim of chaptor three was to investigate the effects of different intensity of treadmill exercise on repair of full-thickness defects of articular cartilage, to provide experimental evidence for the clinical weight-bearing and exercise timing after treatment for cartilage damage.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with lmm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), low-intensity exercise group (LIR), intensity exercise group (MIR) and high-intensity exercise group (HIR).
2. Treadmill exercise intervention:Low, medium and high intensity exercise group were conducted corresponding intensity treadmill exercise respectively four weeks after the modeling. Low-intensity treadmill exercise is incline0°, speed8.2m/min; moderate-intensity treadmill exercise of slope5°, speed15.2m/min; high-intensity treadmill exercise treadmill slope of10°, speed of26.7m/min; each exercise group exercise daily40min, training Monday to Friday, Saturdays and Sundays with no exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; Immunohistochemical determination of type II collagen content in repair tissue; qPCR determination collagen II, glycoside polysaccharide, BMP and GIF content in repair tissue.
Results:General concept and staining tissue sections showed that defects in MIR group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in MIR group, and the lowest score appeared in HIR group. qPCR results showed that the highest collagen II, glycoside polysaccharide, BMP and GIF content was in MIR group, and the minimum content appeared in HIR group, the difference was statistically significant.
Conclusion:The results of this section indicate that moderate mild treadmill exercise can promote the repair of full-thickness articular cartilage defects in rats, low intensity exercise for full thickness articular cartilage defects in rats has limited role in the promotion of full-thickness articular cartilage repair, and high-intensity treadmill exercise has damaging effects. Mechanisms of full-thickness articular cartilage defect was promoted by movement maybe the joint movement increased the content of IGF and BMP, thus contributing to bone mesenchamal stem cells into cartilage cells, cartilage cells and promotes the secretion of matrix and eventually promoted cartilage defect repair.
目前软骨缺损的治疗方法很多,有药物疗法,生物疗法,外科手术方法、组织工程化软骨的合成与替代等。随着生物医学的发展,生物治疗越来越受到重视,软骨细胞移植、细胞因子移植、组织工程化软骨等方法的研究不断深入,目前多限于基础研究,临床研究及应用尚未成熟,其长期疗效尚有待进一步明确。外科手术方法包括微骨折术、自体软骨细胞移植术、同种异体移植、骨膜及软骨膜移植术等,有报道取得了可喜的疗效,但修复的组织远远达不到正常软骨的组织结构,生物力学性能相差甚远,功能也自然严重欠缺,而且,随着时间的推移,这些组织逐渐出现退变,最终导致关节功能丧失。因此,如何改善关节软骨缺损修复的质量越来越受到研究者的关注,成为软骨缺损治疗的一个重要方面。
成年关节软骨没有软骨下营养通路,软骨的营养供给是通过关节活动产生的压力变化,使滑液在关节腔与软骨基质之间进行交流。很多实验证明,制动可导致关节软骨退变,但对其发生机理不但与关节活动有关,还与关节的负重密切相关。有学者提出两种假设：(1)“软骨泵”营养机制不仅需要关节活动,同时还需要一定量的应力,缺乏足够的应力,即使保留关节活动,软骨仍不能有效地获取营养。(2)软骨细胞存在某种应力感受装置,维持软骨细胞的正常生理活动需要一定量的应力刺激。制动后关节内应力异常,除对合面有持续静止应力存在外,其余的软骨表面缺乏应力刺激,持续低应力本身即可导致软骨退变。
目前普遍认为,关节软骨细胞的正常生理功能与其受到的正常应力刺激密切相关,运动可以改善修复软骨的质量。制动和关节负荷加重均导致关节软骨退变。运动可促进全层软骨缺损的修复。软骨细胞对力学刺激敏感,稳定的压力将抑制软骨细胞对基质蛋白的合成,而周期变化压力刺激其合成。所以关节活动对软骨营养传递极其重要。关节活动造成关节软骨受压与减压交替出现,形成所谓“软骨泵”的软骨营养机制。运动可以降低关节内压力,有利于滑液向软骨表面和细胞间质弥散,提供软骨修复的最佳内环境,促进软骨修复。已有学者研究了运动对膝关节软骨的影响,大部分结果表明运动对软骨的修复是有利的,而也有部分研究表明运动并不能促进软骨的修复。我们仔细研读了这些文献,发现他们对于运动的强度、运动介入的时间以及确定运动强度的标准等方面存在较大差异,这也许是导致最终得出不同研究结果的原因所在,而运动的不同强度及不同介入时间对软骨缺损修复的影响尚未有报道。
软骨细胞合成、分泌软骨基质前体物质的正常生理过程一定程度上受到应力大小的调控,软骨细胞在缺乏足够应力刺激时,就会出现合成与分泌功能下降和软骨细胞退变。全层软骨缺损修复的机理为：软骨下骨的出血导致缺损区内血肿形成,同时基底部松质骨内有多种功能的间充质细胞增殖,缺损区为含红细胞、白细胞和未分化细胞的纤维素凝块填充,凝块进一步形成肉芽组织,肉芽组织化生成纤维软骨,后再转化为类透明软骨。全层小软骨缺损的修复分为几个阶段：术后2天即见间充质细胞长入,4周出现纤维软骨,8周出现透明软骨,12周开始退化。目前的研究多集中在被动运动对软骨缺损修复的影响,主动运动对软骨缺损修复的作用尚未见报道。本实验制作大鼠膝关节股骨滑车全层软骨缺损模型,在不同时间、采用不同强度的跑台运动,观察跑台运动对大鼠膝关节全层软骨缺损修复重塑的影响,以期为临床软骨损伤的治疗和康复提供实验依据。
本实验包括三部分,第一部分探讨不同强度的跑台运动对大鼠膝关节软骨全层缺损早期修复的影响,并探讨其修复的机制；第二部分将探讨不同运动介入时间对大鼠膝关节软骨全层缺损修复重塑的影响；第三部分探讨不同强度跑台运动对大鼠膝关节软骨全层缺损修复重塑的影响。
第一部分跑台运动对大鼠膝关节软骨全层缺损早期修复的影响
目的：探讨不同强度运动对大鼠髌股关节软骨全层缺损修复早期的影响；探讨不同强度运动对大鼠髌股关节软骨缺损修复早期血清MMP-3、TIMP-1表达的影响。
材料与方法：
1、造模：6周龄清洁级雄性SD大鼠24只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为lmm的全层软骨缺损模型,随机分为安静组(SED)、低强度运动组(LIR)、中强度运动组(MIR)和高强度运动组(HIR),每组6只。
2、跑台运动干预：造模后一周创口愈合后,低、中、高强度运动组分别进行相应强度的跑台运动。低强度跑台运动为跑台坡度0。,速度8.2m/min;中等强度跑台运动为坡度5。,速度15.2m/min;高强度跑台运动为跑台坡度10。,速度26.7m/min;各运动组每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：实验前即初次跑台运动前24小时采用断尾取血法取血,末次运动后采用心脏取血法取血,低温离心,收集血清于EP管,-80℃保存。运动后以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察：组织切片染色；修复组织O'Driscoll评分；ELISA法测定血液MMP-3、TIMP-1浓度及TIMP-1/MMP-3比值。
5、统计学分析：采用SPSS13.0统计软件进行统计分析,实验数据以均数±标准差表示,组间差异比较采用单因素方差分析,若方差齐,用Bonferroni法进一步两两比较,组内实验前后比较采用配对样本t检验,浓度比值采用Kruskal-Wallis H秩和检验；当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示安静组修复情况最好,细胞及基质较多；O'Driscoll评分显示安静组得分最高,各运动组均低于安静组,差异有统计学意义(F=234.349,P=0.000),高强度运动组得分最低；ELISA结果显示实验前各组之间MMP-3浓度差异无统计学意义(F=0.065,P=0.978),实验前各组之间TIMP-1浓度差异无统计学意义(F=0.580,P=0.635),实验后各组之间血液MMP-3浓度比较差异有统计学意义(F=32.469,P=0.000),HIR组显著高于其他3组,SED组最低；TIMP-1浓度各组间有显著性差异(F=20.195,P=-0.000)；实验后,TIMP-1/MMP-3浓度比值组间差异有统计学意义(χ2=15.380,P=0.002)。
结论：本部分实验证实,对于大鼠膝关节全层软骨缺损,低、中、高强度运动均抑制大鼠髌股关节全厚软骨缺损的早期修复,高强度运动进一步破坏软骨下骨,MMP-3、TIMP-1表达与运动相关,TIMP-1/MMP-3值与软骨修复的程度一致。而不同运动时机及不同运动强度对软骨全层缺损修复的影响值得进一步深入的研究。
第二部分不同跑台运动介入时间对大鼠膝关节软骨全层缺损修复重塑影响的实验研究
目的：观察不同时期跑台运动对软骨缺损修复的影响；为临床软骨损伤后关节负重及功能锻炼时间提供实验依据。
材料与方法：
1、造模：8周龄清洁级雄性SD大鼠40只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为1mm的全层软骨缺损模型,随机分为安静组(SED)、造模后2周运动组(2W)、造模后4周运动组(4W)和造模后8周运动组(8W),每组10只。
2、跑台运动干预：造模后笼内饲养,2W组、4W组、8W组分别在造模后2周、4周、8周进行中等强度跑台运动：坡度5。,速度15.2m/min,每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：造模后10周和14周,每组分别处死5只大鼠。以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察；组织切片染色；修复组织O'Driscoll评分；免疫组织化学法测定修复组织的Ⅱ型胶原含量；qPC测定修复组织Ⅱ型胶原及糖苷多糖含量。
5、统计学分析：实验数据分析采用SPSS13.0统计学软件,对于计量资料采用均数±标准差表示,对于多组之间的比较采用析因设计资料的方差分析,方差齐时整体的比较采用F检验,多重比较采用Bonferroni法,方差不齐时采用Friedman M检验,多重比较采用对应的非参数多样本q检验。当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示4W组修复情况最好；O'Driscoll评分显示4W组得分最高,2W组得分最低,总体比较,各组间有统计差异(F=31.394,P=0.000),不同时间有统计差异(F=22.347,P=0.000)；所有组合的交互效应无显著性差异(F=0.287,P=0.835)；4W组O'Driscoll组织学评分造模后14周与造模后10周有显著性差异(t=-6.500,P=0.003)；免疫组织化学结果显示4W组修复组织Ⅱ型胶原含量最高,2W组含量最低,各组间有统计差异(F=249.063,P=0.000),不同时间有统计差异(F=41.519,P=0.000)；所有组合的交互效应均有显著性差异(F=28.512,P=0.000),2W组显著低于SED组,差异有统计学意义(P=0.000),8W组高于SED组,但差异无统计学意义(P=0.497)。qPCR结果显示4W组修复组织Ⅱ型胶原及糖胺多糖含量最高,2W组含量最低,各组间有统计差异(F=236.601,P=0.000),不同时间有统计差异(F=3.150,P=0.000)；所有组合的交互效应均有显著性差异(F=3.601,P=0.024)。造模后10周4W组显著高于SED组和2W组,差异有统计学意义(P=0.000),造模后14周4W组显著高于SED组和2W组,差异有统计学意义(P=0.000)。
结论：本实验结果表明,在正确的时机进行中等强度的运动能够促进软骨缺损的修复,提高修复组织的质量,缩短修复时间；过早的运动不但不能促进软骨缺损的修复,还会对软骨修复过程造成负面影响；而延迟进行运动则对软骨修复的作用有限。本实验为临床软骨缺损患者的治疗和康复提供了实验依据,但由实验到临床的路还很长,运动影响软骨修复的机制及远期效果需要进一步的深入研究。
第三部分不同强度跑台运动对大鼠全层软骨缺损修复重塑影响的实验研究
目的：探讨不同强度跑台运动对大鼠膝关节软骨全层缺损修复重塑的影响；探讨运动对软骨全层缺损修复重塑影响的作用机制；为临床软骨损伤的运动疗法提供实验依据。
材料与方法：
1、造模：8周龄清洁级雄性SD大鼠40只,在大鼠膝关节股骨远端髌股关节面距离髁间窝2mm处做一直径为1mm的全层软骨缺损模型,随机分为安静组(SED)、低强度运动组(LIR)、中强度运动组(MIR)和高强度运动组(HIR),每组10只。
2、跑台运动干预：造模后4周,低、中、高强度运动组分别进行相应强度的跑台运动。低强度跑台运动为跑台坡度0。,速度8.2m/min;中等强度跑台运动为坡度5。,速度15.2m/min;高强度跑台运动为跑台坡度10。,速度26.7m/min;各运动组每天运动40min,周一至周五训练,周六、日休息。安静组笼内饲养,自由活动。
3、标本取材：造模后10周和14周,每组分别处死5只大鼠。以软骨缺损区为中心,在股骨中下段冠状位切取股骨远端,将其中一侧做石蜡包埋切片,另一侧膝关节用消毒的手术刀切下软骨缺损区的修复组织,-80℃保存。
4、指标检测：修复组织的大体观察；组织切片染色；修复组织O'Driscoll评分；免疫组织化学法测定修复组织的Ⅱ型胶原含量；qPCR测定修复组织Ⅱ型胶原及糖苷多糖含量,BMP及GIF含量。
5、统计学分析：实验数据分析采用SPSS13.0统计学软件,对于计量资料采用均数±标准差表示,对于多组之间的比较采用析因设计资料的方差分析,方差齐时整体的比较采用F检验,多重比较采用Bonferroni法,方差不齐时采用Friedman M检验,多重比较采用对应的非参数多样本q检验。当P≤0.05时认为差异有统计学意义。
结果：大体观及组织切片染色显示MIR组修复情况最好,细胞及基质较多；O'Driscoll评分显示MIR组得分最高,HIR组得分最低,各组间有统计差异(F=214.901,P=0.000),不同时间有统计差异(F=17.695,P=0.000)；所有组合的交互效应均有显著性差异(F=11.041,P=0.000)。进一步进行多重比较显示,造模后10周,MIR组与SED组有显著性差异(P=0.002),造模后14周,MIR组与SED组有显著性差异(P=0.000)。修复软骨组织Igf-1的基因表达,各组间有统计差异(F=546.877,P=0.000),不同时间有统计差异(F=13.287,P=0.000)；所有组合的交互效应均有显著性差异(F=10.769,P=0.000)。修复软骨组织BMP-2的基因表达,各组间有统计差异(F=894.898,P=0.000),不同时间有统计差异(F=12.927,P=0.000)；所有组合的交互效应均有显著性差异(F=12.157,P=0.000)。修复软骨组织糖胺多糖的基因表达统计显示,各组间有统计差异(F=916.688,P=0.000),不同时间有统计差异(F=45.748,P=0.000)；所有组合的交互效应均有显著性差异(F=55.689,P=0.000)。修复软骨组织Ⅱ型胶原的基因表达,各组间有统计差异(F=709.227,P=0.000),不同时间有统计差异(F=20.641,P=0.000)；所有组合的交互效应均有显著性差异(F=27.526,P=0.000)。
结论：本部分实验结果表明,中等轻度跑台运动能促进大鼠膝关节软骨全层缺损的修复重塑,低强度运动对大鼠膝关节软骨全层缺损修复重塑促进作用有限,高强度跑台运动则对大鼠膝关节软骨全层缺损修复具有破坏作用。运动促进关节软骨全层缺损的机制可能为运动使关节内IGF和BMP的含量增多,从而促进了骨髓基质干细胞向关节软骨细胞分化,并促进软骨细胞分泌基质,最终促进了软骨缺损的修复重塑。
Articular cartilage is the important part of the synovial joints, its main function is conducting the distribution of sports load, maintain and withstand the contact stress and the successful completion of joint function activities. Various bone and joint diseases such as cancer and bone trauma can lead to cartilage defects. Articular cartilage has a special structure, it does not contain nerve, blood vessels, lymphatics. Besides, juvenile articular cartilage obtained nutrition by passage of the subchondral, but adult articular cartilage without subchondral nutrition passage, so it has a limited repair capacity, once damaged, it always results in arthritis, what impacts the joint function. Thus the repair of articular cartilage defects has been become highly concerned by the medical professions at home and abroad, and become the urgent problem that needs solve.
There are many kinds of treatment to cartilage defects, such as drug therapy, biological therapy, surgical methods, cartilage tissue engineering and so on. With the development of biomedical, biological therapy attracted more and more attention. The research of chondrocytes transplantation, cytokines transplantation, tissue-engineered cartilage and other methods has been develop quickly, but currently mostly limited to basic research, clinical research and application is not yet mature, and its long-term efficacy remains to be further clarified. Surgical procedures, including microfracture surgery, autologous chondrocyte transplantation, allograft, periosteum and cartilage grafting, have been reported that made gratifying effects, but the repaired tissue is far less than normal cartilage structure, and the biomechanical properties falls far, as well as a serious lack of natural function, and, over time, these organizations began to emerge degeneration, eventually leading to loss of joint function. Therefore, how to improve the quality of articular cartilage defect repair catches more and more attention from researchers.
Adult articular cartilage has no subchondral nutrition pathways, nutrient supply is produced by pressure changes during joint activities which lead to synovial fluid exchange between the joint cavity and the cartilage matrix. Many experiments show that static can lead to degeneration of articular cartilage, but its mechanism occurs not only with joint activities, but also closely related to the weight-bearing of joints. Some scholars have proposed two hypotheses:(1)"cartilage pump" mechanism. The nutritional pathway not only needs joint activities, but also requires a certain amount of stress, lack of adequate stress, even keep joint movement, cartilage is still not effective access to nutrition.(2) there is some cartilage stress sensing means in chondrocytes, to maintain the normal physiological activity of cartilage, chondrocytes need a certain amount of stress stimuli. When joint be fixed, intra-articular stress anomalies, in addition to the occlusal surface static stress sustained presence, the rest of the cartilage surface lacking stress stimuli, sustained low stress itself can lead to cartilage degeneration.
It is generally believed that the normal physiological function of chondrocytes is closely related to normal stress stimulation, exercise can improve the quality of cartilage repair. Static and increased joint loading can leading articular cartilage degeneration. Exercise can promote the repair of full-thickness cartilage defects. Chondrocytes are sensitive to mechanical stimulation, steady pressure can inhibit the synthesis of cartilage matrix proteins, and periodic changes in pressure can stimulate its synthesis. So joints movement is vital to cartilage nutrition supply. Pressure on joints and cartilage caused by alternating pressure to form the so-called "cartilage pump" cartilage nutrition mechanism. Exercise can reduce intra-articular pressure in favor of synovial fluid to the cartilage surface and diffuse interstitial cells provide the best environment to cartilage repair and promoting the repair process. Scholars have studied the effects of exercise on the knee cartilage, most results showed that exercise is beneficial to cartilage repair, but there are also some studies have shown that movement does not promote cartilage repair. We carefully read these documents and found that there are differences among the researches about exercise intensity, exercise timing, exercise intensity criteria and other aspects, which are probably ultimately lead to different findings, the effects of different intensity of exercise and different intervention time for repair of cartilage defects has not yet been reported.
Synthesis of chondrocytes and secretion of cartilage matrix precursor material is regulated to some extent by the normal physiological process, chondrocytes occurs synthesis and secretion of matrix decline in the absence of sufficient stress stimulation. Full-thickness cartilage defect repair mechanisms are:bleeding from subchondral bone to defect region leading to hematoma formation, while the bottom of the base of cancellous bone contains mesenchymal stem cells which have a variety of proliferation functions. The hematoma containing red blood cells, white blood cells and undifferentiated cellulose cells. The clot develops to granulation tissue, then fibrocartilage, hyaline cartilage, finally converted to the cartilage. Small full-thickness cartilage defect repair contains several stages:after two days there are mesenchymal stem cells ingrowth in the defect region, four weeks appeared fibrocartilage in the defect, hyaline cartilage appears at eight weeks, at12weeks the repair tissue began to degenerate. Many studies focus on the effects of passive movement on cartilage defect repair, fewer research the effects of active movement on cartilage repair. Our experiment made a rat knee trochlear full thickness cartilage defect model, different timing and different intensity treadmill exercise was conducted to the rats, in order to observe the effects of exercise on rat knee full thickness cartilage defect repair remodeling, to provide experimental basis for clinical cartilage treatment and rehabilitation.
The experiment consists of three parts, the first part discusses the influence of different intensity treadmill exercise on early repair of rat knee cartilage full-thickness defects, and to explore the mechanisms of repair; the second part will explore the influence of different exercise intervention time on repair of rat knee full-thickness cartilage defect; the third part discusses the impact of different intensity treadmill exercise on repair and remodeling of rat full-thickness articular cartilage defect.
Part One:Effects of treadmill exercise on early repair of cartilage full-thickness defects on rat knee
Purpose:The aim of chaptor one was to investigate the effects of different intensities of exercise on early repair of patellofemoral full-thickness articular cartilage defects of rat, investigate the serum MMP-3, TIMP-1expression.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with lmm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), low-intensity exercise group (LIR), intensity exercise group (MIR) and high-intensity exercise group (HIR).
2. Treadmill exercise intervention:Low, medium and high intensity exercise group were conducted corresponding intensity treadmill exercise respectively one week after the modeling. Low-intensity treadmill exercise is incline0°, speed8.2m/min; moderate-intensity treadmill exercise of slope5°, speed15.2m/min; high-intensity treadmill exercise treadmill slope of10°, speed of26.7m/min; each exercise group exercise daily40min, training Monday to Friday, Saturdays and Sundays with no exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; assay blood MMP-3, TIMP1-concentration and TIMP-1/MMP-3ratio by ELISA.
Results:Macroscopic view and tissue sections staining showed that defects in SED group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in SED group, each exercise group were lower than the SED group, and high-intensity exercise group scored the lowest; ELISA results showed that blood MMP-3, TIMP-1concentration were related to intensity of movement, the higher exercise intensity, the higher the content of MMP-3, while the lower TIMP-1levels, and TIMP-1/MMP-3is consistent with the degree of cartilage repair.
Conclusion:This part of the experiment confirmed that low, medium and high intensity exercise inhibited early repair of patellofemoral full-thickness cartilage defects in rats, high-intensity exercise further damage to the subchondral bone. MMP-3and TIMP-1expression was associated with exercise intensity, and the value of TIMP-1/MMP-3ratio was consistent with the degree of cartilage repair. And further study about the effects of different exercise timings and intensities on the repair of full-thickness cartilage defect was worth.
Part Two:Effect of treadmill exercise timing on repair of full-thickness defects of articular cartilage
Purpose:The aim of chaptor two was to investigate the effects of different of treadmill exercise timing on repair of full-thickness defects of articular cartilage, to provide experimental evidence for the clinical weight-bearing and exercise timing after treatment for cartilage damage.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with1mm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), two weeks after modeling exercise group (2W), four weeks after modeling exercise group (4W) and eight weeks after modeling exercise group (8W).
2. Treadmill exercise intervention:Rats were rearing in cage after modeling.2W group,4W group,8W group were conducted moderate-intensity treadmill exercise after2weeks,4weeks,8weeks after modeling respectively. The moderate-intensity was defined as:the slope of5°, speed15.2m/min, sports40min a day, training from Monday to Friday, Saturdays and Sundays without exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; Immunohistochemical determination of type II collagen content in repair tissue; qPCR determination collagen II and glycoside polysaccharide content in repair tissue.
Results:General concept and staining tissue sections showed that defects in4W group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in4W group, and the lowest score appeared in2W group. Immunohistochemistry showed that the highest collagen type II content of repair tissue was in4W group, the lowest was in2W group. qPCR results showed that the highest GAG and collagen Ⅱ content was in4W group, and the minimum content appeared in2W group, the difference was statistically significant.
Conclusion:The results show that at the right time moderate-intensity exercise can promote cartilage defect repair, improve the quality of repair tissue and shorten the repair time; premature movement will not be able to promote the repair of cartilage defects, but also for cartilage repair negative impact on the process; rather limited role is to delay the movement of cartilage repair. This study provides an experimental basis the clinical treatment of patients with cartilage defects and rehabilitation, but there is a long way to go from experiments to clinical, and the long-term effects of exercise on cartilage repair mechanism needs further study.
Part Three:Experimental study of effect of different intensity treadmill exercise on rat full-thickness cartilage defect repair
Purpose:The aim of chaptor three was to investigate the effects of different intensity of treadmill exercise on repair of full-thickness defects of articular cartilage, to provide experimental evidence for the clinical weight-bearing and exercise timing after treatment for cartilage damage.
Materials and methods:
1. Modeling:A full-thickness cartilage defect model with lmm in diameter was made in the rat knee patellofemoral articular surface of the distal femur, and2mm from the intercondylar fossa. Rats were randomly divided into sedentary group (SED), low-intensity exercise group (LIR), intensity exercise group (MIR) and high-intensity exercise group (HIR).
2. Treadmill exercise intervention:Low, medium and high intensity exercise group were conducted corresponding intensity treadmill exercise respectively four weeks after the modeling. Low-intensity treadmill exercise is incline0°, speed8.2m/min; moderate-intensity treadmill exercise of slope5°, speed15.2m/min; high-intensity treadmill exercise treadmill slope of10°, speed of26.7m/min; each exercise group exercise daily40min, training Monday to Friday, Saturdays and Sundays with no exercise. Rats in sedentary group were rearing in cage, and they can move freely in the cage.
3. Index detection:Generally observed; tissue sections stained; O'Driscoll score; Immunohistochemical determination of type II collagen content in repair tissue; qPCR determination collagen II, glycoside polysaccharide, BMP and GIF content in repair tissue.
Results:General concept and staining tissue sections showed that defects in MIR group repaired best, and the repair tissue contains more matrix; O'Driscoll scores showed the highest score was in MIR group, and the lowest score appeared in HIR group. qPCR results showed that the highest collagen II, glycoside polysaccharide, BMP and GIF content was in MIR group, and the minimum content appeared in HIR group, the difference was statistically significant.
Conclusion:The results of this section indicate that moderate mild treadmill exercise can promote the repair of full-thickness articular cartilage defects in rats, low intensity exercise for full thickness articular cartilage defects in rats has limited role in the promotion of full-thickness articular cartilage repair, and high-intensity treadmill exercise has damaging effects. Mechanisms of full-thickness articular cartilage defect was promoted by movement maybe the joint movement increased the content of IGF and BMP, thus contributing to bone mesenchamal stem cells into cartilage cells, cartilage cells and promotes the secretion of matrix and eventually promoted cartilage defect repair.
引文
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