体外冲击波促进肩袖损伤修复的实验研究
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摘要
第一部分体外冲击波促进大鼠冈上肌止点重建后腱-骨愈合的组织学观察
目的观察体外冲击波(ESW)治疗大鼠冈上肌腱止点重建后腱-骨愈合的影响。
方法健康SD大鼠(365.6±20.5克)35只,随机分为2组:冲击波干预组(16只)、模型组(16只),其余3只备用(以备手术失败、感染、意外死亡使用)。干预组、模型组建立大鼠冈上肌损伤后止点重建动物模型。其中,干预组术后第2天始给予连续一周、一次/天的冲击波治疗,冲击波能量密度流为0.16mJ/mm2,单次冲击量为500次(5Hz,2kPa);模型组不给予冲击波治疗。两组分别于术后1周、2周、4周、8周各取4只大鼠标本进行组织切片HE染色,观察大鼠冈上肌腱骨结合部组织病理学改变情况。
结果组织切片HE染色示:冈上肌止点重建术后1、2周标本示:腱-骨间组织结构连接较为松散,以富含炎细胞、毛细血管的肉芽组织填充;成纤维细胞、胶原蛋白含量少。术后4周炎细胞数目较2周时减少,见较多细胞核呈长梭形的成纤维细胞、类圆形的软骨细胞增生,胶原含量增多,胶原纤维排列较前规则,干预组标本胶原纤维排列更为规则,炎细胞数目减少幅度较大。术后8周腱骨界面由致密结缔组织连接,胶原纤维大量合成,呈垂直纵向规则排列,在干预组中,可见部分区域出现胶原纤维-纤维软骨组织-骨组织的移行带改变,形成类似韧带直接止点样结构。总体上,干预组腱骨界面愈合过程较模型组愈合快(约2-4周)。
结论1、SD大鼠是建立动物肩袖损伤模型的理想选择。2、体外冲击波对SD大鼠冈上肌止点重建模型的腱-骨早期愈合具有明显的促进作用。
第二部分体外冲击波促进大鼠冈上肌止点重建后腱骨愈合的生物学机制
目的探讨ESW促进SD大鼠冈上肌止点重建后腱骨愈合的生物学机制。
方法健康SD大鼠(383.6±22.75克)70只,随机分为2组:冲击波干预组(32只)、模型组(32只),其余6只备用。冲击波干预方法同第一部分。两组分别于术后1周、2周、4周、8周各取8只大鼠标本,其中的一半(4只)大鼠采用实时PCR技术检测,另外一半大鼠采用免疫组织化学技术检测,观察体外冲击波对SD大鼠冈上肌止点重建后血管内皮生长因子(VEGF)、血管性血友病因子(vWF)、骨钙素(OC,BGP)及Ⅱ型胶原蛋白(Collagen-Ⅱ)表达的影响。
结果实时PCR:模型组VEGF的表达于术后1周明显增高,至2周时达到最高点,随后表达逐渐减少,至术后4周时降至1周时水平,至8周时降至损伤前水平。干预组表达趋势同模型组。术后1、2、4周,干预组VEGF上升速度和峰值都明显高于模型组;vWF于术后2周开始表达逐渐增高,至4周时达到最高点,随后表达逐渐减少,至8周时降至2周时水平。干预组表达趋势同模型组,术后4、8周,干预组上升速度和峰值都明显高于模型组;Collagen-Ⅱ于术后2周时的表达明显增高,至4周时达到最高点,至8周时有所下降,仍维持在较高的表达水平。在术后2、4、8周,干预组Collagen-Ⅱ上升速度和峰值都明显高于模型组;骨钙素OC(BGP)从术后2周开始表达增多,至4周时增速较快,至8周时仍保持增高趋势。术后4、8周,干预组OC(BGP)上升速度和峰值都明显高于模型组,差别有统计学意义。免疫组织化学:各检测指标均随着时间的推移表达量逐步增加。vWF、胶原蛋白-Ⅱ、骨钙素均于术后4、8周时表达明显增多,与模型组相比,差别有统计学意义。
结论体外冲击波可通过调节SD大鼠冈上肌止点重建后腱骨结合部血管内皮生长因子、血管性假血友病因子、骨钙素及Ⅱ型胶原蛋白表达,增强腱骨结合部血管形成、软骨组织的再生及成骨作用,促进大鼠冈上肌止点重建后腱骨结合部的早期愈合。
Part one Histological analysis of extracorporeal shock wave promoting the bone-to-tendon healing after the rat supraspinatus tendon insertion reconstruction
Objective To observe the effects of extracorporeal shock waves therapy (ESWT) on tendon-bone healing after the rat supraspinatus tendon reconstruction.
Method 35 SD rats (365.6±20.5g) were randomly classified into 2 groups: ESW group (n=16), model group (n=16) and the other 3(reserved for use in case of death and wound infection). The ESW group and model group were used to establish the rat supraspinatus tendon reconstruction models. The ESW group was given extracorporeal shock wave therapy on the 2nd day after the operation, with a frequency of once a day for 7 days on end. The energy flux density was 0.16 mJ/mm2 and the impact of volume 500 (5Hz, 2kPa)) and the model group was not given ESWT. 4 animals from ESW group and the other 4 from model group were sacrificed at 1、2、4 and 8 weeks after operation for general and histological examinations. The specimens were stained with hematoxylin-eosin to investigate pathological change of the tendon-bone healing tissue.
Result The histological examination revealed that at 1 and 2 weeks after operation, within the model group and ESW group, tendon-bone interface was observed to be comparatively loose and filled with granulation cells、inflammatory cells、fibroblasts and angiogenesis etc, and the content of chondrocytes and collagen fibers was obviously not enough. At 4 weeks after reconstruction, the number of inflammatory cells decreased. Instead, more fibroblasts and chondrocytes, the content of collagen fibers were increased and the fiber arrangement more regular than before. The ESW group, the fiber arranged more regular and the number of inflammatory cells reduced rapidly. At 8 weeks after operation, the healing tissue at the bone-tendon interface had developed into dense connective tissue and the collagen fibers were formed in abundance and the perpendicular collagen fibers were regularly and longitudinally arranged. As for the ESW group, a change of these issues occurred in the transition zones of collagen fibers, fibrocartilage and bone (a structure like a direct ligamentous insertion). On the whole, the histological observations showed that the tissue healing at tendon-bone interface of the ESW group were quicker than those of the control group(about 2-4weeks).
Conclusion 1.SD rat is the ideal choice for establishing animal model of rotator cuff injury. 2. The treatment of ESW can significantly enhance the tendon-bone healing in the early stage after reconstruction of the rat supraspinatus tendon.
The mechanism of extracorporeal shock wave promoting the bone-to-tendon healing after the rat supraspinatus tendon insertion reconstruction
Objective To investigate the mechanism of tendon-bone healing of SD Rats supraspinatus muscle that ESWT promates after the rat supraspinatus tendon reconstruction.
Method 70 SD rats (383.6±22.75g) were randomly classified into 2 groups: ESW group (n=32), model group (n=32) and the other 6 (spared). The ESW group and model group were used to establish the rat supraspinatus tendon reconstruction models. A half of the 8 specimens was used to evaluate VEGF、vWF、OC(BGP) and Collagen-ⅡmRNA expression of the tendon-bone healing tissue by real-time PCR method; another half of the 8 specimens was used to investigate vWF、OC(BGP) and Collagen-Ⅱprotein expression of the tendon-bone healing tissue by immunohistochemisty..
Result Real time PCR data showed that the expression levels of VEGF in the model group went up at 1 week after the operation, and reached to the highest level at 2 weeks after the operation, then gradually dropped after 8 weeks, it recovered to the pre-injury level. The expression levels of VEGF in the ESW group seemed better than that of model group.vWF of ESW group went up rapidly at 2 weeks, and reached its peak at 4 weeks, then dropped gradually to the same level as that was at 2 weeks after the operation. The ESW group had similar results as the model group, the expression levels of vWF in the ESW group seemed better than that of model group at 4 and 8 weeks. The expression level of Collagen-Ⅱincreased rapidaly at 2 weeks after the operation and reached to the peak at 4 weeks and continued with a steady growth until 8 weeks, the expression levels of Collagen-Ⅱin the ESW group seemed better than that of model group at 2、4 and 8 weeks. At 4 and 8 weeks, the expression levels of BGP in ESW group were higher than that of model group. There were significant differences between the model and ESW groups in this experiment. Immunohistochemistry results: The detection index expression gradually increased over time. vWF, Collagen-Ⅱand BGP all increased substantially 4 and 8 weeks after surgery and compared with model group, the difference was statistically significant.
Conclusion The treatment of ESW can enhance the tendon-bone healing in the early stage after reconstruction of the rat supraspinatus tendon by regulating the VEGF, vWF, BGP and typeⅡcollagen expression, promoting angiogenesis, cartilage regeneration and bone formation.
目的观察体外冲击波(ESW)治疗大鼠冈上肌腱止点重建后腱-骨愈合的影响。
方法健康SD大鼠(365.6±20.5克)35只,随机分为2组:冲击波干预组(16只)、模型组(16只),其余3只备用(以备手术失败、感染、意外死亡使用)。干预组、模型组建立大鼠冈上肌损伤后止点重建动物模型。其中,干预组术后第2天始给予连续一周、一次/天的冲击波治疗,冲击波能量密度流为0.16mJ/mm2,单次冲击量为500次(5Hz,2kPa);模型组不给予冲击波治疗。两组分别于术后1周、2周、4周、8周各取4只大鼠标本进行组织切片HE染色,观察大鼠冈上肌腱骨结合部组织病理学改变情况。
结果组织切片HE染色示:冈上肌止点重建术后1、2周标本示:腱-骨间组织结构连接较为松散,以富含炎细胞、毛细血管的肉芽组织填充;成纤维细胞、胶原蛋白含量少。术后4周炎细胞数目较2周时减少,见较多细胞核呈长梭形的成纤维细胞、类圆形的软骨细胞增生,胶原含量增多,胶原纤维排列较前规则,干预组标本胶原纤维排列更为规则,炎细胞数目减少幅度较大。术后8周腱骨界面由致密结缔组织连接,胶原纤维大量合成,呈垂直纵向规则排列,在干预组中,可见部分区域出现胶原纤维-纤维软骨组织-骨组织的移行带改变,形成类似韧带直接止点样结构。总体上,干预组腱骨界面愈合过程较模型组愈合快(约2-4周)。
结论1、SD大鼠是建立动物肩袖损伤模型的理想选择。2、体外冲击波对SD大鼠冈上肌止点重建模型的腱-骨早期愈合具有明显的促进作用。
第二部分体外冲击波促进大鼠冈上肌止点重建后腱骨愈合的生物学机制
目的探讨ESW促进SD大鼠冈上肌止点重建后腱骨愈合的生物学机制。
方法健康SD大鼠(383.6±22.75克)70只,随机分为2组:冲击波干预组(32只)、模型组(32只),其余6只备用。冲击波干预方法同第一部分。两组分别于术后1周、2周、4周、8周各取8只大鼠标本,其中的一半(4只)大鼠采用实时PCR技术检测,另外一半大鼠采用免疫组织化学技术检测,观察体外冲击波对SD大鼠冈上肌止点重建后血管内皮生长因子(VEGF)、血管性血友病因子(vWF)、骨钙素(OC,BGP)及Ⅱ型胶原蛋白(Collagen-Ⅱ)表达的影响。
结果实时PCR:模型组VEGF的表达于术后1周明显增高,至2周时达到最高点,随后表达逐渐减少,至术后4周时降至1周时水平,至8周时降至损伤前水平。干预组表达趋势同模型组。术后1、2、4周,干预组VEGF上升速度和峰值都明显高于模型组;vWF于术后2周开始表达逐渐增高,至4周时达到最高点,随后表达逐渐减少,至8周时降至2周时水平。干预组表达趋势同模型组,术后4、8周,干预组上升速度和峰值都明显高于模型组;Collagen-Ⅱ于术后2周时的表达明显增高,至4周时达到最高点,至8周时有所下降,仍维持在较高的表达水平。在术后2、4、8周,干预组Collagen-Ⅱ上升速度和峰值都明显高于模型组;骨钙素OC(BGP)从术后2周开始表达增多,至4周时增速较快,至8周时仍保持增高趋势。术后4、8周,干预组OC(BGP)上升速度和峰值都明显高于模型组,差别有统计学意义。免疫组织化学:各检测指标均随着时间的推移表达量逐步增加。vWF、胶原蛋白-Ⅱ、骨钙素均于术后4、8周时表达明显增多,与模型组相比,差别有统计学意义。
结论体外冲击波可通过调节SD大鼠冈上肌止点重建后腱骨结合部血管内皮生长因子、血管性假血友病因子、骨钙素及Ⅱ型胶原蛋白表达,增强腱骨结合部血管形成、软骨组织的再生及成骨作用,促进大鼠冈上肌止点重建后腱骨结合部的早期愈合。
Part one Histological analysis of extracorporeal shock wave promoting the bone-to-tendon healing after the rat supraspinatus tendon insertion reconstruction
Objective To observe the effects of extracorporeal shock waves therapy (ESWT) on tendon-bone healing after the rat supraspinatus tendon reconstruction.
Method 35 SD rats (365.6±20.5g) were randomly classified into 2 groups: ESW group (n=16), model group (n=16) and the other 3(reserved for use in case of death and wound infection). The ESW group and model group were used to establish the rat supraspinatus tendon reconstruction models. The ESW group was given extracorporeal shock wave therapy on the 2nd day after the operation, with a frequency of once a day for 7 days on end. The energy flux density was 0.16 mJ/mm2 and the impact of volume 500 (5Hz, 2kPa)) and the model group was not given ESWT. 4 animals from ESW group and the other 4 from model group were sacrificed at 1、2、4 and 8 weeks after operation for general and histological examinations. The specimens were stained with hematoxylin-eosin to investigate pathological change of the tendon-bone healing tissue.
Result The histological examination revealed that at 1 and 2 weeks after operation, within the model group and ESW group, tendon-bone interface was observed to be comparatively loose and filled with granulation cells、inflammatory cells、fibroblasts and angiogenesis etc, and the content of chondrocytes and collagen fibers was obviously not enough. At 4 weeks after reconstruction, the number of inflammatory cells decreased. Instead, more fibroblasts and chondrocytes, the content of collagen fibers were increased and the fiber arrangement more regular than before. The ESW group, the fiber arranged more regular and the number of inflammatory cells reduced rapidly. At 8 weeks after operation, the healing tissue at the bone-tendon interface had developed into dense connective tissue and the collagen fibers were formed in abundance and the perpendicular collagen fibers were regularly and longitudinally arranged. As for the ESW group, a change of these issues occurred in the transition zones of collagen fibers, fibrocartilage and bone (a structure like a direct ligamentous insertion). On the whole, the histological observations showed that the tissue healing at tendon-bone interface of the ESW group were quicker than those of the control group(about 2-4weeks).
Conclusion 1.SD rat is the ideal choice for establishing animal model of rotator cuff injury. 2. The treatment of ESW can significantly enhance the tendon-bone healing in the early stage after reconstruction of the rat supraspinatus tendon.
The mechanism of extracorporeal shock wave promoting the bone-to-tendon healing after the rat supraspinatus tendon insertion reconstruction
Objective To investigate the mechanism of tendon-bone healing of SD Rats supraspinatus muscle that ESWT promates after the rat supraspinatus tendon reconstruction.
Method 70 SD rats (383.6±22.75g) were randomly classified into 2 groups: ESW group (n=32), model group (n=32) and the other 6 (spared). The ESW group and model group were used to establish the rat supraspinatus tendon reconstruction models. A half of the 8 specimens was used to evaluate VEGF、vWF、OC(BGP) and Collagen-ⅡmRNA expression of the tendon-bone healing tissue by real-time PCR method; another half of the 8 specimens was used to investigate vWF、OC(BGP) and Collagen-Ⅱprotein expression of the tendon-bone healing tissue by immunohistochemisty..
Result Real time PCR data showed that the expression levels of VEGF in the model group went up at 1 week after the operation, and reached to the highest level at 2 weeks after the operation, then gradually dropped after 8 weeks, it recovered to the pre-injury level. The expression levels of VEGF in the ESW group seemed better than that of model group.vWF of ESW group went up rapidly at 2 weeks, and reached its peak at 4 weeks, then dropped gradually to the same level as that was at 2 weeks after the operation. The ESW group had similar results as the model group, the expression levels of vWF in the ESW group seemed better than that of model group at 4 and 8 weeks. The expression level of Collagen-Ⅱincreased rapidaly at 2 weeks after the operation and reached to the peak at 4 weeks and continued with a steady growth until 8 weeks, the expression levels of Collagen-Ⅱin the ESW group seemed better than that of model group at 2、4 and 8 weeks. At 4 and 8 weeks, the expression levels of BGP in ESW group were higher than that of model group. There were significant differences between the model and ESW groups in this experiment. Immunohistochemistry results: The detection index expression gradually increased over time. vWF, Collagen-Ⅱand BGP all increased substantially 4 and 8 weeks after surgery and compared with model group, the difference was statistically significant.
Conclusion The treatment of ESW can enhance the tendon-bone healing in the early stage after reconstruction of the rat supraspinatus tendon by regulating the VEGF, vWF, BGP and typeⅡcollagen expression, promoting angiogenesis, cartilage regeneration and bone formation.
引文
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