【作者】 付国建；

【导师】 靳安民；

【作者基本信息】 南方医科大学 ， 骨科学， 2010， 硕士

【摘要】 研究背景：随着社会的发展,交通创伤、运动损伤以及过度负重等导致的关节周围各种急慢性损伤越来越多见。据调查发现,慢性肩关节疼痛目前已经成为继慢性头痛、慢性下腰痛之后的第三大疼痛。引起肩关节疼痛的原因是多方面的,但大多数与肩袖疾病有关,肩袖损伤的发病率占肩关节疾患的17%～41%。肩袖撕裂是肩关节疼痛和功能障碍的主要原因,严重影响患者的生活和工作。肩袖损伤最早是由Smith在1834年发现并命名的,但在当时并未引起重视,直到1931年Codman和Akerson指出本病是引起肩疼的一个重要原因,并对其诊断和治疗作了初步研究之后,许多学者开始了有关肩袖疾病的病因,病理生理机制的探讨。肩袖损伤的治疗分为保守治疗和手术治疗。多数研究表明保守治疗只适用于非巨大撕裂的早期患者,而且有部分患者通过非手术治疗症状缓解不明显,甚至导致肩袖损伤加重。越来越多研究表明,手术治疗在肩袖损伤的治疗中占重要地位。手术目的在于修补撕裂的肩袖而重建力偶平衡,清除不稳定的撕裂缘,扩大间隙、去除撞击因素等。但是随着年龄增长,肩袖组织的变性或因累计性损伤,肌腱组织变脆,失去弹性和伸展性,以至在轻微的外力作用下即可造成肩袖挫伤或完全性肌腱断裂,且老年病人肌腱与骨的附着能力下降,影响手术疗效。受伤的肌腱不能恢复原有的结构和力量,在创伤修复的起始阶段,由肉芽组织(主要是Ⅲ型胶原)填充伤口,纤维细胞产生Ⅰ型胶原替代。实验表明肌腱的生物修复是很缓慢的,猴的实验证实,修补术后2个月肌腱强度恢复55%,1年恢复80%。因此,手术修复肩袖损伤后主要存在以下几个方面问题：1.腱-骨界面愈合时间长,需长时间肩关节制动,影响肩关节活动及术后肩关节功能恢复；2.肩袖损伤手术修复后腱-骨界面抗拉强度难以恢复到正常水平；3.腱-骨界面不愈合,或发生再撕裂。为了克服手术治疗肩袖损伤的不足,恢复肌腱-骨界面的正常复合结构对于肩袖损伤的重建至关重要。一些动物和临床人体的组织学研究发现,尽管现有的外科技术对重建肌腱-骨界面复合结构有极大的促进作用,但无论在修复近期还是晚期,肌腱和骨之间仍不能形成成熟的连接复合体,要么是肌腱与骨之间缺乏正常的过渡连接层,要么是肌腱末端纤维粗细不均,排列紊乱,其抗张力强度与正常组织相比仍有较大差距。因此,需要进一步寻找新的治疗方法。从长远角度来说,用生物学方式调节肌腱的自身修复比单纯手术缝合肌腱意义更大。关于兔髌骨-髌腱复合体中骨-骨愈合和肌腱-骨愈合的比较研究显示软骨组织可能成为一种有潜力的腱-骨愈合的植入材料,有利于增强腱-骨连接点的愈合和纤维软骨层的重建。在骨缺损修复实验中已经证实骨形态发生蛋白(BMP)通过诱导间充质细胞分化为成软骨细胞,然后进一步分化为成骨细胞,启动软骨内成骨过程。在这一过程中可以见到未钙化纤维软骨、钙化纤维软骨、骨组织有序排列形成类似腱-骨界面止点区后三层结构。由此,我们设想在腱-骨界面中加入BMP干预措施,有可能促进腱-骨界面的愈合,在关节内生物学环境、肌腱牵张负荷力共同作用下,有可能诱导腱-骨界面形成类似正常腱-骨界面止点的直接愈合。BMP于1965年被Urist发现,是广泛存在于骨基质中的一种酸性糖蛋白。BMP有诱导成骨的生物活性,是目前认为唯一有异位成骨能力的生长因子。最近又发现它有较强的成软骨特性,已广泛应用于骨缺损治疗。在BMP家族中,BMP-2是最重要的生长因子。由于天然的BMP-2在动物的骨骼中含量极低,制备困难、产量少、价格高,因此利用人重组骨形态发生蛋白(rhBMP-2)来替代BMP-2,近年来美国FDA也批准了rhBMP-2作为骨科应用于骨修复的选择,证明rhBMP-2的疗效及安全性等各方面已得到了权威的认可。目前研究发现rhBMP-2局部应用时代谢快、用量大,早期呈爆发式释放,随后浓度很快下降至治疗水平以下,同时rhBMP-2的半衰期短,难以持续发挥作用,因此本实验拟以纤维蛋白胶(FG)作为rhBMP-2的载体,通过FG封闭rhBMP-2形成缓释体,在FG逐渐降解过程中,rhBMP-2逐步释放出来,发挥稳定、持久的成骨诱导作用,可以在一定时间内维持rhBMP-2局部浓度,避免流失,以达到更好、更快的修复效果。研究目的：1.建立急性肩袖损伤的动物模型；2.通过组织形态学观察及生物力学检测的方法来评价rhBMP-2对兔肩袖损伤重建术后腱-骨界面愈合的修复效果；3.为临床肩袖损伤患者提供可靠的治疗方案。材料与方法：1.健康的雄性新西兰大白兔,八月龄,体重2.42±0.4kg,0.6%戊巴比妥钠水溶液5ml/kg取耳缘静脉注射行全身麻醉。行冈上肌腱行急性损伤后重建其在肱骨大结节上的止点,作为兔肩袖损伤愈合模型。2.第一部分：18只八月龄雄性新西兰大白兔,随机分为3组,每组6只,取双侧肩袖,共12侧。实验组在腱-骨界面注射以FG为载体的rhBMP-2;实验对照组仅填充FG；空白对照组术后不给予任何干预。分别于术后2、4、8周,分批随机应用过量戊巴比妥钠处死各组实验动物,取出各组肩袖标本,对标本进行固定、脱钙、石蜡包埋、切片、行苏木素-伊红染色,光镜观察。第二部分：54只八月龄雄性新西兰大白兔,随机分为3组,每组18只,取右侧肩袖,共18侧。实验组在腱-骨界面注射以FG为载体的rhBMP-2;实验对照组仅填充FG；空白对照组术后不给予任何干预。分别于术后2、4、8周,分批随机应用过量戊巴比妥钠处死各组实验动物,取出各组肩袖标本,用双层塑料袋装好,将标本放入-20℃保存,实验前解冻,将标本固定在MTS858生物力学试验机上做拉伸试验,行腱-骨界面最大抗拉强度(N)及刚度(N/mm)检测。结果：第一部分：光镜观察结果分析表明,术后2周实验组腱-骨界面宽度较宽,主要由软骨组织构成。而实验对照组及空白对照组腱-骨界面以肉芽组织为主,腱-骨界面宽度较大,且分布不均匀。术后4周实验组腱-骨界面连接较前紧密,宽度均一,腱-骨间形成类似直接止点的四层结构。而实验对照组及空白对照组腱-骨界面宽度较前有所减小,但宽度仍不均一,可见成骨反应及大量成纤维细胞。术后8周实验组腱-骨界面出现有明显的Sharpey纤维连接,出现类似直接止点的四层结构。而实验对照组及空白对照组腱-骨界面以结缔组织为主,出现部分Sharpey纤维结构及新骨形成。第二部分：生物力学测试显示,术后2、4、8周各组腱-骨界面最大抗拉强度差异均有显著性(P=0.000),术后各个时间点实验组腱-骨界面的最大抗拉强度均显著高于实验对照组及空白对照组,差异有统计学意义(P=0.000)。实验组最大抗拉强度分别是实验对照组的170.76%、176.53%、179.55%。术后2、4、8周各组腱-骨界面刚度差异均有显著性(P=0.000),术后各个时间点实验组腱-骨界面的刚度均显著高于实验对照组及空白对照组,差异有统计学意义(P=0.000)。实验组刚度分别是实验对照组的140.99%、162.90%、209.99%。结论：1.兔肩袖损伤动物模型可以作为研究rhBMP-2对腱-骨界面损伤后愈合影响的实验研究模型。2. rhBMP-2能够在术后早期诱导腱-骨界面形成类似直接止点的特有结构,显著提高腱-骨界面的抗拉载荷强度及其刚度,促进腱-骨界面的愈合。3.本研究表明rhBMP-2对腱-骨界面损伤的愈合可能起到一定的促进作用,在临床有一定的应用价值。更多还原

【Abstract】 Background:With the social development, transport trauma, sports injuries and excessive weight-bearing, the incidence of joints acute and chronic injuries has also increased. According to the survey, chronic shoulder pain, has now become the third-largest pain, following chronic headache and chronic low back pain. Shoulder pain was caused by many reasons, but most with rotator cuff diseases. The incidence of rotator cuff injury in shoulder disorders accounts for 17%～41%. The major cause of shoulder pain and dysfunction is rotator cuff tear, which seriously impacts patient’s life and work. Rotator cuff injury was first discovered and named by Smith in 1834, but there had been no attention until 1931. In 1931, Codman and Akerson pointed out that rotator cuff injury was an important cause of shoulder pain, and made a preliminary research on diagnosis and treatment of this disease. Then, many scholars began to discuss on the etiology and pathophysiology of this disease.There are two ways to treat rotator cuff injuries:nonsurgical management and surgical management. Studies showed that nonsurgical management was only applicable to non-significant tear in the early stage, but symptoms of some patients through non-surgical treatment were not obviously relieved, even aggravated. More and more research showed that surgical treatment accounted for an important position in the treatment of rotator cuff injuries. The aim of surgery is to repair torn rotator cuff and reconstruct couple balance, remove the torn edge of instability, expand space, remove the impact factors.With the growth of one’s age, degeneration of the rotator cuff or cumulative injury, tendons become brittle, losing its flexibility and extensibility, as well as the rotator cuff contusion or complete rupture can be caused by minor external force. Meanwhile, tendon in elderly patients with reduced ability of bone attachment influences the surgery effect. The injured tendon cannot restore the original structure and strength. Granulation tissue (mainly collagen typeⅢ) fills the wound in the initial stage of healing, and then is replaced by typeⅠcollagen which is produced by fibrocyte. Experiments showed that biologic restoration of tendon injury was very slow. Monkey experiments confirmed that tendon strength recovered up to 55% after 2 months of repair and 80% after one year.Therefore, the main post-operative complications of surgical repair of rotator cuff injury are as follows:firstly, because the healing of tendon-to-bone interface should be a long time, shoulder braked lengthy will impact shoulder activities and postoperative functional recovery. Secondly, the tensile strength of tendon-to-bone interface is difficult to restore to normal levels after restoration of rotator cuff injury. Lastly, tendon-to-bone interface is not healing, or re-tear.In order to overcome the shortage of surgical treatment of rotator cuff injury, to restore the histologically normal insertion site of the rotator cuff tendon is essential for the reconstruction of rotator cuff injury. Some animal and clinical human histological study found that, the existing surgical techniques had great role in promoting the reconstruction of tendon-to-bone interface composite structures. But either short-term or long-term in the repair, it is impossible to regenerate a histologically normal insertion site. Either gap formation lacks the normal tendon and bone transition, or the end of tendon fills with uneven and disorganized fibroblastic response lacking any orientation. Compared with normal tissue, tensile strength of gap formation is still very low. Therefore, we have to find new treatments for rotator cuff repair.From the long-term perspective, the use of biological methods of self-regulating tendon suture is more meaningful than surgery repair. Comparative studies of bone-bone healing and bone-tendon healing on the rabbit patella-patellar tendon complex had shown that cartilage could be a potential implant material in bone-to-tendon healing, and played a very important role in the bone-to-tendon healing and fibrous cartilage layer reconstruction. In the bone defect experiments, mesenchymal stem cells can differentiate into cartilage cells, and then further differentiate into bone cells by bone morphogenetic protein (BMP), starting the process of endochondral bone. In this process, we can see the later three histological phases of the regular tendon-to-bone transition:uncalcified fibrocartilage, calcified cartilage, and bone. As a result, we assume that the healing of tendon-to-bone interface can be promoted by adding BMP interventions. In the intra-articular biological environment, and together with tendon stretch, BMP may induce the formation of regular tendon-to-bone transition.BMP was found by Urist in 1965, which is an acidic glycoprotein and widely present in the bone matrix. BMP can induce bone formation. Currently, BMP is considered to be the only growth factor of ectopic bone formation. We found that BMP can induce cartilage formation recently, and had been widely applied in the treatment of bone defects. In the BMP family, BMP-2 is the most important growth factor. As the natural BMP-2 in animal bones is very low, difficulty to produce, low production, and high prices, recombinant human bone morphogenetic protein-2 (rhBMP-2) takes the place of the BMP. Recently the efficacy and safety of rhBMP-2 have been recognized by the authority, because the US FDA has approved rhBMP-2 as the choice to be used in bone repair.The current study found that rhBMP-2 was difficult to play a sustained role in treatment, because rhBMP-2 had a short half-life which was released quickly in early stage, and then quickly dropped below the treatment level. Therefore, this experiment is intended to use fibrin glue (FG) as a carrier of rhBMP-2. The rhBMP-2 slow-release is formed by FG closure, and in the gradual degradation of fibrin glue, rhBMP-2 gradually release, play a stable and lasting role in the bone induction. The concentration of rhBMP-2 can be maintained within a certain period of time, avoiding the loss and achieving better and faster repair effect.Objective:1. To establish the animal model of acute rotator cuff injury.2. To evaluate the effectiveness of rhBMP-2 in the healing of tendon-to-bone interface of rabbit rotator cuff injury after reattachment by histological and biomechanical testing methods.3. To provide a reliable treatment for patients with rotator cuff injury.Methods:l.Male New Zealand rabbits, aged 8 months, and weighting 2. 42±0.4kg. They were anesthetized with a vein injection of 0.6% pentobarbital sodium solution in a dose of 5ml/kg body weight. Supraspinatus tendon was underwent detachment and repair.2.The first part:Eighteen male New Zealand rabbits, underwent bilateral supraspinatus tendon detachment and repair, were randomly divided into 3 groups (n=12):(1) rhBMP-2 group:a mixture of rhBMP-2 and FG was injected into the bone-to-tendon interface; (2) FG control group:only FG was injected;(3) blank control group:untreated after the surgery. The animals were sacrificed in deep general anesthesia with an overdose of Pentobarbital sodium (n=4 per time point and group) at 2,4 and 8 weeks after surgical procedure. The shoulders were harvested and fixed by immediate insertion in a buffered formaldehyde solution, processed, and embedded in paraffin. The specimens were sectioned longitudinally into 5μm sections, stained with hematoxylin-eosin, and examined under microscopy.The second part:Fifty-four male New Zealand rabbits, underwent right supraspinatus tendon detachment and repair. The rabbits were randomly divided into 3 groups (n=18):(1) rhBMP-2 group:rhBMP-2 in a FG carrier was injected into the bone-tendon interface; (2) FG control group:only FG was injected; (3) blank control group:untreated after the surgery. The animals were sacrificed in deep general anesthesia with an overdose of Pentobarbital sodium (n= 6 per time point and group) at 2,4 and 8 weeks after surgical procedure. The shoulders were harvested, wrapped in saline-soaked gauze, and placed in double plastic bags. Fresh shoulders were frozen (-20℃) until biomechanical testing. The specimens were thawed overnight at room temperature before biomechanical testing. The specimen was affixed to an MTS 858 Mini Bionix material testing system to determine the ultimate tensile strength (in Newtons) and stiffness (in Newtons per millimeter).Results:The first part:Histological examination showed that at the 2nd week postoperatively the interface between tendon and bone of rhBMP-2 group was wide and filled with cartilage tissue. the interface of FG control group and blank group, was uneven width and filled with a loose granulation tissue layer. At the 4th week postoperatively the interface of rhBMP-2 group connect closely and uniform, the four layer direct insertion started to show. Compared with the previous, the tendon-to-bone interface width of the FG control group and blank control group was decreased, but the width was still not uniform, bone formation and a large number of fibroblasts started to show. Sharpey-like collagen fiber formation was found in the interface with the formation of four-layer direct insertion in the rhBMP-2 group on the 8th postoperative weeks. In the FG control group and blank group, the tendon-to-bone interface was filled with granulation tissue and part of Sharpey-like collagen fiber formation and the newly generated bone tissue. The second part:Biomechanical analysis displayed that the ultimate tensile strength and stiffness of bone-tendon interface of each group had significant difference at the 2nd,4th,8th week postoperatively (P= 0.000). The ultimate tensile strength and stiffness of bone-to-tendon interface of the rhBMP-2 group are significantly higher than those of the other two groups at any time-points (both P= 0.000). The ultimate tensile strength in the experimental groups was 70.76%, 76.53%,79.55% greater than the control group at the 2nd,4th,8th week respectively. The stiffness in the experimental groups was 40.99%,62.90%,109.99% greater than the control group at the 2nd,4th,8th week respectively.Conclusion:1. The rabbit model of rotator cuff injuries could be a experimental one which was used to study the effet of rhBMP-2 on the healing of tendon-to-bone injuries.2.RhBMP-2 accelerates healing in a rotator cuff injury and also improves the histological and biomechanical properties of the repair tissue so formed. RhBMP-2 can promote the direct insertion formation and enhance the ultimate tensile strength and stiffness in the tendon-to-bone interface in the early postoperative stage.3. This study showed that rhBMP-2 may play a role in promoting the tendon-to-bone injuries, and had a certain value in the clinical application.更多还原