Effects Of RhBMP-2 On The Healing Of Rotator Cuff Tendon-to-bone Injury: A Histological And Biomechanical Study

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.