The Application Of Tissue Engineering Patch In Rabbit Rotator Cuff Repair

Background and ObjectionInjuries in the tendon-bone junctions are common in sports and work places,and affect more than 30 million people annually worldwide.Orthopedic surgeries were the main treatment for rotator cuff injuries.Unfortunately,rotator cuff tendon-to-bone healing is characterized by forming a scar tissue interface after repair,and the healed interface has a very high rate of re-tearing.Failure of rotator cuff tendon healing after repair happened in more than 70%of patients.Because of the high rate of surgical failure,it is needed to develop novel therapeutic approaches to improve the rotator cuff tendon-bone repair.In the present study,we fabricated a chemical extracted allogeneic tendon scaffold and seeded with BMP-12 overexpressing rabbit bone marrow mesenchymal stem cells(BMSCS)on the scaffolds.In vitro studies were performed to determine the proliferation and differentiation characteristics of the BMP-12 overexpressing BMSCs seeded on the chemical extracted allogeneic tendon scaffolds.In addition,the scaffolds in combination with BMP-12-overexpressing BMSCs were applied in a rabbit rotator cuff repair model.MethodsIn vitro experiment:We fabricated the allogeneic tendon scaffolds by chemical extraction and tested the optimal number of chemical extraction treatments.Rabbit BMSCs were isolated and cultured,and identified by the flow cytometry technique.The cell transfection with adenovirus vectors(control)or adenovirus vectors containing BMP-12 gene(Ad-BMP-12)was performed,and then the transfected BMSCs were planted on the chemical extracted allogeneic tendon scaffolds to observe the adhesion of the cells on the chemical extracted allogeneic tendon scaffolds.The cytotoxicity of the chemical extracted allogeneic tendon scaffolds and the proliferation activity of the cells planted on the scaffolds was detected.Then,the mRNA expression levels of BMP-12,SCX,Tnmd,Tnc,collagen Ⅰ and collagen Ⅲ in seeded cells were detected by real-time quantitative PCR,and the protein expression levels of BMP-12,SCX,Tnmd,Tnc,collagen Ⅰ and collagen Ⅲ were detected by Western blot.In vivo experiment:Rabbit rotator cuff injury repair model was prepared with New Zealand rabbits,and the animal models were divided into 2 groups on average with 9 animals in each group.BMSCs transfected with BMP-12 were planted on chemical extracted allogeneic tendon scaffolds used in the experimental group,while BMSCs without BMP-12 transfected were planted on chemical extracted allogeneic tendon scaffolds used in the control group.The animals were sacrificed at 4,8 and 12 weeks after surgery,and the shoulder joints of the experimental animals were separated for biomechanical examination.Then,the rotator cuff tissues were separated,and the mRNA expressions of collagen Ⅰ and Ⅲ in tendon samples were stained histologically,and the tendon maturity was scored according to the staining results.ResultsIn vitro experiments:The chemical extracted allogeneic tendon scaffold has smooth surface,milky long strip,and certain elasticity and toughness.Ultrastructure observation showed that some connective tissues were found between the tendon bands,which were sparsely arranged and the fibers were arranged in parallel bundles.After the twice treatment of tendon by chemical extraction,not only the immunogenicity was significantly decreased,but also the excellent biomechanical properties and original tendon fibers were not significantly changed.Most of the rabbit BMSCs cells were long spindle or polygonal(fibroblast like appearance),and some of the cells were overlapped and crisscrossed,and arranged tightly in a vortex,with high expression of CD29 and CD90.Compared with the negative control group,there was no significant difference in LDH release in the cell supernatant of the allogeneic tendon combined culture group.The results of cell proliferation activity test showed no significant difference in OD value between the allogeneic tendon group and the negative control group(P>0.05).The mRNA expression levels of SCX,Tnmd,Tnc,collagen I and III were also significantly increased in rabbit BMSCs transfected with BMP-12 vector and the corresponding protein levels also increased significantly.In vivo experiments:The modified tendon maturing scores of the transfected BMP-12 group were significantly higher than those of the control group,and their biomechanical strength was also significantly better than that of the control group.The mRNA expressions of type Ⅰ collagen and type Ⅲ collagen in the transfected BMP-12 group were significantly higher than those in the control group.Histological staining showed that more cartilaginous fibers and chondrocytes could be observed at the interface between tendon and bone in the transfected BMP-12 group.ConclusionsWe found that the chemical extracted allogeneic tendon scaffolds had good biocompatible and biodegradable property.Overexpression of BMP-12 increased the mRNA and protein expression of tenogenic genes in BMSCs cultured with DMEM medium and seeded in chemical extracted allogeneic tendon scaffolds.When BMP-12-overexpressing BMSCs-loaded allogeneic tendon scaffolds were implanted into the injured rabbit supraspinatus tendon-bone junctions,the tendon-bone healing was improved.Our results suggest that application of BMP-12 overexpressing BMSCs loaded 3D-printed chemical extracted allogeneic tendon scaffolds promote the healing of tendon-bone interface,improve collagen organization and increase fibrocartilage in the rabbit rotor cuff repair.Rotator cuff regeneration achieved by BMP-12-overexpressing BMSCs-loaded chemical extracted allogeneic tendon scaffolds may represent a novel approach for the management of rotator cuff defect.