Blocking-down Collagen Type V And Proteoglycan Decorin Genes Expression For Injured Tendon Repair And Regeneration

Background:With the improvement of people's pursuit of health, physical exercise and competitions increased significantly. The clinical damage caused by sports is growing more and more, in which tendon and ligament injuries account for over 50%. As the ligament and tendon have poor regenerative capacity, in the healing tendon, a uniform distribution of small diameter collagen fibrils has been found with poorer mechanical properties than native tissue and shows no improvement of these properties. How to promote the injured tendon to regenerate large diameter collagen fibers is currently a big challenge in tendon repair study. Recently, type V collagen and proteoglycans are found to be important regulatory factors for collagen fibrillogenesis. Their expression is abnormally increased in injured tendon, which may cause the formation of samll diameter collagen fibers. Therefore, this study was designed to inhibit the expression of type V collagen and decorin by gene therapy, construct a tissue engineered tendon, and investigate the biology of collagen fibrillogenesis in in vitro and in vivo studies. Our study included four parts. Part I: Construct a tissue engineered tendon for the following studies. Part II: Inhibit the expression of type V collagen by siRNA and detect its regulatory effect on collagen fibrillogenesis; Part III: Inhibit the expression of proteoglycan decorin by antisense and detect its effects on collagen fibrillogenesis in vitro; Part IV:Repair the injured tendon using tissue engineered tendon which was infected with siRNA-inhibited decorin Lentivirus and evulate the efficiency of tendon regeneration in vivo. This study explored the effects of collagen type V and proteoglycan decorin on fibrillogenesis in tendon repair process. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.Part I: Construction of tissue-engineered tendonOBJECTIVE: To conveniently detect the collagen fibrillogenesis in vitro and repairinjured tendon in vivo, a scaffold-free tissue engineered tendon was developed withtendon cell sheets.METHODS: Tendon cells were cultured in high glucose DMEM with 10% (v/v) FBSand 50μg/mL ascorbic acid. Cells proliferated rapidly and formed coherent cellularsheets within 2 weeks. It could be detached from the substratum by applying a smallroll-up force. The cell sheet was cultured for one more week and then a scaffold-freetissue engineered tendon with living cells and collagen matrix was developed. Thetissue engineered tendon was used for histological examination and transmissionelectron microscopy analysis.RESULTHS: The tissue engineered tendon with living cells and collagen matrix had arelatively compact structure. Hematoxylin-eosin and Masson trichrome stainingrevealed a tissue-specific tendon structure: organized bundles of highly crimped fibersand cells oriented in parallel. Transmission electron microscopy analysis showed thatthe in vitro tissue engineered tendon had a more uniform distribution of smallerdiameter collagen fibrils than native tissue.CONCLUSIONS: Tissue engineered tendon is scaffold-free, so there are fewerexogenous factors, It possesses both the simple and controllable properties of cellculture models. It can be easily and efficiently handled with RNAi and antisense techniques which are not convenient for animal models. Moreover, the in vitro tissue engineered tendon has a tendon-specific structure of well-organized collagen fibrils, so the factors regulating collagen content and fibril formation can be investigated conveniently in this model. In addition, the tissue engineered tendon may be an excellent model to investigate the injured tendon because it has a uniform distribution of small collagen fibrils, which is characteristic of injured tendon. Although the tissue engineered tendon lacks a fair bit from the mechanical perspective, it illustrates a novel model and strategy for future tendon biology research.Part II: Regulatory effect of collagen V on the fibrillogenesis in a tissue engineered tendon modelOBJECTIVE: The presence of uniformly small collagen fibrils in repaired tendon is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and related to fibrillogenesis. This study aimed to investigate the effect of particular subunits of collagen V on the fibrillogenesis of tenocytes.METHODS: RNA interference gene therapy was used in this study to knock down the expression of two subunits of collagen type V and a tissue engineered tendon model was used to detect the regulatory effect of collagen V on collagen fibrillogenesis.RESULTS: The results showed that siRNA againstα1 andα2 chains of collagen type V (COL5A1 siRNA, COL5A2 siRNA) had different effects on collagen I and decorin gene expression. Tissue engineered tendon treated with COL5A1 siRNA had smaller collagen fibrils with abnormal morphology, while those formed by coculrure of COL5A1 siRNA treated and cultured tenocytes with a ratio of 1:0.5 or 1:1 had better fibrogenesis and larger collagen fibrils than that of cultured tenocytes.CONCLUSIONS: Our studies demonstrated that tissue engineered tendon is a novel and useful model for collagen fibrillogenesis biological investigation. The type V procollagenα1 andα2 chains have different effects on tendon matrix gene expression. An optimal level of collagen V is vital in regulating collagen fibrillogenesis. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.Part III: Decorin antisense gene therapy improves regeneration of large collagen fibril using tissue engineered tendon modelOBJECTIVE: Injured ligaments heal with scar tissue, which has uniformly smallercollagen fibrils and poor mechanical properties contrast to those of normal ligaments.Small leucine-rich proteoglycan decorin is known to regulate collagen fibrillogenesis.We hypothesized that the down-regulating proteoglycan decorin would improve largecollagen fibril regeneration.METHODS: The sense and antisense oligonucleotides which were against rabbitdecorin were transfected into rabbit tendon cells. Collagen synthesis, fibril organizationand collagen fibril diameter of tissue engineered tendon were analyzed.RESULTS: RT-PCR and immunofluorescence detection indicated that decorin had beenefficiently down regulated by antisense oligonucleotides in mRNA and protein level.TEM examination showed that down regulation of proteoglycan decorin led to anincreased development of larger collagen fibrils.CONCLUSIONS: This study demonstrated that tissue engineering tendon model is anovel and useful model for biological study of tendon. Down regulation of decorin canimprove larger diameter collagen fiber regeneration.Part IV: Decorin RNAi gene therapy improves regeneration of large collagen fiber in rat patellar tendon defect model.OBJECTIVE: Injured tendons heal with uniformly smaller collagen fibrils and poor mechanical properties. We hypothesized that the down-regulating proteoglycan decorin would improve large collagen fibril regeneration in vivo. METHODS: In this study, we constructed lentivirus containing siRNA against decorin. A tissue engineered tendon which was infected with siRNA-lentivirus was used to repaire rat patellar tendon defect. The repair effect was valued by detecting the ability of collagen synthesis, the microstructure of collagen fiber and histological examination.RESULTS: Real time PCR and immunofluorescence indicated that decorin had been efficiently down regulated by lentivirus containing siRNA against decorin in mRNA and protein level. Down regulation of decorin promote cell proliferation and led to an increased development of large collagen fibrils in repaired tendon tissue.CONCLUSIONS: The results demonstrated that RNA interference technology is a powerful tool for silencing genes in mammalian cells and tissue engineered tendon is a novel and useful model for tendon defect repair. Down regulation of proteoglycan decorin can improve large diameter collagen fiber regeneration and increase mechanical property. This may provide a promising approach to improve functional healing of injured tendon.