Experimental Study On Ligament Acellular Scaffold Construction

ObjectiveLigament can not heal itself after severe injury. There are various alternatives used in ligament reconstruction. Current clinical ligament substitutes include autograft, allograft and synthetic materials, but they have not provided a satisfactory effect due to their own inherent shortcomings. Ideal ligament substitutes must have good mechanical properties, biocompatibility, and the functions to achieve the reparation and reconstruction of the severe injury owing to the formation of ligament-like organization during its degradation in the body. Tissue-engineered ligament (TEL) with the above advantages is an ideal substitute ligament. TEL relies on a construct consisting of cells seeded into a scaffold. The ideal scaffold must be biocompatible with the cell source and recipient, while allowing for cell adhesion and proliferation. Furthermore, the scaffold must have sufficient biomechanical integrity to withstand rehabilitation. Additionally, the scaffold must be to minimize inflammatory, disease transmission and host immune response. As is known, acellular ligament scaffold has the properties of the native ligament extracellular matrix (ECM), its immunogenicity is reduced by cellular components extracted and only minimal antigenic ECM itself due to typeⅠ,Ⅲcollagen, et al is preserved. Therefore, the ligament acellular scaffold is expected to be the ideal tissue-engineered ligament scaffold. The existing literature reports a variety of methods of decellularization to ligament, but each of them has its own disadvantages. In this study, the effectiveness of 1% sodium deoxycholate (DCA),1% tributyl phosphate (TBP) and ligament membrane pulsed and scanned carbon dioxide laser treatments to removing cellular materials from rabbit patellar ligament were determined by histological, DAPI-stained, and immunohistochemical analyses, and possible changes in DNA content, collagen content and mechanical properties due to each treatment were investigated. Then, acellular scaffolds were in vitro co-cultured with allogenic ligament fibroblasts and implanted into homogeneous bodies in order to observe the cytocompatibility of acellular scaffolds.Methods1. Acellular methodPatellar ligament taken from New Zealand white rabbits were divided into three groups, and decellularized respectively with DCA (10mM tris 36h+(0.01% trypsin and 0.02% EDTA 8h+1% DCA 24h+ DNaseⅠ200μg/ml and RNaseⅠ200μg/ml 5h+PBS 48h), TBP(10mM tris 36h and 0.02% EDTA 8h+1% TBP 48h+DNaseⅠ200μg/ml and RNaseⅠ200μg/ml5h+PBS 48h) and Patellar ligament membrane pulsed and scanned by CO2 laser with 10% of the area and 5mJ energy(10mM tris 36h and 0.02% EDTA 8h+ PBS 72h). All operations are at room temperature, on rotary shaking (150rpm), contained 5ml/L penicillin/streptomycin solution (10000U/ml 10000mg/ml) to prevent infection, and each step in the use of PBS were rinsed three times.2. Histology, collagen content and biomechanical evaluationsThe effect of decellularization was analyzed by HE staining, Masson staining, DAPI staining, immunohistochemical observation for cellular materials. Using the DNA and hydroxyproline test kit, we measured the content of DNA and hydroxyproline in each samples. The content of hydroxyproline can be calculated the amount of collagen protein according to hydroxyproline accounts for 13.4% of the total proportion of collagen protein. Samples were placed into grips on a uniaxial load frame (Instron 5542, Needham, MA) for ultimate tensile stress (UTS) and elastic modulus testing. Samples were kept moist with PBS during testing. All positive controls for the analysis were fresh patellar ligaments.3. Study of cell seeding into acellular ligament scaffoldsThree-generation allogenic patellar ligament fibroblasts were selected to culture with acellular ligament scaffolds treated by DCA in vitro. With the cell suspension penetrate into inner scaffold structure, patellar ligament fibroblasts also can be followed and seeded into depth of lyophilizated scaffold with osmosis. Co-culture ligaments were obtained in 3 days and 1 week, and observed whether or not the ligament fibroblasts adhesion to and proliferation into the scaffolds.4. Allograft in vivo biocompatibilityAcellular allogenic ligament scaffolds treated by DCA were implanted into rabbit patellar ligaments. The implants were harvested at 21 days post-implantation and examined grossly and histologically for signs of an inflammatory reaction, and histologically for host cell infiltration (H&E).Results1. Histology, collagen content and biomechanical test resultsAfter decellularization, no nuclear material was evident via H&E and Masson staining in all samples, DAPI staining revealed no presence of DNA within the decellularized ligament scaffolds and samples displayed a complete lack of vimentin staining. The contents of DNA in fresh patellar ligments is 4.7 times as much as acellular ligament scaffolds. There were no samples treated with DCA, TBP and CO2 laser displayed a significant decrease in collagen content compared to fresh patellar ligaments. No significant differences in elastic modulus and UTS were found between fresh patellar ligaments and samples treated with CO2 laser in elastic modulus and UTS, but differences compared to samples treated with DCA or TBP.2. Preliminary studies to assess the capacity of the acellular scaffold to support cellular infiltrationFibroblasts were observed in H&E-stained sections of the co-culture decellularized ligament scaffolds and at 3 and 7 days. Cells were found not only at the surface of the scaffolds but also within the depth of the scaffolds as well, which assumed an elongated shape located adjacent to and within individual ligament fascicles oriented longitudinally along the long axis of the ligament.3. In vivo host cell infiltration and inflammatory response to decellularized scaffoldsA few of cells infiltration by mononuclear, lymphocytes and fibroblast-like host cells were observed around and in explanted decellularized ligament scaffolds with intact membrane at 21 days post-implantation in rabbits through H&E-stained sections. In explanted decellularized ligament scaffolds with intact membrane, lots of cells were found to increase in density not only at the periphery of the implant but also within the depth of the implant as well, which located adjacent to and within individual ligament fascicles oriented longitudinally along the long axis of the ligament. Compared with the ligament injury and repair in situ, there were no evidence of a gross inflammatory reaction or capsule formation was observed and no significant evidence of granuloma formation or infiltration by inflammatory cells.Conclusions1. All DCA, TBP and CO2 treatments can effectively remove cell components and destruct ligament collagen composition and tensile mechanical properties to some extent. Intact ligament membrane is the mainly obstacle to cell extraction.2. The tissue-engineered patellar ligament scaffold is cytocompatible in vitro. The patellar ligament fibroblasts can follow cell suspension to be seeded into depth of lyophilizated scaffold with osmosis and show an elongated shape oriented longitudinally along the collagen fibers.3. The tissue-engineered patellar ligament scaffold did not exhibit histologic or gross evidence of an inflammatory reaction at 21 days post-implantion in rabbits. Intact ligament membrane is the mainly obstacle to cell infiltration. However, host cells infiltration by lots of fibroblast-like cells and a few of mononuclear cells and lymphocytes, were observed at the periphery as well as within the inner matrix of the implanted scaffolds.