| Anterior cruciate ligament-Bone (ACL-to-Bone) interface includes four differentareas: ligament, the mineralized area, the tissue of mineralization, and Bone. Differenttissue area contains different material composition and cells. In the region of theligament, fibroblasts locate in the matrix composed of collagen Ⅰ and collagen Ⅲ. Thecircle like chondrocytes are in the area consists of Ⅱ collagen and proteoglycan in themineralized interface regions. Whereas, hypertrophied chondrocytes are the main celltype among the tissue of Mineralization. In addition, the subchondral bone is made upof osteoblasts, osteocytes and osteoclast and collagen Ⅰ matrix.Interface tissue engineering (ITE) depends on gradient scaffold materials andseeded cells which had differentiation ablitiy. Gradient biomaterials are anisotropic inthe chemical composition, structure, mechanics and biological molecules. It becomesessential hot spot gradually concerning tissue engineering and biomedical research.At present, various natural or synthetic biomaterials have been used for thepreparation of tissue scaffold materials, and are widely used in every field. Due to theadvantages of flexible design, hydrability, viscosity and other characteristics, hydrogelcould be an ideal imitation ECM for interface tissue engineering (ITE). The choice ofcell is extremely significant in the process of tissue repair and regeneration because ofthe cell functional diversity. The tendon stem cells (TSPCs) derived from tendon tissuenot only has universality belongs to stem cell, but also are fast proliferative, havingefficient ability for soft tissue like differentiation. It is an ideal seeded cell choice ofinterface tissue engineering (ITE).Our research mainly includes the preparation of phosphate-collagen gradientmaterial, separation and identification of tendon stem cells, biocompatibility analysis ofphosphate-collagen gradient materialThe main methods and the results are as follows:①The extraction, purification and identification of rat tail collagen Ⅰ: The rat tailcollagen crude extract was prepared as the acetic acid extract method, then salted out,purified with semi-permeable membrane, and lyophilized. The finnal products revealsilvery white and fluffy form. SDS-PAGE electrophoresis coomassie brilliant bluestaining method, infrared spectrum analysis (FITR) was used for identification of the rattail collagen. Results exhibit the products are high purity. ②Preparation of phosphate-collagen gradient material: the combination materials wereprepared as gradient solution mineralization method. Scanning electron microscopy(SEM) analysis exhibits that phosphate composition and collagen I combined into aobvious interface formation, the upper layer calcium phosphate had less precipitateswhile the substratum was opposite.③Biological compatibility analysis of rats and the gradient materials: SD rat was usedfor transplant recipient, HE staining analysis was used for degradation propertiesevaluation of materials, DAPI staining was inspected for cell infiltration. Results show:the materials did not fully degraded, additionally were infiltrated by a great amount ofcells after two weeks transplantation.④Isolation, cultivation, and identification of Tendon stem cells (TSPCs): SD ratAchilles’s tendon tissue was digested as collagenase type I, clones were selected andtested by three-way differentiation, flow cytometry detection. The tendon derived cellswere TSPCs.⑤Analysis of TSPCs compatibility concerning the gradient materials: the TSPCs wereseeded in phosphate-collagen gradient materials, DAPI staining were used forevaluation of cell viability in the materials in time points during two weeksIn conclusion, the phosphate-collagen gradient materials have characteristic microstructure and good biocompatibility. Combined with the tendon stem cells (TSPCs), thegradient materials provide foundation and reference for further interface tissueengineering (ITE) research. |
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