Preparation And Characterization Of Mineralization Of Acellular Tendon In Domestic Pigs

Objective: to prepare and characterize a mineralized acellular tendon scaffold.Methodology:1.Obtaining of acellular tendons: samples were obtained from the anterior hoof tendons of domestic pigs.The obtained samples were dehydrated,embedded,sectioned and stained with HE and Dapi.The effect of acellular was evaluated by hematoxylin and eosin(H & E)staining.Dapi staining and HE staining were performed on the acellular tendons.2.Preparation of the mineralization model of acellular tendon: after the successful preparation of the mineralized fluid,the middle part of the acellular tendon was immersed in the mineralized fluid at low temperature,and then sectioned,the longitudinal section and surface of the tendon were examined by Micro-CT and transmission electron microscope.3.The decellularized tendon 0.2 g/ml was co-placed with the medium(DMEM),and the extracted medium was used as the extracting solution.The MSCs cells were diluted with DMEM gradient extracting solution,cytotoxicity and Histocompatibility of acellular tendons were detected by CCK-8 and livedead staining.Results: H & E staining indicated that the acellular artificial tendon was successfully prepared.Hematoxylin and eosin staining indicated that the acellular effect of the tendon was good.After decellularization,the tendon appears to be looser than its natural structure,but retains the main structure of the tendon.The acellular bone and fibrous tissue showed no difference from the natural structure.2.Dapi staining showed that the fibrous tissue of the artificial tendon was intact and only immunogenic cells were lost.3.The mineralized artificial tendon can be analyzed by micro CT and projection electron microscope,and the mineralized area ratio can be calculated by image software after 4 weeks’ immersion in different mineralized solution,the mineralization area of 0.5% acetic acid + 100 m M citric acid is the highest after 4 weeks mineralization.4.After the same acellular tendon was mineralized,the muscle fibers with mineral-rich deposits between the muscle fibers,a small amount of mineral deposits,and no mineral deposits can be seen in different positions,it is proved that it is possible to deposit minerals in muscle fibers on the same acellular tendon after four weeks immersion in our improved mineralized solution.5.To prepare the successful mineralized artificial acellular tendons,CCK-8 method was used to measure the cytotoxicity of the mineralized acellular tendons,and two groups of tendons were cultured in vitro with different concentrations of extract and DMEM alone,live/dead staining was used to show that the mineralized acellular ligaments had no effect on the survival of MSCs.Conclusion: The successful preparation of acellular artificial tendons with mineralized bands and no immunogenicity may be a key step to realize the regeneration of soft tissue-bone interface in future tissue engineering.