| Objective: As a biomaterial,Extracellular matrix(ECM)can promote tissue repair and regeneration of mice digits,but the effect of ECM powder on the regeneration of mice digit,the characteristics of histomorphological changes and the rule are not clear.The purpose of this study was to investigate the effects of ECM powder on mice digit regeneration and the changes of bone morphology as well as the location,number and time of CD206 positive cells,a marker of M2-type Macrophages(M(?)),in order to provide theoretical basis and reference for clinical treatment of human finger injury.Methods: 1.The preparation of the ECM: The ECM powder derived from porcine bladder was prepared by improved and optimized acellular method,and characterized by HE staining.The biosafety of ECM was tested by cytotoxicity test,acute systemic toxicity test and liver and kidney toxicity test.2.Modeling and grouping: Male kunming mice aged 6 to 8 weeks were used to establish the model of amputation digit with and without the preservation of soft tissue by amputation of the second Phalange2(P2).The experimental animals were randomly divided into ECM group and blank control group.3.Morphological evaluation of mice digit regeneration: The digits of mice were taken from day 1 to day 50(daily),day 90 and day 100 after digit amputation to make paraffin tissue sections and stained:(1)inflammatory cell infiltration,vascular differentiation and mesenchymal cell recruitment were observed by HE staining;(2)The growth of cartilage was evaluated by toluidine blue staining;(3)The growth of nucleus,erythrocyte,muscle fiber and collagen fiber was analyzed by Mallory trichromatic staining.(4)Immunohistochemical staining was used to observe the expression of M2 macrophage marker CD206.Results: 1.The results of ECM characterization experiments showed that the nucleus of the prepared ECM was almost completely removed,meeting the standard of acellular,and had no cytotoxicity,systemic toxicity and hepatorenal toxicity.2.There was no significant difference in cartilage production between the ECM group and the control group.3.In a model of a amputation digit without preserving soft tissue.(1)HE staining showed that at 3 days post amputation(3 DPA),the wound began to contract along the differentiated epidermis structure and truncated tendon at the stump.Compared with the control group,at 5 DPA,a small number of mesenchymal cells began to accumulate in the dermis and skeletal residues at the end of the severed digit.Vascularized fibrous connective tissue was found at the stump of the 7 DPA amputation.The granulation tissue at the end of the 22 DPA amputation digit was gradually mature,inflammatory cells decreased and disappeared gradually,capillaries decreased,and collagen fibers were arranged parallel to the surface of the end of the amputation digit.(2)The results of toluidine blue staining showed that the amount of cartilage was gradually increased at 8 DPA,and the ECM group was more than the control group.The cartilage was mainly generated and distributed on the lateral side of bone and near the periosteum.At 12 DPA,cartilage growth basically reached the peak,and at 21 DPA,intrachondral ossification reaction began to appear,and many cartilages differentiated into braided bone.(3)Mallory’s trichromatic staining results showed that production of collagen fiber was more active in the outer side of bone at 12 DPA,muscle fiber production began to increase at 22 DPA,and bone plug formed in the digit stump at 38 DPA.(4)immunohistochemical results showed that the appearance level of CD206-positive cells increased significantly at about 14 DPA,then gradually decreased and recovered to the initial level at about 21 DPA.Conclusion: In the model of amputation digit without retaining soft tissue,ECM powder from porcine bladder can promote regeneration of amputation digit.The location and time of cartilage formation and cd206-positive staining cells during regeneration were relatively consistent. |
PDF Full Text Request