| Objective: Decellularized cartilage matrix(DCM)is a promising biomaterial for cartilage regeneration,because it provides natural matrix environment and potentially chondroinductive nature.The immunogenicity of DCM as a xenograft biomaterial is currently unclear.In this study,we explore the immunogenicity of DCM and its effect on the chemotaxis and/or function of immunoreactive cells to evaluate its biosafety,with the aim of laying a theoretical foundation for the clinical application of DCM xenograft.Method: a)DCM was prepared from porcine articular cartilage by repeated freeze-thawing and low temperature differential centrifugation;b)agarose gel electrophoresis,HE and DAPI staining were used to assess the decellularized degree of DCM;c)the content of Col-Ⅱ in DCM was measured and its structure was characterized;d)thirty male wistar rats(6-8 weeks)were randomly divided into 5 groups: saline group,low concentration DCM emulsification(1mg/ml),medium concentration DCM emulsification(2mg/ml),high concentration DCM emulsification(3mg/ml)and Col-Ⅱ emulsification group.The rats were immunized subcutaneously at the base of the tail and a booster injection after one week,fed and observed until the sixth week.The severity of arthritis was evaluated by macroscopic view,arthritis index score,gait analysis,flow cytometry,radiology and histopathological;e)subcutaneous immune rejection assay in rats: DCM and undecellularized cartilage were implanted subcutaneously,and three time points(1,2,4)were set for gross and histopathological analysis.Result: a)the ds DNA content in DCM was less than 50 ng/mg,agarose gel electrophoresis results showed that the length of most residual DNA fragments was below 300 bp,and lack of visible nuclear material in tissue stained with H&E or DAPI;this indicated that the DCM prepared by this method met the decellularization standard;b)the content of Col-Ⅱin DCM was 84.82%,and in undecellularized cartilage was 71.81%;fourier transform infrared(FTIR)spectroscopy suggested that Col-Ⅱ in DCM basically retained its natural triple helix structure;c)in the Col-Ⅱgroup,obvious redness and swelling of the hind paw appeared in the second week after immune injection,unable to bear weight and even claudication.The swelling was most obvious in the fourth week,then subsided slightly,and the joint deformity and ankylosis gradually appeared.In the low concentration DCM emulsification group,there was slightly swelling in the second week,but the swelling gradually subsided after the fourth week and no ankylosis appeared.The medium concentration and high concentration DCM emulsification groups reacted less than the low concentration group in turn,and the saline group did not change;d)radiology(X-ray,Micro-CT)showed that soft tissue swelling,narrowing of joint space,blurred and uneven joint surface,and some joint appeared ankylosis with osteophyte hyperplasia in Col-Ⅱ group.Only soft tissue swelling was found in different concentration DCM emulsification group;e)the histological staining showed that in Col-Ⅱ group,synovial tissue proliferated and thickened,synovial cells arranged disorderly,a large number of inflammatory cells infiltrated and articular cartilage was destroyed.Mild synovial hyperplasia,normal joint space and no obvious cartilage destruction were observed in low and medium concentration emulsification groups.High concentration emulsification group and saline group were normal.Conclusion: DCM has low immunogenicity and is not enough to cause grafted-specific immune rejection.Moreover,DCM can induce the polarization of M2 macrophages and play the role of anti-inflammation and promote tissue repair.Therefore,DCM is a safe and promising cartilage repair biomaterial. |
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