| Articular cartilage has limited self-repair ability after damage due to its special structure without blood vessels and lymph,that is an important research field in clinical and regenerative medicine.A large number of approaches have been developed and used in clinical practice for treatment of articular cartilage injury,however there is no satisfactory method to repair articular cartilage injury.In particular,due to the lack of structural and mechanical integration between the newly formed tissue and the injury-surrounding native one,various treatments always lead to failure of the articular cartilage regeneration.Therefore,the neo-cartilage integration is a key issue required to investigate for treatment of articular cartilage injury.It is suggested that maintaining the activities and numbers of the cells surrounding the cartilage defect may be an effective strategy to promote tissue repair and improve the neo-cartilage integration.Galectin-3(Gal-3),a proinflammatory factor,plays important roles during articular cartilage injury and osteoarthritis,and therefore is recognized as a potential target for treating these diseases.Modified citrus pectin(MCP)is a natural antagonist of Gal-3,which can competitively inhibit Gal-3 by binding to the Gal-3 receptors on cell surface.In addition,MCP can directly inhibit the expression of Gal-3.However,the effect of MCP on articular cartilage repair is still unclear,and therefore a preliminary study on this was made in this paper.Firstly,the effect of MCP on chondrocytes were investigated.Next,collagen-based composites loading with MCP were prepared,and then the roles of these composites in repair of full-thickness articular cartilage defects in rabbits were studied.Finally,the molecular mechanism of MCP during the articular cartilage repair was analyzed according to the transcriptome sequencing data of the MCP-treated chondrocytes.The main research contents and findings are as follows:1.Effect of MCP on chondrocytes:Chondrocytes were isolated from rabbit knee and cultured in vitro.The chondrocytes were treated with MCP at different concentrations with or without Gal-3 and interleukin-1β(IL-1β).After treatments,the cell proliferation,gene expression profiles and synthesis of type Ⅱ collagen of the chondrocytes were examined.It was found that MCP could maintain the phenotype of chondrocytes,promote cell proliferation,up-regulate the expression of anabolism related genes and down-regulate the expression of catabolism related genes.Importantly,MCP could inhibit the expression of Gal-3 in chondrocytes,and block the effect of Gal-3 on chondrocytes.In addition,MCP could partially change the osteoarthritis-like phenotype of chondrocytes induced by IL-1β and slow down the degeneration of chondrocytes.2.Preparation and characterization of collagen-MCP composites:At first,the interaction between MCP and collagen was detected by turbidimetry and circular dichroism as well.Furthermore,collagen-MCP composites were constructed via physical adsorption approach.The properties of the composites and the amount of MCP on the composites were characterized by morphology,infrared,XPS,thermal behaviors and enzymatic degradation.It was found that MCP did not affect the triple helix structure of collagen,however it could affect the self-assembly of collagen due to the interaction between collagen and MCP.Collagen-MCP composites could be prepared by simple adsorption methods.In particular,the amount of absorbed MCP,the physicochemical properties and degradation of the collagen-MCP composites could be controlled by adjusting the initial concentration of MCP.It worthy to note that the absorbed MCP could induce the decrease of the degradation rate of collagen-based membrane.3.The effects of collagen-MCP composite on repair of full-thickness articular cartilage defect:The optimized collagen-MCP composites were constructed according to the findings of the previous two parts.Then the amount of MCP on the composites and the release behavior of MCP from the composites were determined.Subsequently,the collagen-MCP composites were implanted into the full-thickness articular cartilage defects of knees in rabbits surgically.8 weeks after implantation,the repair effects of collagen-MCP composites on the articular cartilage defect were evaluated by histology,immunohistochemistry and histological scoring.It was found that MCP could be absorbed into the composite,and then could be released from the collagen-MCP in one day effectively.The collagen-MCP composite showed good histocompatibility and obvious chondro-protective effects.Unexpectedly,the composite could promote the repair of articular cartilage defect,especially the composite(MCP500-C)with MCP effective release amount of 500 μg significantly promoted the regeneration of cartilage and subchondral bone,and improved tissue repair outcomes.Notably,due to the chondro-protective effect of the collagen-MCP composite,it significantly enhanced the neo-cartilage integration.4.Preliminary study on the mechanism of MCP on articular cartilage repair:The mechanism of MCP in articular cartilage repair was investigated by transcriptome sequencing of the chondrocytes and RT-qPCR verification after treatment with MCP.It was found that the expressions of genes of transcription factors and growth factors related to chondrogenesis were up-regulated significantly in chondrocytes with MCP treatment compared with normal chondrocytes,especially the expression of BMP4.These results indicated that TGFβ/BMP4 signal pathway might has important roles during MCP promoting chondrocyte proliferation,differentiation and repair of articular cartilage defect.At the same time,the expressions of genes related to cartilage degeneration,catabolism and inflammation,such as TGFβ1,MMP1,MMP3 and MMP13 in chondrocytes after MCP treatment were significantly down-regulated compared with normal chondrocytes.These results indicated that glycolysis,HIF-1 and IL-17 signal pathways might play crucial roles for the chondro-protective effects of MCP to maintain the cartilage homeostasis,reduce the cartilage catabolism and inflammatory responses as well.To sum up,this paper investigated the effect and mechanism of MCP on repair of articular cartilage defect.The results showed that MCP could maintain the phenotype,promote the proliferation,increase the anabolism and decrease the catabolism of chondrocytes.Collagen-MCP composites could be constructed by adsorption method.The collagen-MCP composites showed good histocompatibility.The collagen-MCP composites,especially the composite MCP500-C promoted the repair of articular cartilage defects and improved the neo-cartilage integration significantly.The transcriptome sequencing data indicated that TGFβ/BMP4 pathway might play important roles during MCP promoting the chondrogenesis and tissue repair,while the glycolysis,HIF-1 and IL-17 signal pathways might have important functions for chondro-protective effects of MCP.Taken together,the results in this study demonstrate the potential application of MCP in articular cartilage repair,tissue engineering and osteoarthritis,and is helpful for rational application of MCP in articular cartilage repair. |
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