| Articular cartilage has limited ability to repair itself due to its special structure without vascular,lymphatic and neural,so the treatment of cartilage defects still faces great challenges.Tissue engineering,especially combined with 3D bioprinting technology to construct tissue-engineered cartilage,provides the most promising therapeutic strategy for articular cartilage injury repair,and is an important research hotspot for articular cartilage injury repair.However,the current 3D bioprinting bioinks used for cartilage tissueengineered scaffolds are mostly deficient in bioactivity,and improving the bioactivity of bioinks is a key issue that needs to be addressed urgently to construct tissue-engineered cartilage by 3D bioprinting.Modified citrus pectin(MCP)is a natural antagonist of the pro-inflammatory factor galectin-3(Gal-3),which has been found to be biologically active in promoting chondrocyte proliferation and maintaining chondrocyte phenotype,and has clear application prospects in cartilage tissue engineering.Based on this,this paper intends to improve the bioactivity of hydrogel scaffolds by MCP composite,and initially investigate the role of MCP composite hydrogels in the construction of tissue engineering cartilage through 3D bioprinting and its mechanism.The main content and conclusions of the research are:1.Effect of MCP on successive passaged chondrocytes:Rabbit knee chondrocytes were isolated and successive passaged in vitro with or without MCP-containing medium to investigate the effects and mechanisms of MCP on the proliferative activity,gene expression and type Ⅱ collagen synthesis of chondrocytes.It was found that MCP could promote the proliferative activity,up-regulate the expression of anabolic-related genes and down-regulate the expression of catabolic-related genes of chondrocytes in successive passages,and maintain the phenotype of chondrocytes.2.The effects of collagen-MCP composite on repair of partial-thickness articular cartilage defect:Referring to the previous research work,we constructed collagen-MCP composites and constructed partial-thickness articular cartilage defect in rabbit knee joints and implanted the composite in the defect to investigate the effect of composite on cartilage defect repair by histology and other means.It was found that collagen-MCP composites did not significantly contribute to the injury of partial-thickness articular cartilage,but collagen-MCP composites compounded with high doses of MCP demonstrated significant chondroprotective effects,preventing the degeneration of cartilage tissue around the injury.3.Preparation and characterization of 3D bioprintable composite hydrogel tissue engineering scaffolds containing MCP:MCP composite GelMA/HAMA/MCP hydrogel bioink was prepared by co-blending,and the printability and other properties of the bioink were characterized by physicochemical and rheological tests;The composite hydrogel scaffolds were constructed by 3D bioprinting,and the physicochemical properties of the composite scaffolds were characterized by means of morphological structure,IR,swelling rate,compressive strength,thermal and enzymatic degradation;the biological activity of the composite scaffolds was investigated by chondrocyte inoculation and culture.It was found that MCP composite hydrogel bioink has good printability and can be used for the preparation of composite hydrogel scaffolds by 3D bioprinting;GelMA/HAMA/MCP composite hydrogel scaffolds have good bioactivity and can be applied to the construction of tissue-engineered cartilage.4.In vitro construction of tissue-engineered cartilage based on MCP composite hydrogel scaffold:We prepared composite hydrogel scaffolds containing different concentrations of MCP and inoculated rabbit articular chondrocytes onto the scaffolds to construct tissue-engineered cartilage,and investigated the effects of the composite hydrogel scaffolds on the formation of tissue-engineered cartilage and its mechanism by means of histology and transcriptomics.It was found that chondrocytes were uniformly distributed in the constructed tissue-engineered cartilage and cartilage-specific extracellular matrix was synthesized;the scaffolds with different MCP concentrations caused differences in chondrocyte gene expression,and the scaffold with MCP content of 1000 μg/ml was more favorable to the differentiation of chondrocytes and the maintenance of chondrocyte phenotype.In summary,this paper initially investigated the feasibility of MCP composite hydrogel bioink in 3D bioprinting for constructing tissue-engineered cartilage.The results revealed that:MCP has the effect of maintaining the phenotype of successive passaged chondrocytes and promoting the proliferation of successive chondrocytes,which helps to obtain a large number of phenotypically stable chondrocytes;In vivo,MCP also has a clear chondroprotective effect;the GelMA/HAMA/MCP composite hydrogel prepared by the co-blending method has good 3D printability,and the composite hydrogel scaffold constructed by 3D bioprinting has good biological activity and may promote the formation of chondrocytes by regulating the HIF-1 signaling pathway,PI3K/AKT signaling pathway and MAPK signaling pathway,etc.However,there are some differences in the effects of different concentrations of MCP composite hydrogel scaffolds on the proliferation,gene expression and phenotype maintenance of chondrocytes.The preliminary results showed that the composite scaffold with MCP concentration of 1000 μg/ml is most favorable for chondrocyte proliferation,differentiation and chondrocyte phenotype maintenance,and has good prospects for application in the field of articular cartilage tissue engineering. |
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