The Preparation And Characterization Of Cartilage Tissue Engineering Scaffolds Functionalized With Active Ingredients Of Traditional Chinese Medicine

The slow regeneration rate of cartilage and the inability to repair cartilage injury in the inflammatory environment greatly limit the usage of cartilage tissue engineering in clinical treatments.In order to solve this problem,tanshinone IIA and quercetin were used as exogenous phytomolecules to prepare functional cartilage tissue engineering scaffolds,which were combined with poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)(PHBV)and silk fibroin respectively,and their material and biological properties were systematically studied and analyzed.(1)Quercetin modified PHBV fibrous scaffolds were prepared by electrospinning combined with chemical grafting.It was found that the hydrophilicity of the scaffolds was significantly improved after grafting with methacrylic acid(MAA)and quercetin,and the micromorphology of the fibrous scaffolds would not be damaged during the modification process,the prepared fibrous scaffold had good degradation performance.The results of cell biology and molecular biology showed that quercetin modified PHBV fibrous scaffolds facilitate the growth of chondrocytes,and can maintain the phenotype of chondrocytes and promote the synthesis of cartilage matrix by up-regulating the transcription of SOX9,ACAN,COL II,and down-regulating the transcription of MMP1,MMP3 and MMP13.It also can inhibit cell apoptosis and reduce oxidative stress by regulating the transcription of related genes.The results of animal experiments show that quercetin modified PHBV fibrous scaffolds can significantly improve the synthesis and secretion of cartilage matrix,promote the formation of cartilage lacunae,and then facilitate the formation of cartilage tissue.(2)Silk fibroin scaffolds were prepared by freeze-drying combined with self-assembly of proteins.When the concentration of silk fibroin was 40 mg/ml,the freezing temperature was-20℃,the cross-linking temperature was-2℃,and the cross-linking storage time was 48 h,the prepared three-dimensional porous scaffolds with suitable pore size and stable structure.Then,tanshinone IIA was mixed with silk fibroin solution to prepare tanshinone ⅡA delivery silk fibroin scaffolds,the cross-sectional pore size of the scaffolds was about 184 μm,the porosity was more than 90%.The scaffolds have a stable structure,good water absorption capacity and degradation performance,and can continuously release tanshinone IIA to the solution.Experiment results of cell biology and molecular biology showed that all scaffolds have good biological compatibility.Tanshinone ⅡA delivery silk fibroin scaffolds,especially when the drug loading concentration is 10 μg/ml(SF/T10),can improve the activity of chondrocytes and maintain the functional expression of chondrocytes by up-regulating the transcription of cartilage matrix formation related genes in chondrocytes,and can alleviate oxidative stress and inhibit apoptosis by regulating the transcription of related genes.In vitro and in vivo cartilage regeneration results indicated that the SF/T10 scaffolds can significantly promote cartilage regeneration.Compared with the pure silk fibroin(SF)group,the cartilage matrix in SF/T10 group was richer and denser,the glycosaminoglycans content was higher,the cartilage tissue was more mature,and the cartilage lacuna structure was formed earlier.Finally,by treating rabbit articular cartilage defects with SF and SF/T10 scaffolds,the results showed that only the defect of the SF/T10 group filled with hyaline-cartilage-like tissue that integrated with the surrounding cartilage perfectly,and the cartilage matrix content and mechanical properties of the new tissue were close to those of normal cartilage tissue.The SF/T10 group yielded better cartilage repair outcomes than those of the nontreated group and SF group.(3)TNF-α and IL-1β were used to simulate the inflammatory environment,the effects of quercetin modified PHBV fibrous scaffolds and tanshinone ⅡA delivery silk fibroin scaffolds on chondrocytes and cartilage regeneration under the stimulation of proinflammatory cytokine were investigated.The results show that quercetin modified PHBV fibrous scaffolds can effectively reduce the damage of chondrocytes caused by inflammatory stimulation,promote the proliferation of chondrocytes,maintain the phenotype of chondrocytes,alleviate the inhibitory effect of inflammatory factors on the synthesis of cartilage extracellular matrix,enhance the functional expression of chondrocytes by regulating the transcription of related genes,and alleviate oxidative stress and apoptosis induced by proinflammatory cytokine stimulation.Tanshinone ⅡA delivery silk fibroin scaffolds can inhibit the dedifferentiation process of chondrocytes stimulated by proinflammatory cytokine,maintain the functional expression of chondrocytes,and reduce oxidative stress and apoptosis.In vitro cartilage regeneration results showed that tanshinone ⅡA delivery silk fibroin scaffolds significantly enhance cartilage regeneration under inflammatory stimulation,and it might promote cartilage regeneration under an inflammatory environment by inhibiting the expression of JNK and p38 and inhibiting the inflammatory signal transduction of MAPKs signaling pathway.Furthermore,the in vitro inflammatory simulation model was constructed by the inflammatory synovial fluid of osteoarthritis patients.The cell biology,molecular biology experiments and in vitro cartilage regeneration results verified that the tanshinone ⅡA delivery silk fibroin scaffolds can maintain the chondrocyte phenotype,promote the synthesis and secretion of cartilage matrix,and inhibit the oxidative stress and cell apoptosis induced by inflammatory synovial fluid,and significantly promote cartilage regeneration under inflammatory synovial fluid stimulation.In conclusion,in this paper,tanshinone ⅡA and quercetin were used as biofactors to construct functional cartilage tissue engineering scaffolds,and it was confirmed that the prepared quercetin modified PHBV fibrous scaffolds and tanshinone ⅡA delivery silk fibroin scaffolds can significantly promote the formation of cartilage tissue and enhance the ability of cartilage regeneration under inflammatory environment,which can be potentially applied in the clinical treatment of cartilage defects via the cartilage tissue engineering approach.