The Preparation And Characterization Of Functional Cartilage Tissue Engineering Scaffolds For Osteoarthritis Cartilage Defects Repair

Articular cartilage,a highly organized tissue with substantial durability,has a limited intrinsic healing capacity.Patients who lost their articular cartilage because of ageing,trauma or degenerative diseases need surgery intervention.Among the various techniques,cartilage tissue engineering(CTE)has been largely explored and proved as a promising tool for repairing cartilage defects.Scaffolds play important role in CTE,however,currently few tissue engineered scaffolds were designed to be applied in pathological microenvironment of cartilage damage caused by degenerative diseases,which will greatly limit the effectiveness of cartilage regeneration.In this thesis,the key scientific problem of how to construct scaffold materials to resist inflammation and hypertrophy pathological microenvironment in degenerative diseases caused cartilage injury was studied.The functional electrospun fiber scaffolds and functional silk fibroin composite scaffolds were developed to repair cartilage injury of partial cartilage injury and full-thickness cartilage injury caused by degenerative diseases.To solve the issue of inflammatory microenvironment existed in partial cartilage defects caused by osteoarthritis,PHBV fiber scaffold was prepared by electrospinning method,and ginsenoside Rg1,an effective component of traditional Chinese medicine,was grafted on its surface to resist the threat of inflammatory microenvironment induced by IL-1β.The results showed that ginsenoside Rg1 can help maintain the chondrocytic phenotype of chondrocytes under normal condition.Ginsenoside Rg1-loaded PHBV films prepared by solvent casting method can promote the proliferation of chondrocytes.The PHBV fiber scaffolds prepared by electrospinning were successfully grafted with ginsenoside Rg1 by using BPO initiation and EDAC crosslinking system.Ginsenoside Rg1 modification did not change the morphology of PHBV fiber scaffolds significantly,but improved the hydrophilicity and degradation rate of PHBV fiber scaffolds.Through cell experiments,it was found that ginsenoside Rg1 modified PHBV fiber scaffolds were beneficial to chondrocyte adhesion.Under the inflammatory environment,the ginsenoside Rg1 modified scaffold can promote the proliferation of chondrocytes,maintain the phenotype of chondrocytes and reduce the inflammatory reaction,which may through restoring chondrocyte metabolic balance of chondrocytes.To solve the issue of inflammatory microenvironment in repairing full-thickness cartilage defects caused by osteoarthritis,newly developed berberine-oleanolic acid complex salt(HL9)was chemically grafted onto hyaluronic acid and further combined with silk fibroin to prepare HL9 grafted hyaluronic acid /silk fibroin(HL9-g-HA/SF)composite scaffold.The results showed that HL9 has a significant protective effect on chondrocytes under inflammatory environment.The FTIR,UV-Vis absorption and thermal degradation results proved that HL9 was successfully grafted onto hyaluronic acid.HL9-g-HA/SF composite scaffold was successfully prepared by freeze-drying method and adding HL9-g-HA significantly increased the swelling ratio and degradation rate of silk fibroin scaffold.Compared with pure silk fibroin and HA/SF composite scaffolds,HL9-g-HA/SF composite scaffolds significantly reduce the IL-1β-caused dedifferentiation to articular chondrocytes via enhancing the transcription of chondrocytic phenotype related genes and reducing the transcription of matrix degradation related genes.Further,adding HL9-g-HA to silk fibroin scaffold can increase the deposition of cartilage specific extracellular matrix under inflammatory environment in vitro,which may be realized by regulating the transcription of oxidative stress related gene of chondrocytes.Moreover,HL9-g-HA/SF composite scaffold can provide a protective environment for cartilage regeneration under inflammatory environment by inducing macrophage polarization through its degradation products.To deal with the issue of hypertrophy pathological environment of full-thickness cartilage injury,decellularized sturgeon extracellular matrix(Stur-ECM)was obtained and further combined with silk fibroin to prepare composite scaffolds with antihypertrophy property.The results showed that Stur-ECM was successfully prepared with combination of cyclic freezing and chemical treatment.And Stur-ECM scaffolds with interconnected pore structure were successfully prepared by freeze-drying and chemical cross-linking method.Chondrocytes displayed a round shape and aggregated to form cellular spheroids within Stur-ECM scaffolds,which is similar to their chondrocytic phenotype within cartilage in vivo,while chondrocytes spread and flattened on type I collagen(COL I)scaffolds.Higher transcriptional level of chondrogenic related genes were observed in chondrocytes incubated with Stur-ECM scaffolds instead of COL I scaffolds.Furthermore,it confirmed that,compared with COL I scaffolds,Stur-ECM scaffolds significantly reduced the transcription level of chondrocyte hypertrophy related genes in chondrocytes following the hypertrophic induction treatment.To test the Stur-ECM scaffold in vivo,chondrocytes with SturECM scaffolds and COL I scaffolds were cultured with hypertrophic media and implanted into nude mice respectively.Following 4 weeks implantation,interestingly,only the specimens derived from COL I scaffolds displayed consequences of chondrocyte hypertrophy like calcification deposition,demonstrating that chondrocyte hypertrophy is ceased by the Stur-ECM scaffolds following hypertrophic induction.It suggests that the Stur-ECM scaffolds can be potentially applied in clinical treating cartilage defects via the CTE approach to avoid the risk of chondrocyte hypertrophy.Then the Stur-ECM was mixed with silk fibroin to prepare composite scaffold,the swelling and degradation rate of scaffolds were improved with the adding of Stur-ECM.The result of cell experiments showed that adding Stur-ECM can improve the transcription level of cartilage matrix generation-related genes and reduce the transcription of hypertrophy-related genes significantly.Furthermore,the composite scaffolds promote chondrocytes to secrete extracellular matrix,which indicated that Stur-ECM/SF composite scaffold can be potentially applied in clinical treating cartilage defects via the CTE approach to avoid the risk of chondrocyte hypertrophy.In conclusion,the ginsenoside Rg1 modified PHBV fiber scaffold,HL9 modified hyaluronic acid /silk fibroin composite scaffold and decellularized sturgeon extracellular matrix /silk fibroin composite scaffold were developed and well characterized in this thesis.Results showed that functional scaffolds developed in this study can protect chondrocytes under pathological environment,which were expected to be applied to repair cartilage injury caused by degenerated diseases.The biological mechanisms of which were further clarified to build the theoretical foundation for developing functional scaffold applied in CTE under pathological microenvironment.