Gas-foamed And Electrosprayed Threedimensional Nanofiber Scaffolds For The Reparation Of Articular Cartilage Injuries

Articular cartilage injuries are extremely prevalent in clinics and are a major cause of limb disability.Trauma or degenerative disease often leads to progressive degeneration of intra-articular tissues,causing joint pain,functional impairment and arthritis.However,articular cartilage has no blood vessels or lymphatic vessels with limited ability to heal itself,and chondrocytes are a mature,highly differentiated cell type with a very slow renewal of the extracellular matrix（ECM）.In addition,inflammation is an important factor in the progression of joint disease,and without effective treatment,inflammation can lead to deterioration of the joint environment.Current therapeutic approaches for articular cartilage injuries are faced with the problem that regenerated cartilage tissue is biochemically and biomechanically inferior to natural hyaline articular cartilage.Therefore,the treatment of joint disease is still one of the challenges facing clinicians.The development of new techniques to effectively counteract cartilage damage and alleviate joint inflammation is important for the clinical management of joint disease.Tissue engineering-based scaffolds to repair articular cartilage provide new strategies for the treatment of joint damage.Electrospinning,a versatile technique for constructing ECM mimetic nanofiber scaffolds,is widely used in the field of tissue engineering.However,the tightly-packed structure of normal two-dimensional（2D）electrospun scaffolds is not conducive to cell infiltration,so it is necessary to improve the 2D electrospun membranes with high porosity,suitable biomechanical properties and considerable bioactivity to achieve the repair and regeneration of articular cartilage.Therefore,to address the current challenges in cartilage tissue engineering scaffolds such as limited cell infiltration,insufficient bioactivity and lack of injectability,current dissertation proposed the construction of porous three-dimensional（3D）nanofiber scaffolds by using gas foaming and electrospraying technology,and chemically modify the 3D nanofiber scaffolds with natural cartilage ECM composition and metal phenolic networks（MPNs）to explore the functions of 3D scaffolds in cartilage injury repair and joint inflammation relief.The main research content can be divided into four parts as follows:（1）Although electrospun nanofibers can emulate ECM architecture;however,small pore size and tightly-packed structure impede cell infiltration.In this study,poly（L-lactide-ε-caprolactone）/silk fibroin（PLCL/SF）nanofiber membrane（2DNFS）was prepared by electrospinning,and then we exploited in situ gas foaming to obtain 3D nanofiber scaffold（3DNFS）using aqueous sodium borohydride（Na BH₄）as the foaming agent.The addition of SF improved the hydrophilicity of the scaffold.The gas foaming scaffolds exhibited a multilayered structure,and the porosity increased from 67.5±4.8%for 2DNFS to 84.1%±4.1%for 3DNFS.3DNFS exhibited faster cell proliferation and cell growth into the scaffolds than that of 2DNFS.Subcutaneous implantation results showed that 3DNFS promoted cell infiltration,collagen infiltration and neo-vessel regeneration,and also reduced the thickness of the fibrous capsule.After 2 weeks of subcutaneous implantation,3DNFS exhibited a higher CD163⁺（M2 phase macrophages）/CCR7⁺（M1 phase macrophages）cell ratio compared to2DNFS,indicating that the gas foamed scaffolds promoted the polarization of macrophages to M2 phenotype and accelerated tissue regeneration.（2）Based on the advantages of gas foamed scaffolds in promoting cell infiltration and tissue repair,the gas foamed scaffolds were chemically modified to promote their bioactivity,and exploited for cartilage tissue engineering.2D PLCL/SF scaffolds（2DS）were fabricated by a dynamic liquid support electrospinning system,then modified with hyaluronic acid（HA）,followed by gas foaming and freeze-drying to successfully afford HA modified 3D PLCL/SF scaffolds（3DHAS）.Compared with the gas foamed scaffold without HA（3DS）,3DHAS exhibited a multi-layered structure with a honeycomb networks.3DHAS also demonstrated a more regular shape and higher compression modulus than 3DS.In vitro cytocompatibility experiments revealed that 3DHAS showed faster proliferation of chondrocytes compared to other scaffolds,and upregulated collagen type II（COLⅡ）and aggrecan（ACAN）gene expression of chondrocytes.Histological analysis of cell-scaffold constructs explanted 8 weeks after subcutaneous implantation in nude mice showed that both 3DS and 3DHAS scaffolds formed cartilage-like tissue,but 3DHAS were superior in promoting chondrocyte maturation and matrix secretion.In addition,3DHAS showed well-integrated boundary between scaffolds and the host tissues after implantation in the full-thickness articular cartilage defect model in vivo.3DHAS also promoted the secretion of COLⅡand reduced the formation of collagen type I（COLⅠ）.（3）From the perspective of introducing anti-inflammatory biochemical signals into the gas foamed scaffold,a chondroitin sulfate（CS）-functionalized gas foamed scaffold was proposed in this study,which was expected to relieve inflammation at joint injury sites and promote cartilage tissue regeneration.2D PLCL/SF scaffolds（2DS）were prepared by conjugated electrospinning,and then modified with CS to further enhance their bioactivity.Subsequently,3D PLCL/SF scaffolds（3DS）and CS modified 3D scaffolds（3DCSS）with tailored size were successfully fabricated by in-situ gas foaming in a confined mold followed by freeze-drying.Gas-foamed scaffolds displayed rapid water absorption and stable mechanical properties.3DCSS promoted chondrocytes proliferation and infiltration,and showed better chondroprotective effects compared to other scaffolds.In the interleukin-1β（IL-1β）induced inflammatory condition,3DCSS reduced the expression of IL-1β,tumor necrosis factor-α（TNF-α）and matrix metalloproteinases-13（MMP13）genes of chondrocytes.Besides,3DCSS scaffolds formed more mature cartilage-like tissues along with the best repair outcome in a rabbit articular cartilage defect model in vivo as well as reduced the expression of pro-inflammatory cytokines,including IL-1βand TNF-α.（4）Based on the above studies in cartilage tissue repair and inflammation relieving,in this study,injectable gelatin/poly（L-lactic acid）nanofiber microspheres（MS）were prepared via electrospraying technology,MS was further modified with tannic acid（TA）named TMS,or metal phenolic networks（MPNs）composed of TA and strontium ions(Sr²⁺)named TSMS.The potential of the different microspheres for osteoarthritis（OA）therapy was subsequently evaluated.All microspheres exhibited nanofibrous morphology and stable structure.Compared to TMS,TSMS exhibited better free radical scavenging ability and showed a sustained release of TA and Sr²⁺,whereas TMS exhibited a burst release of TA.TA-modified microspheres reduced cell apoptosis and the expression of TNF-α,MMP13,a disintegrin and metalloproteinase with thrombospondin motifs 5（ADAMTS5）,and tachykinin-1（TAC1）genes of chondrocytes in hydrogen peroxide（H₂O₂）-induced inflammatory environment.In addition,TSMS could promote the expression of cartilage matrix-related genes COLⅡand ACAN due to the release of Sr²⁺.In the OA rabbit model,TSMS showed considerable repair outcome by reducing intra-articular expression level of inflammatory cytokines（e.g.,TNF-α&IL-1β）and alleviating cartilage degeneration as well as promoting cartilage matrix secretion.In summary,in this study,PLCL/SF 3D nanofiber scaffolds were prepared by gas foaming method.Gas foamed scaffolds with high porosity and multilayered structure can promote cell infiltration and accelerate tissue regeneration.Based on the advantages of gas foamed scaffolds in tissue regeneration,HA and CS were incorporated with 3D scaffolds to improve the mechanical stability and bioactivity of the scaffolds for considerable cartilage regeneration.Finally,injectable nanofiber microspheres were prepared by electrospraying and modified with MPNs for OA treatment.The mechanism of functional injectable microspheres in alleviating OA was also explored.These studies enriched the strategies of constructing functional tissue engineering scaffolds,and provided new ideas for the fabrication of biomimetic scaffold for cartilage tissue regeneration as well as proposed an injectable biomaterial scaffold for OA treatment,which served as a valuable guidance for facilitating the application and translation of 3D engineered nanofiber scaffolds in clinics.