Influence Of Physical And Chemical Properties Of Thermosensitive Hydrogel Scaffold On In Situ Cartilage Repair And Ectopic Cartilage Tissue Regeneration

Successful cartilage tissue regeneration requires scaffolds with particular mechanical stability,biodegradability,good biocompatibility,appropriate size and porosity to provide a desirable microenvironment for the sufficient cell-cell interaction,cell migration,proliferation,and differentiation.Hydrogel,as a kind of three-dimensional scaffold,can swell and keep a certain amount of water with similar biochemical properties as biological tissue.Thermosensitive hydrogel can finish liquid-gel transition with the change of the temperature,and polypeptide,gene,protein,cell and small molecule drugs can be evenly mixed with gel solution.These properties make thermosensitive hydrogel a broad application in the field of tissue engineering.However,the composition,gel transformation,pore structure,mechanical properties,degradation rate of hydrogel are closely associated with cartilage regeneration,and researches on these aspects are desired especially in vivo function process.In this paper,thermosensitive hydrogels based on poly（amino acid）polylactide（PLA）were synthesized,and modified by phenylalanine and cholesterol,respectively.Influences of modifications on the gelation behavior,mechanical properties,pore structure,the degradation behavior as well as the cartilage tissue regeneration were investigated.Section 1.Component effect of stem cell-loaded thermosensitive polypeptide hydrogels on cartilage repairThermosensitive hydrogels based on polypeptides possess the advantage that there is no acid accumulation during the degradation of the polymers.Additionally,the polyalanine-based thermosensitive hydrogel,as three-dimensional culture matrix of chondrocytes,was demonstrated possessing the improved capacity of cell proliferation and biomarker expression of articular cartilage,such as collagen type II（Col II）and sulfated glycosaminoglycans（GAGs）.However,as a physical crosslinked hydrgel,poor mechanical properties limited the effect of cartilage repair.To build a cartilage tissue engineering scaffold with appropriate pore structure and mechanical properties,we introduced a hydrophobic amino acid,namely phenylalanine,intopolyalanine-basedthermosensitivehydrogel.The poly（L-alanine）-block-poly（ethylene glycol）-block-poly（L-alanine）(PA-PEG-PA,EG₉₁A₃₈)andpoly（L-alanine-co-L-phenylalanine）-block-poly（ethylene glycol）-block-poly（L-alanine-co-L-phenylalanine）(PAF-PEG-PAF,EG₉₁A₂₈F₄ and EG₉₁A₂₄F₉)triblock copolymers were successfully synthesized by the ring-opening polymerization of NCA monomers.The sol-to-gel transition temperature and the critical gelling concentration decreased as the increased number of phenylalanine groups.Meanwhile,the synthetic copolymers were detected possessing different gelation,pore size,and mechanical strength,which further influenced the degradation,and cell adhesion and proliferation.Neo-cartilage at 12 weeks post-implantation generated by EG₉₁A₂₄F₉ and EG₉₁A₂₈F₄ hydrogels carrying bone marrow mesenchymal stem cells（BMMSCs）possessed higher levels of GAGs and Col II,and lower levels of collagen type I than that of the EG₉₁A₃₈ and control groups.The results indicated that larger pore sizes and enhanced mechanical strength mediated by higher phenylalanine content could not only promote proliferation of BMMSCs in vitro,but also facilitate hyaline-like cartilage regeneration with reduced fibrous tissue formation in vivo.The thermosensitive PAF-PEG-PAF hydrogels may be promising injectable scaffolds for cartilage tissue engineering.Section 2.Injectable stereocomplex polylactide/cholesterol stereocomplex thermogel for cartilage tissue engineeringPLA is a biodegradable and biocompatible polymer,which is widely used in the realms of tissue engineering and drug delivery.The stereoselective interaction between two complementary enantiomeric poly（_L-lactide）（LLA）and poly（_D-lactide）（DLA）can enhance physical properties such as mechanical properties,thermal resistance and hydrolytic stability compared with the parent polymers.Cholesterol,which heterogeneously distributes in cellular membranes,has good thermodynamic affinity for the cell membrane and the ability to insert into phospholipid membranes regulating the structure and dynamic behavior for improving cell attachment.Moreover,the modification of poly（ethylene glycol）-block-polylactide（PEG-PLA）with cholesterol has been verified to enhance the structural stability and improve the stability of the corresponding assemblies.Furthermore,the hydrgoels formed by cholesterol functionalize PEG-PLA has been demonstrated to improve the chondrocyte proliferation and GAGs production in terms of chondrocyte culture.In this study,the copolymers of 4-arms poly（ethylene glycol）-poly（_L-lactide）（4-arms PEG-PLLA）,4-arms poly（ethylene glycol）-poly（_D-lactide）（4-arms PEG-PDLA）were fabricated by ring opening polymerization of LLA or DLA with Sn（Oct）₂ as catalyst,and the cholesterol modificated 4-arms PEG-PLLA（4-arms PEG-PLLA/Chol）and 4-arms PEG-PDLA（4-arms PEG-PDLA/Chol）were fabricated by condensation reaction with cholesterol chloroformate.The enantiomeric copolymers were assembled into scPLA and scPLA/Chol through stereocomplex.While,the scPLA/Chol_gel was demonstrated possessing enhanced mechanical property,enlarged pore size and more permanent degradability compared with scPLA_gel.Meanwhile,the modification of cholesterol into PEG-PLA thermogel realized improved chondrocytes adhesion.Contributed to those features,the morphology,cartilaginous specific matrix and biomechanical property as well as cartilaginous gene expressions of engineered cartilage from PLA/Chol_gel were superior to those of scPLA_gel,indicating the great potential of PLA/Chol_gel as cell carrier for cartilage tissue engineering.