Preparation And Characterization Of The Biomimetic And Injectable Hydrogels For Cartilage Tissue Engineering

Cartilage tissue engineering?CTTE?provides a new treatment strategy for cartilage repair.The scaffold matrial play a very important role for CTTE.Recently,the CTTE scaffolds,including polymers and inorganic materials,were successfully applied cartilage regenaration.Specifically,for imitating key features of cartilaginous ECM,the biomimetic CTTE scaffold has become a hot research.In this dissertation,based on the summarization of previous research works of CTTE scaffolds,we finds that there are several difficulties lying for the CTTE scaffolds.Firstly,some scaffolds such as synthetic polymers and inorganic materials are not biodegradable and bioactive,and lack specific binding with cells or native tissue,which can not better integrate with the surrounding native tissue;Secondly,some scaffolds can not more effectively encapsulate and deliver desired cells or bioactive molecules to targeted sites of cartilage regeneration via a simple minimally invasive procedure,along with easy fill large and irregular complex defects;Finally,developing scaffolds empolyed no-biocompatible crosslinking approaches in harsh chemical conditions,and thus some potential cytotoxic molecules,like photosensitizers and catalytic agents are inevitably introduced,which are the major obstacles using the scaffold systems.The natural polysaccharides with rich resource and low price,resemble the macromolecular structure of tissue ECM,and thus are able to mimic many features of tissue ECM,and have the potential to direct cellular behaviour during tissue regeneration.They not only are excellently biocompatible and bioactive as well ascontrolled biodegradability,but also can easy introduce crosslinking segments or bioactive molecules that enhances combining capacity with the surrounding cartilage tissue.Therefore,the injetable hydrogel from the natural polysaccharides are able to solve the above difficulties.The major research work and conclusions are as follows:1.Preparation and characterization of the enzymatically crosslinked pullulan/ chondroitin sulfate injectable hydrogel;In this study,an enzymatically cross-linked injectable and biodegradable hydrogel system comprising carboxymethyl pullulan-tyramine?CMP-TA?and chondroitin sulfate-tyramine?CS-TA?conjugates was successfully developed under physiological conditions using horseradish peroxidase?HRP?as a catalyst and hydrogen peroxide?H₂O₂?as an oxidant for CTTE application.The HRP crosslinking method makes this injectable system feasible,minimally invasive and easily translatable for regenerative medicine applications.The physicochemical properties of the mechanically stable hydrogel system can be modulated by varying the weight ratio and concentration of polymer as well as the concentrations of crosslinking reagents.Specifically,the gelation rate and mechanical properties of hydrogels can be tuned independently through HRP and H₂O₂,respectively.In addition,the hydrogel has suitable gelation time,controlled mechanical property and biodegradation.2.Cell culture and in vivo study of the pullulan/chondroitin sulfate hydrogel;When chondrocytes were encapsulated into CMP-TA/CS-TA hydrogels,it was found that cellular functions of chondrocytes,including cell viability and proliferation,gene and protein expression levels of these markers?collagen type I,collagen type II and aggrecan?as well as accumulation of synthesis ECM?total collagen?,were remarkably affected by the content of CS-TA in the hydrogels.Furthermore,CS-TA-mediated microenvironment induced an enhanced cell proliferation,chondrogenetic differentiation and cartilaginous ECM accumulation compared to the CMP-TA hydrogel.In addition,in vivo testing confirmed that the hydrogels used in this system were well tolerated within a mouse subcutaneous implantation model.Overall,the injectable pullulan/chondroitin sulfate composite hydrogels presentedhere are expected to be useful biomaterial scaffold for regenerating cartilage tissue.3.Preparation and characterization of self-crosslinking and injectable hyaluronic acid/RGD-functionalized pectin hydrogel;In the present study,Initially,HA and pectin were chemically modified to bear hydrazide and aldehyde functional groups,obtaining the accelerated degradation of HA-ADH and PAD.The PAD was further modified with the synthetic oligopeptide G4 RGDS sequence though carbodiimide chemistry to promote integrin mediated cell adhesion.Subsequently,we developed a biomimetic injectable hydrogel system based on HA-ADH and PAD-RGD,in which their hydrazide and aldehyde-derivatives enable covalent hydrazone crosslinking of polysaccharides.The hydrazone crosslinking strategy is simple,while circumventing toxicity,making this injectable system feasible,minimally invasive and easily translatable for regenerative purposes.By varying the weight ratios and concentrations of HA-ADH/PAD-RGD,the physicochemical properties of the mechanically stable hydrogel system such as gelation time,mechanical properties and degradation behavior were easily adjustable.4.Cell culture and in vivo study of the hyaluronic acid/RGD-pectin hydrogel;When chondrocytes were encapsulated into the HA-ADH/PAD-RGD and HA-ADH/PAD hydrogels,it was found that cellular functions,including cell viability and proliferation,protein expression levels of these markers?collagen type II and aggrecan?as well as ECM accumulation?total collagen and GAGs?,were remarkably affected by HA/pectin composition and the presence of integrin binding moieties.Furthermore,the oligopeptide G4RGDS-mediated microenvironment within HA-ADH /PAD-RGD hydrogels might well maintain chondrocyte phenotype and increase biosynthesis of cartilaginous ECM.Additionally,the in vivo result confirmed that the HA-ADH/PAD-RGD hydrogels were tolerated within a mouse subcutaneous implantation model.We believe the as-prepared biomimetic injectable hydrogels based on polysaccharides are very promising scaffolds for CTTE application.