Chitosan-based Thermosensitive Composite Gels And Their Applications In Bone Tissue Engineering

Polymer hydrogel is a type of three-dimensional network material with high water content.A variety of synthetic polymers and natural polymers have been used in the preparation of hydrogels so far and many gels have been used in the diverse biomedical field.Nowadays,various kinds of naturally sourced hydrogels have attracted increasing attention in the repair and reconstruction of skin,nerve,cartilage and bone tissue since they have good biocompatibility and porous structure,enabling them to be suitable for cell adhesion,growth,migration,and transport of nutrients,and thus,to mimic the extracellular matrix.Some of them have now shown clinical transformation potential.Nevertheless,desirable hydrogels that are made from biodegradable and biocompatible natural polymers while meeting the different requirements involving varied structures,properties and functions for in vivo applications are still few.Typically,many hydrogels produced with natural polysaccharide or protein materials often show poor mechanical properties,rapid degradation,and insufficient functions in vivo.Therefore,there are pressing needs to significantly improve the mechanical properties,enhance the degradation tolerance and increase the versatility of these natural polymer hydrogels while making the most of their advantages.Chitosan/glycophosphate?GP?composite can form into a type of thermosensitive hydrogel in vivo with good safety.When used for tissue repair,chitosan/GP hydrogel usually shows low strength and rapid degradation,which severely limits its applications.In this study,some efforts were made to improve its mechanical properties,degradation resistance and functionality by combining newly synthesized materials.Some dextran-polylactide?Dex-PLA?copolymers were synthesized and the optimal Dex-PLA was used together with chitosan/GP to improve mechanical properties and degradation resistance of the resulting gels.Another effort was made to endow the gel with certain features such as osteoinductivity,osteoconductivity and in-situ mineralization,silk fibroin and hydroxyapatite were combined with chitosan/GP for developing a new type of injectable gel.In addition,copper-containing bioactive glass nanoparticles were also embedded into chitosan/silk fibroin/GP for preparing a new type of composite gel with increased strength and enhanced degradation resistance while showing osteogenic and angiogenic activity.Based on the injectability and thermosensitivity of chitosan/GP hydrogel,these newly developed novel composite gels can be still injectable while gelling under physiological temperature and pH.These composite gels have been shown to have good safety and can effectively repair bone defects,evidenced by in vitro and in vivo experimental results.Major investigations conducted in the present study were briefly descrined as follows.?1?Preparation and characterization of thermosensitive chitosan/dextran-polylactide/glycerophosphate composite hydrogelPolylactic acid oligomers?PLA?were synthesized and grafted onto the dextran?Dex?backbone by a coupling reaction to obtain a series of different dextran-PLA?Dex-PLA?copolymers.The Dex-PLA with a PLA content of about 50 wt%was selected for the preparation of chitosan/Dex-PLA/GP hydrogel.The optimized ternary composite solutions were found to show the ability to form hydrogel near physiological pH and temperature.Rheological measurements confirmed that the Dex-PLA content in the gel had a certain effect on the initial gelling temperature and gelling time,and the elastic modulus of gels increased significantly with the increase of Dex-PLA content.Compression measurement results showed both E and?₁₀ of chitosan/Dex-PLA/GP gels were around 8 times higher than that of the chitosan/GP gel,respectively.In addition,it was found that in vivo degradation rate of chitosan/Dex-PLA/GP gel became significantly slower when compared with that for chitosan/GP.Thus,a new type of gel with strong mechanical strength and resistance to degradation was obtained.?2?Preparation and characterization of thermosensitive chitosan/silk fibroin/hydroxyapatite/glycerophosphate composite hydrogelSilk fibroin?SF?and nanohydroxyapatite?HA?were combined with chitosan?CH?/GP composite to prepare a series of chitosan/silk fibroin/nanohydroxyapatite/GP?CH/SF/HA/GP?composite gels to impart the resulting gels with improved strength and enhanced degradation resistance while having osteoinductive and osteoconductive nature.These gels were able to gelatinize at physiological temperatures and pH.SEM results showed that CH/SF/HA/GP gels had three-dimensional porous structure with interconnected features.In comparison to the CH/GP gel,the rheological and mechanical measurements reveal that the elastic modulus of the optimized CH/SF/HA/GP gel could be increased by more than 80 times,and the matched compressive modulus and?₁₀ were increased by about 20 times,respectively.The results obtained from in vivo degradation testing showed that the weight retention of the optimized CH/SF/HA/GP gel was higher than 70%after 7 weeks of in vivo degradation.These composite gels have significantly improved mechanical properties and degradation resistance,and can be used as a type of injectable scaffold material for bone repair and reconstruction.?3?Preparation and characterization of chitosan-based hydrogel loaded with copper-containing bioactive glassThe composite gels were prepared by embedding copper-containing bioactive glass nanoparticles into chitosan-based gel to endue the resulting gels with osteogenic and angiogenic activity.Bioactive glass nanoparticles?BG NP?and copper-containing bioactive glass nanoparticles?Cu-BG NPs?were first synthesized by a modified St?ber method.The obtained Cu-BG NPs were combined with the CH/SF/GP gel to prepare functionalized Cu-BG/CH/SF/GP composite gels.SEM images showed that the composite gel had well-defined three-dimensional network structure.Rheological results confirm that these gels had shear-thinning features,and their elastic modulus and the viscous modulus increased with the increasing amount of the incorporated Cu-BG NPs.Silicon ions,calcium ions and copper ions were found to be released in sustained manners and the concentration of Cu ions released from the optimal gel showed a nearly linear trend.After being treated in a simulated body fluid for a period of 4 weeks,the optimal composite gel had a large amount of cauliflower-like apatite deposited on its surface.It was found that the composite gel well supported the adhesion,growth and proliferation of seeded MC3T3-E1 cells.Results obtained from q-PCR analysis and Western blotting confirmed that the optimized composite gel could significantly promote the up-regulation of related angiogenesis genes and increase the synthesis of vascular endothelial growth factor.Based on the standard model of rat calvarial bone defects,it was found that the optimized composite gel showed abilities to completely repair the defect with formation of neovascularizated bone tissues during about 8 weeks without using any seed cells.These results demonstrate that the newly developed composite gels have promising potential for bone repair and reconstruction.