Fabrication Of Injectable Hydrogels Based On Chondroitin Sulfate And Their Application As Bone Repair Scaffolds

In recent years,injectable hydrogel as bone tissue engineering scaffold for bone repair has become a hot spot in tissue regeneration medicine field.Injectable hydrogel as biomedical carrier material has many advantages,such as its structure mimics the extracellular matrix(ECM),can be applied via the minimally invasive injection method,and the hydrogel precursor solution could be filled in irregular tissue defects,etc.However,there still exist some problems need to be solved for injectable hydrogel that applied in bone repair study.For instance,the gelation time is not easy to be controlled,the mechanical strength is insufficient,and the water absorption capacity is limited.To address the problems,in the study,natural polysaccharide chondroitin sulfate(ChS)was chosen as the base material and a series dual/triple crosslinked injectable hydrogels were fabricated via Diels-Alder(DA)click covalent crosslinking with other noncovalent crosslinking or dynamic covalent crosslinking.The structure of polymers was characterized by nuclear magnetic resonance(NMR)spectrometer and Fourier transform infrared(FTIR)spectrometer.The gelation time of the injectable hydrogels was determined by the inversion method and the rheometer.The inner morphology of the injectable hydrogels was observed by scanning electron microscopy(SEM).The mechanical properties of the injectable hydrogels were investigated by a dynamic thermomechanical analyzer(DMA).The water absorption capacity of the injectable hydrogels was determined by weighing method.In vitro cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT)assay.The degradation behavior of the injectable hydrogels was investigated by in vitro and in vivo degradation experiments.Finally,the feasibility of the injectable hydrogels as bone scaffolds was confirmed through animal experiments.The detailed research contents and the results are as follows:1.Thermosensitive dendrimer supramolecular PAMAM@PNIPAM was synthesized via hostguest recognition.Macromolecular diene based on ChS(ChS-F)and macromolecular dienophile crosslinker based on PEG(PEG-AMI)were synthesized via EDC and DCC condensation,respectively.A dual crosslinked injectable hydrogel by noncovalent crosslinking and DA click covalent crosslinking was fabricated based on the three synthesized polymers.The effects of the two kind crosslinking methods on the gelation time,mechanical strength and degradation performance of injectable hydrogel were investigated.The inner morphology was observed using SEM.In vitro cytotoxicity was tested by MTT method.The effect of the dual crosslinked injectable hydrogel on bone repair was investigated by mice tibial defect repairing experiments.The study shows that the noncovalent crosslinking formed from the sol-gel phase transition of PAMAM@PNIPAM induced by thermo stimuli and the covalent crosslinking formed from the DA click reaction between ChS-F with PEG-AMI synergistically make the injectable hydrogel with both high water content(98 %)and mechanical strength(25 MPa).The hydrogel could induce bone growth under the condition that no cells or growth factors were encapsulated.2.Dienophile F127-AMI was synthesized based on another thermosensitive polymer pluronic F127.Thermosensitive copolymer F127@ChS was synthesized via in vitro DA click reaction between F127-AMI and macromolecular diene.Thermosensitive behavior of F127@ChS was investigated via vial inversion method,rheometer and differential scanning calorimeter(DSC).The morphology of F127@ChS was observed using transmission electron microscopy(TEM).A series dual crosslinked injectable hydrogels based on F127@ChS-PEG were fabricated.The effect of PEG-AMI crosslinker amount on water absorption,inner morphology and mechanical strength of the injectable hydrogels was investigated.Inner morphology of the injectable hydrogels was observed using SEM.In vitro cytotoxicity was tested by MTT method.Rat cranial bone defect model was established and bone morphogenetic protein(BMP-4)was encapsulated into the injectable hydrogel for bone repair.Bone regeneration effect was evaluated using X-ray imaging system and histopathology analysis.The study shows that the gelation concentration of F127@ChS is much lower than F127.The thermo induced sol-gel transition of F127@ChS forms the noncovalent crosslinking,while the DA click reaction between F127@ChS with PEG-AMI forms covalent crosslinking.The dual crosslinked injectable hydrogel can effectively encapsulate BMP-4 for bone repair.3.Based on the above work,a dual crosslinked injectable hydrogel based on F127@ChS-PEG was fabricated.Inner morphology of the injectable hydrogel was observed using SEM.Water absorption of the injectable hydrogel was determined by weighing method.The effect of the DA click covalent crosslinking by F127-AMI crosslinker was investigated using a rheometer.In vitro cytotoxicity was tested by MTT method.Rat cranial bone defect model was established and BMP-4 was encapsulated into the injectable hydrogel for bone repair.Bone regeneration effect was evaluated using X-ray imaging system and histopathology analysis.The study shows that compared to F127@ChS-PEG dual crosslinked injectable hydrogel,the fabricated F127@ChS-F127 injectable hydrogel has higher mechanical strength,and the bone repair effect is better.4.Based on the above works,copolymer F127@ChS/furan/ADH containing furan and hydrazide groups was synthesized based on F127@ChS.ChS that containing aldehyde groups(OChS)was synthesized via sodium periodate oxidation.F127@ChS-PEG-OChS injectable hydrogel based on noncovalent crosslinking,DA click covalent crosslinking and dynamic acylhydrazone bond crosslinking was fabricated via a double barred syringe.The effect of different crosslinking method on water absorption capacity,mechanical strength,self-healing and rheological properties was systematically investigated.The degradation behavior of the injectable hydrogel was determined both in vitro and in vivo.In vitro cytotoxicity was tested by MTT method.Rat cranial bone defect model was established and BMP-4 was encapsulated into the injectable hydrogel for bone repair.Bone regeneration effect was evaluated using X-ray imaging system and histopathology analysis.The study shows that the physiochemical properties of the injectable hydrogel could be controlled through regulating the crosslinking method.The introduction of acylhydrazone bond crosslinking afforded the injectable hydrogel self-healing ability,and the F127@ChS-PEG-OChS injectable hydrogel is competent as a bone repair scaffold.5.Based on the above works,we selected another polysaccharide alginate sodium(SA)to synthesize polymers than containing hydrazide group(SA-ADH)and aldehyde group(OSA).A triple crosslinked injectable hydrogel based on noncovalent crosslinking,DA click covalent crosslinking and dynamic acylhydrazone bond crosslinking was fabricated via a double barred syringe.The introduction of SA by dynamic acylhydrazone bond crosslinking on water absorption,mechanical strength and degradation behavior was investigated.Inner morphology of the injectable hydrogel was observed using SEM.Self-healing behavior of the injectable hydrogel was investigated through macroscopic observation.In vitro cytotoxicity was tested by MTT method.Rat cranial bone defect model was established and degradable inorganic Bio-glass(BG)was encapsulated into the injectable hydrogel for bone repair.Bone regeneration effect was evaluated using X-ray imaging system and histopathology analysis.The study shows that the introduction of SA by dynamic acylhydrazone bond crosslinking decreased the water absorption ability,but the mechanical strength was increased and degradation time was prolonged.Animal experiments indicated the triple crosslinked injectable hydrogel with BG encapsulated is favor for bone regeneration.