Polysaccharides/Human-like Collagen Injectable Hydrogels:their Microstructure,Engineering Process And Biocompatibility

New locally injectable biomaterials that are suitable for use as soft tissue fillers are needed to address a significant unmet medical need.In this study,we used pullulan and human-like collagen(HLC)-based hydrogels with various molecular weights(MWs)in combination therapy against tissue defects and facial depressions.Briefly,pullulan was crosslinked with NaIO4 to form a pullulan hydrogel and then coupled with HLC using the reaction between the-NH2 end-group of HLC and the-CHO group present on the aldehyde pullulan to form the collagen/pullulan hydrogel,wherein the NaIO4(0.5 mol/1)acted as the crosslinking and oxidizing agent.The good miscibility of pullulan and HLC in the hydrogels was confirmed via Fourier transform infrared spectroscopy(FT-IR),scanning electron microscopy(SEM),compression testing,enzyme degradation testing,cell adhesions and proliferation and subcutaneous filling assays.Limitations of the pullulan hydrogels included inflammation,poor mechanical strength,and degradation.By contrast,the properties of the collagen/pullulan hydrogels strongly enhanced and stabilized facial depression repair.Here,pullulan-based hydrogels with various MWs were fabricated and physicochemically characterized.The efficacy of these hydrogels was evaluated both in vitro and in vivo.Our results indicate that collagen/pullulan hydrogels may have therapeutic value as efficient soft tissue fillers,with reduced inflammation,improved cell proliferation and delayed hydrogel degradation.Therefore,improve the degradation time and anti-inflammation,a new crosslinking agent was choose to fabricate hydrgels.In this study,we designed multifunctionalized hydrogel scaffolds and injectable particles based on high-molecular-weight(MW)pullulan and human-like collagen(HLC)crosslinked with 1,4-butanediol diglycidyl ether(BDDE)for combination therapy tissue restoration.The properties of the pullulan/BDDE(PB)and pullulan/BDDE/human-like collagen(PBH)hydrogels were characterized via swelling ratio measurements,mechanical tests,and enzymatic degradation in vitro and via subcutaneous injections in vivo.The results demonstrate that the dry hydrogels completely returned to their original state in deionized water.The elastic modulus of the PBH53 dry hydrogels is higher than that of the other hydrogels after exposure to bending stress and compression stress.In addition,the in vitro transfection efficiency and cell adhesion of the PBH hydrogels were exhibited superior fibroblast morphology without high levels of cell death much better than those of the PB hydrogels.In vivo,PB and PBH particles with good biocompatibility and anti-biodegradation were successfully prepared via granulation of wet PB and PBH hydrogels for efficient subcutaneous injection in Kunming mice and New Zealand rabbits.Therefore,the PB and PBH hydrogels were found to be acceptable,safe,soft materials for use in skin restoration,cartilage treatment,and lacrimal dryness therapy.Novel hydrogels based on carboxyl pullulan(PC)and human-like collagen(HLC)crosslinking with 1,4-butanediol diglycidyl ether(BDDE),with their highly tunable properties,are promising soft fillers for tissue engineering.Recent studies,however,have shown that incorporating hyaluronic acid and BDDE results in hydrogels with a microporous structure,a large pore size and high porosity that reduces the cell adhesion and improve the degradation in vivo.To better improve the biocompatibility and prevent biodegradation,the use of carboxyl pullulan to replace hyaluronic acid to fabricate PC/BDDE(PCB)and PC/BDDE/HLC(PCBH)hydrogels was investigated.It is a most promising strategy to prepare gels with carboxyl pullulan owing to its high reactivity,superb selectivity,and mild reaction conditions.The Schiff base reaction of HLC and PC produces the novel functional group RCHONR' in PCBH hydrogels.After 24 weeks of PCB and PCBH hydrogels subcutaneous injection in mice,the hydrogls-tissue inflammation,enzymatic-response and cell attachement was excellent than hyaluronic acid hydrogels.The biocompatibility,cytotocompatibility and anti-biodegradble of PCBH were milder than PCB hydrogels in vivo and in vitro.These results show that the proposed carboxyl pullulan and HLC composition for the fabrication of hydrogels is a promising soft filler for tissue engineering.The CS-HLC-HA/?-GP(chitosan-human like collagen-hyaluronic acid/?-sodium glycerophosphate)hydrogels were prepared based on the self-assembly of the CS-HLC-HA(CCA)fibres.The effects of fibres on the synthesis,characteristics and biomedical applications of the CS-HLC-HA/?-GP(CCAG)hydrogels were studied at different HA contents.The mechanism of the synthesis of the novel CCAG hydrogel was explored by Fourier transform infrared spectroscopy(FT-IR)and X-ray photoelectron spectroscopy(XPS).The hydrogels were characterized by swelling test,gelling time and enzymatic treatments.The results showed that a new amide bond(-CONH)and-NRH2+.were formed.The gelling time and swelling behaviors were dependent on the intertwining,overlap and adsorption of the polymer chains at various temperatures and pH.Furthermore,biomedical applications were evaluated by transmission electron microscopy(TEM),immunohistochemical analysis and haematoxylin and eosin(H&E)staining.The effect of fibres on the histocompatibility of hydrogels revealed that the fibres inside the hydrogel pores reduced the quantity of macrophages,decreased the degree of inflammation,and improved the anti-degradation of the modified hydrogels.This type of new hydrogels emerges an interesting injectable filling biomaterial for tissue engineering.