Fabrication And Properties Of Self-reinforced Hyaluronan Hydrogel With A Double-crosslinked Network

Hydrogel is a three-dimensional polymer network containing a large number of water.Hyaluronan （HA） hydrogel has recently been much concerned in biomedical fileds because of itsinherent biocompatibility, high water adsorption, injectability and mimicking the elastic structureof natural extracellular matrix. However, the application of conventianl HA hydrogel has beenalso limited in a certain extend for its poor mechanical properties and uncontrollable releasingbiomacromolecules. Therefore, in this work, a self-reinforced HA hydrogel with a doublecrosslink network was newly developed. HA microgels with varying crosslink densties （firstcrosslink） were firstly prepared via a inverse emulsion polymerization technique, followed byglycidylmethacrylation to introduce functional double bonds to the microgels. Then, aself-reinforced HA hydrogel with a double crosslink network was built under ultravioletradiation using glycidyl methacrylated HA as the matrix phase and glycidyl methacrylated HAmicrogels as the reinforcement phase, where valent bonding between matrix phase andreinforcement phase was formed. Various analytical methods, including¹H nuclear magneticresonance spectroscopy （¹H NMR）, dynamic laser scattering （DLS）, scanning electronmicroscope （SEM）, transmission electron microscope （TEM）, water swelling test, rheologicalproperties, enzymatic degradation, bovine serum albumin （BSA） loading and delivery, and cellviability, were used to investigated the effects of crosslink density and the mass ratio ofreinforcement phase and matrix phase on the morphology, water swelling ratio, mechanicalproperty, degradability, protein loading and release, as well as the cytocompatibility of thedouble crosslinked HA hydrogel.In the study of matrix phase, the¹H NMR results showed that the increase ofglycidylmethacrylate feeding raised the glycidylmethacrylation degree of HA molecular chain soas to enhance the crosslink density and mechanical properties as well. In the study ofreinforcement phase, for all the particles（HGPs）, spherical shape with smooth surface wasobtained by SEM and TEM analyses, which was independent of the crosslink density. DLSanalysis indicated that before swelling in water, the crosslink density had little influence on themicrogel size and distribution （about1⁷μm）, whereas after being swollen in water the increaseof crosslink density dramatically decrease the water swelling ratio, enzymatic degradation rate aswell as the BSA delivery profile. Based on these studies, a series of double crosslink HA hydrogels （DCNs） were constructed by tailoring the feeding ratio and the crosslink density ofmatrix phase and reinforcement phase. By SEM observation, all DCNs hydrogels displayedinterconnected porous structure with an approximate pore size of20-50μm, and HGPsdistributing in the pores covalently connected with the matrix. The water swelling test revealedthat DCNs-GMA50-HGPs1.5exhibited the lowest swelling ratio of21.64±0.79which was muchlower than38.18±2.33of HA bulk gel. The rheological analysis showed that compared with HAbulk gel, the mechanical properties of DCNs hydrogels were significantly improved, especiallyfor DCNs-GMA50-HGPs1.5possessing the highest storage modulus of5610Pa as opposed tothe lowest storage modulus of1560Pa for HA bulk gel. The loading capacity and deliveryprofile of BSA implied that all DCNs hydrogels presented a high BSA loading ability of over381±41μg.mg^-1and a slower delivery profile than HA bulk gel. Among all these samples,DCNs-GMA50-HGPs1.5exhibited the longest sustained delivery profile. Mouse fibroblast cellculture confirmed a high cellular viability in all HA-based hydrogels, indicating a goodcytocompatibility of these hydrogel samples. In a word, these results demonstrate that DCNshydrogels not only offer unique structural hierarchy, strong mechanical properties and goodcytocompatibility, but also prolong the sustained delivery of protein, will be a promising scaffoldmaterial for soft tissue engineering.