Characterization Of Gelatin-hydroxyapatite Composite Cryogel Material For Bone Regeneration

In bone tissue engineering,the mechanical strength of traditional hydrogel materials is usually insufficient to meet the requirements of bone defect repair,which prompts people to actively develop new gel-based materials.Among them,high-strength cryogel is attractive and considered as a suitable candidate material.Cryogel is a kind of high-strength three-dimensional(3D)macroporous scaffold,which is made from a crosslinkable hydrophilic polymer solution with the molecules in the solution being arranged in an order manner before the occurrence of cross-linking.Gelatin,a product of partial hydrolysis of collagen,has high chemical activity and excellent biological properties.And it is widely used in the field of bone repair.In view of the organic-inorganic compositions of natural bone,hydroxyapatite(HA)is not only the preferred inorganic component for obtaining bionic natural bone composition,but also an excellent reinforcement for further improving the mechanical properties of cryogels.Compared with HA nanoparticles,one-dimensional HA nanowires are theoretically a reinforcing material with more significant effects benefiting from their bridging ability and network-forming ability.In addition,if the interaction between the HA nanoparticles and the polymer matrix can be enhanced by chemical bonding,the mechanical stability of the gelatin-HA composite can also be effectively improved.Therefore,in this study,three kinds of HA(HA nanorods,HA nanowires and double bond modified HA nanorods)were prepared,and combined with methacrylated gelatin,gelatin-HA composite cryogels with different amounts of HA were prepared and comprehensively characterized in terms of pore morphology,swelling ratio,degradation profile,mechanical property,biocompatibility and osteogenic performance to evaluate the feasibility in developing suitable gelatin-HA cryogel scaffolds for bone regeneration.The main work is as follows:(1)Methacrylated gelatin(GelMA)was prepared by the reaction of gelatin and methacrylic anhydride,and the ratio of double bond substitution was?80%determined by formaldehyde titration.(2)HA nanorods(HANRs)with a length of about 100-150 nm and a width of about 20-30 nm were prepared by chemical precipitation method and XRD results showed that the material had a good crystal form.Gelatin cryogels(CNR)with different HANRs contents were successfully prepared by freeze-drying and UV curing techniques,and the pure GelMA cryogel was used as control.Through a series of characterization,it was found that all the obtained cryogels had interconnected porous structure,water retention,shape memory properties and rapid resilience.With the increase of HANRs content,the mechanical strengthes of the composite cryogels were significantly improved,but the crosslinking density was decreased,and the degradation rate was also accelerated.The in vitro biological evaluation results showed that the composite cryogels had good biocompatibility and could support the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs),and the ability of promoting bone differentiation was positively correlated with the content of HANRs.(3)HA nanowires(HANWs)were prepared by hydrothermal method,and the aspect ratio was larger than 80,which could be compared with HANRs to improve the mechanical stability of gelatin cryogels.Compared with the performance of GelMA-HANRs(CNR)cryogels at the same HA content,HANWs were distributed along the pore wall in the GelMA-HANWs(CNW)cryogels and the CNW cryogels had smaller pore sizes and swelling ratios,exhibiting shape memory properties and rapid resilience.Comparing the results of compression test and rheological analysis,it was found that the mechanical properties of CNW composite cryogels were slightly better than those of CNR cryogels and the difference between the two kinds of cryogels was more significant at higher HA content(e.g.50 wt%);CNW composite cryogels also had good biocompatibility and could support adhesion,proliferation of rBMSCs and promote osteogenic differentiation,and the three-dimensional porous structure of cryogels were conducive to cell migration and growth.(4)The double-bonded HANRs(MHANRs),modified by double-bonded disodium pamidronate by chemical precipitation,could maintain the original morphology and crystal form of HANRs.MHANRs could form chemical bonds with GelMA under the ultraviolet light.Compared with the properties of CNR cryogels,due to the increase of crosslinking density,GelMA-MHANRs(CMNR)cryogels had smaller pore size and swelling ratio,and their degradation rates were slowed down,while their mechanical properties were improved.CMNR composite cryogels had good biocompatibility and facilitate bone formation ability.In summary,HA can be used as an inorganic reinforcement to be filled into gelatin cryogels.Compared with HANRs,one-dimensional HANWs with a certain aspect ratio play a bridging role in the gelatin matrix,which can enhance the mechanical strength and structure stability of composite cryogels;MHANRs with double bonds,improves the crosslink density of the composite cryogels through chemical bonding with GelMA,which is more conducive to improving the mechanical stability of gelatin-HA composite cryogels.GelMA-HA composite cryogels with interconnected macroporous structure have excellent biocompatibility and cell affinity,and have the ability to promote osteogenic differentiation.As a kind of good bone repair material,it is expected to have broad prospects in the research and application of bone tissue engineering.