Preparation And Characterization Of Gelatin Hydrogel Reinforced By Bacterial Cellulose Nanofiber For Bone Repair

Natural polymeric materials applied in bone tissue engineering have inherent advantages in adhesion and recruitment to cells in comparison with synthetic polymers.Gelatin is a product of partially hydrolyzed collagen.The RGD sequence in the gelatin backbone provides a specific binding site for cells,which benefits cell adhesion and promotes new tissue regeneration.To form crosslinked network,gelatin is often modified by reacting with methacrylic anhydride.The hydrogel made of methacrylated gelatin(GelMA)has structural features similar to those of extracellular matrix,which mimic the natural three-dimensional microenvironment to support cell adhesion and proliferation,and facilitates tissue generation.However,the mechanical properties of GelMA hydrogels are normally not high enough to meet the requirements of bone tissue engineering.Therefore,composite hydrogels are often prepared by incorporating other materials.In addition,due to the crosslinked network structure of the hydrogels,cells are generally difficult to spread and migrate within hydrogels.Bacterial cellulose(BC)has a unique supramolecular structure and a large number of intermolecular hydrogen bonds.It has a natural nanofibrous structure and thus exhibits excellent mechanical properties,which makes it a suitable reinforcement for hydrogels.In view of these,this thesis combines BC with the GelMA hydrogel in order to improve its mechanical strength;at the meantime,it is expected that BMSC cells can use the BC network as scaffold to spread and migrate along the BC fibers inside the hydrogels.These attempts may be helpful in developing composite hydrogels with strong ability in promoting bone regeneration.The main work of this thesis is briefly described as follows:(1)The preparation of GelMA/BC composite hydrogels was explored and optimized.Firstly,the purified BC fibers were prepared into a kind of suspension and mixed with GelMA aqueous solution to obtain GelMA/BC composite hydrogels after photocrosslinking.However,it was found that the BC fibers could not be uniformly dispersed in the hydrogels,and the mechanical strengthes of the hydrogels were not enhanced significantly.Secondly,the composite hydrogels were prepared by preforming BC fibers into scaffolds,followed by GelMA solution impregnation and photocrosslinking,which significantly improved the dispersion of BC fibers.Compared with the pure GelMA hydrogel,the compressive strengths and strains at break of the composite hydrogels were significantly improved.As the amount of added BC fibers increasing,the compressive strengths of the composite hydrogels were further improved,and at the same time,their mechanical stability was also improved.The results of water swelling test showed that the composite hydrogels had high water absorption capacity and had strong shape retention ability after swelling.As the BC fibers content increases,the hydrogel swelling rate remained stable due to dimensional limit resulting from the BC network.Accordingly,with the increase in the amount of BC fibers,the degradation rates of the composite hydrogels became slower.The incubation of cells on the hydrogels showed that the GelMA/BC composite hydrogels were biocompatible and noncytotoxic.(2)Cell culture was carried out by embedding BMSCs within the GelMA/BC composite hydrogels,however,no cell spreading was observed.The cells were still entrapped within the hydrogel network,and could not adhere to the BC fibrous network and spreading.It was speculated that the stiffness of BC fibers was not high enough to provide sufficient affinity to BMSCs.Then,biomineralization on BC fibrous scaffolds was attempted,coating the fiber surface with a layer of hydroxyapatite,intending to improve affinity for cell adhesion.Though the BMSCs were observed able to adhere and proliferate well on the biomineralized BC fibrous scaffold,no cell spreading was observed if BMSCs were carried by the GelMA hydrogel and infiltrated into the BC fibrous scaffold.The biomineralized BC fibers were stiffer than the pristine BC fibers,and were able to further improve the mechanical properties of the resulting composite hydrogels.Finally,BMSCs were seeded and cultured on the biomineralized BC fibrous scaffold at first,and then impregnated with GelMA hdrogel.It turned out the cells could spread,proliferate and migrate well.These results revealed that the crosslinking network within hydrogels,even a natrual polymer with high cell affinity being used,did not favor the cell spreading and migrating.In summary,the feasibility of BC fibers used as reinforcement for GelMA hydrogel was investigated.The resulting GelMA/BC composite hydrogels demonstrated improved mechanical properties and mechanical stability in comparison with the pure GelMA hydrogel.After the BC fibers were coated with inorganic component by biomineralization,the mechanical properties of the corresponding composite hydrogels were further improved.All GelMA/BC composite hydrogels had good biocompatibility,no obvious cytotoxicity,and support cell growth,which can be further studied targeting bone tissue engineering.However,the issue of cell spreading and migration within the hydrogels should be kept in mind,which still needs further optimization.