Preparation And Properties Of Alginate Composite Gel As Bone Tissue Engineering Scaffold Material

Alginate,as a natural and renewable biomacromolecule,has shown great potential in the field of tissue engineering due to its excellent biocompatibility,biodegradability,no immunogenicity and gelation.However,how to use alginate to produce homogeneous and well-defined tissue engineering scaffold and meet all the performance requirements of the ideal scaffold material is still a difficult task in current tissue engineering.For this reason,in this paper,the homogeneous cross-linking of alginate was achieved using hydroxyapatite-gluconolactone(HAP-GDL)complex as the crosslinking system.The activation of the scaffold surface was realized by the layer-by-layer electrostatic assembly of biomacromolecules.Furthermore,the objective of our study was to fabricate the ideal alginate composite gel scaffold by the optimized combination of the composite materials,using the bioactive nanoparticles as the fillers.Firstly,hydroxyapatite(HAP)was used as endogenous crosslinking agent to prepare homogeneous alginate hydrogel under the hydrolysis of gluconolactone(GDL).And then,alginate-chitosan-gelatin composite gel(Alg-CS-GT)scaffolds were prepared by combining freeze-drying with layer-by-layer electrostatic assembly of biomacromolecules.Comparative analysis of the element composition,XPS spectrum,morphological structure,mechanical properties,swelling,biodegradability and cytocompatibility of alginate gel(Alg Gel),alginate-gelatin composite gel(Alg-GT),Alg-CS-GT and alginate/gelatin-chitosan composite gel(Alg/GT-CS)was performed to verify the feasibility of the method that used the endogenous crosslinking of HAP and layer-by-layer electrostatic assembly of biomacromolecules to fabricate the composite scaffold.The experimental results showed that the prepared alginate scaffolds exhibited good 3D morphology and uniform pore structure.The formation of polyelectrolyte complex during the layer-by-layer assembly has reduced the fragility of ion crosslinking,and improved the mechanical properties and swelling stability of the scaffold materials.In particular,the Alg-CS-GT scaffold showed the best results in controlling swelling and degradation.The gelatin wrapped in the outermost layer of materials via layer-by-layer assembly technology,contained the RGD polypeptide that could effectively improve the cell adhesion of the scaffold material,which revealed that the layer-by-layer electrostatic assembly of biomacromolecules can effectively improve the surface activity of the scaffolds.Furthermore,Alg-CS-GT scaffold exhibited the best results on the adhesion,proliferation and differentiation of MG63 and MC3T3-E1 cells,indicating that it was feasible to construct the ideal composite scaffolds with the endogenous crosslinking of HAP and layer-by-layer electrostatic assembly of biomacromolecules.Then,in order to compensate for the lack of mechanical properties of Alg-CS-GT,thus making it meet the stress requirement of the ideal scaffold material,based on the above method,the bioactive nano-materials,such as bacterial cellulose nanocrystals(BCNs),silica nanoparticles and titanium dioxide nanoparticles,were incorporated into the alginate matrix by physical blending method to prepare alginate/bacterial cellulose nanocrystals-chitosan-gelatin composite gel(Alg/BCNs-CS-GT)scaffold,alginate/silica-chitosan-gelatin composite gel(Alg/SiO2-CS-GT)scaffold and alginate/titania-chitosan-gelatin composite gel(Alg/TiO2-CS-GT)scaffold.The effects of these nano-materials on the pore structure,mechanical properties,swelling property,degradability,bio-mineralization ability and biological activity of the composite materials were investigated by SEM,material testing machine,swelling and degradation test in PBS,in vitro biomineralization test and in vitro cell culture test.The results showed that the obtained alginate composite gel scaffolds exhibited regular 3D morphology and good pore structure.With the increase of nanomaterial content,the pore size of the composite scaffolds decreased and the porosity reduced,resulting in the reduction of the mechanical properties.FT-IR,XRD and thermogravimetric analysis showed that the intermolecular hydrogen bonding between the nanomaterial and the alginate in the composite scaffolds was generated.The incorporation of nano-materials into the composite scaffolds made their swelling and degradation under the control.Meanwhile,SiO2 and TiO2 nanoparticles could enhance the bio-mineralization ability of the composite scaffolds.The results of in vitro cytocompatibility test showed that the addition of nanomaterials could promote the adhesion,proliferation and differentiation of cells on the composite scaffold.However,when the nanomaterial content was too high,the pore size of the composite scaffold was significantly reduced,which was not beneficial to the penetration and growth of cell as well as the transfer of metabolic wastes and nutrients,thus reducing the vitality of cell on the scaffold materials.Finally,in order to broaden the application of alginate scaffold in hard tissue or muscle tissue,high strength alginate/polyacrylamide-chitosan-gelatin composite gel(Alg/PAAM-CS-GT)scaffold was prepared by interpenetrating network(IPN)method.The composite scaffolds constructed by the ionic crosslinks of alginate and the covalent crosslinks of polyacrylamide exhibited edges and corners clear and regular 3D morphology.Along with the PAAM content increased from 40% to 80%,the compressive strength of the composite scaffold exhibited a trend of exponential growth.The swelling and degradation of Alg/PAAM-CS-GT were mainly controlled by PAAM,which was covalently cross-linked in IPN.The higher the PAAM content,the lower the swelling rate and the biodegradability of the composite scaffold.When PAAM content did not exceed 60%,good biocompatibility of the composite scaffold promoted cell proliferation and differentiation.However,with the increase of PAAM content,the pore size of the composite scaffold decreased significantly,resulting in the reduction of cell viability.The above work indicated that the regulation and control of the physicochemical properties and biological activities of alginate composite gel scaffold could be achieved by adjusting the component content of the composite materials.