3D Printing Of SA/GeIMA Hydrogel Scaffold For Bone Repair Applications

There is a growing demand for bone repair materials and devices as the number of patients increases.Recently,tissue engineered scaffold for bone defect repair has attracted tremendous research attention.Compared with traditional fabrication methods,three dimensional printing technology has advantages in high efficient and accurately tailoring the customized scaffold composition and structure.Sodium alginate and gelatin are two common biodegradable natural polymer materials for three-dimensional printing of tissue engineering scaffolds.Sodium alginate（SA）can be crosslinked quickly by calcium ions through the chelation.Methacrylated gelatin（GelMA）not only maintains the good biocompatibility and the low temperature gelation characteristics of gelatin,but also exhibits the ultraviolet light cross-linking feature.Therefore,it is possible to obtain excellent printability of SA/GelMA scaffold by optimizing the slurry composition and the gelation and crosslinking processes of the two materials through tuning the printing parameters.Here,the effects of composition and pore size on the bone repair performance of the printed SA/GelMA tissue engineering scaffold were studied,and the main conclusions are as follows.（1）Optimization of the printing materials composition and printability of three-dimensional printing.First,the effect of the ratio of SA and calcium ions on the printability of the composite hydrogel scaffold was studied;then the rheological behaviors of the printing slurry were measured by a parallel plate rheometer,and the relationship between the printability and rheological properties was analyzed to guide the preparation of printing materials and to optimize the printing process.It was found that calcium ion concentration is the key factor affecting the rheological properties and printability of the SA/GelMA slurry.With the increase of SA concentration（1w/v%-5w/v%）,the range of calcium ion concentration suitable for printing is increased from 0.02M.to 0.05M.Considering the overall printability and mechanical properties of the scaffold,the optimized ratio of the printing slurry is 10w/v%GelMA,5w/v%SA,and 0.02M CaCl₂.（2）The influence of nano-hydroxyapatite（nHA）particles on the mechanical properties and cell behaviors of SA/GelMA scaffolds.In order to improve the mechanical and bone repair properties of the scaffold,nHA particles were compounded with the SA/GelMA slurry as the printing material.The results show that the elastic modulus and compressive strength of the composite scaffold with 20w/v%nHA reach 174KPa and 7.3MPa,which are about 10times and 4.6 times higher than those of SA/GelMA scaffold without nHA,respectively.The osteogenic differentiation ability of bone marrow mesenchymal stem cells（BMSCs）on the SA/GelMA/nHA scaffold is slightly improved.However,the adhesion and proliferation of BMSCs are relatively lower after adding nHA.（3）The influence of gradient pore size and surface mineralization on the mechanical and biological properties of SA/GelMA scaffold.Pore structure is one of the important factors affecting the scaffold performance.Here,the mechanical and biological properties of SA/GelMA scaffolds with uniform pore size and gradient pore size were compared.The results indicate there is no significant difference in the mechanical properties between the two scaffolds with the pore size in range of 400-700μm.However,the gradient pore size scaffold exhibits higher cell loading,and thus promotes cell proliferation and osteogenic differentiation.Meanwhile,the in vitro test shows surface mineralization enhances the osteogenic differentiation of SA/GelMA scaffold.The in vivo test was carried out by implanting the 3D printed SA/GelMA scaffolds in New Zealand rabbit orbital defects with diameters of 3.5mm.Twelve weeks after implantation,the samples were removed,processed,sectioned,and stained with H&E and Masson for histological observation.The results revealed that the mineralized SA/GelMA scaffold with gradient pore structure exhibits excellent bone repair performance comparable to the scaffold loaded with BMP-2,which is much better than the samples without surface mineralization and the scaffolds with uniform pore size.In summary,the SA/GelMA hydrogel scaffolds were fabricated by improving the printability through optimizing the slurry composition,crosslinking process and printing parameters.The composite hydrogel scaffolds with uniform and gradient pore structures were prepared,and calcium phosphate coating was deposited on the scaffold surface by biomimetic mineralization.In vitro cell biology and in vivo implantation experiments showed that the scaffold has excellent bone repair performance.This work may provide insights into 3D printing of hydrogel scaffold with a large number of pore structure gradient for the high throughput screening of pore size effects on mechanical and bone repair properties.In addition,it is valuable to explore surface mineralization and loading growth factors or drugs to improve the bone repair performance of hydrogel scaffolds.