Enhancement Of Osteogenic Properties By 3D Bioprinting Silk Fibroin-Gelatin-Propanediol Scaffold

Due to factors such as the aging of the population,increased incidence of trauma,and the adverse effects of various diseases,including cancer,the number of people suffering from bone defects has grown.Bone tissue engineering enables the restoration and repair of bone defects.This field has attracted great interest and attention from scholars in the past few decades.As a new printing technology,3D bio-printing has a high-precision,low-cost,flexible design that creates extremely complex structures in a short period of time based on patient-specific geometries,consistent with the diversity of bone defects.It has been widely used to solve some of the problems in tissue engineering and regenerative medicine currently.However,as the core technology of 3D bio-printing,it is necessary to find a viable bio-ink material that needs to meet printability and good biocompatibility(innocuous to cells and promote cell attachment,growth,proliferation,and metabolism),better mechanical properties and controllable biodegradability and other characteristics.In bone tissue engineering applications,biomaterials must be biocompatible so as not to trigger an immune response in the host.Degradable polymers classified as natural polymers are more advantageous than synthetic polymers and have attracted more scholars’ attention.SF is a natural fiber polymer with a high level of biocompatibility and degradability,as well as suitable mechanical properties and wide range of sources.It is suitable for a wide range of biomedical applications and is considered as a good material for preparing 3D bioprinting bio-ink.In this study,we used SF,gelatin and safe non-toxic polyols as raw materials,SF acts as a structural matrix to regulate material degradation and mechanical properties,polyols act as additives to induce cross-linking,and gelatin acts as a filler to produce patient-specific implants for soft tissue reconstruction.In the experiment,we observed that the rheological properties of bio-ink varied with different concentrations of polyols and components,and SF/Gel/PG bio-ink had advantages in storage modulus(G’)and β-sheet.We used propylene glycol to physically crosslink overnight to obtain the scaffold material.all scaffolds showed regular and porous microstructures after incubation in theα-MEM medium,and scaffolds exhibited more hydrophilicity and degradation behavior with the increase of incubation time.In vitro,we found that cell adhesion,proliferation,differentiation,and mineralization was enhanced in MC3T3-E1 cells cultured on S-Gel-PG25 and S-Gel-PG30(S-Gel-PG25/30)scaffold compared with MC3T3-E1 cells cultured on S-Gel-PG20,S-Gel-Gly25 and S-Gel-Ery25 scaffolds.Intriguingly,it was showed by Western blotting experiments that Smad1/5/8 was phosphorylated when MC3T3-E1 cells were cultured on S-Gel-PG25/30 scaffold,and Runx2 was also activated.Consistent with this result,the expression level of osteogenic specific genes(Runx2,Osx,Bmp2,Ocn,Type Col-Ⅰ,Opn)were also increased after MC3T3-E1 cells cultured on S-Gel-PG25/30 scaffold.Taken together,we concluded that cell osteogenesis was promoted in MC3T3-E1 cells cultured on S-Gel-PG25/30 scaffold through Smad activated Runx2 pathway.Our findings provide a potential biomaterial for the treatment of bone tissue regeneration.