Research On The Osteogenesis Of 3D-printed Bioglass Scaffolds Loaded With Extracellular Matrix

Background As an important basis for supporting the face,Oral and maxillofacial bone tissue will delayed or poor repaired once the maxillofacial bone defects beyond the limit of the body.It needs implanting bone materials to promote bone healing.As a traditional method of repairing bone defect,bone transplantation can be divided into autogenous bone transplantation,allogenic bone transplantation and artificial synthetic bone transplantation according to the source.Because of excellent bone conduction and mechanical properties,autogenous bone transplantation is regarded as the gold standard of bone defect repair.but its application can not spread on a large scale because of its limited supply and extra trauma to the bone removal site.Allogeneic bone consists of potential risk of disease transmission and the possibility of immune rejection,so it is difficult to be popularized in practical application.Artificial bone graft which solved these problems is one of the hotspots recently,among which bone tissue engineering is the most representative.As a refined and controllable manufacturing technology,3D printing can not only fabricate tissue engineering scaffolds matching the irregular shape of the defect,but also can form a uniform pore structure inner the scaffolds to improve the quality of the tissue engineering scaffolds,which is able to support the defect site,for cell adhesion and proliferation to provide space for new blood vessels,bone trabeculae and so on.Bioactive Glass(BG)is a kind of biodegradable material for bone tissue repair with good biocompatibility and bioactivity.When BG is implanted into the body,it can exchange ions with body fluid and form a layer of apatite(HA)on its surface similar to that of natural bone,then form a firm chemical bond with the surface of bone and induce bone regeneration.BG can release silicon(Si)and calcium(Ca)ionsand so on,which influence on osteoblast-related cells at gene level to accelerate the growth of new bone.Based on 58 s bioactive glass(58S BG),Gel/SA/58 S BG composite scaffolds were prepared by 3D printing in combination with gelatin(Gel)and Sodium Alginate(SA)as raw materials.It was found to have good osteoinduction and biological safety in vitro and in vivo.Extracellular matrix(ECM)is secreted from cells to the outside of the cell.Its main components are collagen,fibronectin,fibronectin and glycosaminoglycan.Both ECM itself and combination with other materials,ECM can be used as biomaterials for tissue engineering or to promote endogenous healing and regeneration.As a reservoir of a variety of cytokines and growth factors,ECM can mimic the physical and chemical conditions present in the microenvironment in vivo,which contribute to the directional differentiation of stem cells.In bone tissue regeneration,adequate blood supply is the key to improve the quality of bone defect repair.Therefore,we seeded rat aorta endothelial cells(RAOECs)and rat bone mesenchymal stem cells(RBMSCs)onto the previous Gel/SA/58 S BG composite scaffolds by 3D printing,were seeded onto the scaffolds,and then rat aortic endothelial extracellular matrix scaffolds(RAOECs-ECM scaffolds)and rat bone marrow mesenchymal stem cell extracellular matrix scaffolds(RBMSCs-ECM scaffolds)were prepared by decellularization in vitro.On this research,we study the biosafety and osteoinduction of two kinds of ECM-loaded scaffolds and pure scaffolds in vitro and in vivo.Objective We firstly prepared bio-glass by sol-Gel process and constructed Gel/SA/58 S BG composite scaffolds by 3D printing loaded with extracellular matrix.We investigate the biological safety and material characteristics of RAOECs-ECM scaffolds and RBMSCs-ECM scaffolds.We also investigate their effects on the proliferation of stem cells in vitro and potential to promote the differentiation of osteogenic and angiogenic vessels.Besides,the effect of stem cell recruitment and the expression of osteogenic vascular factors in the subcutaneous tissue of nude mice was investigated,which will provide the preliminary basis for the study of oral and maxillofacial bone defect repair researh.Materials and methods 1.Preparation of 58 S BG: The raw material was hydrolyzed to form Sol with tetraethyl orthosilicate,Triethyl Phosphate and calcium nitrate as precursors using Sol-gel process,and then it was stored,aged,drying,and sieving.The BG of Ca O-P2O5-Si O2 ternary system was prepared.2.Fabrication of 3D printed Gel/SA/58 S BG scaffolds: A proper amount of gelatin and BG powder was added to deionized water and SA powder is added and mixed enough.The mixture was formed by 3D-printing and soaked in Ca Cl2 and glutaraldehyde solution for cross-linking.The scaffolds were freeze-dried.3.Fabrication of 3D printed Gel/SA/58 S BG scaffolds loaded with ECM.The scaffolds were co-cultured with RAOEC cells and RBMSC cells for 14 days,then decellularized with 0.5% Triton X-100 and 0.1% ammonia solution.4.Detection of decellularization.DAPI staining was used before and after decellularization,and then cell adhesion was observed under Fluorescence microscope light before and after decellularization.Besides,the decellularization efficiency was measured by detecting ds DNA.5.Characterization and detection of scaffolds(1)Surface morphology and elemental analysis : The surface morphology of the scaffold was observed by scanning electron microscope,and the distribution and content of Ca,P,O on the surface of the scaffold were analyzed by EDS.(2)Pore size and porosity: The pore size of scaffolds was measured by SEM,and the porosity of three kinds of scaffolds was measured by media substitution method.(3)Water absorption and swelling rate: the scaffolds were soaked in pure water to fully swell.The water absorption was calculated by comparing the mass and the swelling rate was calculated by comparing the volume before and after swelling.(4)Fourier transform infrared spectroscopy: The molecular structure of the material is characterized and the chemical reactions of the components in the mixture are compared and analyzed.6.Detection of mechanical properties of scaffolds(1)Stress-strain trend: The universal material testing machine was used to carry out the slow load on the support material,and the stress-strain trend chart was drawn according to the relationship between stress and deformation.(2)Elastic modulus: According to the stress-strain trend,we choose the elastic deformation part of the front section to draw the load curve and calculate the elastic modulus.7.Degradation test: Three kinds of scaffolds were placed in simulated body fluid,washing lyophilized every week,recorded after drying weight and measure the mass changes.8.Biosafety test: According to GB/T 14233.2-2005,the scaffold extract was cocultured with L929 cells.The cytotoxicity of the scaffold was determined according to the number of deformable cells.At the same time,the scaffolds were implanted subcutaneously in nude mice to observe the healing of the operation area,and the material tissues of each group were collected and fixed in 4% paraformaldehyde solution for 24 hours.Then HE staining was used to observe the histopathologic changes.9.The local reaction test was carried out in the subcutaneous tissue of nude mice.The inflammation of the surrounding tissues was observed by gross observation and histological staining,and the samples were taken for histological and immunohistochemical staining at 4 weeks and 8 weeks after implantation,local inflammation and osteogenesis and angiogenesis were observed.Results 1.58 S bio-glass was successfully prepared by sol-gel method.2.The composite scaffolds prepared by 3D printing have relatively regular pore structure and good mechanical properties,and the pore size porosity has not changed obviously after ECM loading.3.The shape of the three scaffolds was stable in the first 6 weeks.The obvious dissolution deformation began to appear after 8 weeks in the ECM-loaded scaffolds,while appeared after 12 weeks in the pure scaffolds group.4.CCK-8 test showed that there were a lot of viable cells attached to the three scaffolds after culturing with RBMSC cells,and the number of cells increased with the increase of time in the 7d,the ECM-loaded scaffold group showed a more obvious promoting effect than the simple scaffold group,in which the number of loaded RAOECs-ECM scaffold cells was better than that of the RBMSCs-ECM scaffold group(p <0.05);5.Observed by scanning electron microscopy,The RBMSCs adhere to three kinds of scaffolds after culture.The endothelial-like cells were more common with less minerals on the scaffolds loaded with RAOECs-ECM.However,there were a lot of minerals around cells on RBMSCs-ECM scaffold.6.RT-PCR showed that the expressions of vascular genes CD31 and VEGF,and osteogenic genes BMP-2 and RUNX-2 were significantly increased in the scaffolds loaded with RAOECs-ECM(p<0.05).Compared with the pure scaffolds,the expressions of vascular gene CD31,osteogenic genes BMP-2 and RUNX-2 were increased in the early stage(7d)(p<0.05).7.Biosafety test: The deformation rate of L929 cells in the three scaffold groups all met the toxicity requirements of the national standard GB/T 14233.2-2005 for implantable medical devices.The skin tissue of animals in each group healed well,Histopathological staining showed no pathological abnormality.8.After three groups of scaffolds were implanted subcutaneously in nude mice,CD31 and VEGF were highly expressed on the surface of RAOECs-ECM scaffolds,and OCN and RUNX-2 expression on RBMSCs-ECM scaffold performed best.Conclusion The 3D Gel/SA/58 S BG composite scaffolds loaded with RAOECs-ECM and RBMSCs-ECM had good biosafety and biocompatibility.Scaffolds loaded with ECM were superior to Pure scaffolds in promoting proliferation of RBMSCs.Scaffolds loaded with RAOECs-ECM and RBMSCs-ECM showed certain advantages in promoting angiogenesis and expression of osteogenic-related factors.