In Vitro Induced Cartilage Induction Of 3D Printed Methacrylate Gelatin Sodium Alginate/Hyaluronic Acid Hydrogel Scaffold

Objective: Explore the possibility of preparing composite methacrylate gelatin(gel),alginate(alg)and hyaluronic acid(Ha)hydrogel material scaffolds based on 3D printing technology.The scaffolds with certain mechanical properties and high degree of discrimination were screened and prepared.To study its ability to induce bone marrow mesenchymal stem cells to differentiate into chondrocytes in vitro.Methods: The first part,based on 3D printing technology to prepare hydrogel stent.First,sodium alginate and hyaluronic acid were physically crosslinked using polyethylene glycol glycidyl ether(PEGDG)as a crosslinking agent,and then mixed with gel in different proportions to obtain a gel/alg-Ha composite hydrogel,Finally,the stent is prepared using 3D printing technology.The tensile strength and compressive strength were measured to verify the mechanical properties of the stent.The swelling ratio and water absorption were measured to verify the water absorption performance of the stent.The degradation rate in vitro was determined and the degradation performance of the stent was verified.In the second part,we verified the in vitro induction of cartilage differentiation of bone marrow mesenchymal stem cells into scaffolds.Gel+(alg+Ha)4,gel+(alg+Ha)6gel+(alg+Ha)8 groups were set as experimental groups according to different concentrations of alg-Ha containing 4%?6%?8%(g/ml).The gel group,gel+alg group and alg+Ha group were used as the control group.The rabbitbone marrow mesenchymal stem cells were cultured in vitro and inoculated into each experimental group and control group.After 7 days of culture,the adhesion morphology of the cells and the scaffold was observed by scanning electron microscopy(SEM),and the growth ability of the support cells of the scaffold was detected by calcein/propidium iodide staining;after 14 days of culture,hematoxylin-eosin staining was performed to observe the cells.Morphology and proliferation,type II collagen immunohistochemical staining was used to detect cartilage differentiation characteristics;The samples of 3,7and 14 days were selected to detect the expression of cartilage-related characteristic genes(fluorescence quantitative PCR)and glycosaminoglycan(GAG)secretion,and the ability to differentiate into chondrocytes was verified.Results: A series of cartilage scaffolds were successfully prepared by 3D printing technology.A series of characterizations of the printed stents showed that the strongest mechanical properties were gel+(alg+HA)8 1.1 MPa in the experimental group and 0.179 MPa in the simple gel group.In the swelling rate and in vitro degradation experiments,it was found The swelling rate and biodegradation of the gel+(alg+HA)6 group in the experimental group were closest to the cartilage tissue.In the experiment of inducing chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro,calcein/propidium iodide staining was used to detect the proliferation and activity of cells,which indicated that gel +(alg + HA)6 cells in the experimental group had the best proliferation and activity.Scanning electron microscopy was used to observe the adhesion and growth of cells in the materials.Cells in the scaffolds containing gel could adhere and grow better in the control group alg+Ha.In the detection of GAG secretion,immunohistochemical staining of type II collagen and PCR results showedthat the stent containing Ha had a certain tendency to promote chondrogenic differentiation of mesenchymal stem cells compared with the gel group and gel+alg group;The expression levels of cartilage-characterized genes such as SOX9 and ACAN were significantly higher.In general,the cartilage-inducing properties of the experimental group gel+(alg+ha)6 group were the best.Conclusions: A series of modifications and cross-linking of gelatin,sodium alginate and hyaluronic acid are carried out;they are mixed together to form an interpenetrating network hydrogel and a highly accurate stent is printed by3 D printing.Through the characterization of scaffolds and in vitro induction of chondrogenic differentiation of bone marrow mesenchymal stem cells,it was shown that 3D printing technology can print composite gelatin,sodium alginate and hyaluronic acid complementary complementary scaffolds.Through this research,we can provide direction for innovative composite biomaterials,and provide theoretical basis for the design of cartilage-inducing materials based on 3D printing technology.