Preparation Of Bio-Gel Scaffolds Containing Sodium Tanshinone ⅡA Sulfonate And Its Experimental Study On Bio-3D Printing

Background and aims In recent years,patients with coronary artery disease,especially most acute coronary syndromes,have demonstrated features such as endothelial dysfunction and elevated inflammatory factors,and endothelial dysfunction has become an important predictor of this type of adverse event.Stent systems used to treat coronary stenosis have shown some recent efficacy,but still have many drawbacks.3D Bioprinting(3DP)technology is at the forefront of the life sciences and its application in the medical industry is already a major trend.Tanshinone ⅡA sodium sulfonate is a water-soluble derivative of Tanshinone ⅡA,the main active ingredient of the Chinese medicine Danshin,which has been shown in many studies to have cytoprotective and pro-vascularizing effects.The STS was mixed with gelatin methacrylate(GelMA)hydrogel to enhance the bioactivity of the printing ink,and human umbilical vein endothelial cells(HUVECs)were prepared using 3D Bioprinting technology.The biogel membrane and scaffold were prepared using 3D Bioprinting technology.The results were used to evaluate whether STS can protect cells and promote vascularization of 3D scaffolds in the process of 3D Bioprinting,and to provide new ideas and methods for the application of Salvia miltiorrhiza in the field of bio-3D printing.METHODS Human umbilical vein endothelial cells(HUVECs)were selected as the experimental subjects.(1)In vitro viability assays were performed using purchased HUVECs,and the CCK-8 viability assay was used to explore the concentration range of sodium tanshinone ⅡA sulfonate that is most suitable for cell growth;(2)Gelatin-methacrylic anhydride(STS/GelMa)bioink containing sodium tanshinone ⅡA sulfonate was prepared by mixing different concentrations of STS with GelMa hydrogel,and cellular bio-3D The final concentration of STS was selected for subsequent experiments by evaluating its degradability,injectability and surface characteristics in combination with(1);(3)the GelMa bioink containing the final concentration of STS was mixed with HUVECs in a certain ratio and the 3D printer was used to prepare the STS/GelMa bioink containing HUVECs.STS/GelMa biogel film containing HUVECs,and the activity and pro-vascularization effects were evaluated by CCK-8 viability assay and fluorescent PCR in vitro amplification method,respectively;(4)the final concentration of STS,GelMa hydrogel and HUVECs cell suspension were used as printing materials,and these three materials were co-mixed in a certain ratio to form STS/GelMa/HUVECs mixed bio ink,the STS/GelMa biogel-like scaffold containing HUVECs was prepared using a 3D printer,and the biocompatibility of the scaffold was evaluated by CCK-8 viability assay and fluorescence PCR in vitro amplification method.Results(1)Endothelial cell viability: compared to the control group,the activity of STS showed an increasing trend in the range of 25-75ug/m L(P < 0.05);when the concentration of STS was greater than 75ug/m L,the cell viability showed a decreasing trend.Therefore,25-75ug/m L of Tanshinone ⅡA sodium sulfonate was selected for the follow-up experimental study.(2)Evaluation of the degradability of STS/GelMa gel films: STS/GelMa gel films degraded in all groups as time increased;the addition of STS accelerated the degradation of STS/GelMa biohydrogels compared to the control group,and the higher the concentration the faster the degradation rate.Evaluation of injectability: The injectability of the biohydrogels was good within the optimum concentration range and was not affected by the concentration.Evaluation of surface appearance: Under the microscope,the addition of STS caused voids and depressions on the surface of the gel compared to the control group,and the higher the concentration,the more obvious the phenomenon.The final concentration of STS at 50ug/m L was selected as the final concentration of STS.At this concentration,the injectability of STS/GelMa biohydrogel was good and the degradation rate was moderate.(3)Cell viability in STS/GelMa gel membranes containing HUVECs: The cells in both STS/GelMa/HUVECs and GelMa/HUVECs gel membranes showed benign proliferation and maintained good activity;compared with the control group,the cells in the STS-containing gel membranes were more active with increasing incubation time after the addition of STS(P < 0.05).Fluorescence PCR in vitro amplification experiments showed that the expression signals of PECAM-1,eNOS and VEGFA genes were up-regulated in both STS/GelMa/HUVECs and GelMa/HUVECs gel membranes;compared to the control group,the expression of PECAM-1,eNOS and VEGFA genes were higher in HUVECs in STS-containing gel membranes with the increase of incubation time after the addition of STS.The expression of PECAM-1,eNOS and VEGFA genes was higher in HUVECs in the STS-containing gel membranes compared with the control group(P < 0.05).The above experimental results suggest that the addition of STS can promote cell proliferation and increase the possibility of their pro-neovascular growth.(4)The biocompatibility of STS/GelMa gel-like scaffolds containing HUVECs showed that the cells in both STS/GelMa/HUVECs and GelMa/HUVECs gel-like scaffolds could grow benignly;compared with the control group,the expression of cell activity and angiogenesis-related genes in the STS-containing gel-like scaffolds increased with the increase of culture time after the addition of STS(P < 0.05).The cell activity and expression of genes related to blood vessel formation were higher in the STS-containing gel-like scaffolds with increasing culture time compared with the control group(P < 0.05).The results of this experiment are consistent with the results of the biogel film,indicating that the addition of STS does facilitate the growth and proliferation of cells in STS/GelMa/HUVECs,and also has a beneficial effect on the vascularisation of the scaffolds.Conclusion This experiment applied STS to 3D Bioprinting experiments,which not only increased the bioactivity of GelMa hydrogel,but also reduced the damage to cells during the printing process and increased the cell proliferation activity;most importantly,the addition of STS improved the biocompatibility of the printed samples,providing new ideas and methods to study the application of Chinese herbal medicine Salvia miltiorrhiza in the field of 3D Bioprinting.