3D Bioprinting Tissue Engineering Blood Vessels And Study On Its Cytotoxicity

Vascular diseases represented by middle-aged and elderly cerebrovascular diseases and infant congenital heart disease have always been one of the major medical problems threatening human health.The treatment of such diseases largely relies on 3D printing of tissue engineering blood vessels and complex vascular network.This paper introduces a method to build the bioactive vascular tissue engineering in vitro.This method use gelatin-sodium alginate-carbon nanotubes as raw material,introduces stepping motor components in the extrusion biological 3D printer,and prints out the vascular scaffold horizontally by means of the combination of the directional extrusion hydrogel of the printing nozzle and the rotating cylinder model.Subsequently,mouse epidermal fibroblasts were inoculated onto the inner and outer walls of the hollow tubular scaffold.Firstly,gelatin-sodium alginate-carbon nanotubes mixed bio-ink was prepared quantitatively by blending method.It is printable when SA is above 2%and Ca²⁺in the crosslinking agent is between 1%and 5%.Viscoelastic and steady-state viscosity tests show that the introduction of gelatin can provide good formability in the early printing stage,and the cross-linking process of sodium alginate is crucial to the mechanical properties and stability of the printing structure.Temperature sensitivity experiment shows that the pure sodium alginate composition has no obvious temperature sensitivity,and gelatin base ink viscosity increases obviously with the decrease of temperature,while the gel critical temperature of 18?.The infrared spectrum of SA and its Ca²⁺cross-linked products show that the characteristic absorption peaks of 2940 cm^-1,890 cm^-1 and 950 cm^-1 wave-number decreased with the increase of Ca²⁺concentration,indicating the site where the cross-linking occurred,while the appearance of the characteristic absorption peaks of 1530 cm^-1 in the gelatin based mixed ink group shows the presence of gelatin.Then the modification process of biological 3D printer and the scaffold printing process are introduced.Stepper motor suit assembly to Bio-Architect^?-Pro biological 3D printers to achieve the scaffold printing.Crosslinked in the 4%CaCl₂ crosslinking bath for 30 min.The Red dye perfusion experiment shows that the structure of scaffold is continuous and complete without any gaps.The inner diameters of the scaffold are 3 mm,4 mm and 5 mm,the average wall thickness is 0.5 mm,and the length is up to 7¹0 cm.The porosity of Gel-SA,Gel-SA-0.5%C and Gel-SA-1%C are 89.25%,55.55%and 19.01%,the water absorption rate are 334.02%,272.28%and 149.01%respectively,the equilibrium time of swelling are6h,12h and 18h respectively.Among the three groups,Gel-SA-0.5%C group has the strongest tensile strength,indicating that the right amount of doped carbon nanotubes effectively increases the mechanical properties of the scaffold.The three systems show certain tolerance and strong degradation ability in both PBS buffer solution and I collagenase solution.SEM observation shows that the presence of cross-dimensional pores after freeze-drying are conducive to the cell adhesion spreading,the transport and infiltration of nutrients,and the increase of the roughness of the inner and outer walls of the tubes.The fibroblasts are inoculated into the 3D printing rectangular scaffold and then the dead/live staining shows that the scaffold and the cell extension are in good shape within two weeks.Quantitative experiment shows that the mixed ink system has better biocompatibility and less cytotoxicity after 3D printing.Next,tissue mass method is used to conduct primary and subculture of epidermal fibroblasts of 24 h newborn Balb/c rat.According to the growth curve,fibroblasts have a strong ability to divide and could be amplified by an order of magnitude within a week.HE staining shows that the cells are plump in morphology and the cell coverage rate is about 90%on the seventh day of culture.Finally,multilayer bioactive blood vessels are constructed.The SEM and dead/live staining show that the vascular construction cells are evenly spread on the inner and outer wall of the vessel.The cells adhere to each other in good shape.CCK detect shows that the cell viability of the three groups at 7th day are 86.64±4.50%,80.58±6.70%,and 65.23±5.50%,the toxicity levels of which are class I,I and II respectively,indicating that the proliferation rate of the cells is fast and the cells show a relatively healthy growth trend after 7 days of inoculation,the biocompatibility of the materials is good,and the cytotoxicity of the system is less affected by the right amount of doped carbon nanotubes,and the construction meets the biological activity requirements of bionic vascular.