Preparation Of Grass-derived Tracheal Scaffold Composites And Its 3D Bioprinting Process

Cellulose nanocrystals(CNC)is a nano material with excellent mechanical properties,good hydrophilicity and good degradability prepared from cellulose.At the same time,it is also a material that can effectively improve the properties of polymers.Cellulose is widely used as raw material of biomaterials because it exists in a large number of grass plants.Polycaprolactone(PCL)is generally regarded as an ideal material source for 3D printing,and has the potential to be applied to tissue engineering and biomedicine.CNC/PCL tissue engineering scaffolds with different loading amount of CNC were prepared by using CNC prepared by acid hydrolysis as reinforcement material.The mechanical properties of PCL scaffolds were enhanced and the thermodynamic properties of PCL were improved.In this paper,the feasibility of CNC/PCL composite as tissue engineering tracheal stent material and the preparation process of 3D printing tissue engineering tracheal stent are systematically studied.A new method of preparing nanocomposites by combining 3D bioprinting and electrospinning technology is proposed,and some innovative results are obtained.(1)CNC was extracted from Humulus japonicus stems(HJS)by sulfuric acid hydrolysis,and then CNC with high aspect ratio was prepared by high temperature and high pressure.As the reinforcing material of nano materials,3D printing materials with different ratios were prepared by compounding with PCL,and the chemical properties of the scaffolds were analyzed by SEM,XRD,FTIR,DSC and other characterization methods,Then the biocompatibility and mechanical properties of the material were tested,which proved that the material is feasible as tissue engineering scaffold material.The results show that compared with pure PCL,the microstructure of CNC-5/PCL composites is more compact and orderly,and the stability is also very high.DSC and XRD tests show that with the continuous increase of CNC,the melting point and crystallinity of CNC/PCL composites increase first,reach the maximum when the content of CNC reaches 3%,and then decrease.With the increase of CNC content,the mechanical properties of the scaffolds were significantly improved.When the CNC content reached 5%,the tensile strength reached the maximum,which was about 19%higher than that of PCL scaffolds.The biocompatibility of CNC/PCL nanocomposites was tested by MSCs cell culture.The cells on the composites with 3%CNC content could grow normally and had good biocompatibility.Therefore,as a tissue engineering scaffold material,CNC/PCL meets the requirements of chemical stability,good mechanical properties and biocompatibility.(2)A theoretical model of rheological properties of CNC/PCL composite material was established,and rheological parameters of the composite material were measured by rheometer.A series of exploration work on CNC/PCL printing process was carried out by combining simulation and experiment,and finally a high precision 3D printing of CNC/PCL composite scaffold was successfully achieved.The results show that:according to the rheological test results,in the theoretical model,CNC/PCL composites show the characteristics of shear thinning,that is,the viscosity of PCL decreases with the increase of temperature.Then,the fluid state,velocity field and pressure field of the composites are simulated by Fluent software,The flow characteristics and printing forming mechanism of composites produced by 3D printing technology are analyzed.Through the analysis of temperature field and velocity field,it can be obtained that the 3D printing process of composites belongs to the stage of changing from solid separated materials to molten mixing,and gradually forming solid mixtures after cooling.In the flow channel,the temperature of the composite varies from 40℃ to 100℃,and the flow rate is mainly determined by the screw speed.Finally,the experimental analysis of 3D printing technology is carried out,and the relevant parameters are optimized.It is found that printing temperature,printing speed and other related parameters affect the tensile strength and surface accuracy of composites.Through orthogonal test,range analysis and optimization experiment,the most suitable printing parameters for 3D printing PCL/CNC composites are determined:the printing temperature is 100℃,the screw speed is 0.8r/s,and the printing speed is 5mm/s.(3)The measurement of the mechanical properties of biocomposite materials is a measurement of small deformation.At present,it is difficult to have professional measuring instruments on the market to meet the accuracy requirements of the test,and the commonly used hardness testers or tensile testers are also difficult to achieve the mechanical properties of the scaffold.This problem is required,and a test system with automatic hardness and toughness is developed to solve the problem of testing the mechanical properties of biological scaffolds.This test system uses two sets of differential bridges to realize the output of stress and strain respectively,and conducts mechanical tests in different ways under constant temperature,humidity and strain rate to obtain various mechanical properties of the material.Aiming at the particularity of composite biomaterials,through the equipment testing of biocomposite materials composed of nanocellulose and polycaprolactone,to verify whether this test system can meet the requirements.The results show that,through experiments,the test system developed and studied can accurately reflect the mechanical properties of the biomaterial scaffold.The slope of the "force-displacement" loading curve of the tested scaffold is used as a characterization of the toughness/hardness of the scaffold,and the scaffold’s performance can be intuitively reflected.The mechanical properties are good or bad,and the accuracy of the test system can reach 0.01,which has theoretical basis and application value for the establishment of 3D bioprinting technology to tissue repair.(4)According to the influence of the filling structure of the 3D printing inner scaffold on the pore parameters and mechanical properties of the scaffold,a new tracheal scaffold 3D printing concave hexagonal classification algorithm is proposed,and the different fillings are established by CAD software The structure of the scaffold model has evaluated its porosity and elastic modulus,and compared the mechanical properties of the 3D printed tracheal scaffolds with different filling structures in different directions.The results show that the proposed concave hexagon filling model can ensure the porosity and reduce the lap length during the printing process.In terms of mechanical properties,compared with orthogonal and triangular filling models,when the porosity is 60%,the elastic modulus of the bracket printed by concave hexagon algorithm is 1.04 MPa,while the modulus of the bracket printed by triangular and orthogonal filling model is 0.72 MPa and 0.81 MPa respectively,which means that when the three filling patterns with the same porosity are printed,The concave hexagon bracket has the highest elastic modulus and has better elasticity to deal with the problem that the trachea needs to resist transverse deformation.(5)A new type of tracheal scaffold is designed to overcome the shortcomings of traditional scaffolds.It is used to prepare bioabsorbable tracheal scaffolds.The existing equipment and technology combine 3D bioprinting and electrospinning to prepare tracheal composite scaffolds.3D bioprinting can obtain the inner layer of the tracheal scaffold.The use of CNC/PCL mixture through electrospinning can form the nanofiber film used as the outer layer.The results show that according to the mechanical property test and biocompatibility test,the tensile strength of the scaffolds prepared by 3D bioprinting combined with electrospinning technology is 19.5 ± 1.2MPa,which is significantly better than that of a single 3D bioprinting scaffolds,and the number of cell adhesion and growth cultured on the tracheal scaffolds is also higher than that of the scaffolds prepared by 3D bioprinting alone.