| With the development of industrial mechanization and transportation,people have more and more opportunities to obtain high-risk jobs.The incidence of spinal cord and peripheral nerve injury has greatly increased.Peripheral nerve injury has become one of the most serious health problems in the world.Peripheral nerves are important tissues of the human body,distributed in various parts of the human body,and have the function of connecting the central nervous system and other organs.The nervous system has limited ability to repair its own nerve damage,and usually requires external adjuvant therapy.At present,the main method of clinical repair of peripheral nerve defects is to reconnect intermittently,but excessive tension during intermittent may cause secondary neuronal necrosis.Allogeneic or xenogeneic nerves are ideal nerve sources,but treatment failure often results in severe immune rejection.Therefore,repairing nerves is one of the most challenging areas.Tissue engineering is an interdisciplinary field of life science and material science,and provides a potential alternative platform for the implantation of nerve cells.3D scaffold bioprinting regenerates tissue by depositing biomaterials and cells "layer by layer" on a small unit size,creating an organized biological construct.3D bioprinting can accurately construct cell-bearing biological materials into functional structures with complex structures.Compared with the traditional bionic scaffold construction method in tissue engineering,it has a richer and diverse scaffold form,which can support a wider range of applications.Because of its rapid prototyping and control capabilities in material preparation,3D bioprinting opens up new prospects for the development of different types of neural scaffolds that can be used to treat specific patients.3D bioprinting technology has huge potential in medical tissue engineering potential.The characteristics of bio-ink are crucial.The bio-ink based on the combination of scaffold and cells should be able to meet the performance and biological characteristics of biomaterials at the same time.In this paper,graphene is dispersed by ionic liquid to prepare graphene dispersion system,which greatly reduces the agglomeration effect of graphene and facilitates the subsequent printing of hydrogels.Then the Gr/Gel/SA mixed bio-ink was quantitatively prepared using the blending method,and a series of physical and chemical performance analyses were performed on the mixed bio-ink.A printability analysis was conducted and the best printability conditions were: Gel =6%,SA=2% and Ca2+=2%.As far as the rheology of the material is concerned,the thixotropy of the gel after crosslinking is significantly enhanced in the thixotropy detection of the ink,and the addition of the gelatin component observably improved the thixotropy performance of the mixed gel system,indicating that the introduction of gelatin It can provide a good predetermined shape effect in the early stage of printing;the elasticity and steady-state viscosity of the mixed ink after Ca2+ cross-linking in the viscoelastic and steady-state viscosity testing are significantly enhanced,indicating that After crosslinking,the material had great stability and mechanical strength.It is critical that temperature sensitivity testing showed that the temperature sensitivity of the material depends on gelatin,and the viscosity of gelatin-based ink increases significantly with decreased temperature.Its gel temperature is about 22 ℃,indicating that the mixed ink system is temperature sensitive The temperature of the printing platform can be controlled to be lower than the critical gel temperature to achieve the predetermined shape at the initial stage of printing.The results of cross-linking reaction were detected by Fourier infrared spectrometer,and three characteristic absorption peaks of 2940 cm-1,890 cm-1 and 950 cm-1wave number were found,and the intensity of these absorption peaks will vary with the cross-linking agent The concentration increases and weakens,which fully shows that the cross-linking reaction occurs at these sites on the macromolecule.By adjusting the parameters of temperature,printing speed,air pump pressure and filling distance and other parameters in combination with the fluid characteristics of biological ink,the most suitable printer conditions for the printing bracket are determined.This paper further introduces the preparation and characterization of the composite scaffolds.The prepared scaffolds were arranged in a multi-layer regular well-shaped structure with uniform pore size and regular overall shape of the scaffold.With the increase of graphene composition,there was no significant effect on the pore size of the stent.The scaffold had large pores with a pore size of about 300 microns and micropores with a diameter of about 50 microns.This porous structure of the scaffolds could transfer nutrients and metabolic wastes,which was conducive to subsequent cell culture.Graphene was evenly distributed throughout the scaffolds,and there was no obvious graphene agglomeration,indicating that graphene was well dispersed and the scaffolds material was uniform.In addition,with the increase of graphene,the color of the scaffolds was getting darker and darker.The highest graphene scaffolds was 0.2%,and the interior of the scaffolds was almost completely covered with graphene.In each group of 0% Gr/Gel/SA scaffolds,0.02%Gr/Gel/SA scaffolds,0.08% Gr/Gel/SA scaffolds and 0.2% Gr/Gel/SA scaffolds,the mass percentage of carbon element gradually increased,and The change of nitrogen element decreased slightly,and the content of carbon element gradually increased,which can indicate that graphene had been successfully doped into each scaffold.Adding graphene will affect the mechanical properties of the scaffolds,excessively high graphene content would reduce the mechanical strength of the scaffolds,which may cause agglomeration due to high graphene content.The phenomenon of agglomeration will lead to a decrease in the volume fraction of graphene,which will also lead to a reduction in the mechanical properties of the stent.Thewater absorption rate of the scaffold decreases slightly with the increase of graphene,so the water absorption capacity of the scaffold containing graphene decreases.After 96 hours,the water absorption rates of Gels/SA,0.02% Gr/Gel/SA,0.08% Gr/Gel/SA and 0.2% Gr/Gel/SA scaffolds were 1100.5 ± 18.96%,1041.2 ± 11.21%,1019.5 ± 29.65 % And 1031.8 ± 15.54%.Adding a small amount of graphene did not cause a significant change in the porosity of the scaffolds.However,as the graphene content increases,the pore walls of the scaffolds become thicker,which results in a slight decrease in the porosity of the stent.The porosities of Gels/SA,0.02% Gr/Gel/SA,0.08% Gr/Gel/SA and 0.2% Gr/Gel/SA scaffolds were 89.01 ±1.02%,89.98 ± 0.56%,76.99 ± 2.41% and 65.47 ±,respectively.3.15%.The addition of graphene increases the inorganic content of the material and makes the material less hydrophilic,so the porosity became smaller,which led to slower degradation and ultimately reduced the degradation rate of the scaffolds.The degradation rates of Gel/SA,0.02%Gr/Gel/SA,0.08% Gr/Gel/SA and 0.2% Gr/Gel/SA scaffolds after 15 days of degradation were 70.32%,62.79%,58.25% and 52.54%,respectively.As the graphene content increased,the contact angle of the scaffolds became larger and larger,indicating that the hydrophilicity of the material became worse and worse.The contact angles of Gel/SA,0.02% Gr/Gel/SA,0.08% Gr/Gel/SA and 0.2% Gr/Gel/SA stents were 56.21 ± 1.24 °,64.38 ± 2.45 °,96.20 ±0.25 ° and 116.37 ± 3.54 °,respectively.The PC12 cells studied in this paper grow well and have stable cell conditions,which could meet the experimental needs.After the cells were seeded on the scaffold for 7 days,the cell-scaffold complex was fixed,and the use of scanning electron microscopy showed that the cells in each group of scaffolds proliferated well.A large number of cells adhered to the scaffold,and there was a layered growth between different cells.The single-photon fluorescence microscope was used to observe the cells grown on the composite scaffold for 3days.The results showed that the cells were in good condition,the number of live cells was large,the number of dead cells was small,the scaffold could be fully spread on the scaffold,and had crawled into the scaffold;By Calcein-AM staining,PI staining and Hochest 33258 staining,it was found that the cells were evenly distributed on the scaffold and had good activity;as the number of culture days increased,the number of cells growing on the scaffolds of each group also gradually increased.The cells on the Gels/SA scaffolds had a smaller number of cells than the Gr/Gel/SA scaffolds.The cells on the Gr/Gel/SA scaffolds were more dense.After a long period of culture,excessive graphene content would have an increasingly negative effect on the cells.The cytotoxicity of the scaffolds would increasingly be seen from the CCK test.Among the composite scaffolds,the scaffolds with a graphene content of 0.08% were the most conducive to cell growth,and the number of cell survival and proliferation are much higher than those of other scaffolds. |
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