Preparation Of CS/Gel/Gr Hybrid Cartilage Scaffold By 3D Bioprinting And Study Its Performance

Cartilage is one of the important tissues found in humans and animals.Most cartilage tissues in the body are subjected to large mechanical loads,so cartilage tissue is easily injured.Unlike most tissues,cartilage has no blood vessels,nerves,and lymphatic vessels.Its primary function is to provide a smooth,lubricated surface so that the surface friction coefficient is low to deliver mechanical loads.There are currently several repair methods,but the repair effect is very limited.The rapid development of tissue engineering technology in recent years has provided new solutions for cartilage repair.Three-dimensional scaffold-based bioprinting creates tissue-organized biological constructs to regenerate tissue by "layer-by-layer" deposition of biomaterials and cells on small unit sizes.Compared to traditional tissue engineering material preparation methods,this technique shows important advantages in simulating natural cartilage by finely controlling cell distribution and regulation of mechanical and chemical properties.Extrusion-based 3D bioprinting has been used to make multiphase scaffolds for osteochondral regeneration,and most existing commercial bioprinters are based on this technology.The nature of bio-ink is critical to developing functional living tissue through 3D bioprinting.Bio-ink based on stent and cell combinations should be able to simultaneously satisfy biomaterial properties and biological characteristics.In addition,in order to simulate the physiological structure of cartilage,it is also necessary to meet specific characteristics such as printability,bioabsorbability,and biodegradability to ensure good performance of key biomaterials.Based on this,this study uses chitosan,gelatin,hyaluronic acid,etc.to adjust the rhythm of different materials by different ratios,and select the most suitable bio-ink combination and ratio.The ratio of the finally printed bio-ink suitable for printing was Cs: Gel: HA = 1: 8: 0.02.In order to improve the mechanical properties of the composite scaffold material,the effect of doping trace graphene on the physical properties and biocompatibility of the scaffold was observed.The composite ratio of Cs: Gel: HA=1: 8: 0.02 was respectively doped with 0.024%.,0.06%,0.1% of graphene,and bio-ink not doped with graphene was used as a control group.The parameters of the temperature,printing speed,air pump pressure and filling pitch of the 3D bioprinter are comprehensively adjusted by combining the fluid characteristics of the bio-ink to determine the printing conditions optimum for printing the bio-composite holder.In this study,Cs,Gel,and HA,which can complement each other,were selected as the basic materials.On the basis of this,trace graphene was added,and after 3D bioprinting technology,it was freeze-dried and then prepared to have a size of 8 mm × 8 mm × 2 mm and 8 mm × 8 mm × 5mm composite bracket of two specifications.The conclusions drawn mainly include: the different proportions of the prepared stents have both large pores of about 300 ?m and small pores of about 50-70 ?m.The porous structure of the composite stent is beneficial to the growth of subsequent cells on the stent.Cell behavior such as stretching and proliferation.After adding graphene,the porosity of the composite scaffold did not change significantly.The composite scaffolds of each group had higher porosity and could reach more than 80%.Only the wall of the hole becomes thicker and smoother,and the aperture is slightly reduced.Through semi-quantitative analysis of EDS and observation by polarized light microscopy,it can be seen that graphene is successfully doped into each group of composite scaffolds according to the added ratio and can be evenly distributed inside the scaffold.In terms of water absorption,the hydrophilicity of the composite scaffold with graphene was lower than that of the control scaffold,but each scaffold had strong water absorption and could be used for subsequent cell research.The degradation rate of each group was detected within 2 weeks.The results showed that the composite scaffold had the highest degradation rate,and the degradation rate of the composite scaffold with graphene was lower than that of the control group.However,the degradation rate of composite scaffold does not change linearly with the amount of graphene added.Therefore,the content of graphene is not the main factor determining the degradation rate of composite scaffold.The bone marrow mesenchymal stem cells(BMSCs)used in this study have good growth state and strong proliferation ability,and have the ability to differentiate into cartilage.BMSCs were inoculated on a composite scaffold for culture,and cell-scaffold complexes were constructed and the growth of BMSCs cultured within 11 days was examined.First,the proliferation of cells in each group was observed in one week.Cell survival was observed on days 1,3,5 and 7 by Calcein-AM staining,Propidium Iodide staining and Hochest 33342 staining,respectively.It was found that with the increase of culture time,the cells proliferated well on the scaffold,the number of viable cells on the scaffold was large,and the number of dead cells was small.The cells were kept on the scaffold by single photon fluorescence microscopy on the third day of scaffold culture.It is biologically active and evenly distributed on the scaffold.There are living cells adhering and stretching on the surface of the scaffold.The cell-scaffold complex is fixed when the cells are inoculated into the scaffold for 11 days.The internal pores of the composite scaffold are found by scanning electron microscopy.A large number of cells adhered,stretched and proliferated on the outer surface of the stent.All the tests indicated that the biocompatibility of each group was good,but the state of BMSCs on the composite scaffold containing graphene was better than that of the control group.