Preparation Of Grass-derived Nanocellulose And Gelatin Composite And Study On The Forming Mechanism Of Bio-3D Printing Skin Scaffold

Gelatin(GEL),which has the same biocompatibility as collagen(COL),has the advantages of no antigenicity,low price,wide range of sources,etc.,and thus becomes a potential materials for 3D bio-printing tissue engineering skin scaffold.However,the poor mechanical properties limit its application.The mechanical properties of GEL can be improved by filling cellulose nanocrystals(CNC)into gelatin.Based on the analysis of mechanical properties of CNC/GEL,this topic systematically studied the feasibility of CNC/GEL composite hydrogel as a scaffold material for skin tissue engineering and the printability of tissue-skinned skin scaffolds by bio-3D printing.Some innovative achievements have been made,mainly including:(1)The CNC was isolated from Humulus japonicus stems(HJS),corn husks(CH),and wheat straws(WS)by sulfuric acid hydrolysis.HT-CNC was isolated from bleached HJS by sulfuric acid hydrolysis combined withhigh temperature and high pressure(HT)pretreatment.The properties of the samples were characterized by SEM,TEM,FITR,XRD and TGA.The effect of HT pretreatment on CNC performance was studied.The results showed that HJS was a better source of CNC extraction than CH and WS.During the HT pretreatment process,HT-HJS was more fully swelled,and the hydrogen bond was recombined to increase the order of molecular arrangement in the HT-HJS fiber.During the acid hydrolysis process,the full swell of HT-HJS in the transverse direction was beneficial to the increase of the lateral diffusion rate of H+;the hydrogen bond recombination was beneficial to the increase of the longitudinal crystallinity along the molecular chain of the cellulose.Finally,HT140-CNC with high aspect ratio(63.40)and high crystallinity(83.34%)was obtained,which was much higher than the aspect ratio(32.00)and crystallinity(70.50%)of the CNC directly isolated without HT pretreatment.(2)The CNC/GEL composite hydrogel was prepared by using two CNC with different aspect ratios.The elastic modulus and fracture toughness of the composite gel were measured by compression test and indentation test.The effects of the aspect ratio and content of the CNC on the mechanical properties of the composite hydrogel were studied.The prediction model of the elastic modulus and fracture toughness of the composite hydrogel were established.The results showed that the CNC with higher aspect ratio couldmore significantly improve the elastic modulus and fracture toughness of the composite hydrogel compared with the CNC with lower aspect ratio.When the CNC filling amount reached 10%,the elastic modulus and fracture toughness of CNC/GEL were up to the maximum values,which was 8.90 times of the single GEL elastic modulus and 8.31 times of the fracture toughness.When the CNC content was more than 10%,the CNC in the GEL matrix was agglomerated,and the elastic modulus and fracture toughness of the CNC/GEL started to decrease.Through comparison and analysis,the COX model and Jintegral model were respectively corrected.The error value of the modified model for CNC/GEL elastic modulus prediction was within ±2%,and the error value of fracture toughness prediction was within ±6%,which were all better than the original model.(3)The chemical stability of the 10%-CNC/GEL-5 as a skin scaffold was analyzed by SEM,XRD,FTIR,DSC,swelling ratio and other characterization methods.The biocompatibility of the 10%-CNC/GEL-5 as a skin scaffold material was analyzed by light microscopy,SEM,CCK-8 and Live/dead cells staining methods.The results showed that the microstructure of the10%-CNC/GEL-5 composite hydrogelwas more compact and orderlyand the stability was significantly better compared with the GEL-5 hydrogel.The stability of the 10%-CNC/GEL-5 was further enhanced after cross-linking treatment,and the swelling balance could be achieved without dissolving in the PBS solution at 37℃.Biological experiments confirmed that the10%-CNC/GEL-5 after cross-linking treatment still had good biocompatibility.Therefore,the 10%-CNC/GEL-5 met the chemical stability and biocompatibility requirements as a skin scaffold material.(4)The theoretical model of rheological properties of the CNC/GEL composite hydrogel was established.The rheological parameters of the hydrogel were measured using a rheometer,and the extrusion of the hydrogel in the cylinder was simulated using FLUENT.The theoretical model showed that "shear thinning" occured when the hydrogel flowed under the shear stress in the nozzle,and the shear stress and velocity are nonlinearly distributed along the diameter direction in the circular section of the barrel and the nozzle.The rheological test results verified that the CNC/GEL composite hydrogel had the characteristics of "shear thinning" and"viscosity recovery" in the theoretical model.The FLUENT simulation further proved that the shear stress and velocity were nonlinearly distributed along the diametrical direction in the circular section of the barrel and the nozzle,and the fiber was extruded out of the nozzle with the phenomenon of "extrusion swell".The results of three different ways all confirmed the printability of CNC/GEL hydrogel.(5)The printability of the CNC/GEL hydrogel was verified by 3D bioprinting experiments.The forming mechanism of the scaffld during 3D bioprinting and the influence of printing parameters on the forming effect were analyzed.The results show that:"shear thinning" allowed the hydrogel to flow out of the nozzle smoothly."Extrusion swell" made the actual width of the extruded fiber larger than the nozzle diameter."Viscoelastic recovery" enabled the scaffold to be stably formed after printing.The viscoelastic recovery time was 30 s.Changes of parameters in 3D printing process such as print pressure,print temperature,and nozzle diameter could cause a change in the amount of "shear thinning" and "extrusion swell" of the hydrogel inside the cylinder,thereby affecting the actual molding effect of the extruded fiber.After the CNC was added,the interactions between the molecules inside the hydrogel were enhanced,the viscosity and modulus of the hydrogel were increased,and the resistance to deformation was enhanced,which was beneficial to improve the forming effect of the scaffold.