Stereolithography Printed Tunable Poisson's Ratio Nanocellulose Scaffolds For Research On Cartilage Induction Of Stem Cells

Cellulose nanofibers(CNFs),as an important natural polymer material and new nanomaterial,has high mechanical properties,low density,good biocompatibility,biodegradability,nanostructure effects and other characteristics.And the CNFs as the three-dimensional(3D)printing biomaterials for constructing tissue engineering scaffolds with complex topology structure has become a hot research in domestic and foreign.Poisson's ratio material has a special pore structure and the negative ratio of the transverse shrinkage strain of the material to the longitudinal elongation strain is Poisson's ratio.Most of the biological tissues exhibit Poisson's ratio effect,and therefore the scaffolds with tunable Poisson's ratio may be more suitable for simulating the mechanical behavior of natural tissues.As one of 3D printing technologies,Stereolithography(SLA)has the characteristics of rapid molding speed and high precision.Therefore,the preparation of CNFs biological scaffolds with variable Poisson's ratio by SLA technology will have great development potential and application prospect.In this paper,CNFs/PEGDA mixtures with photocurable properties were prepared by mixing the water-based photo-curing resin polyethylene glycol diacrylate(PEGDA)with CNFs and the rheological properties and photocurable properties of CNFs/PEGDA blending resin were studied.The AutoCAD drawing software was used to design different geometrical structure with different Poisson ratio to construct the optical mask for SLA.The CNFs/PEGDA blending resin was cured by SLA to prepare hydrogels with different Poisson's ratio structures.And then the aerogel scaffolds with microspores and nanopore structures were obtained by freeze-drying the hydrogels and the morphology of the scaffolds was characterized.The aerogel scaffolds with different Poisson's ratio structure were used to cultivate stem cell and then induce stem cell to Chondrocytes.The effects of the different Poisson's ratio structure on cell adhesion,cell morphology,growth,proliferation and differentiation were studied.The main conclusions are as follows:1.The CNFs and PEGDA can be well mixed and the obtained CNFs/PEGDA blending resin are transparent,stable and without agglomeration phenomenon.The viscosity of CNFs/PEGDA blending resin decreases obviously with the increase of shear rate,displaying a shear thinning characteristics,which belongs to pseudo-plastic fluid.In the case of 10 wt.% PEGDA content,when CNFs content is more than 2.5wt%,the static viscosity of CNFs/PEGDA blending resin is higher than ~ 3Pa.s,which meets the requirement of ink-jet photo-curing printing for the viscosity of the material;When CNFs content is is less than 2.0wt%,the static viscosity of the system is lower than ~ 3Pa.s,which meets the requirements of SLA printing for the viscosity of the material.In the case of 2.0 wt.% CNFs content,when PEGDA content is less than 20wt%,the static viscosity of the system meets the viscosity requirement of SLA material;When PEGDA content is more than 30wt%,the static viscosity of the system conforms to the viscosity requirement of inkjet printing material.2.The CNFs/PEGDA blending photo-curable resin have good photo-curable properties.The CNFs content has no effect on the curing depth of CNFs/PEGDA blending photo-curable resin,while the initiator content and PEGDA content have a great influence on the curing depth of the blending photo-curable resin.In the case of 20 wt.% PEGDA content and 1.0 wt.% CNFs content,when the content of initiator increased from 0.05 wt.%,0.10 wt.% to 0.20 wt.%,the penetration depth of the blending photo-curable resin decreased from 1.27 mm,1.03 mm to 0.95 mm,and the critical exposure increased from 91.16 mJ/cm~2,100.86 mJ/cm~2 to 125.65 mJ/cm~2.In the case of 0.1wt% initiator content and 1.0 wt% CNFs content,when the content of PEGDA increased from 10wt%,20wt% to 30wt%,the penetration depth of the blending photo-curable resin increases from 0.93 mm,1.03 mm to 1.89 mm,and the critical exposure increased from 76.68 mJ/cm~2,100.86 mJ/cm~2 to 165.27 mJ/cm~2.When the PEGDA content increased from 10wt%,20wt% to 30wt%,the compressive modulus of CNFs/PEGDA photo-curable hydrogel increased from 0.52 Mpa,1.73 MPa to 3.65 Mpa.Therefore,considering the penetration depth,the critical exposure and the compressive modulus,the suitable amount of the initiator and PEGDA is 0.10 wt.% and 20 wt.%,respectively.3.The CNFs/PEGDA blending photo-curable resin was used as raw material to prepare the CNFs/PEGDA hydrogel scaffolds with positive Poisson's ratio,zero Poisson's ratio and negative Poisson's ratio by SLA technique and photo mask method.The influence of exposure time on the clarity of hydrogel structure was analyzed.And the hydrogel structure was simulated with Matlab software.Optical microscopy showed that clear hydrogel structurre can be obtained when the exposure time was between 60 s and 70 s at a light intensity of 11mw/cm~2.The Matlab simulation results show that the Fresnel diffraction theory can explain the overcuring phenomenon under the condition of mask curing.4.The CNFs/PEGDA aerogel with positive,zero and negative Poisson ratio were prepared by freeze-drying of the hydrogels and the compressive modulus and morphology of the aerogel were characterized.The compressive modulus of CNFs/PEGDA aerogels with positive Poisson's ratio,zero Poisson's ratio and negative Poisson's ratio is 2.63 MPa,2.86 MPa and 2.94 MPa respectively.SEM showed that the aerogel scaffolds have both micron and nanoscale pore structures,and the micropores and nanopores penetrated each other with well developed nanopores and abundant surface area.5.The mouse bone marrow mesenchymal stem cells(BMSCs)was cultured and induced to chondrocytes on the aerogel scaffolds with positive,zero and negative Poisson ratio structures respectively.And the effects of structure and Poisson's ratio of the scaffold on cell adhesion,cell morphology,growth proliferation and differentiation were investigated.Confocal Laser Scanning Microscopy(CLSM)showed that mouse BMSCs have a strong vitality after three days of culture on the scaffolds and were able to adhere to the material for growth;the proliferation test showed that the proliferation rate of BMSCs cultivated on negative Poisson's ratio structure material was higher than that of other Poisson ratio materials.The results of Alcian Blue and Toluidne Blue stains showed that CNFs/PEGDA aerogels could promote the formation of proteoglycan and glycosaminoglycans and promote the differentiation of BMSCs into chondrocytes after Mice BMSC being induced two weeks on negative Poisson's ratio structure CNFs/PEGDA aerogels.The results of Hematoxylin-Eosin stains,CLSM and Actin-Tracker Green showed that BMSCs were mostly round and single,while chondrocytes were clustered together to form obvious cartilage cavities structure.CLSM showed that a greater number of chondrocytes were induced on negative Poisson's ratio structure material than that of other Poisson ratio materials,demonstrating negative Poisson's ratio structure aerogel is more favorable for of BMSCs differentiation.