Regulation Of Multi-Scale Pore Structure Of Nanocellulose Scaffolds And Their Poisson’s Ratio Effect

As a carrier for the adhesion and growth of cells,the material properties and pore structure of tissue engineering scaffolds have important effects on the growth and proliferation of cells.Different biological tissues exhibit different Poisson’s ratio effects under the action of external forces.Therefore,scaffolds should match the strength and stiffness of biological tissues and simulate the mechanical behavior of biological tissues under external forces.The preparation of multistage pore tissue engineering scaffolds with adjustable Poisson’s ratio is of great significance for cell culture.CNFs（Cellulose nanofibers）derived from plants fibers,with good biocompatibility,hydrophilicity and high mechanical properties is an excellent material for the preparation of multistage pore tissue engineering scaffolds.The scaffolds can be prepared via combining the photocuring and freeze-drying with the incorporation of CNFs into a polyethylene glycol diacrylate（PEGDA）matrix as raw materials.For one thing,CNFs can be used as the skeleton of aerogel precursor hydrogels to maintain the structure of the scaffolds,which contribute significantly to micropore formation in aerogel after freeze-drying.For another,PEGDA can be cross-linked,which endowed the hydrogels with customized pore structure.In theory,this strategy can realize the complementarity of advantages and obtain the multistage pore tissue engineering scaffolds with adjustable Poisson’s ratio.In this paper,according to the material,structure and performance requirements for tissue engineering scaffolds,the fabrication and application of CNFs aerogels with multi-stage pores and different Poisson’s ratio were studied.CNFs with high aspect ratio were prepared by combining neutral TEMPO medium oxidation and high pressure homogenization,which were mixed with PEGDA to prepare the photocurable resin mixtures.The controllable preparation of CNFs/PEGDA hydrogels with tunable Poisson’s ratio was achieved through masking-photocuring.On the basis of adjusting the preparation conditions of CNFs hydrogels,the pore structure of CNFs/PEGDA aerogel was regulated by changing the freeze-drying process conditions so that the aerogels pores with high porosity,evenly distributed interconnection and nanoscale fiber structure were obtained.Then,CNFs/PEGDA aerogels with tunable Poisson’s ratio and multi-stage pore structure was prepared.The Poisson’s ratio and elastic modulus of CNFs/PEGDA aerogels were determined by finite element software ABAQUS.Finally,stem cell cultivated on CNFs/PEGDA aerogel scaffolds with multi-stage pore structure and tunable Poisson’s ratio to study the effects of Poisson’s ratio structure and pore size on cell growth and proliferation.The main conclusions are as follows:1.Nanocellulose with a length to diameter ratio of more than 80 was prepared by TEMPO/NaClO/NaClO₂mediated oxidation system combined with high pressure homogenization,which was mixed with PEGDA in a high-speed mixer to produce a high viscosity CNFs/PEGDA photocurable resin mixtures.Columnar CNFs/PEGDA hydrogels were fabricated by solution casting-template method combined with photocuring.The water retention value of the hydrogels increased as the CNFs dosage increased,but decreased as the PEGDA dosage increased.The compression modulus increased slowly as the CNFs dosage increased,and greatly increased as the PEGDA dosage increased.The result showed that the addition of CNFs could improve the water retention performance of CNFs/PEGDA hydrogels,and PEGDA contributed more to the compression modulus of CNFs/PEGDA hydrogels.2.CNFs/PEGDA blend photocuring mixtures was used as raw material to obtain CNFs/PEGDA hydrogel with clear profile and tunable Poisson’s ratio through masking-photocuring.The influence of curing time on the clarity of the pore structure of hydrogels was studied by optical microscopy,and the optimal curing time of CNFs/PEGDA hydrogels with different Poisson ratios was determined to realize the controllable preparation of CNFs/PEGDA hydrogels.3.The columnar CNFs/PEGDA aerogels were prepared from hydrogels by freeze-drying.By changing the preparation conditions and freeze-drying process of the hydrogels,the pore structure of the aerogels can be adjusted in the micron scale,and the compression modulus and tensile modulus of the aerogels can also be regulated accordingly.The porosity of aerogel increased with the increase of the dosage of CNFs.When the dosage of CNFs was 1wt%,the porosity of aerogel reached the highest of 74.1%and aerogel had uniformly distributed pores.The average aperture of CNFs/PEGDA aerogels increased from 83μm and 99μm to 107μm when the aspect ratio of CNFs increased.When the molecular weight of PEGDA increased from 575 to 1000,the average aperture of aerogel decreased.When the relative molecular weight is 700,the compressive modulus and tensile modulus of aerogel are 0.42 MPa and0.545 MPa,respectively,which meet the requirements of mechanical properties of scaffolds in cartilage tissue engineering.When the dosage of PEDGA increased from 15 wt%to 30wt%,the porosity of the columnar aerogel decreased sharply from the highest 74.1%to 28.6%.When the dosage of PEGDA was 15 wt%,the porosity of the columnar aerogels was the highest and had a nanometer filaments structure.The lower the pre-freezing temperature,the smaller the porosity and average pore size of aerogel.Considering the average pore diameter,porosity,pore structure and compression modulus of aerogels,the optimal process conditions for preparing aerogels with tunable Poisson ratio were determined as follows:CNFs aspect ratio of 85.8,dosage of 1 wt%;The molecular weight of PEGDA was 700 and the dosage was15 wt%.The pre-freezing temperature is﹣20℃.4.Under the above optimal conditions for aerogel preparation process,CNFs/PEGDA hydrogels with tunable Poisson’s ratio was freeze-dried to obtain CNFs/PEGDA aerogels,and the pore structure of CNFs/PEGDA aerogels was regulated on the micron and nano scale.The results presented that the CNFs/PEGDA aerogel scaffolds with tunable Poisson’s ratio had micron scale pores,and the large pore walls of the scaffolds were evenly distributed with nanoscale pores,and the inner walls of the large pores were connected with the overlapping nanoscale fibers.The models of different honeycomb structures were established by Auto CAD,and the mechanical simulation of CNFs/PEGDA aerogel with different Poisson’s ratio was carried out by using finite element software ABAQUS.The results indicated that the aerogels with positive Poisson’s ratio structure had the Poisson’s ratio of>1.9,and the aerogels with negative Poisson’s ratio structure had the Poisson’s ratio of<-0.188.The Poisson’s ratio of aerogels with zero Poisson’s ratio structure were close to zero.The elastic modulus of PPR400,PPR600 and PPR800,was 0.183 MPa,0.190 MPa and 0.183 MPa respectively.And the elastic modulus of NPR400,NPR600 and NPR800 was 0.088 MPa,0.086 MPa and 0.086 MPa.As for ZPR400,ZPR600 and ZPR800,the measured elastic modulus was 0.158 MPa、0.159 MPa、0.158 MPa,respectively.It can be seen that the elastic modulus of PPR aerogels is the largest,while the elastic modulus of aerogels with different Poisson’s ratio is not related to the grid size of the aerogels.5.Human umbilical cord mesenchymal stem cells（MSCs）were cultured on 9 groups of CNFs/PEGDA aerogels with tunable Poisson’s ratio.The results of field emission scanning electron microscopy suggested that the MSCs cultivated for four days were mainly adhered to the scaffold in the form of single cells.And the fibers on the inner wall of the scaffold contributed to the adhesion and growth of MSCs.Laser confocal microscopy results showed that the MSCs on the different structure of aerogels cultured for 7 days still has high activity.On the pore wall of aerogel scaffolds,MSCs mainly proliferated outward along the pore wall centered on the inoculated cells.The MSCs deposited on the inner wall of the aerogel scaffold migrated and grew along the fibrils on the inner wall during inoculation.The result of cell proliferation test showed that the Poisson’s ratio and pore size of the scaffold had influence on the growth and proliferation of MSCs.Under the same pore size,the aerogel scaffold with zero Poisson’s ratio structure can provide a better growth environment for MSCs,and the cell proliferation rate on the aerogel scaffold with 400μm pore size is the fastest.6.This study contributes to theoretical significance for the fabrication of nanocellulose scaffolds with variable Poisson’s ratio and their application in tissue engineering.