Viscoelastic Regulation Of Collagen-based Hydrogel Scaffolds And Its Effect On Chondrocyte Growth Behavior

Osteoarthritis（OA）is a common chronic disease characterized by degeneration of articular cartilage,which is more likely to occur in sliding joints with heavy load and large amount of activity,such as knee joint.It can be caused by many factors,including aging,obesity,overuse,trauma and joint deformity.As articular cartilage has no vascular,nerve and lymphatic distribution,once damaged,it can’t initiate inflammatory response and does not have self-repair ability.Thus,a variety of therapeutic methods have been proposed to repair cartilage defects,including arthroscopic debridement,microfracture,autologous cartilage transplantation,allogeneic cartilage transplantation and autologous chondrocyte transplantation.Although these repair strategies have some advantages,the actual effect is not ideal.Therefore,how to repair cartilage defects has always been a scientific problem in the field of clinical medicine.In recent years,with the development of cell biology and material science,tissue engineering technology is recognized as a new solution for repairing cartilage defects.Researchers usually use hydrogels as scaffolds seeded with chondrocytes,which are transplanted to defect sites for inducing cartilage tissue regeneration.It is well known that articular cartilage has complex composition and structure,and has obvious viscoelastic characteristics,which plays a role in absorbing load and protecting joints.In addition,it has been found in recent years that the viscoelastic microenvironment has a significant impact on the growth of stem cells,fibroblasts,chondrocytes and other cells.Thus,the development of viscoelastic hydrogel scaffolds has been focused by researchers.At present,the methods to regulate viscoelasticity of hydrogels mainly include changing monomer concentration,molecular weight and crosslinking density.However,the method of controlling pore orientation to regulate viscoelasticity of hydrogels is rare and the related mechanism is not clear.Therefore,the design and preparation of biomimetic cartilage scaffolds with tunable viscoelasticity,the influencing mechanism of pore structure on the viscoelasticity of scaffolds,and the role of scaffold viscoelasticity in the behavior of chondrocytes are the key issues to be solved in the treatment of cartilage defects by cartilage tissue engineering technology.For this,this paper studied the viscoelasticity regulation of hydrogel scaffolds and the growth behavior of chondrocytes,revealed the influencing mechanism of pore structure on scaffold viscoelasticity and explored the role of viscoelasticity in the behavior of chondrocytes.The main contents are as follows:Firstly,cellulose nanocrystal（CNC）and collagen（COL）were selected as raw materials to prepare CNC/COL composite hydrogel scaffolds.The viscoelasticity of the hydrogel scaffolds was regulated by designing pore structure and changing CNC component concentration.The scaffolds were characterized from the morphology,structure and viscoelasticity.The results show that CNC is a short rod.The CNC and COL in the scaffolds were bonded by hydrogen bond.The average pore size of the horizontal scaffold was 396±24μm,while that of the random scaffold was 209±21μm.The pore size was affected by the difference of pore structure,but wasn’t affected by the variance of CNC concentration.The porosity of the anisotropic scaffold was slightly higher than that of the isotropic scaffold,which was～80%.The porosity wasn’t influenced by CNC concentration.Besides,the swelling rate of the scaffold wasn’t affected by pore structure,but was affected by CNC concentration.Moreover,the horizontal oriented pore structure showed obvious anisotropic swelling deformation behavior.The rheological results show that the horizontal oriented pore structure and the incorporation of CNC component can shorten the stress relaxation time(τ_1/2)and creep time(γ_1/2),reduce the stress relaxation degree（SD）,increase the deformation recovery degree（RD）,and increase the storage modulus（G’）and loss modulus（G"）of the scaffold.Then,based on the bionics principle,mimicking the hierarchical structure of natural articular cartilage,the CNC/COL hydrogel scaffold with biomimetic hierarchical structure was prepared by iterating the horizontal,random and vertical structures using EDAC/NHS chemical crosslinking combined with freeze-drying technology.The pore structure,interlayer bonding strength and viscoelasticity of the scaffold were studied.The SEM result confirmed that the pore orientation in each layer of the caffold was consistent with the orientation of collagen fibers within the cartilage tissue,and there was no obvious gap between the layers.The result of microcomputer controlled electronic tensile testing machine shows that the interfacial bonding strength of the scaffold is approximately 70 k Pa.The rheological results show that the viscoelastic properties of the biomimetic hierarchical structure are all between those of the horizontal and vertical.Meanwhile,Maxwell-Weichert and Variable Network Density models were used to fit the the curves of stress relaxation and G’dependence on strain amplitude.The theoretical models fitted well with the experimental curves,and the experimental phenomenon was explained from the perspective of the microscopic interaction between materials.The horizontal and vertical which have the highest and lowest values of G’were extracted to clarify the resistance to torsional deformation mechanism of oriented pore structures in rheological tests.Finally,the random porous CNC/COL hydrogel scaffolds with different viscoelastic levels were selected to culture rat chondrocytes for studying the effects of scaffold viscoelasticity on chondrocyte behavior.The results show that the shortening ofτ_1/2 andγ_1/2,and the increase of G’and G"promote the spreading and proliferation of chondrocytes,and the deposition of type II collagen（COL II）and glycosaminoglycan（GAGs）,and inhibite the secretion of rat interleukin-1β（IL-1β）.In addition,the weight values ofτ_1/2,γ_1/2,G’and G"in the progression of chondrocyte behavior was assessed using Principal Component Analysis（PCA）by SPSS software.The weight value ofτ_1/2 is as high as 80.662%,indicating that the stress relaxation plays a key role in chondrocyte behavior regulated by viscoelastic hydrogel scaffolds.Based on the above results,it is proposed that FAK/YAP,as mechanically sensitive factors,mediate the response behavior of chondrocytes to scaffold viscoelasticity.FAK and YAP were proposed to participate in and mediate the response behaviors of chondrocytes to the viscoelasticity of hydrogel scaffolds.In summary,by studying the viscoelastic regulation of CNC/COL composite hydrogel scaffolds and the behavior of chondrocytes,the influencing mechanism of pore structure on the scaffold viscoelasticity was revealed,and the viscoelasticity as a mechanical clue plays an important role in the growth of chondrocytes was proved.This study is of great significance to the development and optimization of scaffold materials for cartilage tissue engineering and cartilage tissue repair.