The Effect Of PCL Fiber Diameter And Surface Roughness On Macrophages Behavior

When tissue dysfunction is caused by disease or trauma and the body cannot be repaired by itself,regenerative medicine implants biomaterials into the body to trigger the body’s innate regeneration mechanism to assist restore the damaged tissue.However,when the materials is implanted,it inevitably causes surgical wound,triggering an inflammatory response.Macrophages are the dominant immune cells in the host immune reaction to implanted materials.Its phenotype transition is the key to determining the direction of the inflammatory response,and this phenotype change is highly dependent on the topographical structure of the surrounding tissues.Therefore,rational design of the physical topography and structure of the implanted biomaterials,so that the biomaterials and the host immune system interact positively,and direct the immune response into benefiting for tissue regeneration,which is the core of achieving tissue repair.The fibrous scaffold prepared by electrospinning has the advantages of easy-to-adjust size（fiber diameter,pore size）,controllable structure（roughness,fiber orientation,stiffness）,and high simulation of extracellular matrix in vivo.It has distinct advantages in regenerative medicine.Based on this,this subject employ electrospinning and solution-induced crystallization method to prepare micro-nanostructure PCL fibrous scaffolds to explore the effects of pore size,roughness and stiffness changes on the macrophages polarization and the osteogenic differentiation of rBMSCs.At the same time,the rat subcutaneous transplantation test was used to explore the inflammatory response and tissue regeneration of the PCL fibrous scaffold with micro-nano multilevel structure in vivo.This subject aims to explore the relationship between the micro-nano multilevel structure of electrospinning fibrous scaffolds and inflammation,and provide new ideas for the structure design of tissue regeneration fiber scaffolds.（1）Preparation and physical and chemical properties of PCL micro-nano multilevel structure fibrous scaffold.Three kinds of smooth fiber scaffolds（P10,P12,P15）with various diameters were fabricated by electrospun process,and then nano-protruding crystal structures（P10-SK,P12）were further constructed on the surface of the smooth fiber scaffolds by solutioninduced crystallization（P10-SK,P12-SK,P15-SK）.Scanning electron microscope observation showed that with the increase of the mass fraction of the solution,the fiber diameter increased from 0.45 μm（P10 group）to 11.57 μm（P15 group）,making the pore size of the fiber scaffold increased from 1.78 μm（P10-SK）to 12.50 μm（P15）.Atomic force microscopy tests showed that the surface roughness（Sa）of the fiber scaffold with a string crystal structure is in the submicron level.The contact angle test shows that the crystal structure can improve the hydrophobicity of the PCL fibrous scaffold.The mechanical test results show that as the fiber diameter and pore diameter increase,the elastic modulus of the fibrous scaffold decreases.Among them,the P15 group has the lowest stiffness,which is 5.62 MPa of P15 and 6.68 MPa of P15-SK respectively.（2）The influence of PCL fiber scaffold with micro-nano multilevel structure on macrophages behavior.Cell live/dead fluorescent staining showed that the six fiber scaffolds had good biocompatibility.ELISA analysis showed that the SK structure can enhance the production of IL-4,and the P15-SK can reduce the production of IL-6.Cellular immunofluorescence staining analysis showed that compared with the small-pore and highstiffness fibrous scaffolds,the large-pore and low-stiffness P15 showed the most M2 macrophages polarization.（3）The effect of macrophages on the PCL fiber scaffold with micro-nano multilevel structure on the osteogenic differentiation of stem cells.The ALP activity and mineralized nodule staining showed that compared with the smooth structure,the SK structure showed higher ALP activity and could promote the osteogenic differentiation of stem cell.（4）Animal experiment analysis.Hematoxylin-Eosin（HE）staining showed that fiber scaffolds（P15,P15-SK）with large pore size and low stiffness（P15,P15-SK）can reduce the accumulation of macrophages and the formation of FBGCs.Masson tricolor（MT）staining shows that collagen is deposited less on the large-pore,low-stiffness fiber scaffolds（P15,P15SK）,and the collagen structure is distributed in a network with low density,showing a good tissue regeneration effect.The results of qPCR analysis showed that fibrous scaffolds with large pore size and low stiffness（P15,P15-SK）support the production of IL-10 and restrain the production of IL-6;compared to the smooth fibrous scaffold,the fibrous scaffolds（P10-SK,P12-SK,P15-SK）with SK structure have active impact on inflammation subsides...