Preparation Of Polycaprolactone/polyethylene Glycol/Hydroxyapatite Electrospun Nanofibers And Its Effect On Macrophage Behavior In Vitro

Objective:Tissue engineering scaffolds as foreign implants not only have excellent physical and chemical properties,but also pay attention to the interaction between scaffolds and macrophages.Polycaprolactone(PCL)has low immunogenicity,nano-hydroxyapatite(n HAP)has good bone induction,and polyethylene glycol(PEG)has high hydrophilicity.Nanofiber scaffolds can be prepared based on electrospinning technology.In this experiment,PCL/PEG/n HAP electrospun composite scaffolds were constructed to explore the effects of changes in material composition and composition ratio on the micro morphology.And focusing on the effect of such scaffolds on macrophage polarization aims to provide a new research idea for the development of a kind of biological scaffolds with immune regulation function.Methods:In this study,the PCL,PCL/PEG and PCL/PEG/n HAP nanofiber scaffolds were prepared by electrospinning technology.The morphology,pore size,wettability and mechanical properties of the scaffolds were characterized by scanning electron microscope(SEM),water contact angle tester and universal material testing machine,and the effects of the addition of n HAP and PEG on the properties of the scaffolds were discussed.Mouse bone marrow mesenchymal stem cells(m BMSCs)and mouse monocyte macrophage leukemia cells(RAW264.7)were inoculated on the scaffolds of each group.The distribution and morphology of cells were observed by laser scanning confocal microscope(LSCM)and SEM,and the proliferation of cells was detected by CCK-8 method,to evaluate the cytocompatibility of the materials in vitro and the effect of the materials on the polarization of macrophages.Results:1.Scanning electron microscopy showed that the fiber surface of PCL/PEG/n HAP nanofiber scaffold was smooth and the size was mainly distributed in 100～300nm,indicating that the solution obtained from the blending of these three materials can successfully prepare nano-sized composite scaffold by electrospinning technology.Moreover,the diameter of nanofibers increased with the addition of PEG and n HAP,from96.06±36.13 nm in PCL group to 243.62±93.57 nm in PCL/PEG/n HAP group.FTIR results showed that the surface chemistry of PCL/PEG/n HAP composite scaffolds was effectively improved compared with PCL group.The test results of universal material testing machine show that the addition of PEG and n HAP significantly improves the tensile strength of PCL based nanofiber scaffolds,making the scaffolds have better toughness and processability.The results of water contact angle test showed that the hydrophilicity of the scaffolds added with PEG increased significantly from 108°±3.80° to 61.93°±2.64 °.2.In vitro cell experiments showed that the scaffolds in each group had good cell compatibility,RAW264.7 and m BMSCs can adhere and proliferate on the scaffold.The results of TRITC Phalloidin-DAPI staining of two kinds of cells inoculated on the scaffolds showed that both kinds of cells could stably adhere and grow on the scaffolds of each group.The results of CCK-8 cell proliferation experiment showed that the scaffolds were inoculated with RAW264.7 the cell proliferation rate increased steadily with time in the single culture group,the scaffold inoculated m BMSCs single culture group and the scaffold inoculated co-culture group.In each culture group,the PCL/PEG/n HAP scaffold group had the highest cell proliferation rate(P < 0.05).Scaffolds inoculated with RAW264.7,the experimental results also verified that the scaffolds in each group can effectively promote RAW264.7 adhesion and growth.The results of ELISA in the co-culture group of two kinds of cells inoculated with scaffolds showed that after implant scaffolds,the expression level of TNF-α in each group was significantly lower than that in the control group,the expression level of IL-10 in PCL/PEG/n HAP group was higher than that in PCL group and PCL/PEG group(P < 0.05).Conclusion:In this study,a kind of PCL/PEG/n HAP nanofiber composite scaffolds were successfully constructed by electrospinning technology.By adjusting the components of the scaffolds and adjusting the parameters of the electrospinning machine,the physical and chemical properties of PCL based scaffolds were optimized,and the micro-morphology,mechanical properties and hydrophobicity of the scaffolds were improved.m BMSCs and RAW264.7 were inoculated into scaffolds in each group,the results of cell experiment in vitro verified that PCL/PEG/n HAP scaffold group was conducive to cell adhesion and proliferation and had good biocompatibility.At the same time,ELISA results showed that macrophages cultured on the scaffold showed a high level of IL-10 expression,suggesting that PCL/PEG/n HAP scaffold had the potential to promote the polarization of macrophages and was more conducive to tissue repair.