| Due to long-term exposure to the external environment,skin is easy to cause different types of damage,which will have a serious impact on the physical and mental health of patients.At present,the treatment methods of skin injury include autologous transplantation,allograft,wound dressing,etc.,but the difficult wound healing and scar tissue formation of wound healing are still clinical difficulties.Macrophages and fibroblasts play an integral role in skin healing.M1-type macrophages can trigger inflammatory responses and kill pathogens,while M2-type macrophages are beneficial for inflammation resolution and skin tissue formation.The balance of M1-type and M2-type polarization of macrophages plays an important role in rapid scarless wound healing.One of the main cells in the dermis of the skin is fibroblasts.After skin injury,fibroblasts are recruited to the skin wound in large numbers and differentiated into myoblasts under the stimulation of related growth factors and cytokines.Myoblasts can secrete a large amount of collagen at the wound site and provide a strong tension to promote the process of tissue fibrosis,thus accelerating wound contraction and healing.However,the constant presence of myoblasts will cause scar formation.The construction of wound repair materials that can regulate inflammatory response and fibroblast differentiation during wound healing is expected to realize rapid scarless healing of refractory wounds.Silk fibroin（SF）of silkworm silk is a natural protein,which has great potential as a biological material in the field of skin repair due to its non-toxicity to organisms,superior biocompatibility and good cell adhesion.Previous studies have shown that the micro-nano structure of scaffold materials can regulate the polarization,cell morphology,adhesion,migration and differentiation of macrophages.In this study,multimicron structure of polydimethylsiloxane PDMS template was prepared by soft lithography and femtosecond laser etching,and then multimicron structure of PDMS elastic impression was re-engraved onto SF membrane by solution casting method to prepare multimicron structure of SF membrane.It is expected that the physical microenvironment formed by the multilevel micron structure can regulate the polarization of macrophages and the differentiation of fibroblasts to achieve rapid scarless healing of wounds.The main results obtained in this study are as follows:（1）Multistage silk fibroin（SF）films with uniform and regular morphology were preparedMultimicron structure PDMS templates were prepared by soft lithography and femtosecond laser etching,and multimicron structure SF membranes were obtained by using the templates and solution casting method.The micron grooves on the silicon wafer are copied onto the PDMS template by soft lithography,and the sub-micron ridges are machined on the PDMS template with micron grooves by femtosecond laser.These orientated ridges are parallel to the grooves.SF membranes were divided into two experimental groups of 100-50-25 and400-400-200 according to different sizes of micron groove structure for further study.The results of scanning electron microscopy（SEM）showed that the surface of the unstructured SF film was smooth,flat and without scratches,while the surface of the experimental group had a uniform and regular multilevel ordered structure.The water vapor transmittance of all silk fibroin films was 700-850 g·m-2·d-1,which was within the normal skin water vapor transmittance of 240 g·m-2·d-1 to 1920 g·m-2·d-1.The results of contact Angle test show that the silk fibroin film material is hydrophilic material with proper swelling property,which can absorb part of water and resist the swelling of water well.The results of infrared spectroscopy,X-ray diffraction（XRD）and thermal properties show that there is no significant difference in chemical groups,crystallographic properties and thermal properties between the unstructured SF films and the multimicron structured SF films.The above results show that SF film has good air permeability,water swelling resistance and hydrophilic properties,and is suitable for dressing.The multimicron structure SF membrane prepared by PDMS template and solution casting only changed the surface structure of silk fibroin membrane,but did not significantly affect the chemical properties,which is conducive to the study of the effect of multimicron structure on wound healing in SF membrane.（2）Regulation of macrophage behavior by multimicron SF membranesThe results of CCK-8 showed that macrophages could proliferate on multimicron structured SF membranes,but the proliferation rate was lower than that on unstructured SF membranes.The results of SEM and cytoskeleton staining showed that the unstructured SF membrane could only promote the formation of dispersed pseudopods in a small number of cells,showing M1 morphology.Multimicron SF membranes can not only promote some cells to extend out of dispersed pseudopods to groove walls and other positions,showing the morphology of dispersed polypseudopods of M1 type,but also induce some cells to extend in a stretching-oriented way,showing the elongated spindle of M2 type.Flow cytometry results also showed that the unstructured SF membranes could promote M1-type polarization in about5%of macrophages,and multimicron structured SF membranes could promote M1-type polarization in some cells and M2-type polarization in other cells.The results of RT-q PCR detection on the expression of M1 type characteristic genes（i NOS,TNF-αand IL-1β）and M2type characteristic gene（Arg-1）were also confirmed.Among them,400-400-200 experimental group had a more significant effect.（3）Regulation of fibroblast behavior by multimicron structured SF membranesThe results of CCK-8 assay showed that fibroblasts could proliferate normally on multimicron structured SF membranes,but their proliferation rate was lower than that on unstructured SF membranes.SEM and FITC experiments showed that the multimicron structure SF membrane increased the surface roughness of the membrane,which was conducive to the adhesion of L-929 fibroblasts.The experimental component fibroblasts generated a large number of filamentous pseudopods,and the cells extended to both ends.Immunofluorescence detection showed that multilevel micron structure SF membrane promoted fibroblast differentiation into myofibroblast at the early stage of wound healing,and the promotion effect was more significant in the 400-400-200 group.RT-q PCR results showed that multimicron structure SF membrane inhibited the formation of myoblasts at the later stage of wound healing,and the inhibitory effect was more significant in the 400-400-200 group.（4）Regulation of macrophage and fibroblast co-culture by multi-micron SF membranesCCK-8 results showed that the co-culture system delayed the cell proliferation time.The results of SEM and FITC also showed that the formation time of pseudopodia was delayed in the co-culture system.The results of immunofluorescence showed that the multilevel micron structure SF membrane promoted the differentiation of fibroblasts into myofibroblasts more significantly in the co-culture system.The results of RT-q PCR showed that the 400-400-200group was more conducive to promoting the expression of Col-I,TGF-β1,FN andα-SMA in the co-culture system.（5）Study on the efficacy of multi-micron structure SF membrane in the repair of full-layer skin defect wounds in ratsFurther,the effect of multimicron structure SF membrane on wound healing was tested by the full-layer skin defect model of rats.The untreated unstructured SF membrane was used as the control group,while the multimicron structure SF membrane was used as the experimental group.The results showed that there was no significant difference in the wound healing rate between the experimental group and the control group 7 days after surgery.14 days after surgery,the healing effect of the experimental group was significantly higher than that of the control group.H&E staining showed that there was no significant difference between the experimental group and the control group at 7 days after surgery.14 days after surgery,a small number of inflammatory cells and a large number of granulation tissue formation were observed in the skin of the 100-50-25 and 400-400-200 groups compared with the control group.Masson staining showed that a large number of collagen fibers were formed in the skin lesions of rats in the experimental group compared with the control group 7 days after surgery.At 14 days after surgery,a large number of collagen fibers were produced in the skin lesions of both the experimental group and the control group,but the promoting effect of the experimental group was more obvious.Immunohistochemical results showed that the expression of proinflammatory protein i NOS（M1 type）and anti-inflammatory Arg-1 protein（M2 type）in macrophages of the control group and the experimental group was positive 7 days after operation.At 14 days after operation,the expression of i NOS（type M1）was lower and the expression of Arg-1（type M1）was higher in the experimental group than in the control group.There was no significant difference inα-SMA expression between the control group and the experimental group at 7 and 14 days after surgery.Compared with the control group,the expression levels of TGF-β1 and Col-I were higher at 7 and 14 days after surgery,but no significant expression was found in the control group.These results indicate that multimicron structured SF membranes can promote full-skin wound healing better than raw SF membranes.In conclusion,multilevel micron materials constructed on the surface of silk fibroin protein membrane can promote phenotype and functional changes of macrophages and fibroblasts,improve cell microenvironment and promote cell differentiation.This study systematically explored the regulatory mechanism of morphology-induced macrophages and its influence on the physiological function of fibroblasts,providing a new idea for improving skin healing,and having important guiding significance for finding materials for skin wound healing and optimizing wound healing. |
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