Study On The Effect Of Silk Nanofiber Hydrogel Loaded Bone Marrow Mesenchymal Stem Cells On Deep Wound Healing

Objective:Stem cells have potential in wound therapy,but are limited by injury in injection process and inferior cell retention at wounds.In this study,silk nanofiber hydrogels with mass fractions of 0.5wt%,1wt%and 2wt%were prepared to explore their feasibility as a carrier for SD rat BMSCs and influence on stem cells.Then the optimal SFN concentration was selected to study the effect of SFN hydrogels loaded with BMSCs on wound healing.Methods:Firstly,pure SFN hydrogels were prepared from raw silk using a simple non-cross-linking method.To provide the nutrients and ionic environment required by cells,high concentra-tion medium was mixed with pure SFN hydrogels by ultrasonication in proportion.The obtained SFN hydrogels with medium were used for subsequent experiments.Material experiments were performed in inversion test,injectable property test,and rheolog-ical experiment to observe the morphology,injectable properties,and physical characteristics of SFN0.5、SFN1、SFN2 hydrogels.Cell viability,proliferation,and migration assay were performed on BMSCs encapsulated in SFN hydrogels in vitro to assess material biocompatibility.A cellular PCR experiment was con-ducted to detect the sternness and trauma-related gene expression of BMSCs,so we could observe the effect of three-dimensional SFN hydrogel microenvironment on stem cells.Cell viability assay after injection with a needle was perforrmed to verify the protection of cells by hydrogels.Through the above experiments,the most suitable SFN concentration hydrogel was selected as stem cell carrier to carry out animal experiments.The bioluminescence imaging experiment was performed in vivo to observe the survival of BMSCs in wound microenvironment.Then whole skin excision wound healing experiments were carried out in SD rats.The healing rate was monitored by photographing the wounds at different time points,tissue PCR experiment was performed to analyze the gene expression,hematoxylin-eosin staining was performed to observe the new epidermis,CD68 and CD206 fluorescence stain-ing was performed to detect inflammation,and CD31 and α-SMA fluorescence staining was per-formed to assess vascularization,Masson’s trichrome staining was performed to measure collagen thickness and sirius scarlet stain was performed to analysis scarring.Results:According to material experiments,hydrogels with low SFN content were more fluid and the hydrogels with high SFN content were less fluid.SFN2 is more advantageous for adhesion to the wound surface.The three concentrations of SFN hydrogels selected in this study all had good shear thinning properties and injectable performance.The modulus increased with the in-crease of SFN content,and the mechanical properties were stable.SFN2 were more suitable for the differentiation of BMSCs to skin tissue.In vitro experiments,the BMSCs cultured in hydrogels for 3 days in the three groups always maintained high activity.SFN could load cells efficiently without obvious cytotoxicity.The mi-gration rates of SFN0.5,SFN1,and SFN2 decreased sequentially.The slow migration rate pro-longed the retention time of the interosseous stem cells in the target tissue.The cell PCR results showed that compared to the control group,the expression of SOX2 was increased,the expression of OCT4 was close,and the expression of KLF4 was decreased in the experimental group of stem-ness genes.The expression of ANGPT1 and HGF were increased,the level of SFN2 was the high-est,and the expression of SDF-1and VEGF-α were decreased in trauma-related genes.Also the content of SFN could change the physical properties of hydrogel and thus affect the expression of stem cells.SFN2 was selected as the next object of study since the previous results showed that SFN2 is most suitable carrier.The cell viability results after injection showed that SFN2 hydrogel could reduce the loss of stem cells during injection.In vivo experiments,the results of bioluminescence imaging experiment showed that SFN2 could significantly improve the survival rate of BMSCs on the wound surface.The wound healing experiment was divided into four groups:Blank group,SFN2 group,BMSCs group and SFN2+BMSCs group.The results showed that SFN2+BMSCs had the shortest healing time,the fastest proliferation of new epithelium,the lowest number of CD68+ cells,the highest conversion rate of macrophages from M1 to M2,the highest number of blood vessels,the largest vascular area,the closest collagen deposition thickness to normal skin,and the highest type Ⅲ/type Ⅰ collagen ratio.Tissue PCR results showed that the expressions of ANGPT1,HGF and VEGF-α in SFN2+BMSCs group were increased,and SDF-1 was slightly decreased.Considering the com-plexity of the wound environment,the gene expression in vivo and in vitro is not completely con-sistent.Combined with the healing rate and angiogenesis.the decrease of SDF-1 did not affect the advantage of SFN2 hydrogel-loaded stem cells in wound repair.Conclusion:The injectable SFN hydrogels with shear thinning property had good biocom-patibility and could be used to load interbone stem cells and regulated their gene expression.SFN hydrogels effectively protect stem cells during the injection process,reduce migration loss and prolong the retention time of cells on the wound surface.SFN2 hydrogel loaded with BMSCs could shorten healing time,accelerate epithelialization,inhibit inflammatory response,promote vascularization,increase collagen deposition and prevent scar hyperplasia.In sum,the silk nano-fiber hydrogels with suitable cues are effective carriers for BMSCs and have promising future in skin regeneration.