| Objective:SF-MA-BG accelerates diabetic wound healing through restored HIF-1a pathwayMethods:1.Borosilicate glass was prepared by sol-gel route.Then,the BG powders were chemically modified,and methacryloyloxy groups were grafted on its surface using TMSPMA.Preparation of SF-MA-BG composite hydrogels（SF-MA-BG）:SF-MA was dissolved in water to prepare a SF-MA aqueous solution（20%w/v）,then photoinitiator LAP（0.2%w/v）and BG-MA powders were dissolved or dispersed in SF-MA solution.2.In order to verify Characterization of SF-MA-BG,BG,BG-MA,SF,SF-MAwere analyzed by 1H nuclear magnetic resonance spectroscopy,ATR-FTIR spectra,XRD,XPS and SEM.3.The effect of SF-MA-BG composite hydrogel on HUVECs viability was test by cck8 and live/dead Kit.4.The effect of SF-MA-BG composite hydrogel on HUVECs migration was analyzed using a transwell assay and scratch assay.The impact of the SF-MA-BG hydrogel on the tube formation potential of HUVECs was studied by tube formation assay.5.The effect of SF-MA-BG composite hydrogel on Angiogenesis-related gene and protein expression by q PCR and Wb.6.The effect of SF-MA-BG composite hydrogel on Macrophage regulation in vitro by q PCR and flow cytometry.7.This study was initiated to develop a rat model of type 2 diabetes.Then two full-thickness wounds of 15mm x 15mm were performed on the back of each rat after anesthetizing by gaseous isofluorane and the infection was caused by inoculation of 1×107 CFU/ml of Staphylococcus aureus（S.aureus）.The wound bearing rats were treated with（1）nothing as blank control（2）SF-MA and（3）SF-MA-3BG.The SF-MA or SF-MA-3BG was applied onto the wound and exposed to UV light for 30 seconds form a gel covering the wound.At day 0,3,10 and 21,the wound was photographed by a digital camera.Then diabetic wound healing was analysed by hematoxylin and eosin（H&E）,Masson and Immunofluorescence staining of CD80,CD206,HIF-1a,VEGF,i NOS,TGF-β,a-SMA and CD31.Results:1.The structure of SF and SF-MA were confirmed by 1H nuclear magneticresonance（NMR）.The characteristic peaks of NMR spectra were assigned by referring to previous papers（34,37,38）.The peaks at chemical shift 5.3 ppm and 5.6 ppm indicate the existence of methacryloxy groups.And the characteristic resonance peak of themethyl group of GMA（δ=1.8 ppm）has appeared by addition of the GMA.In addition,the characteristic peak of lysine methylene group（δ=2.9 ppm）decreases significantly with the addition of GMA,which indicated successful modification of the lysine residues in SF.Meanwhile,the structure of SF,SF-MA,BG,BG-MA and SF-MA-BG wereexamined by ATR-Fourier-transform infrared spectroscopy（ATR-FTIR）.The characteristic peaks of amide I,II,III and V at 1636,1516,1232 and 674 cm-1respectively were found in SF,SF-MA,and SF-MA-BG,and the peak at 1060 cm-1 is attributed to the random conformation of SF.The broad peaks at 1460 and 1050 cm-1found in BG,BG-MA and SF-MA-BG were attributed to the B-O-B and Si-O-Si vibration of borosilicate glass.The properties of borosilicate glass were furtherlyinvestigated by XRD.The broad peaks suggest that the prepared material was amorphous in structure.The diameter distribution analysis showed that the size of BG particles ismainly around 1μm.And BG-MA was successfully obtained through the modification of BG by silane coupling agent according to the results of XPS which showed the presence of C-C-C,C-C-O and C-C=O.2.Then tensile strength and adhesive strength of SF-MA-BG were measured by a mechanical testing machine.The addition of BG-MA can further improve the mechanical strength,and the maximum tensile strength can reach up to 40kpa（Fig.3B）,and theadhesive strength between SF-MA-BG and pigskin can also reach up to 23kpa（Fig.3C）,which provides the necessary strength guarantee for future clinical use.The degrading behavior and ion releasing of SF-MA-BG were illustrated by ICP-OES（Fig.3D-3F）.Immersed in the PBS,SF-MA-BG can steadily release various therapeutic ionsrepresented by Si,B and Cu ions which are the basis of the physiological functions of SF-MA-BG hydrogel.3.The proliferation of HUVECs up to 5 days was quantified by CCK-8 kit,the optical density（OD）values in all groups were similar and the differences were not statistically significant,and the OD values increased over time,which suggested cellsmaintained good vitality and ability to continue to proliferate.In the Live/Dead assay we found that the HUVECs cells in the groups of SF-MA,SF-MA-1BG,and SF-MA-3BG displayed the same morphology as that of the positive control,but relatively fewer green cells were found in the SF-MA-6BG group.4.For the evaluation of HUVECs migration,a transwell assay was conducted and the results showed that HUVECs migrated significantly faster in the SF-MA,SF-MA-1BG,SF-MA-3BG and SF-MA-6BG,while SF-MA-3BG group took the lead.In the scratch assay,faster wound closure of HUVECs was observed in the SF-MA-3BG group after 12h.Quantified data clearly demonstrated the above trend.The proangiogenic potential of SF-MA-BG hydrogel in vitro were evaluated by tube-formation assays.After incubating on Matrigel substratum for 6h,SF-MA-3BG group exhibited more junctions and longer tube lengths（8.105±0.123mm）than other groups,indicating higherangiogenesis capability.5.It was found that all these genes（VEGF,HIF-a,PDGF,e NOS,ang,and SDF-1）were upregulated in the SF-MA-3BG group.The results of immunofluorescence staining showed that the activity of HIF-a in the SF-MA-BG group was higher compared toControl and SF-MA group.The results of Western Immunoblotting also confirmed that cells cultured with the extract medium of SF-MA-3BG can express more VEGF andHIF-a proteins.6.After culturing macrophages with the extract medium of different materials,it was found the expression of the pro-inflammatory genes TNF,IL-1,i NOS and IL-6 in the SF-MA-BG group was elevated compared to control group,indicating SF-MA-BG promoted the normal inflammatory responses of macrophages,but the expression of these genes in SF-MA-BG group was lower than those in LPS and SF-MA groups,while the expression of anti-inflammatory genes including IL-10,Arginase（arg）and TGF-b and CD206 was substantially upregulated,indicating that SF-MA-BG won’t cause an expanding inflammation which is harmful to wound repairing.The flow cytometryresults showed that in comparison with LPS group,the expression of M1 marker CD86in SF-MA and SF-MA-BG groups were reduced.Furthermore,the expression of M2marker CD206 were improved after treated with the extract medium of SF-MA-BGcompared to SF-MA and LPS group.7.The SF-MA-BG group had a faster healing rate than the SF-MA and control groups,according to HE staining results and optical images,and the SF-MA-BG group had a faster rate of fibrous repair than the other groups,according to Masson’s trichrome staining.When a SMA and CD31 were co-labeled,it was discovered that the SF-MA-BG party had more vessels and greater vessel diameters at 3 and 10 days than the SF-MA and control groups.The SF-MA-BG group attracted more macrophages at 3 and 10 days than the SF-MA and control groups,according to immunofluorescence staining findings.There were even more M2-directed macrophages.Conclusion:Both angiogenesis inhibition and inflammatory dysfunction due to the impairment of HIF-1a make diabetic wounds difficult to heal.To address the issue,a compositesystem of biocompatible silk fibroin and versatile bioactive borosilicate glass wassuccessfully synthesized and applied to improve the activity of HIF-1a and promote the regeneration of diabetic wounds.This composite system can infiltrate the entire wound surface and be in vivo photocrosslinked to form an integral SF-MA-BG gel binding with the wound,and effectively inhibit bacterial growth by releasing antibacterial copper ions.Meanwhile,SF-MA-BG hydrogel can improve HIF-1a activity via chelation betweenHIF-1a and Cu2+to restore HIF-1a pathway that regulates angiogenesis and inflammation.With the improved HIF-1a activity,angiogenesis was enhanced,and inflammatoryfunctions of macrophages were recovered.Furthermore,SF-MA-BG can regulate theimmune response of macrophages and promote quick conversion of macrophages to M2type,enabling macrophages to participate in tissue repairing.The in vivo experiments confirmed that this newly developed SF-MA-BG hydrogel accelerated the diabeticwound healing with improved HIF-1a activity,enhanced angiogenesis and regulatedinflammation,thus holding promising application prospects in biomedical fields such as diabetic wound healing. |
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