| Diabetic foot is a kind of inflammatory disorder complication caused by hyperglycemia at the diabetic wound area and also a serious medical problem that has not been solved so far,therefore,it is one of the effective and feasible solutions to design and develop a kind of biomaterials dressing with immunoregulatory activity for healing chronic inflammation.Carrageenan is a type of sulfated linear polysaccharide extracted from marine red algae.Due to the difference in the content of sulfate groups and the structural characteristics,it can produce different immunomodulatory activities.In this study,we desulfurized commercial-grade κ-carrageenan to prepare κ/β-carrageenan oligosaccharides with different sulfate degree,which were analyzed by Fourier transform infrared spectrometer(FT-IR),1D and 2D nuclear magnetic resonance(NMR),elemental analysis(EA)and gel permeation chromatography(GPC)methods were used to determine the structure,sulfate content and molecular weight of carrageenan before and after degradation and desulfation;the cytotoxicity of κ/β-carrageenan oligosaccharides to RAW264.7 macrophages and NIH-3T3 fibroblasts was detected by cell counting kit-8(CCK-8);confocal laser scanning microscope(CLSM)was used to observe the morphology of cells after rhodamine-phalloidin staining;quantitative real-time PCR(q PCR)and flow cytometry(FCM)were used to detect the effect of κ/β-carrageenan oligosaccharides on the secretion of immune factors and the distribution of M1 and M2 macrophages after stimulating M1-type RAW264.7 macrophages;the transwell culture system and cell scratch simulation experiment were used to determine the effect of macrophage stimulation on fibroblast proliferation and collagen secretion.Finally,the material was blended with collagen and freeze-dried to make a sponge dressing with uniform holes and suitable pore diameter.After the establishment of the model on the full-thickness excisional wound of diabetic rats,general observation,H&E staining,and Masson’s staining were used to analyze the effect of the materials on wound repair.The experimental results show that κ-carrageenan is successfully desulfurized and degraded to obtain κ/β-carrageenan with low sulfate content and low molecular weight.Moreover,the desulfurization process is accompanied by partial methylation reaction;the material with high concentration(400μg/m L)has no obvious toxicity to RAW264.7 and NIH-3T3 cells;low-degree of sulfated-κ/β-carrageenan oligosaccharide(L-DSKOS)can stimulate M1-like RAW264.7 macrophages to secrete large amounts of the anti-inflammatory factor interleukin(IL)-10,transform growth factor(TGF)-β2,and reduce the secretion of pro-inflammatory factors IL-6 and tumor necrosis factor(TNF)-α.The FCM results show that L-DS-KOS can effectively accelerate the transformation of macrophages from M1 to M2-phenotype,and then produce anti-inflammatory effects;Transwell co-culture results show that L-DS-KOS can accelerate the growth rate of lower fibroblasts by regulating M1-like macrophages,and at the same time increase the expression of type Ⅰ and type Ⅲ collagen;a faster gap healing rate in the scratch simulating experiment also confirms its accelerating effect on the growth of fibroblasts.The general observation of the repair process of the full-thickness excisional wound in diabetic rats shows that the sponge dressing with the ratio 3: 7 of L-DS-KOS: collagen can effectively accelerate the wound healing;through H&E and Masson staining observation,it can be seen that the L-DS-KOS/collagen dressing can better shorten the inflammation period of the wound site,and improve the process of collagen deposition and epithelial formation,thereby promoting the repair of skin wounds in diabetic rats.Considering that L-DS-KOS can effectively regulate the secretion of immune factors and accelerate the conversion of macrophage phenotype from M1 to M2 to improve the special long-term inflammatory microenvironment in the diabetic wound site,we believe that it is expected to be used as a new type of immunomodulatory active biomaterial for the design of diabetes wound dressing. |
