A method for harvesting viable cells from wound dressings.

Wound fluid has been well studied for exploring protein biomarkers contained in it. However, cells in wound fluid have not received much attention due to the difficulty in their collection. Our study aimed to establish a method for collecting viable cells from discarded wound dressings. A protocol was designed to wash out nonadherent cells and detach adherent cells from silicone-faced foam wound dressings using trypsin-EDTA. The optimal concentration and incubation time of trypsin-EDTA for collecting equivalent proportions of different cell types to the original cell population were determined in vitro. Cell composition and gene expression changes in monocytes, lymphocytes, neutrophils, fibroblasts and keratinocytes were confirmed using immunocytochemistry and RNA-sequencing ex vivo. Full-thickness wounds were created on 9-week-old male C57BL/6J mice. Wound fluid was collected, and half of it was applied to the wound dressings. The original cell population in the wound fluid and the cell population collected from wound dressings were compared. In the in vitro study, 0.25% trypsin-EDTA and 2.5-min incubation time were considered optimal for collecting adherent cells from wound dressings. In the ex vivo study, among all cell types, only CD3+ lymphocytes showed a significantly higher cell proportion in the collected group. The relative gene expression of the five selected cells showed no significant changes (p-value >0.05, |log2 fold change| < 1.5, differential gene expression analysis). Viable nonadherent and adherent cells were collected from wound dressings without altering gene expression and could be used in future studies for cellular analysis of wound fluid.

Efficacy of wearable vibration dressings on full-thickness wound healing in a hyperglycemic rat model.

Local low-frequency vibration promotes blood flow and wound healing in hard-to-heal diabetic foot ulcers (DFUs). However, vibration treatment is challenging in patients with DFUs due to wound management difficulties and low adherence. Consequently, developing wearable self-care devices becomes imperative for effective wound healing. This study introduces a wearable vibration dressing and assesses its impact on wound healing in hyperglycemic rats. Low-frequency vibration at 52 Hz was applied to the wound for 40 min/day in awake rats. Relative wound areas on post-wounding days (PWDs) 4-7 were significantly smaller and the wound closure rate was significantly higher in the vibration group than in the control group (p < 0.05, respectively). The total haemoglobin at baseline and after vibration on post-wounding day 7 was significantly larger in the vibration group than in the control group (p < 0.05). On PWD 7, the thickness of the granulation tissue was significantly higher in the vibration group than in the control group (p < 0.05). Moreover, the number of blood vessels at the wound site and vascular endothelial growth factor A protein expression were significantly higher in the vibration group than in the control group (p < 0.05, respectively). The ratio of (CD68+ /iNOS+ )/(CD163+ ) macrophages in the vibration group was significantly lower than that in the control group (p < 0.05). These results indicate the potential of wearable vibration dressings as new self-care devices that can promote angiogenesis and blood flow, improve inflammation, and enhance wound healing in DFUs.

Which biomarkers predict hard-to-heal diabetic foot ulcers? A scoping review.

Diabetic foot ulcers (DFUs) often develop into hard-to-heal wounds due to complex factors. Several biomarkers capable of identifying those at risk of delayed wound healing have been reported. Controlling or targeting these biomarkers could prevent the progression of DFUs into hard-to-heal wounds. This scoping review aimed to identify the key biomarkers that can predict hard-to-heal DFUs. Studies that reported biomarkers related to hard-to-heal DFUs, from 1980 to 2023, were mapped. Studies were collected from the following databases: MEDLINE, CINAHL, EMBASE, and ICHUSHI (Japana Centra Revuo Medicina), search terms included "diabetic," "ulcer," "non-healing," and "biomarker." A total of 808 articles were mapped, and 14 (10 human and 4 animal studies) were included in this review. The ulcer characteristics in the clinical studies varied. Most studies focused on either infected wounds or neuropathic wounds, and patients with ischemia were usually excluded. Among the reported biomarkers for the prediction of hard-to-heal DFUs, the pro-inflammatory cytokine CXCL-6 in wound fluid from non-infected and non-ischemic wounds had the highest prediction accuracy (area under the curve: 0.965; sensitivity: 87.27%; specificity: 95.56%). CXCL-6 levels could be a useful predictive biomarker for hard-to-heal DFUs. However, CXCL6, a chemoattractant for neutrophilic granulocytes, elicits its chemotactic effects by combining with the chemokine receptors CXCR1 and CXCR2, and is involved in several diseases. Therefore, it's difficult to use CXCL6 as a prevention or treatment target. Targetable specific biomarkers for hard-to-heal DFUs need to be determined.

Local low-frequency vibration accelerates healing of full-thickness wounds in a hyperglycemic rat model.

AIMS/INTRODUCTION: Local low-frequency vibration (LLFV) promotes vasodilation and blood flow, enhancing wound healing in diabetic foot ulcers with angiopathy. However, vibration-induced vasodilation does not occur, owing to chronic hyperglycemia and inflammation. We hypothesized that LLFV improves glycometabolism and inflammation, leading to vasodilation and angiogenesis in diabetic wounds. Therefore, this study investigated the effect of LLFV on wound healing in hyperglycemic rats, primarily focusing on glycometabolism, inflammation, vasodilation, and angiogenesis. MATERIALS AND METHODS: Streptozotocin-induced hyperglycemic Sprague-Dawley rats were used in this study. We applied LLFV to experimentally-induced wounds at 50 Hz and 0, 600, 1,000 or 1,500 mVpp for 40 min/day from post-wounding days (PWD) 1-14. RESULTS: The relative wound areas in the 600 and 1,000 mVpp groups on PWD 5-7 were significantly smaller than those at 0 mVpp. The expression of Glo-1 (1,500 mVpp) and Slc2A4 (1,000 and 1,500 mVpp) was upregulated on PWD 4 and 14, respectively. However, there was no difference in methylglyoxal expression levels in any group until PWD 14. At 1,000 mVpp, the expression of Tnfa on PWD 4, and that of Ptx3 and Ccl2 on PWD 14 was downregulated. Furthermore, the M1/M2 macrophage ratio was considerably decreased on both days. The expression of Nos3, Vegfa and vascular endothelial growth factor A was upregulated on PWD 4. In addition, vasodilation and angiogenesis were more obvious on PWD 14 with 1,000 mVpp. CONCLUSIONS: The results suggest that LLFV promotes wound healing, improves glycometabolism and inflammation, and enhances vasodilation and angiogenesis in hyperglycemic wounds.

Low-frequency vibration promotes AMPK-mediated glucose uptake in 3T3-L1 adipocytes.

Delayed healing of diabetic foot ulcers (DFUs) is one of the major consequences of angiopathy caused by hyperglycemia stemming from insulin resistance. Interventions that improve blood supply and hyperglycemia are essential for treating DFUs. Low-frequency vibration (LFV) promotes peripheral blood flow and wound healing in DFUs, regardless of hyperglycemia. We hypothesized that LFV promotes non-insulin-mediated glucose uptake, which is also referred to as AMPK-mediated glucose uptake, in adipocytes at wound sites, thereby alleviating hyperglycemia, which, in turn, accelerates wound healing. The objective of this in vitro study was to identify LFVs that optimally promote glucose uptake in adipocytes and investigate the mechanism underlying enhanced glucose uptake caused by LFV. 3T3-L1 adipocytes were used in this study. LFV was applied at 50 Hz for 40 min/d to investigate the most effective vibration intensity (0-2000 mVpp) and duration (0-7 d) of glucose uptake. We comparatively assessed 2-deoxyglucose (2-DG) uptake in control and vibration groups. To elucidated the mechanism underlying 2-DG uptake induced by LFV, wortmannin and compound C were used to inhibit insulin-mediated GLUT4 translocation and AMPK activation, respectively. Additionally, GLUT4 translocation to the plasma membrane was assessed using immunofluorescence image analysis. Our results indicated that 2-DG uptake in the 1000 and 1500 mVpp groups was higher than that in the control group (p = 0.0372 and 0.0018, respectively). At 1000 mVpp, 2-DG uptake in the 5- and 7-d groups was higher than that in the non-vibration group (p = 0.0169 and 0.0452, respectively). Although wortmannin did not inhibit 2-DG uptake, compound C did. GLUT4 translocation to the plasma membrane was not observed in the vibration group adipocytes treated with compound C. Thus, our results indicated that an LFV of 50 Hz, 1000 mVpp, 40 min/d, over 5 d was optimal for accelerating AMPK-mediated GLUT4 translocation and glucose uptake in adipocytes.

Chronological changes in rat heel skin following depressurization of pressure ulcer-like dermal lesions.

In our previous study, we proposed an animal model in which pressure ulcer-like dermal lesions can be produced by denervation of the sciatic nerve and application of a pressure load to rat heel skin. In the present study, we divided these animals into non-treated and pressure loading groups, and initiated hindlimb unloading (depressurization) by tail suspension at 1, 3, 5, 7, and 14 days after inflicting lesions (1-14d pressurization groups). Chronological changes in heel lesions were examined morphologically in all treatment groups after 1, 3, 7, 14, 28, and 40 days. Open dermal lesions were formed by 14 days in the loading group and numerous macrophages were present. In the 14d pressurization group, numerous macrophages were still distributed in and around lesions and Vascular endothelial cell growth factor (VEGF) expression was strongly detected by 3 days, but a thin germinal layer began to appear and CD68-positive macrophages and VEGF immunoreactions decreased gradually by 7 days later. By 14 days after depressurization, the germinal layer was repaired, and macrophages and immunoreactions of VEGF were similar to those of non-treated skin. These chronological changes were similar to those in human pressure ulcers, but from 5d after depressurization, different chronological changes were observed. Specifically, epidermis was thickened and macrophages were hardly detected at 5 days in the loading group, but the epidermis disappeared by 1 day in the 5d pressurization group. Subsequently, numerous macrophages aggregated and VEGF expression was increased by 3 days, and the remaining healing process was similar to that in the 14d pressurization group. Even when unloading was performed during the early stages (5d pressurization group), the epidermis disappeared and macrophages were then distributed before repair of the lesion was observed. These results suggest that earlier migration of macrophages to skin lesions might be associated with rapid wound healing.

Inhibition of the migration of MCP-1 positive cells by icing applied soon after crush injury to rat skeletal muscle.

Migration of the macrophages to the injured site soon after the skeletal muscle injury is crucial for subsequent regeneration of the muscle fibers. The Monocyte chemoattractant protein-1 (MCP-1) is important chemokine for regulating migration of the monocytes/macrophages. Earlier reports have discussed that icing applied soon after muscle crush injury retards muscle regeneration through retardation of macrophage migration. The MCP-1+ cells and neutrophils might promote the migration of the macrophages. To test the hypothesis that icing soon after the skeletal muscle injury affects MCP-1+ cells and neutrophils, we examined the effect of icing on MCP-1+ cells and neutrophils after crush injury to skeletal muscle in rats. Owing to the icing application for 20 min soon after the injury, accumulation of the macrophages was inhibited until 12 h after injury. Numbers of the neutrophils at 3 h after the injury and the MCP-1+ cells at 6 h and later after the injury in the icing group were significantly lower than those in the non-icing group, suggesting that these phenomena contribute to the retardation of macrophage migration.

Constant existence of the sensory branch of the nerve to the pyramidalis distributing to the upper margin of the pubic ramus.

Twenty-one sides of 11 adult Japanese cadavers were investigated, and 2 of 21 sides exhibited absence of the pyramidalis. We observed that all of the nerves to the pyramidalis included the sensory nerve branch, which distributed to the aponeurotic tissue in the upper area of the pubic ramus. To investigate the clinical relevance and developmental process of the pyramidalis, detailed innervation patterns of the pyramidalis and the lumber plexus were examined and compared with the case of absent pyramidalis. The nerves to the pyramidalis could be classified into five types by the derivative nerves and two subtypes by their courses associated with the funiculus spermaticus. In the cases of absent pyramidalis, similar sensory branches distributed close to the upper area of the pubic ramus. We deduced that the sensory branch extended along with the muscular branch to the pyramidalis after development of the pyramidalis and that only the sensory branch remained in cases in which the pyramidalis disappeared. The two subtypes might associate with descensus testis. Surgeons performing inguinal hernia repair using a mesh and tension-free surgical technique should preserve the nerves around the funiculus spermaticus to avoid diminished proprioception in the lower abdominal wall.

Morphological study on the pressure ulcer-like dermal lesions formed in the rat heel skin after transection of the sciatic nerves.

Due to transection of bilateral sciatic nerves, pressure ulcer-like dermal lesion occurred in the hairy skin covering of the heel skin in almost all rats. In the present study, chronological changes of the rat heel skin after the transection were morphologically and immunohistochemically examined. In the heel skin, redness and swelling began by 3days after the operation, and open wound formed by 17days. At the redness and swelling stage, edema extensively occurred in the dermis. At the thickening stage, the epidermis at the pressed site became transiently thicker, and at the whitening stage, rapidly thinner. At these stages, the epidermis in the skin surrounding the pressed site became gradually thicker. At the yellow scar stage, the skin was covered only by necrotic tissues and horny layer. These layers were scratched during walking and turning, and the yellow scar stage became the open wound stage. Inflammatory reaction began at the thickening stage, and at the yellow scar and open wound stages, necrosis, infiltration of inflammatory cells and dilation of small blood vessels were observed. These morphological features are quite similar to those in the human pressure ulcer. These findings suggest that these dermal injuries could compare the human pressure ulcer for medical treatment and depressurization in future study.