1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D₃ (1,25 (OH)₂ D₃) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)₂D₃ or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)₂D₃, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)₂D₃ restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.     

基质金属蛋白酶9对糖尿病足溃疡愈合影响的研究进展

在糖尿病足患者溃疡创面分泌物中,基质金属蛋白酶9 (matrix metalloproteinase-9,MMP-9)过高是预测糖尿病足的发生及足溃疡难愈的主要指标,其可能的机制包括:高水平MMP-9降低VEGF的表达、抑制成纤维细胞的生物学行为影响糖尿病足溃疡愈合;失衡的MMP-9/TIMP-1比值影响糖尿病足溃疡愈合。选择性MMP-9抑制剂(包括小分子抑制剂、高级伤口辅料抑制剂、基于干扰基因水平表达的RNA抑制剂)可以作为促进糖尿病足溃疡愈合的手段,但仍需大样本、多中心随机对照试验以及长期随访进一步验证其疗效及安全性。现查阅近年来涉及MMP-9和糖尿病足溃疡相关的文献,综述MMP-9对糖尿病足溃疡愈合的影响及其机制研究进展。     

Inhibition of exosomal miR‐24‐3p in diabetes restores angiogenesis and facilitates wound repair via targeting PIK3R3

Diabetic foot ulcer (DFU) is one of the common ailments of elderly people suffering from diabetes. Exosomes containing various active regulators have been found to play a significant role in apoptosis, cell proliferation and other biological processes. However, the effect and the underlying mechanism of action of diabetes patients derived from circulating exosomes (Dia‐Exos) on DFU remain unclear. Herein, we aim to explore the potential regulatory role of Dia‐Exos. First, we attempted to demonstrate the harmful effect of Dia‐Exos both in vivo and in vitro. miRNA‐24‐3p (miR‐24‐3p) was found enriched with Dia‐Exos. Hence, inhibition of this miRNA could partially reverse the negative effect of Dia‐Exos, thus, in ture, accelerates wound repair. Luciferase assay further verified the binding of miR‐24‐3p to the 3′‐UTR of phosphatidylinositol 3‐kinase regulatory subunit gamma (PIK3R3) mRNA and the PIK3R3 expression enhanced human umbilical vein endothelial cells functionality in vitro. Hence, the findings of this study reveal the regulatory role of Dia‐Exos in the process of wound healing and provide experimental evidence for the therapeutic effects of knocking down miR‐24‐3p in DFU treatment.     

High glucose represses the proliferation of tendon fibroblasts by inhibiting autophagy activation in tendon injury

Diabetic foot ulcer (DFU) is a kind of common and disabling complication of Diabetes Mellitus (DM). Emerging studies have demonstrated that tendon fibroblasts play a crucial role in remodeling phase of wound healing. However, little is known about the mechanism underlying high glucose (HG)-induced decrease in tendon fibroblasts viability. In the present study, the rat models of DFU were established, and collagen deposition, autophagy activation and cell apoptosis in tendon tissues were assessed using Hematoxylin–Eosin (HE) staining, immunohistochemistry (IHC), and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, respectively. Tendon fibroblasts were isolated from Achilles tendon of the both limbs, and the effect of HG on autophagy activation in tendon fibroblasts was assessed using Western blot analysis, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry. We found that cell apoptosis was increased significantly and autophagy activation was decreased in foot tendon tissues of DFU rats compared with normal tissues. The role of HG in regulating tendon fibroblasts viability was then investigated in vitro, and data showed that HG repressed cell viability and increased cell apoptosis. Furthermore, HG treatment reduced LC3-II expression and increased p62 expression, indicating that HG repressed autophagy activation of tendon fibroblasts. The autophagy activator rapamycin reversed the effect. More importantly, rapamycin alleviated the suppressive role of HG in tendon fibroblasts viability. Taken together, our data demonstrate that HG represses tendon fibroblasts proliferation by inhibiting autophagy activation in tendon injury.     

Metformin Induces Cell Cycle Arrest,Reduced Proliferation,Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients

BackgroundSeveral epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation.ResultsMetformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin.ConclusionsMetformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.     

Microenvironment-induced myofibroblast-like conversion of engrafted keratinocytes

Myofibroblasts,recognized classically by-smooth muscle actin(-SMA)expression,play a key role in the wound-healing process,promoting wound closure and matrix deposition.Although a body of evidence shows that keratinocytes explanted onto a wound bed promote closure of a skin injury,the underlying mechanisms are not well understood.The basal layer of epidermis is rich in undifferentiated keratinocytes(UKs).We showed that UKs injected into granulation tissue could switch into-SMA positive cells,and accelerate the rate of skin wound healing.In addition,when the epidermis sheets isolated from foreskin cover up the wound bed or are induced in vitro,keratinocytes located at the basal layers or adjacent sites were observed to convert into myofibroblast-like cells.Thus,UKs have a potential for myofibroblastic transition,which provides a novel mechanism by which keratinocyte explants accelerate skin wound healing.     

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods

We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results

All EPC phenotypes except the kinase insert domain receptor (KDR)⁺CD133⁺ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34⁺KDR⁺ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions

Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34⁺KDR⁺ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.     

Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers

Background

Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.

Methods and Findings

Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.

Conclusions

This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.     

Leptin Promotes Wound Healing in the Skin

Introduction

Leptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.

Methods

Immunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.

Results

Ob-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.

Conclusion

Topically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.     

糖尿病足溃疡的危险因素探讨及亲水性纤维含银敷料促进患者伤口愈合的临床对照研究

摘要 目的：探讨糖尿病足溃疡（DFU）的危险因素,并研究亲水性纤维含银敷料促进患者伤口愈合的应用价值。方法：选取2017年8月-2020年12月期间我院收治的糖尿病患者230例。230例糖尿病患者中发生DFU的79例,纳为DFU组,剩余151例未发生DFU,纳为无DFU组。DFU组的患者采用随机数字表法分为治疗1组39例和治疗2组40例,治疗1组给予传统纱布敷料,治疗2组给予亲水性纤维含银敷料。采用多因素Logistic回归分析DFU发生的危险因素。观察治疗1组、治疗2组的临床疗效和临床指标。结果：DFU组、无DFU组在性别、年龄、户籍地、糖尿病病程、并发下肢血管病变、并发周围神经病变、足底有胼胝、血红蛋白（Hb）、C反应蛋白（CRP）、白蛋白（ALB）、总胆固醇（TC）、纤维蛋白原（FIB）、红细胞沉降率（ESR）、高密度脂蛋白（HDL）、 胱抑C（CysC）、 低密度脂蛋白（LDL）、脂蛋白a[Lp(a)]、糖化血红蛋白（HbAlc）方面对比差异有统计学意义（P<0.05）。男性、糖尿病病程、并发下肢血管病变、并发周围神经病变、足底有胼胝、ALB、ESR、CysC、CRP、Lp(a)、HbAlc均是DFU发生的危险因素（P<0.05）。治疗2组的临床总有效率明显高于治疗1组（P<0.05）。治疗2组的创面愈合时间、出现明显肉芽时间、住院时间短于治疗1组,住院费用高于治疗1组（P<0.05）。结论：DFU的发生受到多种因素影响,包括男性、糖尿病病程、并发下肢血管病变等,因此临床应加强对这些因素的预防和控制。此外,亲水性纤维含银敷料可促进DFU患者创面愈合,疗效显著。     

From a Traditional Medicinal Plant to a Rational Drug: Understanding the Clinically Proven Wound Healing Efficacy of Birch Bark Extract

Background

Birch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models.

Methodology/Principal Findings

We show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases.

Conclusion/Significance

Our results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.     

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.     

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.     

Thrombomodulin Promotes Corneal Epithelial Wound Healing

Purpose

To determine the role of thrombomodulin (TM) in corneal epithelial wound healing, and to investigate whether recombinant TM epidermal growth factor-like domain plus serine/threonine-rich domain (rTMD23) has therapeutic potential in corneal epithelial wound healing.

Methods

TM localization and expression in the murine cornea were examined by immunofluorescence staining. TM expression after injury was also studied. The effect of rTMD23 on corneal wound healing was evaluated by in vitro and in vivo assays.

Results

TM was expressed in the cornea in normal adult mice. TM expression increased in the early phase of wound healing and decreased after wound recovery. In the in vitro study, platelet-derived growth factor-BB (PDGF-BB) induced TM expression in murine corneal epithelial cells by mediating E26 transformation-specific sequence-1 (Ets-1) via the mammalian target of rapamycin (mTOR) signaling pathway. The administration of rTMD23 increased the rate of corneal epithelial wound healing.

Conclusions

TM expression in corneal epithelium was modulated during the corneal wound healing process, and may be regulated by PDGF-BB. In addition, rTMD23 has therapeutic potential in corneal injury.     

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.     

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild-type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen-deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix-degrading proteases, probably in the dissection of the fibrin-rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.     

Identification of the Critical Therapeutic Entity in Secreted Hsp90α That Promotes Wound Healing in Newly Re-Standardized Healthy and Diabetic Pig Models

Chronic and non-healing skin wounds represent a significant clinical, economic and social problem worldwide. Currently, there are few effective treatments. Lack of well-defined animal models to investigate wound healing mechanisms and furthermore to identify new and more effective therapeutic agents still remains a major challenge. Pig skin wound healing is close to humans. However, standardized pig wound healing models with demonstrated validity for testing new wound healing candidates are unavailable. Here we report a systematic evaluation and establishment of both acute and diabetic wound healing models in pigs, including wound-creating pattern for drug treatment versus control, measurements of diabetic parameters and the time for detecting delayed wound healing. We find that treatment and control wounds should be on the opposite and corresponding sides of a pig. We demonstrate a strong correlation between duration of diabetic conditions and the length of delay in wound closure. Using these new models, we narrow down the minimum therapeutic entity of secreted Hsp90α to a 27-amino acid peptide, called fragment-8 (F-8). In addition, results of histochemistry and immunohistochemistry analyses reveal more organized epidermis and dermis in Hsp90α-healed wounds than the control. Finally, Hsp90α uses a similar signaling mechanism to promote migration of isolated pig and human keratinocytes and dermal fibroblasts. This is the first report that shows standardized pig models for acute and diabetic wound healing studies and proves its usefulness with both an approved drug and a new therapeutic agent.     

Hydrocellular Foam Dressing Promotes Wound Healing along with Increases in Hyaluronan Synthase 3 and PPARα Gene Expression in Epidermis

Background

Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin.

Methodology/Principal Findings

We created two full-thickness wounds on the dorsolateral skin of rats. Each wound was covered with either a hydrocellular foam dressing or a film dressing and hyaluronan levels in the periwound skin was measured. We also investigated the mechanism by which the hydrocellular foam dressing regulates hyaluronan production by measuring the gene expression of hyaluronan synthase 3 (Has3), peroxisome proliferator-activated receptor α (PPARα), and CD44. Hydrocellular foam dressing promoted wound healing and upregulated hyaluronan synthesis, along with an increase in the mRNA levels of Has3, which plays a primary role in hyaluronan synthesis in epidermis. In addition, hydrocellular foam dressing enhanced the mRNA levels of PPARα, which upregulates Has3 gene expression, and the major hyaluronan receptor CD44.

Conclusions/Significance

These findings suggests that hydrocellular foam dressing may be beneficial for wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis. We believe that the present study would contribute to the elucidation of the mechanisms underlying the effects of hydrocellular foam dressing-induced moist environment on wound healing and practice evidence-based wound care.