Recombinant Hepatocyte Growth Factor Accelerates Cutaneous Wound Healing in a Diabetic Mouse Model

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.     

Recombinant hepatocyte growth factor accelerates cutaneous wound healing in a diabetic mouse model

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.     

Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes

Chitosan is a copolymer of beta(1 --> 4) glucosamine and N-acetyl-D-glucosamine, which accelerates the infiltration of polymorphonuclear leukocytes (PMN) in the early phase of wound healing. In the granulation tissue treated with chitosan in canine experimental wound, osteopontin (OPN) was strongly positive in PMN immunohistochemically. OPN is a glycosylated phosphoprotein and promotes the attachment or spread of a variety of cell types. In addition, OPN may play a role in granulomatous inflammation. Production of OPN in PMN was therefore investigated in vitro using human PMN in this study. PMN stimulated with granulocyte-colony stimulating factor (G-CSF) and chitosan accumulated OPN mRNA, and released OPN into their culture supernatants. These findings suggest that OPN is synthesized by migrating PMN which plays the novel role of regulating the evolution of wound healing with chitosan treatment at the early phase of healing.     

Chitosan-based dressings loaded with neurotensin—an efficient strategy to improve early diabetic wound healing

One important complication of diabetes mellitus is chronic, non-healing diabetic foot ulcers (DFUs). This study aims to develop and use dressings based on chitosan derivatives for the sustained delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. Three different derivatives, namely N-carboxymethyl chitosan, 5-methyl pyrrolidinone chitosan (MPC) and N-succinyl chitosan, are presented as potential biomaterials for wound healing applications. Our results show that MPC has the best fluid handling capacity and delivery profile, also being non-toxic to Raw 264.7 and HaCaT cells. NT-loaded and non-loaded MPC dressings were applied to control/diabetic wounds to evaluate their in vitro/in vivo performance. The results show that the former induced more rapid healing (50% wound area reduction) in the early phases of wound healing in diabetic mice. A NT-loaded MPC foam also reduced expression of the inflammatory cytokine TNF-α (P < 0.001) and decreased the amount of inflammatory infiltrate on day 3. On day 10 MMP-9 was reduced in diabetic skin (P < 0.001), significantly increasing fibroblast migration and collagen (COL1A1, COL1A2 and COL3A1) expression and deposition. These results suggest that MPC-based dressings may work as an effective support for sustained NT release to reduce DFUs.     

羧甲基壳聚糖膜促进大鼠微粒皮移植创面愈合的实验研究

目的：本研究旨在评估羧甲基壳聚糖膜取代异体皮作为微粒皮移植载体促进创面愈合的作用。方法28只大鼠中,每次随机抽取2只大鼠配对同时手术,在大鼠背部两侧制作直径25 mm、对称圆形全层皮肤缺损创面各1个,两侧创面分别设为实验组和对照组,均移植自体微粒皮,对照组创面覆盖相互配对大鼠的异体皮,实验组创面覆盖羧甲基壳聚糖膜。于术后7、11、14 d 观察记录各组创面愈合时间,并于术后7、14、19 d 采集创面组织行组织病理学检查。结果实验组的羧甲基壳聚糖膜覆盖下微粒皮能够成活,且能修复创面。实验组创面平均愈合时间为（15.6±2.0）d,短于对照组创面平均愈合时间为（18.8±1.9）d,两组比较,差异有统计学意义（t =8.987,P〈0.05）。组织切片结果显示：羧甲基壳聚糖膜覆盖下微粒皮修复的新生表皮层生长较异体皮覆盖下新生表皮厚。结论羧甲基壳聚糖膜作为生物敷料,能够有效地保护创面,提供微粒皮修复创面的微环境。可用于取代异体皮覆盖微粒皮移植的创面。     

Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing

Nonhealing wounds of the lower equine limb represent a challenging model. The platelet is a natural source of a myriad of growth factors and cytokines that promote wound healing. This study evaluates the potential of platelet derived factors to enhance wound healing in the lower equine limb. Platelets were isolated from horse blood and activated with thrombin, a process known to induce growth factor release. This produced a platelet gel composed of platelet-rich plasma (PRP). To test this all-natural wound healant, 2.5-cm(2) full thickness cutaneous wounds were created below the knee and hock of a thoroughbred horse. Wounds were treated with PRP gel or left untreated. Sequential wound biopsies collected at Days 7, 36, and 79 postwounding permitted comparison of the temporal expression of differentiation markers and wound repair. To test the hypothesis that wounds treated with PRP gel exhibit more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls, tissues were stained for cytokeratin 10, a suprabasal differentiation marker, and the reestablishment of collagen was evaluated by trichrome staining. PRP gel-treated wounds at Day 7 expressed intense cytokeratin 10 staining near the wound junction in suprabasal epidermal layers, while staining in control tissues was less intense and restricted to apical epidermal layers distal to the wound junction. By Day 79, the staining was equal in both groups. However, PRP gel-treated wounds at Day 79 contained abundant, dense collagen bundles oriented parallel to each other and to the overlying epithelium, whereas control tissues contained fewer collagen fibers that were oriented randomly. Thus, treatment of wounds with PRP gel induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. This study reveals that this novel all-natural wound healant induced wound repair in injuries previously deemed untreatable.     

富血小板纤维蛋白联合姜黄素纳米颗粒水凝胶促糖尿病小鼠创面愈合

背景:糖尿病难愈性创面目前仍缺乏十分理想的治疗手段,探索促进糖尿病创面愈合的方法意义重大。目的:观察富血小板纤维蛋白联合姜黄素纳米颗粒水凝胶对糖尿病小鼠创面愈合的影响。方法:分别制备富血小板纤维蛋白与姜黄素纳米颗粒水凝胶。从100只成年C57BL/6J小鼠中随机取20只作为正常对照(A组),另外80只建立糖尿病模型。在建模成功的72只小鼠背部制作直径1 cm的圆形全层皮肤损伤创面,随机分4组处理:B组涂抹生理盐水,C组涂抹富血小板纤维蛋白与游离姜黄素溶液,D组涂抹富血小板纤维蛋白与姜黄素纳米颗粒溶液,E组涂抹富血小板纤维蛋白与姜黄素纳米颗粒水凝胶,每组18只。A组小鼠背部制作直径1 cm的圆形全层皮肤损伤创面,涂抹生理盐水。于创面造模当日即开始给药,之后隔日分别给予上述对应药物,直至伤后12 d。处理后第3,9,12天,观察创面愈合及创缘组织病理组织学变化。结果与结论:(1)处理后第12天,A、C、D、E组创面基本愈合完全,B组创面未完全愈合,E组创面愈合率高于B、C、D组(P <0.05);(2)处理后第3天苏木精-伊红染色显示,E组创面愈合情况好于B、C、D组,炎细胞浸润及肉...     

Water-soluble chitin as a wound healing accelerator.

Water-soluble chitin (WSC) was prepared by controlling degree of deacetylation (DD) and molecular weight of chitin through alkaline and ultrasonic treatment. Its accelerating effect on wound healing in rats was compared with those of chitin and chitosan. Full-thickness skin incision was made on the backs of the rats and then three kinds of powders (chitin, chitosan, WSC) and an aqueous solution of WSC were embedded in the wounds. The tensile strength and the hydroxyproline content of the wounded skins were measured and histological examination was performed. The WSC was found to be more efficient than chitin or chitosan as a wound-healing accelerator. The wound treated with WSC solution was completely reepithelialized, granulation tissues in the wound were nearly replaced by fibrosis and hair follicles were almost healed at 7 days after initial wounding. Also, the WSC-solution-treated skin had the highest tensile strength and the arrangement of collagen fibers in the skin was similar to normal skins. The WSC solution is considered to be a suitable wound-healing agent due to its easy application and high effectiveness.     

Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro

Exudates from non-healing wounds contain elevated levels of proteolytic enzymes, like elastase from polymorphonuclear granulocytes (PMN elastase), reactive oxygen species (ROS) and reactive nitrogen species (RNS). The overproduction of proteolytic enzymes leads to reduced concentrations of growth factors and proteinase inhibitors, resulting in an imbalance between degradation and remodelling processes. Thus, the reduction of protein-degrading enzymes and scavenging of ROS and RNS seem to be suitable ways to support the healing process of chronic stagnating wounds. The aim of this study was to test selected wound dressings from different biomaterials (collagen, oxidized regenerated cellulose (ORC) and ORC/collagen mixture), regarding their antioxidative potential in vitro and their influence on the concentration and activity of PMN elastase in chronic wound fluid. Antioxidant capacity of the investigated wound dressing was determined by a pholasin-based chemiluminescent assay. PMN elastase concentration was determined by means of ELISA. Enzyme activities could be measured by a fluorescence assay. As the presented data demonstrates, all tested materials showed antioxidant capacity. In addition, the investigated materials were able to reduce the concentration and activity of PMN elastase. Beside other aspects, such as biocompatibility, biodegradability, fluid absorption and clinical effects (e.g. angiogenesis and microcirculation), the understanding of these properties may help to support the further refinement of wound dressings for improved wound healing.     

Accelerated wound closure in neutrophil-depleted mice

The infiltration of neutrophils into injured tissue is known to protect wounds from invading pathogens. However, more recent studies suggest that neutrophils might inhibit the wound repair process. To investigate the role of neutrophils in wounds, mice were neutrophil-depleted by injection with rabbit anti-mouse neutrophil serum. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in neutropenic than control mice (77.7+14.2% vs. 41.2+0.9%, P<0.02 at day 2). Dermal healing was not affected by neutrophil depletion, as neither collagen deposition nor wound-breaking strength was significantly different between neutropenic and control mice. As the delayed repair of diabetic individuals exhibits robust inflammation, the effect of neutrophil depletion on diabetic wound healing was investigated. Similar to the observations in wild-type mice, wound closure was accelerated by nearly 50% in neutropenic, diabetic mice. The results suggest that although neutrophils may provide protection against infection, they may retard wound closure.     

Accelerated wound healing by smad3 antisense oligonucleotides-impregnated chitosan/alginate polyelectrolyte complex

Smad3 mediates the intracellular signaling of TGF-beta1 superfamily and plays a critical role in the cellular proliferation, differentiation and elaboration of matrix pivotal to cutaneous wound healing. Smad3 antisense oligonucleotides (ASOs) impregnated polyelectrolyte complex (PEC) containing chitosan and sodium alginate was prepared for accelerated wound healing. Physicochemical properties of PEC were characterized by zeta potential, scanning electron microscopy and bioadhesive test. Full-thickness, excisional wounds were made on the dorsum of C57BL6 mice. Then, smad3 ASOs-PEC, PEC alone, smad3 ASOs and gauze dressing were applied to determine concentration of TGF-beta1 and collagen in tissues and observe the wound contraction and histology of tissues. Zeta potentials and bioadhesive strengths of ASOs-PEC were increased as the chitosan ratio in PEC. In smad3 ASOs-PEC, the healing process suggested by wound closure and histological observation was faster than other groups because collagen contents increased and level of TGF-beta1 decreased. These results demonstrate that the smad3 ASOs-PEC composed of chitosan and sodium alginate could be applied for accelerated wound healing.     

Role of collagen cross-linking on equine wound contraction and healing

The present study was carried out to evaluate the effect of collagen cross-linking inhibition on equine wound contraction and healing. In five male adult donkeys, two full-thickness skin wounds (20 × 20 mm in diameter) were created on the lateral aspect of forelimbs, at the mid-point between the carpal and fetlock joints under general anesthesia. Two other wounds were created on the neck of each donkey symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate, 5 mg/ml, added to methyl cellulose gel and only methyl cellulose gel, respectively. Treatment of wounds were started at 24 h after wounding and continued every other day for ten successive days. The wounds were evaluated over a 3-week period. On days 0, 1, 3, 5, 7, 9, 12, 15, 18, and 21, digital photographs were taken of all wounds after careful shaving to visualize the wound margin. Rulers were held vertically and horizontally close to the wound as a reference. Epithelialization and granulation tissue formation were measured for each wound using Scion Image software. Percentage of the wound contraction, epithelialization, and healing were calculated for each wounds. At the end of the study, biopsy was taken from the center of each wound for hydroxyproline measurement and the same corner of each wound for histopathological examination. Macroscopic evaluation revealed significant differences in wound contraction and healing process between test and control groups in wounds located in neck (P < 0.05), but there was no significant difference in percent of epithelialization at the same area (P > 0.05). Significant differences were observed in the percent of wound contraction and healing between the test and control groups in wounds located in the forelimb (P < 0.05), but no significant difference was observed in percent of epithelialization at this area (P > 0.05). There were no significant differences between median of hydroxyproline levels of left and right wounds in forelimb and neck (P > 0.05). Histopathological examination revealed no significant differences between median of epithelialization, inflammatory infiltration, presence of dermal granulation tissue, fibroblast proliferation, arrangement of fibroblasts, collagen deposition, and collagen bundle formation scores in the specimens prepared from left and right wounds in forelimb and neck (P > 0.05). Our data demonstrated that collagen cross-linking could play a key role in equine wound contraction and healing at the limb and neck area.     

Inducible costimulator (ICOS) and ICOS ligand signaling has pivotal roles in skin wound healing via cytokine production

Skin wound healing is mediated by inflammatory cell infiltration of the wound site. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor-ligand pair. Although the ICOS-ICOSL pathway participates in adaptive immunity, its roles in skin wound healing, which is mediated by innate immune responses, remain unknown. To clarify these roles, repair of excisional wounds was examined in ICOS(-/-) mice, ICOSL(-/-) mice, and ICOS(-/-)ICOSL(-/-) mice. Each mutant strain showed similar, dramatic delays in wound healing, especially at early times. Knockout mice showed suppressed keratinocyte migration, angiogenesis, and granulation tissue formation, and diminished T-cell, macrophage, and neutrophil infiltration. The loss of ICOS and/or ICOSL resulted in marked suppression of cytokine expression in wounds, especially the Th2 cytokines interleukin (IL)-4, IL-6, and IL-10. T-cell transfer experiments and T-cell depletion therapy further clarified the important roles of ICOS expressed on T cells and its interaction with ICOSL. Application of IL-6, but not IL-4, to the wounds significantly increased the onset of early wound healing in mutant mice. Thus, our results indicate that ICOS-ICOSL costimulatory signaling has critical roles during wound healing, most likely by inducing IL-6 production.     

Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule-1 (ICAM-1) delayed skin wound healing and mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1(-/-)) show more delayed wound healing. Deficiency of both endothelial selectins (E-selectin or P-selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM-1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L-selectin/ICAM-1(-/-) mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1(-/-) mice with both E- and P-selectin blockade. Wild-type mice with both E- and P-selectin blockade showed delayed wound healing that was comparable with that in L-selectin/ICAM-1(-/-) mice. Combined E- and P-selectin blockade in L-selectin/ICAM-1(-/-) mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor-beta and interleukin-6. Application of basic fibroblast growth factor (bFGF) but not platelet-derived growth factor to the wounds significantly improved wound healing in L-selectin/ICAM-1(-/-) mice with both E- and P-selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1 and may provide critical information for the therapy of skin wounds.     

马桑提取物促进大鼠烧伤创面愈合的作用和机制

 目的：探讨马桑提取物(CSME)对大鼠烧伤创面愈合的影响及机制。方法：随机将40只SD雄性大鼠分成生理盐水（NS）组、白凡士林（WPJ）组、磺胺嘧啶银（SSD）组和CSME组,每组10只。将大鼠麻醉后,制备背部深Ⅱ度皮肤烧伤创面。而后分别用NS敷料、WPJ敷料、SSD敷料和CSME敷料覆盖治疗21 d。在第1 d、3 d、7 d、14 d和21 d,观察动物的临床症状和创面状况（上皮化速率、结痂及被毛等）后,分别取出创面组织,用于组织学观察,检测丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性,检测表皮生长因子（EGF）和碱性成纤维细胞因子（bFGF）蛋白水平及胶原mRNA表达。结果：CSME组新生上皮生长高度活跃,有大量新生被毛生长,创面上皮化速率明显高于其它各组 (P<0.05)。SSD组创面中央新生被毛稀疏而细小,而CSME组创面中央被毛密集,大部分被毛接近正常。CSME组镜下可见全部被多层上皮细胞覆盖,新生胶原纤维充分分化,排列整齐,组织结构明朗,皮脂腺和毛囊增生极其活跃等,显著优于其它各组。本研究显示,烧伤后从第1 d到21 d,CSME组早期EGF和bFGF水平显著高于其它各组,后期迅速下降,低于其它组（P<0.05或P<0.01）。SSD组Ⅰ型与Ⅲ型胶原mRNA表达的比值显著高于其它各组和正常组织,CSME治疗组显著低于其它各组和正常组织(P<0. 05)。结论：本研究表明,CSME可诱导烧伤创面无瘢痕愈合。CSME的这种作用与其早期增强EGF和bFGF蛋白水平,后期抑制两者的水平有关,也可能与其抑制烧伤创面Ⅰ型胶原而增强Ⅲ型胶原mRNA表达密切相关。     

Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair

The bone marrow provides inflammatory cells and endothelial progenitor cells to healing cutaneous wounds. To further explore the bone marrow contribution to skin and healing wounds, we used a chimeric mouse model in which the bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice is transplanted into normal C57BL mice. We found that normal skin is a target organ for bone marrow-derived cells from both the hematopoietic and the mesenchymal stem cell pool. We present evidence that the bone marrow contribution to normal skin and the healing cutaneous wound is substantially greater than the previously recognized CD45+ subpopulation, where 15%-20% of the spindle-shaped dermal fibroblasts were bone marrow-derived (EGFP+). Furthermore, the bone marrow-derived cells were able to contract a collagen matrix and transcribe both collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. Whereas endothelial progenitor cells were found early during the wound repair process, bone marrow-derived endothelial cells were not seen after epithelialization was complete. Our data show that wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow-derived cells and the adjacent uninjured dermal mesenchymal cells for reconstituting the dermal fibroblast population.     

Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages.

Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors.     

烫伤创面愈合后的瘢痕形成与神经支配

背景：既往研究表明神经因素在创面愈合中具有重要调控作用,但有关神经调节与创面愈合后瘢痕形成之间的关系鲜有报道。 目的：观察烧伤创面愈合过程中神经支配与创面愈合质量之间的关系。 方法：将30只大鼠右侧T9~L1阶段脊神经根切断,制作失神经支配皮肤模型;然后在大鼠背部右侧失神经支配皮肤区域制作直径4 cm的深Ⅱ度烫伤创面,设为模型组,左侧对称正常皮肤制作同样的创面,设为对照组,伤后连续观察创面变化,于7,14,21 d采用免疫组织化学法观察Ⅰ和Ⅲ胶原分泌情况,并计算Ⅰ/Ⅲ型胶原比例的变化,探讨创面愈合速度及愈合质量。 结果与结论：模型组Ⅰ,Ⅲ型胶原分泌和Ⅰ/Ⅲ型胶原比值于伤后各时间点均显著低于对照组(P < 0.05)。模型组Ⅰ型胶原分泌与伤后7,14,21 d逐渐增加(P < 0.05),Ⅲ型胶原于伤后21 d时分泌明显增高(P < 0.05),Ⅰ/Ⅲ型胶原比值与伤后21 d时明显降低(P < 0.05)。结果证实,创面早期神经支配可以促进创面愈合,创面重塑期减轻神经支配可能会改善创面重塑质量。