Insights into Peptide Mediated Antibiofilm Treatment in Chronic Wound: A Bench to Bedside Approach.

Chronic wound biofilm infections are a threat to the population with respect to morbidity and mortality. The presence of multidrug-resistant bacterial pathogens in chronic wound renders the action of antibiotics and antibiofilm agents difficult. Therefore an alternative therapy is essential for reducing bacterial biofilm burden. In this scenario, the peptide-based antibiofilm therapy for chronic wound biofilm management seeks more attention. A synthetic peptide with a broad range of antibiofilm activity against preformed and established biofilms, having the ability to kill multispecies bacteria within biofilms and possessing combinatorial activity with other antimicrobial agents, provides significant insights. In this review, we portray the possibilities and difficulties of peptide-mediated treatment in chronic wounds biofilm management and how it can be clinically translated into a product.

Human CD206+ Macrophages Show Antifibrotic Effects on Human Fibroblasts through an IL-6-Dependent Mechanism In Vitro.

BACKGROUND: Pathologic scarring including keloid and hypertrophic scar causes aesthetic and physical problems, and there are clinical difficulties (e.g., posttreatment recurrence) in dealing with pathologic scarring. Understanding the mechanisms that underlie scar control in wound healing will help prevent and treat pathologic scarring. The authors focused on CD206+ macrophages in the wound-healing process, and hypothesized that CD206+ macrophages have antifibrotic effects on fibroblasts. METHODS: The authors established a co-culture system for CD206+ macrophages and fibroblasts (cell ratio, 1:1). The authors examined the CD206+ macrophages' antifibrotic effects on fibroblasts after a 72-hour culture, focusing on fibrosis-related genes. To identify key factor(s) in the interaction between CD206+ macrophages and fibroblasts, the authors analyzed cytokines in a conditioned medium of the co-culture system. RESULTS: Under co-culture with CD206+ macrophages, expression of the following in the fibroblasts was significantly down-regulated: type 1 (fold change, 0.38) and type 3 collagen (0.45), alpha smooth muscle actin (0.24), connective tissue growth factor (0.40), and transforming growth factor-beta (0.66); the expression of matrix metalloproteinase 1 was significantly up-regulated (1.92). Conditioned medium in the co-culture showed a high interleukin (IL)-6 concentration (419 ± 88 pg/ml). When IL-6 was added to fibroblasts, antifibrotic changes in gene expression (as observed under the co-culture) occurred in the fibroblasts. CONCLUSIONS: The authors' in vitro results revealed that CD206+ macrophages have antifibrotic effects on fibroblasts by means of a paracrine mechanism involving IL-6. Understanding these effects, especially in vivo, will help elucidate the mechanism of scar control in wound healing and contribute to the development of new scar treatments.

Topical Application of Virus-Derived Immunomodulating Proteins and Peptides to Promote Wound Healing in Mouse Models.

Immune modulators play critical roles in the progression of wounds to normal or conversely delayed healing, through the regulation of normal tissue regrowth, scarring, inflammation, and growth factor expression. Many immune modulator recombinants are under active preclinical study or in clinical trial to promote improved acute or chronic wound healing and to reduce scarring. Viruses have evolved highly efficient immune modulators for the evasion of host-defensive immune responses that target and kill invasive viruses. Recent studies have proven that some of these virus-derived immune modulators can be used to promote wound healing with significantly improved speed and reduced scarring in rodent models. Mouse full-thickness excisional wound model is one of the most commonly used animal models used to study wound healing for its similarity to humans in the healing phases and associated cellular and molecular mechanisms. This chapter introduces this mouse dermal wound healing model in detail for application in studying viral immune modulators as new treatments to promote wound healing. Details of hydrogel, protein construction, and topical application methods for these therapeutic proteins are provided in this chapter.

Loss of Atg7 in Endothelial Cells Enhanced Cutaneous Wound Healing in a Mouse Model.

BACKGROUND: Emerging evidence has linked autophagy to skin wound healing; however, the underlying cellular and molecular mechanisms remain poorly understood. The present study was designed to determine the role of autophagy in endothelial cell (EC)-mediated skin wound healing in mice. METHODS: Autophagy-related gene (Atg7) in mouse ECs was inactivated by the Cre-loxP system under the control of an EC-specific VE-Cadherin (Cdh5) promoter (Atg7EC-/- mice). Full-thickness skin wounds were created on the dorsum of wild-type (WT), Cdh5-Cre+, floxed Atg7 (Atg7F/F), and Atg7EC-/- mice. Autophagic activity was determined by autophagic flux assay in the primary culture of ECs isolated from these mice. The wound re-epithelialization and angiogenesis was examined by histological analyses. The angiogenic activity of ECs was evaluated by tube formation assay in vitro. EC proliferation was examined by a cell count CCK-8 kit. EC-originated intercellular communication with dermal fibroblasts and keratinocytes was assessed by measuring the effect of EC conditional medium on the growth of keratinocytes and fibroblasts. The levels of VEGF, EGF, bFGF in EC conditional medium were measured by ELISA. RESULTS: Autophagy deficiency in ECs markedly enhanced the re-epithelialization and the wound closure during skin wound healing. However, it has minimal impact on angiogenesis in the wounded skin. Notably, autophagy deficiency in ECs did not affect their proliferation and migration or angiogenic activity per se but enhanced the EC conditional medium-induced proliferation and migration of keratinocytes and fibroblasts. CONCLUSIONS: These results demonstrate for the first time an inhibitory role of autophagy in the EC-originated paracrine regulation of skin wound healing.

The Influence of Microscopic Inflammation at Resection Margins on Early Postoperative Endoscopic Recurrence After Ileocaecal Resection for Crohn's Disease.

BACKGROUND AND AIMS: The pathogenesis and risk factors for early postoperative endoscopic recurrence of Crohn's disease [CD] remain unclear. Thus, this study aimed to identify whether histological inflammation at the resection margins after an ileocaecal resection influences endoscopic recurrence. METHODS: We have prospectively followed up patients with CD who underwent ileocaecal resection at our hospital between January 2012 and January 2018. The specimens were histologically analysed for inflammation at both of the resection margins [ileal and colonic]. We evaluated whether histological results of the resection margins are correlated with endoscopic recurrence of CD based on colonoscopy 6 months after ileocaecal resection. Second, we assessed the influence of known risk factors and preoperative therapy on endoscopic recurrence of CD. RESULTS: A total of 107 patients were included in our study. Six months after ileocaecal resection, 23 patients [21.5%] had an endoscopic recurrence of CD. The histological signs of CD at the resection margins were associated with a higher endoscopic recurrence [56.5% versus 4.8%, p < 0.001]. Disease duration from diagnosis to surgery [p = 0.006] and the length of the resected bowel [p = 0.019] were significantly longer in patients with endoscopic recurrence. Smoking was also proved to be a risk factor for endoscopic recurrence [p = 0.028]. CONCLUSIONS: Histological inflammation at the resection margins was significantly associated with a higher risk of early postoperative endoscopic recurrence after an ileocaecal resection for CD.

Partial thickness wound: Does mechanism of injury influence healing?

Wound healing is a complex multistep process which is temporally and spatially controlled. In partial thickness wounds, regeneration is possible from the stem cells in the edges of the wound and from the remnants of the epidermal appendages (such as hair follicles and sebaceous glands). This study examines whether the mechanism of injury influences healing of wounds of similar depth. Burn and excisional wounds were created on the back of Hampshire pigs and harvested at 7, 14, 28, 44, 57 and 70days after injury and processed for histology and immunohistochemistry. Quantitative analysis of re-epithelialisation, inflammatory response and thickness of the scar and semi-quantitative analyses of the architecture of the resultant scar were performed and subjected to statistical analysis. Results demonstrated a higher number of neutrophils, macrophages and lymphocytes present in the burn on day 7 compared to the excisional wounds. The inflammatory profile of burn wounds was higher than that of excisional wounds for the first month after injury albeit less marked than on day 7 after injury. Re-epithelialisation was markedly advanced in excisional wounds compared to burn wounds at day 7 after injury, corresponding to the higher number of hair follicles in the underlying dermis of excisional wounds at this time point. The thickness of the neo-epidermis increased with time and at day 70 after wounding, the neo-epidermis of the burn was significantly thicker than the neo-epidermis of the excisional scar. Interestingly, following partial thickness excision of skin, there was neo-dermal reformation albeit with an altered architecture, lacking the normal basket-weave pattern of collagen. The thickness of the dermis of partial thickness excisional scar was greater than that of the adjacent unwounded skin. The neo-dermis of the burn scar was even thicker, with the collagen arranged more compactly and disorganised compared to excisional scar and normal skin. This study provides evidence that the mechanism of injury does influence wound healing and the resultant scarring.

The Length of Surgical Skin Incision in Postoperative Inflammatory Reaction.

BACKGROUND AND OBJECTIVES: Surgery provokes inflammatory and immune responses, so efforts have been made to reduce host response by using less invasive techniques. The purpose of this experimental study was to investigate the surgical stress induced by skin incision and the role of liver response in this process. METHODS: Seventy male anesthetized Wistar rats were subjected to a midline incision confined strictly to the skin (dermis) of either 1 cm long (n = 20), 10 cm long (n = 20), or no incision (n = 20) or served as controls (n = 10). Skin trauma was left open for a 20-minutes period, and then was meticulously sutured. At 3 and 24 hours later, laparotomy was performed on half the rats of each group, for blood and liver sampling. In serum and liver homogenates, cytokine-induced neutrophil chemoattractant (CINC)1/interleukin (IL)-8 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assays and nitric oxide (NO) using a Griess reaction. RESULTS: Skin trauma was found to significantly (P < .01) increase all inflammatory mediators tested (CINC1/IL-8, TNF-α, NO) in serum of operated rats versus controls, the increase being proportionally dependent on the length of skin incision. In liver homogenates, CINC1/IL-8 was significantly (P < .01) increased in operated animals versus controls, similarly to serum levels. In contrast, liver TNF-α levels were inversely related to serum levels, and a significant (P < .01) decrease in TNF-α was observed in liver homogenates of operated animals compared with the controls, indicating that the increased TNF-α in blood reflects liver TNF-α secretion. CONCLUSION: Our findings suggest that inflammatory and immune reactions induced by skin-only surgical trauma are closely correlated to the length of skin incision.

Comparative efficacy of immunohistochemical markers in surgical healing. / Avaliação da eficácia de três marcadores imunoistoquímicos envolvidos nos diferentes tempos da cicatrização da ferida cirúrgica.

OBJECTIVE: to evaluate the efficacy of three immunohistochemical markers involved in the wound healing process. METHODS: experimental study of 40 Wistar rats of the markers metalloproteinases and matrix metalloproteinase 9 (MMP-9), beta transforming growth factor (TGF-ß) and myofibroblasts and smooth muscle actin alpha (α-MLA) markers, studied from fragments of surgical scar of abdominal incision involving skin, aponeurosis and peritoneum. The animals were divided into four subgroups of ten according to the day of death, scheduled in three, seven, 14 and 21 days. RESULTS: MMP-9 expression showed a progressive increase of its concentration, more evident from 7th to 14th days, varying the tissue immunoexpression between 2.65% and 11.50% . TGF- ß showed expression at high level on the 3rd day, fell in the 7th, rising again in the 14th, with a small decrease in the 21st day, varying the tissue immunoexpression between 0.03% and 2.92%. The α-AML presented levels with little variation and a slight increase, varying the tissue immunoexpression between 0.88% and 3.23%. CONCLUSION: MMP-9 presented as the best marker, followed by TGF-ß. However, α-AML was not a good indicator of the evolution of tissue repair.

OBJETIVO: avaliar a eficácia de três marcadores imunoistoquímicos envolvidos no processo de cicatrização de ferida cirúrgica. MÉTODOS: estudo experimental em 40 ratos da raça Wistar, dos marcadores metaloproteinases e metaloproteinase da matriz 9 (MMP-9), fator de transformação do crescimento beta (TGF-ß) e miofibroblasto e alfa actina de músculo liso (α-AML), estudados a partir de fragmentos de cicatriz cirúrgica de incisão abdominal envolvendo pele, aponeurose e peritônio. Os animais foram distribuídos em quatro subgrupos de dez de acordo com o dia da morte, programada em três, sete, 14 e 21 dias. RESULTADOS: na expressão da MMP-9 ocorreu aumento progressivo de sua concentração, mais evidente do 7º ao 14º dias variando a imuno-expressão tecidual entre 2,65% e 11,50%.TGF- ß mostrou expressão em nível alto no 3º dia, caiu no 7º, voltando a subir no 14º, com pequena queda no 21º dia variando a imuno-expressão tecidual entre 0,03% e 2,92%. A α-AML apresentou níveis com pouca variação e discreto aumento variando a imuno-expressão tecidual entre 0,88% e 3,23%. CONCLUSÃO: a MMP-9 se apresentou como melhor marcador, seguido pela TGF-ß. Já o α-AML não se mostrou um bom sinalizador da evolução da reparação tissular.

Avaliação da eficácia de três marcadores imunoistoquímicos envolvidos nos diferentes tempos da cicatrização da ferida cirúrgica / Comparative efficacy of immunohistochemical markers in surgical healing

RESUMO Objetivo: avaliar a eficácia de três marcadores imunoistoquímicos envolvidos no processo de cicatrização de ferida cirúrgica. Métodos: estudo experimental em 40 ratos da raça Wistar, dos marcadores metaloproteinases e metaloproteinase da matriz 9 (MMP-9), fator de transformação do crescimento beta (TGF-β) e miofibroblasto e alfa actina de músculo liso (α-AML), estudados a partir de fragmentos de cicatriz cirúrgica de incisão abdominal envolvendo pele, aponeurose e peritônio. Os animais foram distribuídos em quatro subgrupos de dez de acordo com o dia da morte, programada em três, sete, 14 e 21 dias. Resultados: na expressão da MMP-9 ocorreu aumento progressivo de sua concentração, mais evidente do 7º ao 14º dias variando a imuno-expressão tecidual entre 2,65% e 11,50%.TGF- β mostrou expressão em nível alto no 3º dia, caiu no 7º, voltando a subir no 14º, com pequena queda no 21º dia variando a imuno-expressão tecidual entre 0,03% e 2,92%. A α-AML apresentou níveis com pouca variação e discreto aumento variando a imuno-expressão tecidual entre 0,88% e 3,23%. Conclusão: a MMP-9 se apresentou como melhor marcador, seguido pela TGF-β. Já o α-AML não se mostrou um bom sinalizador da evolução da reparação tissular.

ABSTRACT Objective: to evaluate the efficacy of three immunohistochemical markers involved in the wound healing process. Methods: experimental study of 40 Wistar rats of the markers metalloproteinases and matrix metalloproteinase 9 (MMP-9), beta transforming growth factor (TGF-β) and myofibroblasts and smooth muscle actin alpha (α-MLA) markers, studied from fragments of surgical scar of abdominal incision involving skin, aponeurosis and peritoneum. The animals were divided into four subgroups of ten according to the day of death, scheduled in three, seven, 14 and 21 days. Results: MMP-9 expression showed a progressive increase of its concentration, more evident from 7th to 14th days, varying the tissue immunoexpression between 2.65% and 11.50% . TGF- β showed expression at high level on the 3rd day, fell in the 7th, rising again in the 14th, with a small decrease in the 21st day, varying the tissue immunoexpression between 0.03% and 2.92%. The α-AML presented levels with little variation and a slight increase, varying the tissue immunoexpression between 0.88% and 3.23%. Conclusion: MMP-9 presented as the best marker, followed by TGF-β. However, α-AML was not a good indicator of the evolution of tissue repair.

Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds.

BACKGROUND: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentiation is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. MATERIALS AND METHODS: Macrophage Fas-induced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to express green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. RESULTS: GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). CONCLUSIONS: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an increase in neutrophils and M1 macrophages and attenuated collagen deposition.

Tumors Alter Inflammation and Impair Dermal Wound Healing in Female Mice.

Tissue repair is an integral component of cancer treatment (e.g., due to surgery, chemotherapy, radiation). Previous work has emphasized the immunosuppressive effects of tumors on adaptive immunity and has shown that surgery incites cancer metastases. However, the extent to which and how tumors may alter the clinically-relevant innate immune process of wound healing remains an untapped potential area of improvement for treatment, quality of life, and ultimately, mortality of cancer patients. In this study, 3.5 mm full-thickness dermal excisional wounds were placed on the dorsum of immunocompetent female mice with and without non-malignant flank AT-84 murine oral squamous cell carcinomas. Wound closure rate, inflammatory cell number and inflammatory signaling in wounds, and circulating myeloid cell concentrations were compared between tumor-bearing and tumor-free mice. Tumors delayed wound closure, suppressed inflammatory signaling, and altered myeloid cell trafficking in wounds. An in vitro scratch "wounding" assay of adult dermal fibroblasts treated with tumor cell-conditioned media supported the in vivo findings. This study demonstrates that tumors are sufficient to disrupt fundamental and clinically-relevant innate immune functions. The understanding of these underlying mechanisms provides potential for therapeutic interventions capable of improving the treatment of cancer while reducing morbidities and mortality.

Novel skin chamber for rat ischemic flap studies in regenerative wound repair.

BACKGROUND: In plastic surgery, skin flap is an important approach to reconstructive wound repairs. The rat dorsal skin flap is a clinically relevant and popular animal model to investigate and evaluate flap survival and necrosis. Nonetheless, flap survival is often unstable with unpredictable outcomes, regardless of previous attempts at design modification. METHODS & RESULTS: In the present study, we report a novel flap chamber that provides stable and reproducible outcomes by separating the dorsal skin flap from its surrounding skin by in situ immobilization. The flap chamber blocks circulation that disturbs flap ischemia from both basal and lateral sides of the flap tissue. Demarcation of skin necrosis is macroscopically evident on the flap and supported by distinct changes in histological architecture under microscopic examination. The utility of the novel skin flap chamber is further proven by applying it to the examination of flap survival in streptozotocin-induced diabetic rats with an increase in skin necrosis. The flap chamber also affords size modifications where a narrower flap chamber increases ischemia and provides manipulable therapeutic windows for studying cell therapies. Accordingly, intradermal injection of endothelial cells 3 days before flap ischemia significantly increases the survival of skin flaps. CONCLUSIONS: The novel flap chamber not only may stabilize the skin flap and provide reproducible outcomes that overcome the shortfalls of the traditional ischemic flap but also may afford size modifications that support research designs and test therapeutic approaches to regenerative repair.

Nesfatin-1 improves oxidative skin injury in normoglycemic or hyperglycemic rats.

Hyperglycemia is one of the major causes of suppressed angiogenesis and impaired wound healing leading to chronic wounds. Nesfatin-1 a novel peptide was reported to have antioxidant and anti-apoptotic properties. This study is aimed to investigate the potential healing-promoting effects of nesfatin-1 in non-diabetic or diabetic rats with surgical wounds. In male Sprague-Dawley rats, hyperglycemia was induced by intraperitoneal (ip) injection of streptozotocin (55 mg/kg). Under anesthesia, dorsum skin tissues of normoglycemic (n=16) and hyperglycemic rats were excised (2 × 2 cm, full-thickness), while control rats (n=16) had neither hyperglycemia nor wounds. Half of the rats in each group were treated ip with saline, while the others were treated with nesfatin-1 (2 µg/kg/day) for 3 days until they were decapitated. Plasma interleukin-1-beta (IL-1ß), transforming growth factor-beta (TGF-ß-1), IL-6 levels, and dermal tissue malondialdehyde (MDA), glutathione (GSH) levels, myeloperoxidase (MPO) and caspase-3 activity were measured. For histological examination, paraffin sections were stained with hematoxylin-eosin or Masson's trichrome and immunohistochemistry for vascular endothelial growth factor (VEGF) was applied. ANOVA and Student's t-tests were used for statistical analysis. Compared to control rats, skin MPO activity, MDA and caspase-3 levels were increased similarly in saline-treated normo- and hyperglycemic rats. Nesfatin-1 depressed MDA, caspase-3, MPO activity and IL-1ß with concomitant elevations in dermal GSH and plasma TGF-ß-1 levels. Histopathological examination revealed regeneration of epidermis, regular arrangement of collagen fibers in the dermis and a decrease in VEGF immunoreactivity in the epidermal keratinocytes of nesfatin-1-treated groups. Nesfatin-1 improved surgical wound healing in both normo- and hyperglycemic rats via the suppression of neutrophil recruitment, apoptosis and VEGF activation.

Innate Mucosal Immune System Response of BALB/c vs C57BL/6 Mice to Injury in the Setting of Enteral and Parenteral Feeding.

BACKGROUND: Outbred mice exhibit increased airway and intestinal immunoglobulin A (IgA) following injury when fed normal chow, consistent with humans. Parenteral nutrition (PN) eliminates IgA increases at both sites. Inbred mice are needed for detailed immunological studies; however, specific strains have not been evaluated for this purpose. BALB/c and C57BL/6 are common inbred mouse strains but demonstrate divergent immune responses to analogous stress. This study addressed which inbred mouse strain best replicates the outbred mouse and human immune response to injury. METHODS: Intravenously cannulated mice received chow or PN for 5 days and then underwent sacrifice at 0 or 8 hours following controlled surgical injury (BALB/c: n = 16-21/group; C57BL/6: n = 12-15/group). Bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay for IgA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, while small intestinal wash fluid (SIWF) was analyzed for IgA. RESULTS: No significant increase in BAL IgA occurred following injury in chow- or PN-fed BALB/c mice (chow: P = .1; PN: P = .7) despite significant increases in BAL TNF-α and SIWF IgA (chow: 264 ± 28 vs 548 ± 37, P < .0001; PN: 150 ± 12 vs 301 ± 17, P < .0001). Injury significantly increased mucosal IgA in chow-fed C57BL/6 mice (BAL: 149 ± 33 vs 342 ± 87, P = .01; SIWF: 236 ± 28 vs 335 ± 32, P = .006) and BAL cytokines. After injury, PN-fed C57BL/6 mice exhibited no difference in BAL IgA (P = .9), BAL cytokines, or SIWF IgA (P = .1). CONCLUSIONS: C57BL/6 mice exhibit similar airway responses to injury as outbred mice and humans, providing an appropriate model for studying mucosal responses to injury. The BALB/c mucosal immune system responds differently to injury and does not replicate the human injury response.