Nitric oxide synthesis inhibition alters rat cutaneous wound healing

BACKGROUND: Nitric oxide (NO) is an important molecule that participates in wound repair, but its effects on cutaneous wound healing are not well understood. The aim of this study was to investigate the effects of NO synthesis blockade on rat cutaneous wound healing by the administration of N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NO synthases. METHODS: NO synthesis was inhibited by administration of L-NAME (20 mg/kg/day) in drinking water. An excisional wound was done, and the animals were killed 7, 14, and 21 days later. Wound contraction and blood pressure were evaluated. The lesion and adjacent skin were formalin fixed and paraffin embedded. Mast cells were quantified, and vessels were evaluated using stereological methods. RESULTS: L-NAME-treated animals presented delayed wound contraction, alterations in collagen organization, and neoepidermis thickness. The inhibition of NO synthesis increased mast cell migration 7 days after wounding, but decreased 21 days after wounding. Volume density of vessels was decreased in L-NAME-treated animals, 21 days after lesion. Surface density of vessels was frequently smaller in L-NAME-treated animals than in controls. CONCLUSIONS; The blockade of NO synthesis impaired cutaneous wound healing, acting in early and late phases of wound repair.     

Efficacy of Ageratum conyzoides on tissue repair and collagen formation in rats

Background.  Wound healing occurs as a fundamental response to tissue injury. Several natural products have been shown to accelerate the healing process. Aim.  To observe the efficacy of topical administration of an ethanolic extract of Ageratum conyzoides on cutaneous wound healing in rats. Methods.  An ethanolic extract of A. conyzoides was prepared, and its wound‐healing efficacy on rats was studied. An open excision wound was made on the back of each rat, and 200 μL (40 mg/kg body weight) of the A. conyzoides extract was applied topically once daily to the treated wounds. The control wounds were treated with 200 μL of 50% ethanol. The wound tissues formed were removed at 4, 8 and 12 days after wounding, and biochemical parameters such as DNA, total protein, total collagen, hexosamine and uronic acid were estimated. The extent of epithelialization and the tensile strength of the wounded tissues were also measured. Results.  The A. conyzoides extract increased cellular proliferation and collagen synthesis. Wounds treated with the extract were found to heal much faster, based on the improved rates of epithelialization and wound contraction, and on the histopathological results. A 40% increase in the tensile strength of the treated tissue was seen. Conclusions.  Topical application of A. conyzoides accelerates the rate of wound healing.     

Are excessive granulation tissue formation and retarded wound contraction due to decreased collagenase activity in wounds in tight-skin mice?

Wound contraction is delayed in tight-skin mice but the mechanism(s) are unknown. The purpose of this study was to investigate collagenase levels and the formation of granulation tissue in experimental wounds in tight-skin mice. One full-thickness skin excision (20×20 mm) was made on the back of nine tight-skin and eight normal mice. Granulation tissue analyses were performed 7 days post-operatively. The collagenase activity was determined by the use of a radiolabelled telopeptide-free collagen substrate, and the amount of granulation tissue was determined gravimetrically. Wound contraction was delayed (P<0.001) in tight-skin mice (mean 22%) compared with normal mice (mean 46%). The collagenase activity was decreased (P<0.05) by 40%, whereas the quantity of granulation tissue was increased (P<0.001) by 60% in the wounds of tight-skin mice. Decreased collagenase content may provide one explanation for the delayed contraction of full- thickness wounds in tight-skin mice. Furthermore, this animal wound model may prove useful in the understanding of the pathogenesis, and in exploration of treatment, of excessive granulation tissue formation during wound healing.     

Modeling dermal granulation tissue with the linear fibroblast-populated collagen matrix: a comparison with the round matrix model

BACKGROUND: Wound contraction typically is not symmetrical; for example, a square-shaped wound will not yield a square scar. Interestingly, the round fibroblast-populated collagen matrix has been used as a model of wound contraction, even though contraction in this model is mostly symmetrical. OBJECTIVE: We wanted to compare the round versus linear fibroblast-populated collagen matrix to see which would be a better model of dermal granulation tissue. METHODS: Gross and microscopic morphology, contraction kinetics, cytoskeletal architecture, and apoptotic and proliferative indices were compared between the round versus the linear fibroblast-populated collagen matrix. A rat excisional wound model was used as an in vivo standard of healing. RESULTS: The rate of contraction was similar between the two models, although the mode of contraction was grossly asymmetric in the linear while remaining symmetric in the round model. Cellular survival and proliferation were both dependent on matrix attachment in both models; this was analogous to the attachment-dependence of granulation tissue. In the attached (restrained) condition, the level of cellular organization was higher in the linear than in the round matrix; the tissue architecture of the linear matrix, moreover, mimicked that of the excisional wound model. CONCLUSION: The round versus linear fibroblast-populated collagen matrix displayed a similar proliferative and survival response to matrix attachment. The latter model, however, demonstrated tissue organization with attachment and asymmetrical contraction after detachment analogous to that of the in vivo wound model. The linear fibroblast-populated collagen matrix appears to be the better model of dermal granulation tissue.     

Localization of collagen alpha 1(I) gene expression during wound healing by in situ hybridization

The cellular localization of collagen alpha 1(I) chain gene expression during wound healing in rats was investigated using in situ hybridization. Activation of collagen gene expression was first found within fibroblastic cells around the deep layers of the granulation tissue as early as 16 h post wounding. Heavily labeled cells were also detected near the intact wound edge and around hair follicles. At day 6 intense alpha 1(I) collagen gene expression was found within most cells of the granulation tissue and after day 8 most of the activity was localized to cells directly underneath the epidermis. 26 d after the induction of the wounds hardly any alpha 1(I) collagen gene expression could be demonstrated, which indicates a close, time-dependent control of collagen synthesis during repair processes.     

Recombinant human platelet-derived growth factor enhanced dermal wound healing by a pathway involving ERK and c-fos in diabetic rats

BACKGROUND: Platelet-derived growth factor (PDGF) has been shown to promote dermal wound healing, however, the molecular mechanisms responsible for are not fully understood. OBJECTIVE: The present study was undertaken to investigate the possible signaling mechanisms by which PDGF improved healing of cutaneous wound in diabetic rats. METHODS: Four full-thickness skin wounds were created on the dorsum of Wistar diabetic rats. Animals were treated with or without recombinant human PDGF (rhPDGF) at 7.0 microg/cm(2) wound or vehicle daily 1 day after wounding. The animals were then killed after various intervals of wounding, and the wounded skin tissues were used for histological evaluation, analysis of the phosphorylation of extracellular signal-regulated kinases (ERK) and the expression of c-fos protein, as well as the labeling indices of proliferative cell nuclear antigen (PCNA). RESULTS: Topical application of rhPDGF significantly accelerated the rate of reepithelialization compared with vehicle-treated or untreated group at 7 days after wounding. At the histological level, the significant increases in the degree of reepithelialization, the thickness of granulation tissue and the density of capillary bud were observed in the wound sites in rhPDGF-treated group at 7 and 14 days after wounding. Moreover, treatment with rhPDGF increased PCNA labeling indices, c-fos protein expression and ERK phosphorylation in the wounded tissues at the indicated time after wounding. CONCLUSION: These results suggest that application of rhPDGF increases cell proliferation, and enhances dermal tissue repair in diabetic skin lesion of rats, which might be partly mediated by ERK activation and c-fos protein expression.     

Angiogenesis in wound healing

During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-beta, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, alpha(v)beta3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: alpha(v)beta3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of alpha(v)beta3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of alpha(v)beta3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds.     

PDGF supplementation alters oxidative events in wound healing process: a time course study

Platelet-derived growth factor (PDGF), an important stimulant, plays a role in almost all stages of wound healing process. In various studies, it has been shown that PDGF has healing effects in this process. In the present study, we especially focused on investigating the effects of exogenous PDGF administration on oxidative events during cutaneous wound healing process. Experiments were performed on 42 female Wistar-albino rats. Animals were divided into four groups: control, untreated, chitosan-treated and chitosan + PDGF-treated. Two uniform full-thickness excisional skin wounds were made under anesthesia in all animals except control group. In the chitosan + PDGF-treated groups, the wounds were treated topically with a single daily dose PDGF-BB (7 ng/ml) after wounding. In the chitosan-treated groups, the wounds were treated topically with equal amount of blank chitosan gel. After that, on the 3th and 7th days of wound healing, the animals were killed. Thiobarbituric acid-reactive substances (TBARS), nitric oxide (NOx), ascorbic acid (AA), glutathione (GSH) levels and superoxide dismutase (SOD) activity were measured spectrophotometrically in the wound tissues. PDGF significantly increased TBARS levels in early phase of wound healing. In contrast, it significantly decreased TBARS levels in later phase of healing. In the chitosan + PDGF-treated group, NOx levels decreased on days 3 and 7 when compared with the chitosan-treated groups. Non-enzymatic antioxidant levels were increased by PDGF administration and this may have contributed to increase in wound tissue antioxidant capacity. In the light of these findings, PDGF supplementation may have altering effects on oxidative events depending on the time in wound healing process.     

Effect of retinoids on wound healing in diabetic rats

Summary In order to clarify the effect of retinoids on wound healing, diabetic rats were treated with oral acitretin or isotretinoin at 1 mg/kg for 5 days prior to the setting of deep punch biopsies, 6 mm in diameter, and for 14 days thereafter. Wound care was done with either a dry or a wet dressing. Wound surface area and transepidermal water loss were measured twice weekly at the time of the change of dressings. Statistically significant increases of both test parameters were noted after 1 week in retinoid-treated animals, but only with dry dressings. No excessive granulation tissue was observed at any time. The data suggest that under certain unfavorable circumstances, problems in wound healing may arise under retinoid treatment.     

Hyperbaric oxygen therapy mediates increased nitric oxide production associated with wound healing: a preliminary study

OBJECTIVE: The objective of this preliminary study was to document general somatic and wound nitric oxide (NO) levels during and after hyperbaric oxygen therapy (HBOT). DESIGN: The study evaluated 6 chronic wound patients that responded favorably to HBOT treatment (20 treatments; 2.0 atmosphere absolute [ATA] x 90 minutes). Successful HBOT was associated with increased wound granulation tissue formation and significantly improved wound closure. Wound fluid and fasting plasma samples were obtained for measurement of nitrate and nitrite (NOx), the stable oxidation products of NO; plasma L-arginine (L-Arg); and asymmetric dimethylarginine (ADMA). NOx measurements were obtained before treatment (baseline), after 10 and 20 treatments, and at 1 and 4 weeks after therapy. RESULTS: Wound fluid NOx levels tended to increase during treatments, were significantly elevated at 1 and 4 weeks after therapy, and correlated with reductions in wound area. Plasma L-Arg and ADMA were unchanged during and after HBOT. CONCLUSION: This preliminary study documents a significant increase in local wound NO levels (by NOx measurements) after successful HBOT and suggests that this mechanism may be an important factor in promoting enhanced wound healing and wound closure associated with this therapy.     

Wound healing of human skin transplanted onto the nude mouse after a superficial excisional injury: human dermal reconstruction is achieved in several steps by two different fibroblast subpopulations

Abstract It has been established that human skin grafted onto the nude mouse is able to regenerate after being subjected to a full-thickness wound. In the present work, we sought to determine the cells involved in the connective tissue repair process following superficial wounding. Two months after transplantation, superficial wounds were made at the center of the graft using mechanical dermabrasion. At various times thereafter, ranging from 2 days to 6 weeks, healing grafts were harvested and processed for immunohistological study with species-specific and cross-reacting antibodies directed against human or mouse antigens. The grafted human skin regenerated according to the following series of events. First, the human dermis underneath the scab became devoid of human fibroblasts while the surrounding human dermis preserved its own characteristics. The TUNEL reaction on early-phase healing wounds indicated that apoptosis occurred steadily within this area and could be the mechanism by which cells disappeared. Moreover, cell death was reduced when the wound was covered with an occlusive dressing. The human dermis beneath the wound was then invaded by mouse cells which deposited type I collagen on the human extracellular matrix and produced mouse granulation tissue at the surface above it. Human keratinocytes migrated over the mouse granulation tissue to reconstruct the epidermis. Eventually, the mouse granulation tissue was progressively invaded by human fibroblasts, which formed a human neodermis. The overall process appeared to depend upon several successive epithelial-mesenchymal interactions, which were not species-specific. This suggests that myofibroblasts arise from a specific subpopulation of fibroblasts, probably located at the interface between the dermis and adipose tissue, and that the granulation tissue is eventually remodeled by another population of fibroblasts present in the human dermis. Received: 31 March 1999 / Accepted after revision: 15 July 1999 / Accepted: 13 August 1999     

Topical tacrolimus does not negatively impact acute skin wound healing

Despite the increasing use of topical tacrolimus, there is little information about its effect on skin wound healing. To determine effects on acute cutaneous wound healing, two full‐thickness skin wounds were imparted on the backs of 45 hairless mice, which were then divided into vehicle‐, topical tacrolimus‐ and topical steroid‐treated group. Each drug was topically applied once daily. The wound area was assessed by using dermoscopic images every two days after wounding. At 3, 7 and 11 days after wounding, 10 wounds in each group were collected for semi‐quantitative analysis of histological features including re‐epithelialization, polymorphonuclear leucocytes, fibroblasts and collagen. We also checked the mRNA expression levels of EGF, TGF‐β, TNF‐α and IL‐1α. While topical application of clobetasol propionate was found to delay re‐epithelialization and infiltration of polymorphonuclear leucocyte, topical treatment with tacrolimus showed patterns similar to that of the vehicle. In the tacrolimus‐treated group, mRNA expression levels of IL‐1α and TGF‐β were slightly decreased, while the others were similar with the vehicle‐treated group. Unlike steroid, topical tacrolimus, therefore, did not disturb the wound healing process in a murine skin wound model.     

Effects of Human Adipose-derived Stem Cells on Cutaneous Wound Healing in Nude Mice

Background

Despite numerous treatments available for deteriorated cutaneous wound healing such as a diabetic foot, there is still the need for more effective therapy. Adipose-derived stem cells (ASCs) are mesenchymal stem cells, which are self-renewing and multipotent. Mesenchymal stem cells have the potential for tissue repair and regeneration.

Objective

To investigate the effects of human ASCs on the healing of cutaneous wounds in nude mice.

Methods

15-mm round full-thickness skin defects were generated on the back of BALB/c nude mice. The mice were divided into three groups for wound coverage: (i) human ASCs-populated collagen gel, (ii) human dermal fibroblasts-populated collagen gel, and (iii) collagen gel alone. Wound contraction was prevented with a splint method. Wound size was measured 10 days after injury. At 28 days histological analysis was performed.

Results

Both ASCs and dermal fibroblasts accelerated wound closure, but dermal fibroblasts were more effective than ASCs. At 28 days, the dermal portion of ASCs or dermal fibroblasts wound scars were thicker than collagen gel wound scars.

Conclusion

ASCs and dermal fibroblasts stimulate cutaneous wound healing and improve scar thickness.     

Electron irradiation slows down wound repair in rat skin: a morphological investigation

Summary To date, there have been few morphological investigations of the effect of electron radiation on the healing of skin wounds in rats. The present morphological study examines the wound repair process in electron-irradiated rat skin by electron microscopy. Standardized, full-thickness, incisional wounds were made in the lower dorsal skin of animals which had been locally irradiated with 9·6 Gy electron radiation 7 days previously. The irradiation dose was maximal at 3 mm depth. Twenty-four rats were used in the investigation: 12 were irradiated and 12 sham-irradiated. Three rats from each experimental group were killed at 1, 3, 7 and 14-day time intervals after wounding. The morphological effect of electron irradiation on the repair of each wound was investigated by light microscopy (LM) and scanning electron microscopy (SEM). New granulation tissue visualized by SEM was quantified using computerized image analysis. The results suggest that a single, partial-body, controlled depth dose of electron irradiation delays wound repair. LM showed that there is a depression of the inflammatory cell and tissue exudate response, slowing of epithelial migration, and a decrease in fibroblast representation, together with a delay in the formation of collagen bundles. Granulation tissue formation was impaired up to 7 days post-wounding, but was restored to around control values by day 14, indicating that healing was delayed. However, as the healing of normal tissue was not prevented, this study supports a preoperative role for the use of low-dose electron irradiation therapy for the treatment of electron-sensitive superficial pathologies in surgical practice.     

Healing potential of zerumbone ointment on experimental full-thickness excision cutaneous wounds in rat

Zerumbone is a monocyclic sesquiterpene compound. Based on report, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The study was undertaken to evaluate the therapeutic effects of topical zerumbone on excision wounds in rats. A 1% (w/w) simple ointment containing zerumbone was applied topically (100 mg ointment per rat) once a day on full-thickness excision wounds created on rats. The wound tissue was removed and used for estimation of antioxidant activity and to observe histopathological changes. Immunohistochemical staining was performed to study the expression pattern of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1 and collagen IV. Zerumbone exhibited antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Candida tropicali. Zerumbone ointment has potent wound healing capacity as evident from the wound contraction on 15th post wounding day. The histopathological examinations of healed wound sections showed greater tissue regeneration, more fibroblasts and angiogenesis in zerumbone ointment-treated group. VEGF, TGF-β1 and collagen IV expression were also correlative with the healing pattern observed. Zerumbone possesses potent antioxidant activity by increasing superoxide dismutase, catalase, glutathione and decreased lipid peroxidation. The synergistic effects of both antimicrobial and antioxidant activities in zerumbone are deduced to have accelerated the wound repair. The results demonstrate that zerumbone possessed strong wound healing potential and can be exploited to accelerate excision wound healing.     

腐植酸钠促进大鼠伤口愈合的实验观察

目的探讨腐植酸钠对大鼠皮肤创伤愈合的作用。方法建立大鼠皮肤切创模型,以不同浓度的腐植酸钠(0.5%,1%,2%)作用于伤口,分别于术后3,6,9,14,21d时间点观测创面愈合率和平均愈合时间,HE常规染色观察新生肉芽组织及形态学改变。结果腐植酸钠各剂量组的创伤平均愈合时间为(13.7±0.65)d,明显快于空白组的(15.95±1.13)d和阴性组的(15.72±1.24)d,差异均有统计学意义(P均<0.05);术后3,6,9,14d腐植酸钠各剂量组的伤口皮肤平均愈合率显著高于空白组和阴性组,差异均有统计学意义(P均<0.01)。HE染色表明,与空白组和阴性组相比,术后3,6,9d的腐殖酸钠各剂量组的炎症细胞明显减少,新生肉芽组织较厚、新生毛细血管密集、表皮愈合速度较快。结论不同剂量腐植酸钠均能促进实验性皮肤创伤的修复。     

Fibronectin. Localization in normal human skin, granulation tissue, hypertrophic scar, mature scar, progressive systemic sclerotic skin, and other fibrosing dermatoses

The histologic localization of fibronectin (FN) in normal human skin, granulation tissue, hypertrophic scar, mature scar, progressive systemic sclerotic skin, and tissue of other fibrotic disorders was investigated by an indirect immunofluorescence technique using specific antiserum prepared in rabbits against purified human plasma FN. In granulation tissue that developed just after traumatic wounding, FN seemed to increase remarkably in the wound as a fibrillar network. In the hypertrophic scar, one to five years after wounding, FN was detected in a linear or curling arrangement throughout the dermis. On the contrary, FN gradually decreased in the wound of the mature scar five to 20 years after wounding. There were some interesting observations among other diseases. In the skin of patients with progressive systemic sclerosis and morphea, FN was found to be localized faintly on the dermoepidermal junction and papillary dermis. In the involved skin of dermatofibroma, FN was observed in a curling arrangement throughout the dermis.     

Beta-adrenoceptor blockade delays granulation tissue formation in polyurethane sponge implants

Background:  The role of adrenoceptors in granulation tissue formation is not well understood. The aim of this study was to investigate the effects of alpha- and beta-adrenoceptor blockade on granulation tissue development using polyurethane (PU) implants in the rat.
Methods:  Animals were treated orally with propranolol (beta1- and beta2-antagonist), atenolol (beta1-antagonist) or phentolamine (alpha1- and alpha2-antagonist) until euthanasia. The control group received only water. All animals received subcutaneous implants of PU sponges. After 14 days, implants were collected, formalin-fixed and paraffin-embedded. Sections were stained with hematoxylin and eosin and Sirius red and immunostained for CD68 and alpha-smooth muscle actin.
Results:  The number of inflammatory cells and the volume density of myofibroblasts and blood vessels were lower in the control group than in the propranolol- and atenolol-treated groups. The collagen fiber score was greater in the control group than in the propranolol- and atenolol-treated groups. The inflammatory infiltrate, collagen fiber score, blood vessel density or myofibroblast differentiation was not affected by phentolamine. The percentage of fibrovascular invasion was greater in the antagonist-treated groups than in the control group.
Conclusions:  Blockade of beta1- and beta2-adrenoceptors, but not alpha-adrenoceptors, impairs granulation tissue development in PU implants due to interference with the inflammatory response.