Dermal mast cells in scleroderma: their skin density, tryptase/chymase phenotypes and degranulation

To determine the distribution, tryptase/chymase phenotypes and degranulation of mast cells (MCs) in the dermis of patients with scleroderma, we examined MC density in the skin of 22 patients with systemic sclerosis (SSc) and 11 with localized scleroderma (LSc). We used antitryptase and antichymase antibodies after Carnoy's fixation. Detailed reports of two representative patients with SSc and LSc are included. In the scleroedematous stage (grade 1) showing oedema in both papillary and reticular dermis with variable homogenization of collagen bundles in the reticular dermis, MC skin density was variable in each specimen although MC skin density, as a whole, was significantly increased as compared with normal skin ( P  < 0.05). In the sclerotic stage (grade 2) characterized by homogenization of collagen bundles in the entire dermis, MC skin density was significantly decreased as compared with normal skin ( P  < 0.005). LSc showed changes similar to those in SSc. The ratio of MC_TC cells (both tryptase- and chymase-positive MC) to MC_T cells (tryptase-positive but chymase-negative MC) was variable in SSc and LSc. MC_T cells were exclusively dominant in three patients with SSc and two with LSc. In a patient with SSc (patient 1) showing remarkable perivascular and interstitial oedema in the upper dermis, MC skin density was increased in the oedematous portion and tryptase-positive granules were distributed in extracellular locations. In another patient with LSc (patient 2), tryptase positivity increased and chymase positivity decreased in both number and intensity as the skin sclerosis progressed. MCs must have variable interactions with the lesional skin in SSc and LSc. The present study suggests that MCs are involved in the development of interstitial oedema.     

The regeneration of substance P-containing nerve fibers in the process of burn wound healing in the guinea pig skin

The regeneration of substance P (SP)-containing nerve fibers in the process of burn wound healing in the guinea pig skin has been studied by immunohistochemistry. SP-like immunoreactivity, which was specifically localized in neural elements of intact skin, was found to disappear in the burn wound including its margin on day 2 post burn. The SP-containing nerve fibers were first detected in periods later than day 2 post burn, and the regeneration seemed to occur in association with regeneration of blood vessels at the wound margins. These nerve fibers gradually increased in number and acquired maximum density on day 14 post burn. In addition, such renewed fibers showed sprouting to form a dense network, which has never been observed in intact skin, in the upper granulation tissue just beneath the regrowing epidermis. Following that peak period, the density of the fibers gradually decreased to less than that of controls. The characteristic process of regeneration of SP-containing nerve fibers, having a peak period of fiber density at least in burn wound healing, appeared similar to that of the regeneration of sympathetic catecholaminergic nerve fibers reported previously.     

A Histological Study of Cutaneous Thermal Wounds Using a Clostridium perfringens-Derived Wound Healing Substance with Wound Healing Stimulation Activity

We studied the effects of a Clostridium perfringens-derived wound-healing substance (WHS) on the healing of thermal burn wounds. Third-degree burn injuries were inflicted on the back skin of rats. We histologically evaluated the effects of WHS ointments and compared them with those of lysozyme chloride ointment. We observed the formation of dermal collagen fibers and the increase of capillaries in the WHS ointment treated groups. From the results of hematoxylin and eosin staining and silver staining, an increase in capillaries was observed one week after the application of WHS ointment. Three weeks after the application, when the epithelization was in the final stage, capillary formation ceased. In the WHS ointment-applied groups, electron microscopic observation showed that new collagen fibers were regularly formed in the dermis. On the other hand, in the lysozyme chloride ointment-applied groups, new collagen fibers were present, but were irregularly formed. The main wound healing stimulative action of the WHS ointment could be due to its acceleration of new capillary formation.     

Collagen metabolism in granulating wounds of rat skin

Summary Collagen metabolism in granulating wounds of rat skin was studied with biochemical, isotopic and electron microscopical methods. Deposition of collagen in rat skin wounds was not only the result of an increase in collagen synthesis but it was also caused by a decrease in collagen degradation.Our investigations showed significant differences in the collagen turnover at different times of wound healing. Decreased collagen catabolism at the early stages of wound healing contributed decisively to collagen accumulation in the wound area. At later stages, during wound contraction and remodelling of the scar, the rate of collagen degradation rose.The above-mentioned results are discussed in the context of general criteria of scar formation.     

CD109, a novel TGF‐β antagonist,decreases fibrotic responses in a hypoxic wound model

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF‐β co‐receptor and TGF‐β antagonist shown to inhibit TGF‐β‐induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild‐type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post‐wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild‐type mice showed increased levels of HIF‐1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post‐wounding as compared to those in wild‐type mice and to non‐hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.     

Medicinal plants and their natural components as future drugs for the treatment of burn wounds: an integrative review

Burn wound healing is a complicated process including inflammation, re-epithelialization, granulation, neovascularization and wound contraction. Several biochemicals are involved in burn healing process including antioxidants, cytokines and liver and kidney damage biomarkers. Although several preparations are available for the management of burn wound, there is still a necessity of researching for efficacious medicine. The aim of the present study was to evaluate herbal preparations and their phytochemical constituents for burn wound management. For this purpose, electronic databases including Pubmed, Scirus, Scopus and Cochrane library were searched from 1966 to July 2013 for in vitro, in vivo or clinical studies which examined the effect of any herbal preparation on different types of burn wound. Only 3 human studies were found to include in this review. In contrast, there were 62 in vivo and in vitro studies that show the need for more clinical trials to prove the plant’s potential to cure burn wound. Among single herbal preparations, Allium sativum, Aloe vera, Centella asiatica and Hippophae rhamnoides showed the best burn wound healing activity. Flavonoids, alkaloids, saponins and phenolic compounds were active constituents present in different herbs facilitating wound closure. Glycosides including madecassoside and asiaticoside and proteolytic enzymes were among the main active components. Phytochemicals represented positive activity at different stages of burn wound healing process by various mechanisms including antimicrobial, anti-inflammatory, antioxidant, collagen synthesis stimulation, cell proliferative and angiogenic effect. Overall, several herbal medicaments have shown marked activity in the management of wounds—especially burn wounds—and therefore can be considered as an alternative source of treatment. Furthermore, various natural compounds with verified burn-induced wound healing potential can be assumed as future natural drugs.     

Mast cell numbers in incisional wounds in rat skin as a function of distance, time and treatment

The dynamic changes in skin mast cell (MC) numbers around incised wounds were studied, using experimental designs amenable to multiple analyses of variance. Sixty-four Wistar albino rats were shaved in the interscapular region, wounded or not wounded, and then killed 2 or ro days later. During this period, the rats were exposed continually to a cold (2° C) or control (20° C) climate and treated daily over the shaved region with either tap water or a weak sulphuric acid (pH 3.5) solution. The MCs within five adjacent fields of the wound or the control reference and within the superficial and deep halves of the skin were counted (at × 400). The greatest decrease in MC numbers occurred within about 700 μm of the wound. Whereas the paucity of MCs within the wound region was evident at 2 days, near-normal levels were achieved by day 10. Cold exposure produced little effect, but MCs responded differently to the water and acid treatments as a function of distance and skin depth.     

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。     

The role of mast cells in cutaneous wound healing in streptozotocin-induced diabetic mice

Mast cells (MCs) reside in cutaneous tissue, and an increment of MCs is suggested to induce vascular regression in the process of wound healing. To clarify participation of MCs in diabetic cutaneous wound healing, we created an excisional wound on diabetic mice 4 weeks after streptozotocin injections and subsequently investigated the healing processes for 49 days, comparing them with control mice. The rate of wound closure was not markedly different between the diabetic and control mice. In the proliferative phase at days 7 and 14, neovascularization in the wound was weaker in diabetic mice than in control mice. In the remodeling phase at day 21 and afterward, rapid vascular regression occurred in control mice; however, neovascularization was still observed in diabetic mice where the number of vessels in granulation tissues was relatively higher than in control mice. In the remodeling phase of the control mice, MCs within the wound began to increase rapidly and resulted in considerable accumulation, whereas the increment of MCs was delayed in diabetic mice. In addition, the number of fibroblast growth factor (FGF)- or vascular endothelial growth factor (VEGF)-immunopositive hypertrophic fibroblast-like spindle cells and c-Kit-positive/VEGFR2-positive/FcεRIα-negative endothelial progenitor cells (EPCs) were higher in diabetic wounds. In conclusion, neovascularization in the proliferative phase and vascular regression in the remodeling phase were impaired in diabetic mice. The delayed increment of MCs and sustained angiogenic stimuli by fibroblast-like spindle cells and EPCs may inhibit vascular regression in the remodeling phase and impair the wound-healing process in diabetic mice.     

Expression of SKALP/elafin during wound healing in human skin

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a proteinase inhibitor with specificity for polymorphonuclear leucocyte (PMN)-derived elastase and proteinase-3. SKALP is absent in normal human epidermis, but is strongly induced in inflammatory dermatoses such as psoriasis. SKALP is putatively involved in the regulation of cutaneous inflammation by inhibiting PMN derived proteinases. The aim of this study was to investigate SKALP expression and PMN infiltration during wound healing in human skin. This was examined in healing excisional wounds in normal skin and in impaired healing in various types of chronic venous ulcers. Tissues were analysed using immunohistochemistry and Northern blot analysis. Healing of excisional wounds was studied from day 0 to day 14. An influx of PMN was seen rapidly after wounding and was maximal between day 2 and 4 and then subsided. SKALP was induced within 48 h and was expressed in the suprabasal keratinocytes of the wound edge and the migrating epidermal sheet. SKALP expression was maximal on day 4 and was downregulated at the time of complete reepithelialization (7–14 days). In venous ulcers, PMN were abundant in the wound bed and scarce under the wound edge. SKALP was strongly expressed in the keratinocytes of the wound edge in all types of ulcers studied. In the wound bed, SKALP was not detectable. Our results suggest that SKALP plays a role in the acute, inflammatory phase of wound healing. From the kinetics and topology of SKALP expression we surmise that it negatively regulates PMN infiltration. Some of the results reported here were presented at the meeting of the European Society for Dermatological Research in Vienna, September 1994.     

Chitosan and gelatin biopolymer supplemented with mesenchymal stem cells (Velgraft®) enhanced wound healing in goats (Capra hircus): Involvement of VEGF,TGF and CD31

AimCell-based therapy has emerged as promising strategy for chronic and impaired wounds treatment. Current research is focused on developing biomaterial systems that act as a niche for mesenchymal stem cells (MSCs) to promote wound healing through paracrine molecular cascading. This study was aimed to evaluate the wound healing potential of Velgraft, a ready-to-use biodegradable artificial skin substitute, on excision wound in goats.Materials and methodsTwelve male goats were randomized divided in to three groups of four animals each. After infliction of surgical wound, Velgraft and Soframycin were applied on wounds of the animals of Groups II and III while Group I (sham operated) served as control. Wound diameters were measured at pre-defined time-points for determination of progressive wound healing up to 28 days. Skin sections were stained using Hematoxylin and eosin (H&E) for examining the histoarchitectural changes, Masson trichome staining for ascertaining collagen synthesis and immunohistochemistry for expression of CD31, VEGF and TGF-β1 proteins to determine post-treatment angiogenesis in the inflicted wounds.ResultsVelgraft application appreciably enhanced wound closure by day 21 which was confirmed through restoration of the normal skin architecture as evident based on histopathological examination and characterized by complete regeneration of epidermal layers, collagen fibers, blood capillaries and hair follicular formation. Stimulation of angiogenesis markers was also observed at different time-points post-Velgraft application; which is suggestive of the improved angiogenesis and vasculogenesis.ConclusionVelgraft facilitates wound healing by augmenting early wound closure, enhancing collagen synthesis and deposition, trichosis development and promoting revascularization and epidermal layers restoration.     

Human chronic wounds treated with bioengineered skin: histologic evidence of host-graft interactions

Bioengineered skin is being used to successfully treat a variety of wounds. Randomized controlled clinical trials have shown that a living bilayered skin construct (BSC), consisting of human neonatal keratinocytes and fibroblasts in a collagen matrix, was able to accelerate complete closure of both venous and diabetic ulcers. BSC was particularly effective in difficult-to-heal wounds of long duration. In patients treated with BSC, no obvious signs of gross clinical rejection were observed. Testing of these treated patients showed no BSC-specific immune response and no immune response to bovine collagen or alloantigens expressed on keratinocytes and fibroblasts. However, very little is known about the histologic changes that occur after BSC has been placed on human wounds. We report our preliminary histologic observations in this uncontrolled study of a cohort of 11 patients with 14 wounds treated with BSC in whom biopsy specimens of the grafted sites were obtained at least 2 weeks after application of the construct. The etiology of these ulcers varied from arterial or venous disease to an extensively and poorly healing burn wound. Histologically, thickening of the grafted bioengineered skin was seen in all samples where residual BSC could be identified. Mucin deposition was noted in the dermal layer of the wounds and BSC in 13 of the 14 specimens examined. Unexpectedly, and in spite of good clinical outcome, 4 of the 14 specimens exhibited a foreign body-like granulomatous response. There was no history of prior exposure to BSC in the 4 patients who had a granulomatous response. These early histologic observations suggest that stimulatory interactions develop between BSC and the wound. The consistently found deposition of mucin may point to a fetal pattern of wound repair associated with the neonatal cells in BSC.     

The role of skin substitutes in the treatment of burn injuries

Extensive burn wounds are difficult to manage and repair. Several engineered skin substitutes have been developed to aid in this process. These substitutes are designed with particular objectives in mind which dictate the circumstances under which they can, and should, be employed to promote healing or prepare the burn wound for final closure with autograft. This article discusses some of the rationale behind the use of skin substitutes and reviews some of the substitutes in use at the present time. Current perspectives suggest that skin substitute use is still in its infancy and that there is some way to go before their role in clinical practice becomes clear. Nevertheless the prospect of being able to supply new wound repair components and to influence the healing process to modify outcome and improve the quality of the healed burn wound will ensure a continuing high degree of interest in these potentially useful and beneficial medical devices.     

Neuropeptide-containing C-fibres and wound healing in rat skin. Neither capsaicin nor peripheral neurotomy affect the rate of healing

Wound healing in rat skin was studied in standardized wounds inflicted on both hind legs after unilateral sciatic nerve sectioning and/or capsaicin-induced depletion of sensory nerve (C-fibre) neuropeptide content. Daily visual inspection, histological examination and immunohistochemistry with antibodies against substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide, neuropeptide Y and a pan-neuronal marker, protein gene product 9.5 (PGP 9.5) were used to assess wound healing and determine the distribution of dermal nerve fibres. In controls, nerve fibre density in the wound tissue was low during the first few days after wound infliction, but started to increase on day 4, reaching a peak on day 7 when 25% of medial wounds and 70% of lateral wounds were healed. All wounds were healed on day 11, a scar appearing on day 14 followed by a decrease in nerve fibre density. Capsaicin treatment and/or sciatic nerve sectioning reduced the density of CGRP-immunoreactive nerve fibres by 70% and that of PGP 9.5-immunoreactive fibres by 50%. The capsaicin-induced reduction in PGP 9.5-immunoreactive nerve fibre density is attributable to partial destruction of peripheral nerve fibres. CGRP-immunoreactive and PGP 9.5-immunoreactive nerve fibre density was restored both in capsaicin-treated and denervated groups, reaching a maximum, corresponding to the original level, by days 4-10. Neither the reduction in nerve fibre density following sciatic nerve sectioning nor the impairment of sensory nerve functional capacity following capsaicin treatment affected the rate of wound healing, all wounds being closed on day 11. The study shows that it is difficult to knock out all cutaneous sensory innervation. Thirty per cent of C-fibre innervation seems enough to ensure a normal wound healing.     

Non‐invasive assessment of healing of bacteria infected and uninfected wounds using optical coherence tomography

Background/purpose: Bacterial infection is one of the main predisposing factors for the delay in wound healing. To facilitate a timely decision for correct therapy, it is important to accurately monitor the morphological changes in the infected wounds using noninvasive tools. In the present study, we have explored the use of optical coherence tomography (OCT) for monitoring the healing of superficial wounds infected with Staphylococcus aureus in mice under in vitro and in vivo conditions and studied the changes in collagen birefringence in the infected wounds. Methods: The tape stripping method was used for generating superficial skin wounds in mice and wounds were infected with S. aureus. For in vitro studies, infected and uninfected wound tissues were resected, back scattered intensity and birefringence changes in collagen during wound healing were studied on the 2, 4 and 10th day of postinfection using polarization‐sensitive (PS) OCT and images were compared with histology. Real‐time OCT was used for studying the kinetics of healing of infected wounds under in vivo conditions. Results: From the PS‐OCT images, different phases of wound healing such as inflammation, reepithelialization and collagen remodeling could be identified. The edematic regions appeared prominent in infected wounds. Compared with uninfected wounds, reepithelization and collagen remodeling phases of wound healing were delayed significantly in the infected wounds. These changes were comparable with the different stages of wound healing observed under in vivo conditions. Conclusion: OCT imaging can provide a rapid assessment of the morphological changes associated with bacteria‐infected and uninfected wounds and thereby aid in timely treatment planning.     

Nutrition and cutaneous wound healing

Wound healing is a complex and energy-demanding process. The relationship between nutrition and wound healing has been recognized for many centuries. Several studies have indicated that nutritional deficiencies are more prevalent among patients with chronic wounds. Malnutrition may alter the inflammatory response, collagen synthesis, and wound tensile strength, all of which are crucial for wound healing. Although the specific role of nutrition and supplementation in wound care remains uncertain, it is necessary to identify and correct nutritional imbalances to avoid any potential deterioration of the healing process. It is also important to recognize the differences in pathophysiology between acute and chronic wounds. A burn, surgical, or a traumatic wound is different from a diabetic foot ulcer, which is different from a pressure ulcer. Chronic wounds are more prevalent in the aging population, and patients often have underlying comorbidities, such as diabetes mellitus, peripheral vascular disease, connective tissue disease, or other systemic illnesses that may alter energy metabolism and contribute to impaired healing. Management approaches to acute wound care may not apply universally to chronic wounds. In this review, we discuss the available data and possible roles for nutrition in wound healing.     

Comparison of the effects of tretinoin, adapalene and collagenase in an experimental model of wound healing

Adapalene is a new naphthoic acid derivative with strong retinoid agonistic pharmacological properties. We propose that adapalene might contribute to the wound repairing process as is detected with retinoids. In this controlled study, the effects of topical adapalene, tretinoin and collagenase on full-thickness wound healing were compared in an animal model. Thirty-two adult male Wistar-Albino rats were used in the study. Two circular, full-thickness wounds were made for each animal with a standard 8-mm punch biopsy, on both sides of the midline on the back. No treatment was given to Group I rats (n:8) which comprised the control group. Tretinoin cream (0.1%) was applied topically in Group II (n:8), adapalene gel (0.1%) in Group III (n:8), and collagenase ointment in Group IV (n:8) once daily. On day 7, the wounds were photographed to measure the wound surface area. The wounds on the left side of each animal were excised on day 7, for histopathologic and biochemical examination. The treatments were continued for the right side wounds up to 14 days when the same procedure was repeated. In Group II, a significant decrease in hydroxylproline (HP) levels was detected at day 7 (p = 0.018), and an increase at day 14 (p = 0.002) compared to the control group. HP results revealed no difference either in Group III nor in Group IV versus control at day 7 or 14. However, findings of improved healing were more prominent in Groups II and III than the other groups in histopathologic examination. In conclusion, tretinoin and adapalene contributed to the wound healing process resulting in an enhancement of collagen production, angiogenesis and granulation tissue formation.     

Transient production of stem cell factor in dermal cells but increasing expression of Kit receptor in mast cells during normal wound healing

Mast cells are involved in inflammatory skin disorders and wound healing processes, but the mechanism behind mast cell activation is obscure. In this study, we stained the stem cell factor (SCF) and the Kit receptor in tryptase-positive mast cells, since these molecules are essential for mast cell survival, growth, migration and activation. For this purpose, biopsies were taken from the edge of normally healing wounds of 12 patients undergoing skin transplantation on days 0, 1, 3, 7 and 14, and from chronic leg ulcers and psoriatic skin for comparison. In healing wounds, SCF-positive cells rapidly increased in number in the dermis peaking on day 1, but declined thereafter to their baseline values. The percentage of Kit-positive mast cells increased slowly but steadily reaching a maximum (73+/-22%, P=0.02) on day 14. In chronic ulcers, most of the mast cells were Kit-positive both in the wound bed and in the perilesional skin (87+/-9% and 86+/-13%, respectively). The number of SCF-positive cells was higher in the wound bed than in the dermis of perilesional skin. In the psoriatic skin of ten patients, lesional specimens showed significantly higher numbers of SCF-positive dermal cells as well as a higher percentage (88+/-12% vs 46+/-26%, P=0.004) of Kit-positive mast cells than nonlesional skin. In conclusion, our findings show that the expression of SCF increases rapidly in the early stages of wound healing but declines thereafter, whereas the expression of Kit in mast cells is induced slowly in healing wounds. In chronic wounds as well as in psoriatic lesions, both SCF and Kit are intensely expressed. Thus, it seems possible that SCF and Kit receptor interact, and this could lead to persistent mast cell activation and growth in chronic wounds and psoriasis, whereas only temporary mast cell activation is apparently needed in healing wounds.     

Overview of the role for calreticulin in the enhancement of wound healing through multiple biological effects

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production.