The epithelialization of split skin graft donor sites —a test model for the efficacy of topical wound therapeutic agents

Summary Wound areas on the upper thigh following split thickness skin grafts can be standardized. They are, therefore, suitable as wound models for comparative investigations into the kinetics of epithelialization using various wound therapeutic agents. In a prospective clinical study, the epithelialization time in two split thickness graft donor sites was examined in an intraindividual comparison on 16 patients. Zinc oxide and potassium/calcium chloride hydrogel were used as the comparative substances. The results confirm the suitability of the study design for the testing of efficacy of topical wound therapeutic agents. The model can be standardized, is practical, and does not require additional stress to the patient.     

Metallothioneins: potential therapeutic aids for wound healing in the skin

Metallothioneins are a family of low molecular weight proteins containing approximately 30 % cysteine. Expression of the metallothionein gene is up-regulated in the skin following topical application of zinc and copper, and in wound margins particularly in regions of high mitotic activity. This induction of metallothionein in the wound margin may reflect its role in promoting cell proliferation and reepithelialiation. The action of metallothioneins in these processes may result from the large number of Zinc-dependent and copper-dependent enzymes required for cell proliferation and matrix remodeling. In addition, selected growth factors may modulate metallothionein gene expression and hence the ability of cells to proliferate. Recent studies suggest that metallothioneins may be an attractive target for development as vulnerary agents.     

Zinc deficiency and the prolonged accumulation of zinc in wounds

Zinc is required for protein synthesis and therefore for wound healing. Zinc levels were 50 per cent higher in muscle and skin from abdominal wounds of rats during the maturation phase of wound healing, but mild deficiency greatly reduced this accumulation. The results suggest that zinc takes part in the late stages of wound healing when protein is laid down, and that such extra local demands expose otherwise marginal zinc deficiency.     

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear "impaired" wound model. To impair wound healing, macrophages were depleted by pretreatment with iota-carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor-α (TNF-α) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP-2 expression in wounds. In contrast, TNF-α, MMP-9, and TIMP-1 were significantly up-regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF-α production and, to a lesser degree due to release of metalloproteinases.     

Zinc and Skin Health: Overview of Physiology and Pharmacology

Background. Zinc is known to have a critical role in overall human physiology, which likely explains many of its therapeutic uses for the last several thousand years. The specific roles zinc plays in skin health and function are less widely known yet are likely just as critical based on the manifestations of dietary zinc deprivation, which include moderate to severe dermatitis.
Objective. To provide a critical review of the scientific literature as to the physiologic importance of zinc to skin, the biochemical basis for these effects, and pharmacologic aspects of zinc therapeutics.
Results and Conclusions. Skin is in a continual state of renewal, placing a high demand on zinc-based enzymes and proteins that direct this process. The importance of zinc physiologically is especially evident in studies of wound healing and inflammation reduction. During these processes, the high needs for zinc can be supplemented externally, generally increasing the rates of the natural processes. Topical zinc delivery involves the pharmacologic optimization of zinc delivery, often mediated by the solubility of the zinc material and interactions within the product matrix.
DR. SCHWARTZ AND DR. MARSH ARE EMPLOYEES OF PROCTER & GAMBLE, WHICH SUPPORTED THE WORK. PATENT APPLICATIONS FOR TECHNOLOGY DISCUSSED IN THE ARTICLE HAVE BEEN FILED. DR. DRAELOS IS A CONSULTANT TO PROCTER & GAMBLE.     

Influence of zinc deficiency on breaking strength of 3-week-old skin incisions in the rat

The effect of dietary zinc deficiency on the breaking strength of abdominal skin incisions was studied in rats 21 days postoperatively. Zinc deficiency was induced with a low-zinc diet (1.4 mg zinc/kg diet) 14 days preoperatively. Thereafter serum zinc was reduced by 60%, but the zinc concentration in unwounded skin and liver remained similar to that of pair-fed controls given a zinc-adequate diet (33 mg zinc/kg). The wound breaking strength (maximal load until wound disruption) was significantly lower in the zinc-deficient group (75% that of control wounds). The zinc concentration in wound tissue had decreased in the zinc-deficient group, but the wound hydroxyproline concentration was similar in the two groups. The results indicate that zinc is an important trace element during the early remodeling of scar tissue.     

基质金属蛋白酶—1在表皮修复中的生物学作用

目的 阐明有关基质金属蛋白酶－1（MMP－1）在皮肤损伤后,表皮修复过程中的作用。方法 回顾近年来有关MMP－1在皮肤损伤修复中的作用及研究进展,总结其在上皮化局部微小环境内的表达及细胞分子生物学效应。结果 皮肤损伤信号直接引发表皮细胞于特定的部位及时间表达MMP－1;MMP－1通过特异性分解创面基质胶原蛋白,促进表皮细胞的增殖及迁移,由此影响表皮损伤创面的愈合结果。结论 皮肤损伤后表皮细胞表达MMP－1与创面重新上皮化直接相关。     

瘦素介导的细胞内信号途径在创面愈合中的作用

目的综述瘦素(Leptin)介导的细胞内信号途径及其对创面愈合作用的研究进展。方法广泛查阅近年文献,对Leptin理化性质、受体作用机制、受体相关的细胞内信号途径及其在皮肤和黏膜创面愈合中的作用进行综述。结果Leptin是一种由ob基因表达、相对分子质量为16×103的蛋白激素,通过结合其特异性受体,活化Janus激酶/信号传导及转录活化子途径、丝裂原活化的蛋白激酶通路和磷脂酰肌醇-3-激酶通路等主要信号途径,参与机体能量代谢、体重平衡、创伤愈合等多种功能的调控。Leptin受体广泛存在于机体各种组织,提示Leptin功能的多向性。皮肤创伤后局部Leptin表达增多,能促进角化细胞增殖、上皮形成、成纤维细胞增殖和胶原合成,起到加速创伤修复的作用。黏膜损伤或黏膜细菌感染后Leptin表达也显著增高,可通过调节黏膜腺体分泌、改善黏膜血流量以及与内皮素1协同作用来促进黏膜损伤的修复。结论Leptin能够通过活化其受体相关的细胞内信号途径促进创面愈合。     

The mouse dorsal skin fold chamber as a means for the analysis of tissue engineered skin

The therapy of extensive and deep burn wounds is still a challenging task for reconstructive plastic surgery. The outcome is generally not satisfactory, neither from the functional nor from the aesthetic aspect. Several available skin substitutes are used but there is need for optimization of new skin substitutes which have to be tested in vitro as well as in vivo. Here, we show that the dorsal skin fold chamber preparation of mice is well suited for the testing of skin substitutes in vivo. Dermal skin constructs consisting of matriderm^® covered with a collagen type I gel were inserted into full thickness skin wounds in the skin fold chambers. The skin substitutes integrated well into the adjacent skin and got epithelialized from the wound edges within 11 days. The epithelialization by keratinocytes is the prerequisite that also cell-free dermal substitutes might be used in the case of the lack of sufficient areas to gain split thickness skin grafts. Further advantage of the chambers is the lack of wound contraction, which is common but undesired in rodent wound healing. Furthermore, this model allows a sophisticated histological as well as immunohistochemical analysis. As such, we conclude that this model is well suited for the analysis of tissue engineered skin constructs. Besides epithelialization the mode and extend of neovascularization and contraction of artificial grafts may be studied under standardized conditions.     

Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent: a new paradigm in wound healing

HYPOTHESIS: In patients with diabetic foot and pressure ulcers, early intervention with biological therapy will either halt progression or result in rapid healing of these chronic wounds. DESIGN: In a prospective nonrandomized case series, 23 consecutive patients were treated with human skin equivalent (HSE) after excisional debridement of their wounds. SETTING: A single university teaching hospital and tertiary care center. PATIENTS AND METHODS: Twenty-three consecutive patients with a total of 41 wounds (1.0-7.5 cm in diameter) were treated with placement of HSE after sharp excisional debridement. All patients with pressure ulcers received alternating air therapy with zero-pressure alternating air mattresses. MAIN OUTCOME MEASURE: Time to 100% healing, as defined by full epithelialization of the wound and by no drainage from the site. RESULTS: Seven of 10 patients with diabetic foot ulcers had complete healing of all wounds. In these patients 17 of 20 wounds healed in an average of 42 days. Seven of 13 patients with pressure ulcers had complete healing of all wounds. In patients with pressure ulcers, 13 of 21 wounds healed in an average of 29 days. All wounds that did not heal in this series occurred in patients who had an additional stage IV ulcer or a wound with exposed bone. Twenty-nine of 30 wounds that healed did so after a single application of the HSE. CONCLUSIONS: In diabetic ulcers and pressure ulcers of various durations, the application of HSE with the surgical principles used in a traditional skin graft is successful in producing healing. The high success rate with complete closure in these various types of wounds suggests that HSE may function as a reservoir of growth factors that also stimulate wound contraction and epithelialization. If a wound has not fully healed after 6 weeks, a second application of HSE should be used. If the wound is not healing, an occult infection is the likely cause. All nonischemic diabetic foot and pressure ulcers that are identified and treated early with aggressive therapy (including antibiotics, off-loading of pressure, and biological therapy) will not progress.     

Wound healing in denervated rat skin

Recently, several reports have suggested that innervation influences wound healing. However, some investigators have reported that nerve injury prevented wound healing while others have suggested it had no influence on full-thickness skin wound healing. We created denervated skin areas on rats by dissection of the spinal hemicord. Subsequently, 15-mm-diameter skin defects were made symmetrically within the denervated area on the right side of the back and the normal innervated area on the left side. Biopsies were performed at 3, 7, and 14 days after wounding. We measured changes of the wound surface area, the rate of wound contraction, and the rate of epithelialization. The differences were not significant at 3 or 7 days after the operation. However, we could observe significantly delayed wound healing of the denervated skin areas compared to the normal areas at 14 days. Both wound contraction and epithelialization were delayed in the denervated groups. Our results suggest that sensory disturbance is a negative factor for skin wound healing.     

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model

Background and Objective

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross‐links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model.

Materials and Methods

Non‐closing non‐grafted, partial closing non‐grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide‐field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization.

Results

The endogenous fluorescence excitation of cross‐links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non‐closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial‐close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement.

Conclusions

We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin‐digestible collagen cross‐links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678–685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.     

Acceleration of wound healing by a live yeast cell derivative

Acceleration of the normal rate of burn wound healing would serve to decrease the morbidity and possibly the mortality of burn victims. A live yeast cell derivative (LYCD) has previously been reported to stimulate wound epithelialization and this study was designed to evaluate that hypothesis. Twenty-six human skin graft donor sites in nine patients were compared in a double-blind, randomized, single-center inpatient study. Thin donor sites were used as a model for superficial wound healing. Statistically significant earlier angiogenesis and epithelialization occurred in donor sites treated with LYCD ointment as compared with donor sites in the same patients treated simultaneously with ointment base. Stinging pain was noted by seven patients, but in all cases the pain was mild and required no analgesia.     

Buried chip skin grafting for treatment of perianal burns

A technique of buried chip skin grafting for perianal burn injury is described. Small chip skin grafts are buried in the granulating wound around the anus and this procedure achieves epithelialization of the perianal and perineal wound within about 5 or 6 cm laterally from the middle with extremely small amounts of skin graft. Graft survival is not disturbed by stools or gently wiping of the wound to remove stools, as grafts are buried in the holes. If infection occurs soon after the operation, graft survival does not appear to be affected because the grafted holes show good drainage. This procedure is considered to be very useful for treating perianal or perineal granulating wounds in extensively and deeply burned patients who have limited autograft donor sites.     

Vertical (two-layer) skin grafting: new reserves for autologic skin

The main, permanent source of burn coverage continues to be autologic skin. In patients with major burns, the amount of available autologic skin may be insufficient. Consequently, severe wounds are covered after debridement with other biological or synthetic skin substitutes. Another source of skin reserves for wound coverage is the use of cultured keratinocyte sheet graft alone or with any dermal substitute. Some of these materials provide only temporal coverage and are often costly and time-consuming in preparation. These factors can be critical in burned patients. To expand the effective means of wound coverage, the authors sought a new source of autologic skin. The dermal grafts that were the marginal product of skin harvesting were meshed and grafted on the debrided third-degree burn, granulated wound, or muscle. The authors observed good dermal grafts "take" with rapid or slow epithelialization. They saw no the delay in donor site healing where the skin grafts overlapped. The histological difference in usual skin grafts and dermal grafts was studied after their harvesting and "taking."     

The hairless mouse ear: an in vivo model for studying wound neovascularization

Microvascular ingrowth into damaged tissue is an essential component of the normal healing process. In fact, wound therapy is often aimed at promoting neovascularization. However, little is known about the mechanisms that regulate microvascular ingrowth into a healing wound. This limited knowledge is largely due to the lack of adequate models in which microvascular ingrowth can be quantitatively analyzed throughout the healing process. To address this deficiency, we developed a model in which a wound was created on the ear of the hairless mouse-a well established model for directly viewing and measuring skin microcirculation. While the animals were under ketamine and xylazine anesthesia, 2.25 mm diameter full-thickness wounds were created on the dorsum of hairless mouse ears down to but not including the cartilage (0.125 mm depth). With the use of video microscopy and computer-assisted digitized planimetry, the precise epithelial and neovascular wound edge was viewed and measured regularly throughout healing. Therefore, this model can provide objective data on wound epithelialization and neovascularization throughout healing. This model was used to examine the effect of topical wound agents on epithelialization and neovascularization. Differential effects by these anti-microbial agents on these two processes were observed, which suggests clinical implications for their use.     

A review of the Turned-down Onto Pericapsular-tissue Hemisectioned Amputated Toe (TOPHAT) flap for wound coverage during ray amputations of the toes

IntroductionExposure of the adjacent Metatarsal-Phalangeal Joint (MTPJ) commonly occurs after application of Topical Negative Pressure Wound Therapy (TNPWT) for a ray amputation wound. This is due to mechanical soft tissue erosion or trauma to the adjacent digital artery from direct pressure effect. This results in toe gangrene requiring a ray amputation and ultimately a larger wound bed. We describe the use of the Turned-down Onto Pericapsular-tissue Hemisectioned Amputated Toe (TOPHAT) flap – a filleted toe flap to protect the adjacent MTPJ capsule combined with a novel Negative Pressure Wound Therapy with instillation and dwell-time (NPWTi-d) dressing technique. The flap protects the adjacent joint capsule and reduces the wound burden whilst allowing the wound to benefit from TNPWT, thereby accelerating wound healing.Material and methodsA retrospective review was conducted of patients with toe gangrene requiring ray amputation that underwent the TOPHAT flap on in our institution from 2019 and 2020. Complications such as wound dehiscence, hematoma, flap necrosis and secondary infection were recorded. Other outcomes recorded were time taken to final skin grafting and time taken for complete wound epithelialization.Results9 patients underwent treatment with the TOPHAT flap. 2 patients had flap necrosis. 7 patients progressed to definitive skin coverage with skin grafting. One patient subsequently had progressive arterial disease despite successful skin grafting and required above knee amputation. The mean time to final skin grafting and complete wound epithelialization was 49.5 days and 107.5 days respectively. All patients were satisfied with the outcomes and were able to return to their pre-morbid function.ConclusionsThe TOPHAT flap has a consistent vascular supply that provides durable soft tissue coverage. It is a robust and easily reproducible technique to accelerate wound healing after ray amputations even in patients with peripheral vascular disease.     

Digital image analysis versus clinical assessment of wound epithelialization: a validation study

To evaluate the progress in wound healing, wound assessment is mandatory. Epithelialization is traditionally assessed subjectively by the clinician. In a previous study, subjective assessment of epithelialization was shown to be reliable. In this study, reliability of epithelialization measured by digital image analysis was investigated and then, we validated the subjective evaluation by comparing this assessment to measurements with digital image analysis. Clinicians assessed epithelialization in 50 burn wounds that were treated with a split skin graft. Epithelialization of these wounds was also measured by three observers using digital image analysis. Reliability of digital image analysis was tested using the intraclass correlation (IC). To test validity, subjective clinical assessment was correlated with digital image analysis (IC). The results showed that interobserver reliability of epithelialization measured by digital image analysis was good (IC coefficient 0.74). Subjective clinical assessment of epithelialization showed a strong correlation with digital image analysis (IC coefficient 0.80). In conclusion, subjective clinical evaluation of wound epithelialization is as good as an objective measure, in this study digital image analysis. Since digital image analysis is more time-consuming, we recommend the use of the subjective evaluation for daily practice.     

Ketanserin accelerates wound epithelialization and neovascularization

We investigated the acute effects of topical ketanserin, a 5-HT₂ (serotonin) receptor blocker, on wound epithelialization and vascularization with the use of the hairless mouse ear model. Varying concentrations of Ketanserin (0%, 0.2%, 2.0%, 20% weight/volume) were administered to standardized full-thickness skin wounds on the dorsum of the hairless mouse ear immediately after surgery and daily thereafter. With the use of video microscopy and computer-assisted planimetry, vascularization and epithelialization were traced every third day until the wounds were fully healed. Arteriole diameters at selected sites near the skin wound were measured before wound creation and after wounding. It was concluded that topically administered ketanserin significantly accelerates both the vascular ( p < 0.001 at 2% and 20% concentrations) and epithelial ( p < 0.001 at 20% concentration) rates of wound healing in full-thickness nonpathologic skin wounds. Vasodilation of terminal arterioles was not a major response to Ketanserin. Faster epithelialization was possibly due to direct effect of ketanserin on epithelial cells.