Validation of a model for the study of multiple wounds in the diabetic mouse (db/db)

The genetically diabetic db/db mouse exhibits symptoms that resemble human type 2 diabetes mellitus, demonstrates delayed wound healing, and has been used extensively as a model to study the role of therapeutic topical reagents in wound healing. The purpose of the authors' study was to validate an excisional wound model using a 6-mm biopsy punch to create four full-thickness dorsal wounds on a single db/db mouse. Factors considered in developing the db/db wound model include reproducibility of size and shape of wounds, the effect of semiocclusive dressings, comparison with littermate controls (db/-), clinical versus histologic evidence of wound closure, and cross-contamination of wounds with topically applied reagents. The size of wounds was larger, with less variation in the db/db mice (31.11 +/- 3.76 mm2) versus db/- mice (23.64 +/- 4.78 mm2). Wounds on db/db mice that were covered with a semiocclusive dressing healed significantly more slowly (mean, 27.75 days) than wounds not covered with the dressing (mean, 13 days; p < 0.001), suggesting the dressings may splint the wounds open. As expected, wounds healed more slowly on db/db mice than db/- mice (covered wounds, 27.75 days versus 11.86 days, p < 0.001; wounds not covered, 13 days versus 11.75 days, p = 0.39). Covered wounds, thought to be closed by clinical examination, were confirmed closed by histology only 62 percent of the time in the db/db and 100 percent of the time in the db/- mice. Topical application of blue histologic dye or soluble biotinylated laminin 5 to one of the four wounds did not spread locally and contaminate adjacent wounds. Multiple, uniform, 6-mm wounds in db/db mice heal in a relatively short time, decrease the number of animals needed for each study, and allow each animal to serve as its own control. The db/db diabetic mouse appears to be an excellent model of delayed wound healing, particularly for studying factors related to epithelial migration.     

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.     

Skin repair and scar formation: the central role of TGF-beta

Wound healing is a complex process that we have only recently begun to understand. Central to wound repair is transforming growth factor beta (TGF-beta), a cytokine secreted by several different cell types involved in healing. TGF-beta has diverse effects, depending upon the tissue studied. This review focuses on healing in skin, particularly the phases of cutaneous wound repair and the role of TGF-beta in normal and impaired wound-healing models. It also explores TGF-beta activity in scarless foetal wound healing. Knowledge of TGF-beta function in scarless repair is critical to improving healing in clinical scenarios, such as diabetic wounds and hypertrophic scars.     

Healing of burns after treatment with 670-nanometer low-power laser light

Recent investigations have reported contradictory results on the influence of low-power laser light on wound healing. Low-power laser with a power output of 250 mW and an emitted laser light of 670 nm have been insufficiently investigated to date. The effect of a 250-mW/670-nm laser light on the healing of burning wounds in rats was investigated. Thirty rats were burned on both flanks. One wound was irradiated with 670-nm laser light (2 J/cm2), whereas the other side remained untreated. Macroscopic evaluation of the wounds was performed daily; 10, 20, and 30 days after burning, 10 rats were killed and the wounds histologically evaluated. Neither macroscopic nor histologic examination of the irradiated wound showed accelerated wound healing when compared with control wounds. In the present study, irradiation of burns with a 250-mW/670-nm laser light produced no beneficial effects on wound-healing processes.     

Curcumin treatment enhances the repair and regeneration of wounds in mice exposed to hemibody gamma-irradiation

Hemibody irradiation in multiple fractionated doses is frequently used for the treatment of various neoplastic disorders. It produces both acute and late effects on the skin and subcutaneous tissues that have profound implications in the healing of surgical wounds. Because of the crucial practical importance of hemibody radiation exposure associated with skin wounds, it is imperative to investigate the efficacy of cost-effective herbal products in the reconstruction of irradiated wounds. Therefore, the effect of pretreatment of curcumin was studied on the healing of excision wound in mice exposed to 2, 4, 6, or 8 Gy of hemibody gamma-radiation. A full-thickness skin wound was created by removing the skin flap of the dorsum of 8- to 10-week-old Swiss albino mice partially (lower half, below the rib cage) exposed to 2, 4, 6, or 8 Gy of gamma-radiation. The progression of wound contraction was monitored periodically by capturing video images of the wound, where the first image of each wound from different groups was obtained 1 day after wounding and that day was considered as day 0. Eight animals were used in each group at each exposure dose for wound contraction studies. Furthermore, the effect of curcumin on mean healing time after exposure of mice to 2, 4, 6, or 8Gy of hemibody gamma-radiation was also evaluated, where eight animals were used in each group at each exposure dose. Collagen, hexosamine, DNA, nitric oxide, and histologic profiles were also evaluated during the course of healing of excision wounds at days 4, 8, and 12 after irradiation treated or not with curcumin before exposure to 0 or 6 Gy of gamma-radiation. Six animals were used in each group at each interval for each biochemical parameter studied, except for histologic evaluations, where four animals were used in each group at each interval. Exposure of mice to different doses of gamma-radiation resulted in a dose-dependent delay in contraction and wound-healing time of excision wound, whereas curcumin pretreatment caused a significant elevation in the rate of wound contraction and a decrease in the mean wound-healing time. Treatment with curcumin before irradiation enhanced the synthesis of collagen, hexosamine, DNA, nitrite, and nitrate, and histologic assessment of wound biopsy specimens revealed improved collagen deposition and an increase in fibroblast and vascular densities. The authors' study demonstrates that curcumin pretreatment has a conducive effect on the irradiated wound and could be a substantial therapeutic strategy for ameliorating radiation-induced delay in wound repair in cases of radiation-induced skin injuries.     

Immediate closure of traumatic upper arm and forearm injuries with the latissimus dorsi island myocutaneous pedicle flap

This paper reports the authors' experience with latissimus dorsi island pedicle flaps in the acute treatment of massive arm injuries. Seven patients with upper arm injuries and four patients with forearm injuries were treated with latissimus dorsi pedicle flaps. All cases involved massive soft-tissue loss and open fractures. Primary healing of wounds occurred without complications in 10 of 11 patients; the eleventh developed a wound infection. There were no instances of flap loss or vascular complications. This report compares and discusses surgical management options and details the importance of robust, immediate soft-tissue coverage for optimal functional recovery. Contrary to traditional thought, delay in definitive wound closure may be unnecessary when aggressive debridement is followed by acute flap closure.     

Cutaneous wound healing in the sea cucumber, Thyone briareus

Wound healing in the integument of the sea cucumber, Thyone briareus, was studied for up to 50 days after inflicting wide excisional wounds and for 14 days after producing incisional wounds. Rapid re-epithelialization of the wound was effected by the migration of epidermal cells and pigment cells from the periphery of the wound margin. This occurred without apparent evidence of concomitant mitotic activity. Dermal wound healing was completed by the fourteenth day in the incision wounds but occurred very slowly in the broad excision wounds. Morula cells seem to be involved in both epidermal and dermal wound healing, although their precise role is unknown. In excisional wounds the integument was never completely restored to its normal appearance during 50 days of observation.     

Defective macrophage function in wound repair of P/J mice

The strength of healing full-thickness incised dermal wounds in P/J mice was less than that of CD-1 mice although the strength of intact skin was similar for each strain. Five days after surgery, P/J mice had wounds with tensile strengths of 65 +/- 18g while CD-1 mice had wounds with strengths of 85 +/- 15g. The wound breaking strength of P/J mice was restored to normal values (86 +/- 18g) by administering glucan. The consequence of defective monocytes in wound repair is discussed in reference to P/J mice.     

Full-thickness human foreskin transplantation onto nude rats as an in vivo model of acute human wound healing

Current wound-healing models do not fully duplicate the in vivo human environment. The feasibility of grafting human full-thickness foreskin onto nude rats, as a model of acute wound healing, was evaluated. Incisions were then created on the grafted skin, and wound healing was evaluated. Full-thickness human skin was obtained after elective circumcision and was grafted subcutaneously onto the dorsal thorax of nude rats. At 10 days after transplantation, graft beds were judged for graft viability, on the basis of gross appearance, texture, and adherence. Full-thickness wounds were then made in the foreskin. Graft wounds were left to close by secondary intention. The wounds were allowed to heal for 7 days. Wounds were excised and tested for breaking stress. Histological evaluations included proliferating cell nuclear antigen, factor VIII, hematoxylin and eosin, and trichrome staining. Twenty grafts were performed, with 100 percent viability. Upon incision, all grafts bled freely, indicating a rich vascular supply and tissue viability. Graft viability was confirmed by the presence of proliferating cells in the parabasal stratum of the epithelium. Furthermore, there was evidence of angiogenesis, as confirmed by staining for factor VIII. Breaking stress was evaluated by tensiometry, 7 days after wounding. Histological evaluations revealed viable grafts and active wound-healing events. Full-thickness human skin can be successfully transplanted onto nude rats, providing a larger, more physiological model of human wound healing. This model closely parallels the in vivo situation, providing a promising model for study of the complex biological processes of acute human wound healing, in a reproducible manner.     

The Effects of Sericin Cream on Wound Healing in Rats

Sericin has good hydrophilic properties, compatibility, and biodegradation, it can be used as a wound-healing agent. We evaluated the effects of sericin on wound healing and wound size reduction using rats by generating two full-thickness skin wounds on the dorsum. Group 1 animals were treated with Betadine^® on left-side (control) wounds and, with 8% sericin cream on right-side (treated) wounds. Group 2, cream base (formula control) and 8% sericin cream (treated) were topically applied to left-, and right-side wounds respectively. Sericin-treated wounds had much smaller inflammatory reactions, and wound-size reduction was much greater than in the control throughout the inspection period. Mean time in days for 90% healing from sericin-treated wounds was also much less than for cream base-treated wounds. Histological examination after 15 d of treatment with 8% sericin cream revealed complete healing, no ulceration, and an increase in collagen as compared to cream base-treated wounds, which showed some ulceration and acute inflammatory exudative materials.     

The effects of sericin cream on wound healing in rats

Sericin has good hydrophilic properties, compatibility, and biodegradation, it can be used as a wound-healing agent. We evaluated the effects of sericin on wound healing and wound size reduction using rats by generating two full-thickness skin wounds on the dorsum. Group 1 animals were treated with Betadine on left-side (control) wounds and, with 8% sericin cream on right-side (treated) wounds. Group 2, cream base (formula control) and 8% sericin cream (treated) were topically applied to left-, and right-side wounds respectively. Sericin-treated wounds had much smaller inflammatory reactions, and wound-size reduction was much greater than in the control throughout the inspection period. Mean time in days for 90% healing from sericin-treated wounds was also much less than for cream base-treated wounds. Histological examination after 15 d of treatment with 8% sericin cream revealed complete healing, no ulceration, and an increase in collagen as compared to cream base-treated wounds, which showed some ulceration and acute inflammatory exudative materials.     

Angiogenesis Is Induced and Wound Size Is Reduced by Electrical Stimulation in an Acute Wound Healing Model in Human Skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.     

Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces.

The efficacy of electrical fields in soft-tissue repair is unclear. Materials with a charged surface provide a localized charged environment. We examined the effects of surface-charged particles in wound healing in rats with paired dorsal incisions with one side serving as a control. Tensiometry demonstrated that after 10 days, wounds with positively charged particles were 53 percent stronger (p less than 0.001) than controls (10 rats, 30 wound strips), whereas differences with negatively charged (6 rats, 15 strips) or uncharged beads (11 rats, 33 strips) were insignificant. Histologically, wounds with positively charged particles were characterized by large quantities of collagen-rich connective tissue and by prominent bead-associated giant cells. At 94 days, no differences in wound strength were noted. This method of creating charged local environments has potential clinical implications and may add insights into the behavior of cells in response to charged stimuli.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ?mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.     

Intrinsic electric fields promote epithelization of wounds in the newt, Notophthalmus viridescens

The lateral electric fields (LFs) in the vicinity of small wounds made in hindlimb digit tip skin of Notophthalmus viridescens have been measured and manipulated. Healing of these wounds was assessed by crystal violet staining and by histology. Paired experiments were conducted on the animals: the healing of one digit's wound was compared with healing of the contralateral digit's wound whose LF was changed from that of its contralateral wound. When currents were passed through the animal (into one wound and out of the contralateral wound) so that the LF of one wound was zero while the contralateral wound had an enhanced LF, the wounds with the enhanced LF healed more rapidly than the wounds with the zero LF. When digits on one side were treated with 30 microM benzamil in an artificial pond water so that their wound LFs were reduced to approximately zero, and the contralateral wounds were kept in artificial pond water without benzamil so that they had normal wound LFs, there was significantly less epithelization of the benzamil-treated wounds than of the control wounds. This effect on wound healing was reversed by adding currents that restored the normal wound LFs, but not by adding currents that reversed the wound LFs to the opposite polarity. When currents were added to reverse the wound LFs on one side of the animal, leaving the contralateral wounds free of added currents, the wounds with the reversed LFs healed more poorly than the contralateral wounds with normal LFs. These results are consistent with the hypothesis that the intrinsic LFs in the vicinity of wounds promote epithelization of these wounds.     

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.