Shengxian Jia

Fibrin sealant combined with fibroblasts and platelet‐derived growth factor enhance wound healing in excisional wounds

We test the hypothesis that the fibrinogen‐thrombin formulation of fibrin sealant combined with fibroblasts and PDGF‐BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full‐thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 × 105 cells/wound) embedded in the fibrinogen component. At post‐wounding day 7, there was no difference in the diluted vs. non‐diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF‐BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two‐fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.

Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.

A number of in vivo and ex vivo skin models have been applied to human wound healing studies. A reliable skin model, which recapitulates the features of human wound repair, is essential for the clinical and mechanical investigation of human cutaneous wound healing. Full-skin ex vivo culture systems have been used in wound healing studies. However, important structures of the skin, such as the differentiation of keratinocytes and epidermis-dermis junction, are poorly characterized in this model. This study aims to develop an optimized partial-thickness human ex vivo skin culture (HESC) model to maintain human skin characteristics in vitro. During our culture, the basal layer, suprabasal layer, and stratum granulosum layer of epidermis were preserved until day 8. Analyses of hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) and 2 (BP230), showed that the integrity of the basement membrane of the epidermis was well preserved in the HESC model. In contrast, an organotypic culture with human keratinocytes and fibroblasts failed to show an integrated basement membrane. Maintenance of skin structure by histological analysis and proliferation of epidermal keratinocytes by Ki67 staining were observed in our model for 12 days. Complete re-epithelialization of the wounding area was observed at day 6 post wounding when a superficial incisional wound was created. The expression of Ki-67 and keratin 6, indicators of activated keratinocytes in epidermis, was significantly upregulated and new collagen synthesis was found in the dermis during the wound healing process. As control, we also used organotypic culture in studying the differentiation of the keratinocyte layers and incisional wound repair. It turned out that our model has advantage in these study fields. The results suggest that our HESC model retains important elements of in vivo skin and has significant advantages for the wound healing studies in vitro.

Sodium channel Nax is a regulator in epithelial sodium homeostasis.

Blocking Nax expression improves scarring and atopic dermatitis–like symptoms by improving barrier function. Rubbing sodium in a wound The skin serves as a critical barrier to the outside world; however, little is known about how this barrier returns to homeostasis after it is disturbed. Water loss occurs during many skin disorders, resulting in an increase in extracellular sodium concentration. Now, Xu et al. report that the sodium channel Nax functions as a sodium sensor that contributes to epithelial homeostasis. Nax, which is present in multiple epithelial tissues and up-regulated in scars, increases sodium flux and induces the downstream production of mediators of epithelial cell proliferation and inflammation that may lead to scar formation. Indeed, blocking Nax in animal models decreases scarring and atopic dermatitis–like symptoms, suggesting that Nax may contribute to epithelial homeostasis. The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a perturbation of the epidermal barrier, water is lost, resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as a sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin, which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. Nax is present in multiple epithelial tissues, and up-regulation of its downstream genes is found in hypertrophic scars. In animal models, blocking Nax expression results in improvement in scarring and atopic dermatitis–like symptoms, both of which are pathological conditions characterized by perturbations in barrier function. These findings support an important role for Nax in maintaining epithelial homeostasis.

Hydration Status Regulates Sodium Flux and Inflammatory Pathways through Epithelial Sodium Channel (ENaC) in the Skin

Although it is known that the inflammatory response that results from disruption of epithelial barrier function after injury results in excessive scarring, the upstream signals remain unknown. It has also been observed that epithelial disruption results in reduced hydration status and that the use of occlusive dressings that prevent water loss from wounds decreases scar formation. We hypothesized that hydration status changes sodium homeostasis and induces sodium flux in keratinocytes, which result in activation of pathways responsible for keratinocyte-fibroblast signaling and ultimately lead to activation of fibroblasts. Here, we demonstrate that perturbations in epithelial barrier function lead to increased sodium flux in keratinocytes. We identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaCs) and causes increased secretion of proinflammatory cytokines, which activate fibroblast via the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increased sodium concentration, which simulates reduced hydration, in the media in epithelial cultures or human ex vivo skin cultures. Blockade of ENaC, prostaglandin synthesis, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, employing a validated in vivo excessive scar model in the rabbit ear, we demonstrate that utilization of either an ENaC blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrate that the activation of COX-2 in response to increased sodium flux is mediated through the PIK3/Akt pathway. Our results indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators and suggest that the ENaC pathway is a potential target for a strategy to prevent fibrosis.

The Expression of Proinflammatory Genes in Epidermal Keratinocytes Is Regulated by Hydration Status

Mucosal wounds heal more rapidly, exhibit less inflammation, and are associated with minimal scarring when compared with equivalent cutaneous wounds. We previously demonstrated that cutaneous epithelium exhibits an exaggerated response to injury compared with mucosal epithelium. We hypothesized that treatment of injured skin with a semiocclusive dressing preserves the hydration of the skin and results in a wound healing phenotype that more closely resembles that of mucosa. Here we explored whether changes in hydration status alter epidermal gene expression patterns in rabbit partial-thickness incisional wounds. Using microarray studies on injured epidermis, we showed that global gene expression patterns in highly occluded versus non-occluded wounds are distinct. Many genes including IL-1β, IL-8, TNF-α (tumor necrosis factor-α), and COX-2 (cyclooxygenase 2) are upregulated in non-occluded wounds compared with highly occluded wounds. In addition, decreased levels of hydration resulted in an increased expression of proinflammatory genes in human ex vivo skin culture (HESC) and stratified keratinocytes. Hierarchical analysis of genes using RNA interference showed that both TNF-α and IL-1β regulate the expression of IL-8 through independent pathways in response to reduced hydration. Furthermore, both gene knockdown and pharmacological inhibition studies showed that COX-2 mediates the TNF-α/IL-8 pathway by increasing the production of prostaglandin E2 (PGE2). IL-8 in turn controls the production of matrix metalloproteinase-9 in keratinocytes. Our data show that hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing.

Intravenous curcumin efficacy on healing and scar formation in rabbit ear wounds under nonischemic, ischemic, and ischemia–reperfusion conditions

Curcumin, a spice found in turmeric, is widely used in alternative medicine for its purported anti‐inflammatory and antioxidant activities. The goal of this study was to test the curcumin efficacy on rabbit ear wounds under nonischemic, ischemic, and ischemia–reperfusion conditions. Previously described models were utilized in 58 New Zealand White rabbits. Immediately before wounding, rabbits were given intravenous crude or pure curcumin (6 μg/kg, 30 μg/kg, or 60 μg/kg) dissolved in 1% ethanol. Specimens were collected at 7–8 days to evaluate the effects on wound healing and at 28 days to evaluate the effects on hypertrophic scarring. Students t test was applied to screen difference between any treatment and control group, whereas analysis of variance was applied to further analyze for all treatment groups in aggregate in some specific experiments. Treatment with crude curcumin suggested accelerated wound healing that reached significance for reepithelialization in lower and medium doses and granulation tissue formation in lower dose. Purified curcumin became available and was used for all later experiments. Treatment with pure curcumin suggested accelerated wound healing that reached significance for reepithelialization in lower and medium doses and granulation tissue formation in lower dose. Treatment with pure curcumin significantly promoted nonischemic wound healing in a dose–response fashion compared with controls as judged by increased reepithelialization and granulation tissue formation. Improved wound healing was associated with significant decreases in pro‐inflammatory cytokines interleukin (IL)‐1 and IL‐6 as well as the chemokine IL‐8. Curcumin also significantly reduced hypertrophic scarring. The effects of curcumin were examined under conditions of impaired healing including ischemic and ischemia–reperfusion wound healing, and beneficial effects were also seen, although the dose response was less clear. Systemically administrated pure curcumin significantly promotes nonischemic wound healing and reduces hypertrophic scarring. Improvements in wound healing were associated with decreased inflammatory markers in wounds. Further study is needed to optimize dosing in ischemic and ischemia–reperfusion wound healing. In aggregate, the studies strongly support the systemic administration of curcumin to improve wound healing.

Alteration of Smad3 signaling in ischemic rabbit dermal ulcer wounds

Impaired reepithelialization is a hallmark of chronic, ischemic wounds; however, the pathogenesis of the delayed reepithelialization in these wounds remains poorly understood. Transforming growth factor β is involved in both the normal and hypoxic wound‐healing response and exogenous overexpression of Smad3, which has been known to accelerate reepithelialization. Recently, it was shown in the rabbit ear dermal ulcer model that Ad‐Smad3 injection enhanced reepithelialization and granulation tissue formation suggesting a positive effect of Smad3 on wound healing. However, little is known about the role of Smad3 in the ischemic wound healing process. In this study, we examined the effect of Smad3 in an ischemic wound model. Ad‐Smad3 or Ad‐LacZ (108 pfu/wound) was injected into either ear of white New Zealand rabbits. Twenty‐four hours later, these ears were rendered ischemic using an established model, and four 7 mm full‐thickness punch wounds were made on each ear. Histological evaluation showed a significant increase in reepithelialization parameters in Ad‐Smad3–transfected wounds (p<0.01). In contrast, granulation tissue parameters were not affected by Smad3 in ischemia. Smad4 and Smad7 mRNA‐expression was not affected by Smad3 overexpression. Connective tissue growth factor protein was up‐regulated under ischemic conditions but was unaffected by Smad3 transfection in both ischemic and nonischemic wounds. Our results suggest an enhancing effect of Smad3 on reepithelialization in an ischemic wound model that, in turn, might provide novel therapeutic options. Furthermore, the lack of alteration of Smad‐dependent intermediates by Smad3 overexpression suggests the activation of Smad‐independent pathways in ischemia.

The effects of topically applied silicone gel and its silver derivative on the prevention of hypertrophic scarring in two rabbit ear-scarring models.

Topically applied silicone gel is an effective treatment in the management of hypertrophic scars. This early study of silicone gel predates other well-controlled scientific studies that demonstrate these findings. Our well-established rabbit ear scarring model creates 7 mm punch wounds down to the bare cartilage, including the removal of the perichondrium. In this study, we employ a new model that creates 10 mm punch wounds that leaves the perichondrium intact. Both the 7 mm and new 10 mm scar models were used to study the effectiveness of silicone gel and silicone gel silver respectively on hypertrophy and the inhibition of scar formation. All samples were harvested at post-wounding day 35 for histological analysis. Silicone gel significantly reduced scar area (p=0.005), scar elevation index (p=0.03), and epidermal area (p=0.016). Silicone gel silver significantly reduced scar elevation index (p=0.004). The new 10 mm scar model resulted in more hypertrophic scarring than the typical, 7 mm wound scar model (p=0.0001). In conclusion, silicone gel and its silver derivative are effective in preventing hypertrophic scarring and scar models that leave the perichondrium intact causes scars with more hypertrophy.

Systemic administration of hemoglobin improves ischemic wound healing

BACKGROUND Oxygen plays multifaceted roles in wound healing, including effects on cell proliferation, collagen synthesis, angiogenesis, and bacterial killing. Oxygen deficit is a major factor in the pathogenesis of chronic wounds. MATERIALS AND METHODS We present a novel mechanism for oxygen delivery to ischemic wounds by systemic administration of an oxygen carrier substitute derived from bovine hemoglobin (IKOR 2084) in our ischemic rabbit ear wound model. The wound healing indexes, including epithelial gap and neo-granulation tissue area, were histologically analyzed. In situ expression of endothelial cells (CD31+) and proliferative cells (Ki-67+) were examined by immunohistochemistry analysis. The messenger RNA expression of collagen I, III, and vascular endothelial growth factor was measured by quantitative RT-PCR. Sirius Red staining was implemented for detection of collagen deposition, and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis was performed to examine dermal cellular apoptosis. RESULTS Systemic administration of IKOR 2084 significantly improved oxygen tension of ischemic tissue. When compared with saline controls, IKOR 2084 treatment enhanced wound repair as demonstrated by a reduced epithelial gap and increased granulation tissue area. The expression of Ki-67+, CD31+, vascular endothelial growth factor and collagen was also enhanced by IKOR 2084 administration. Moreover, apoptosis analysis in the wounds showed that cell survival in the dermis was increased by systemic IKOR 2084 administration. CONCLUSIONS Our study suggests that systemic delivery of IKOR 2084 ameliorates hypoxic state, subsequently promotes angiogenesis, cellular proliferation, and collagen synthesis, attenuates hypoxia-induced apoptosis, and improved ischemic wound healing.

Evaluating the effects of subclinical, cyclic ischemia-reperfusion injury on wound healing using a novel device in the rabbit ear

ObjectiveThis study aimed to evaluate the effect of cyclic ischemia-reperfusion (IR) injury on wound healing using a novel rabbit ear model. Materials and MethodsA lightweight clamp apparatus was developed for reversible occlusion of the central ear artery. Ventral ear wounds were analyzed postoperatively for epithelialization and granulation as well as gene expression after 3 consecutive days of IR cycling. ResultsBy postoperative day #7, ears showed no gross tissue necrosis, but histologic analysis of wounds confirmed a significant impairment in epithelial and granulation tissue gaps as well as total epithelial and granulation tissue areas (P < 0.001). Quantitative polymerase chain reaction analysis of IR wounds indicated significant up-regulation of heat shock protein-70 and down-regulation of superoxide dismutase 1 relative to sham controls (P < 0.05). ConclusionsA novel rabbit ear model for the induction of subclinical, cyclic IR injury in cutaneous tissue has been developed that will serve as a valuable tool for the testing of new therapeutics.