Enhancing sensitivity of lateral flow assay with application to SARS-CoV-2.

Lateral flow assay (LFA) has long been used as a biomarker detection technique. It has advantages such as low cost, rapid readout, portability, and ease of use. However, its qualitative readout process and lack of sensitivity are limiting factors. We report a photon-counting approach to accurately quantify LFAs while enhancing sensitivity. In particular, we demonstrate that the density of SARS-CoV-2 antibodies can be quantified and measured with an enhanced sensitivity using this simple laser optical analysis.

Tranexamic acid and trauma: current status and knowledge gaps with recommended research priorities.

A recent large civilian randomized controlled trial on the use of tranexamic acid (TXA) for trauma reported important survival benefits. Subsequently, successful use of TXA for combat casualties in Afghanistan was also reported. As a result of these promising studies, there has been growing interest in the use of TXA for trauma. Potential adverse effects of TXA have also been reported. A US Department of Defense committee conducted a review and assessment of knowledge gaps and research requirements regarding the use of TXA for the treatment of casualties that have experienced traumatic hemorrhage. We present identified knowledge gaps and associated research priorities. We believe that important knowledge gaps exist and that a targeted, prioritized research effort will contribute to the refinement of practice guidelines over time.

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 x 10(5) 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.

Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms.

Regulating collagen metabolism can control hypertrophic scars in cutaneous wounds. Hypertrophic scars can be reduced by occlusive dressings such as silicone sheeting; however, their mechanism is still unknown. We hypothesized that hydration of keratinocytes reduces the collagen secretion of fibroblasts by modifying the cytokine levels. Stratified human epidermal keratinocytes and confluent human dermal fibroblasts were co-cultured serum free for 72 hours. Keratinocytes were either kept at the air interface or hydrated. Messenger RNA (mRNA) levels of interleukin-1 (IL-1)alpha, IL-1beta, tumor necrosis factor alpha (TNF-alpha), keratinocyte growth factor (KGF), and procollagen-1 were analyzed by real-time reverse transcription-polymerase chain reaction. Secretion of cytokines into conditioned media was quantified by enzyme-linked immunosorbent assay and collagen content by Western blot. The content of collagen-I decreased by 44% in the presence of hydrated keratinocytes. Co-culture with air-treated keratinocytes decreased collagen-I only by 23%. Co-cultured hydrated keratinocytes had significantly higher TNF-alpha mRNA (172%) than hydrated keratinocytes. At the protein level, there was an overall trend toward increased TNF-alpha levels in hydrated cultures. IL-1beta secretion decreased significantly under hydration (42% monoculture, 58% co-culture). Co-culture stimulated a 240% increase of KGF mRNA in fibroblasts compared with monocultured fibroblasts. Fibroblasts secreted 4.5-fold more KGF in hydrated co-cultures and sixfold more KGF in air-treated co-cultures. Hydration of keratinocytes modifies important paracrine interactions between keratinocytes and fibroblasts and reduces collagen-1, which supports the hypothesis that hydration of the epidermis and restoration of water barrier function play an important role in scar formation.

Delayed wound healing in Mac-1-deficient mice is associated with normal monocyte recruitment.

The Mac-1 integrin is an important mediator of migration and inflammatory activation of neutrophils and monocytes. However, the role of Mac-1 in modulating macrophage emigration and activation and its subsequent impact on cutaneous wound healing have not been fully elucidated. To examine the significance of Mac-1 to murine wound healing, we measured epithelialization and granulation tissue formation in partial-thickness ear wounds and full-thickness head wounds, respectively, in Mac-1-deficient mice. Wounds were histologically analyzed at postwounding days 3, 5, and 7. The gap measured between the leading edges of inward-migrating granulation tissue was significantly increased in knockout mice compared with control animals at day 5 (3.8+/-0.3 vs. 2.6+/-0.5 mm; p<0.001) and day 7 (2.2+/-0.4 vs. 0.96+/-0.73 mm; p=0.005). Epithelial gap measurements were also increased in knockout mice vs. wild-type controls at days 3 (0.62+/-0.02 vs. 0.54+/-0.07 mm; p<0.05) and 5 (0.58+/-0.06 vs. 0.39+/-0.08 mm; p<0.001). Immunohistochemistry showed equal numbers of macrophages in knockout and control wounds. These findings show that Mac-1 is required for normal wound healing but that the attenuation in the deposition of granulation tissue and wound epithelialization in Mac-1 knockout mice is not associated with decreased monocyte migration into the wound.

Reduction of hypertrophic scar via retroviral delivery of a dominant negative TGF-beta receptor II.

Effective blockade of the pluripotent cytokine transforming growth factor (TGF)-beta as a means of cutaneous scar reduction is a strategy with great potential. This desired effect may be achieved through the overexpression of mutant TGF-beta receptors within the wound milieu. Our goal was to examine the effects of dominant negative mutant TGF-beta receptor II (TGFbetaRIIdn) protein expression in a well-established rabbit ear model of hypertrophic scarring. Serial injections of a retroviral construct encoding a truncated TGFbetaRII and the marker green fusion protein (pMSCV-rIIdn-GFP) were performed in 7mm punch wounds at day 10 and day 12 (two-day injection group) or days 8, 10, 12 (three-day injection group) post-wounding. Delivery of an empty vector (pMSCV-GFP) at the same time points served as a negative control. Histomorphometric analysis of wounds harvested at day 28 revealed a modest, though statistically significant reduction (20%, p=0.027) in the scar elevation index (SEI) in two-day treated and a more modest reduction in SEI (12%) in the three-day treated arm compared to null-treated controls. Confocal microscopy confirmed stable, yet variable transfection of the construct in both peri-wound tissue as well as rabbit dermal fibroblasts transfected in vitro. Optimisation of this novel application in retroviral gene therapy could lead to effective anti-scarring strategies.

Adenoviral human telomerase reverse transcriptase dramatically improves ischemic wound healing without detrimental immune response in an aged rabbit model.

Chronic ischemic wounds are major clinical problems, and are especially prevalent in elderly patients. Management of these wounds costs billions of dollars annually in the United States. Because of the severe impairment in tissue repair, ischemic wounds among the aged are major challenges for physicians. For example, transforming growth factor-beta1 stimulates healing of young patients' ischemic wounds, but it is totally ineffective in treating the ischemic wounds of aged patients. Therefore, our goal is to develop a better therapeutic strategy for elderly patient ischemic wounds. Because human telomerase reverse transcriptase (hTERT) has emerged as having a role in promoting cell proliferation, we hypothesized that hTERT overexpression may improve ischemic wound healing in the elderly. We successfully tested this hypothesis by demonstrating for the first time that gene delivery of hTERT by adenovirus (Ad-hTERT) dramatically improved ischemic wound healing in an aged rabbit model. Importantly, our histological data indicate that no deleterious immune response was induced in the aged rabbits. This finding has broad implications for the field of gene therapy because the foremost obstacle in the use of adenoviral vectors for gene therapy is that they provoke strong innate and adaptive immune responses in the host. Moreover, Ad-hTERT significantly improved survival of primary rabbit dermal fibroblasts that were treated with hypoxia and hydrogen peroxide (oxidative stress). This model is clinically relevant because it simulates the ischemia cycle of an ischemia-reperfusion injury, which can lead to stroke, myocardial infarction, and other tissue injuries. We conclude that Ad-hTERT is an effective and novel approach to treating the ischemic wounds of elderly patients.

Inhibition of procollagen C-proteinase reduces scar hypertrophy in a rabbit model of cutaneous scarring.

Hypertrophic scarring, which results from excessive collagen deposition at sites of dermal wound repair, can be functionally and cosmetically debilitating to the surgical patient. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. One of the key steps in collagen stabilization is the cleavage of the C-terminal propeptide from the precursor molecule to form collagen fibrils, a reaction catalyzed by procollagen C-proteinase (PCP). We tested the ability of a PCP inhibitor to reduce hypertrophic scar formation in a rabbit ear model. After the placement of four, 7-mm dermal wounds on each ear, New Zealand white rabbits received PCP inhibitor subcutaneously in the left ear at four time points postwounding: days 7, 9, 11, 13 (early treatment; n=20 wounds) or days 11, 13, 15, 17 (late treatment; n=20 wounds). The right ear of each animal served as a control (vehicle alone). Wounds were harvested on postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. Early treatment of wounds with PCP inhibitor did not reduce scar formation compared with controls (p>0.05). However, late treatment resulted in a statistically significant reduction in the scar elevation index (p<0.01). Our results point not only to the potential use of PCP inhibitors to mitigate hypertrophic scarring but also to the temporal importance of drug delivery for antiscarring therapy.

Age effect on HSP70: decreased resistance to ischemic and oxidative stress in HDF.

BACKGROUND: Heat pre-conditioning results in induction of heat shock proteins including HSP70 that gives a cytoprotective effect against further stress. However, HSP70 induction is attenuated in aged cells. The lower HSP70-levels may contribute to the impaired stress response seen in the aged, and to the higher rates of chronic wounds in aged, which arise from repeated ischemia-reperfusion injury. The aim of this study was to investigate a possible connection by comparing the viability of heat pre-conditioned aged versus young human dermal fibroblasts (HDF) after exposure to stress. MATERIALS AND METHODS: Young (15-28) and aged (61-77) HDF were heat pre-conditioned (42 degrees C, 1 h) and after recovery (1, 2, or 20 h) treated with carbonyl-cyanide-m-chlorophenylhydrazone (hypoxic stress) or with hydrogen peroxide (oxidative stress) for 1 h. HSP70 levels were determined by Western blot. Cell damage was assessed by quantifying lactic dehydrogenase (LDH) in conditioned media. Aged HDF were transfected with HSP70-plasmid, consecutively heat pre-conditioned and exposed to oxidative stress. RESULTS: HSP70 increased in heat pre-conditioned young HDF by 96, 189, and 237% after 1, 2, and 20-h recovery, respectively, and in aged HDF by 27, 61, and 26%. LDH-release was only decreased in young HDF 20-h after heat-treatment compared with non-heat treated cells (P < 0.001). HSP70-transfection of aged HDF with plasmid reduced LDH-release by 29%. CONCLUSIONS: Heat pre-conditioning fails to protect aged HDF to oxidative or hypoxic stress due in part to impaired HSP70 induction compared to young.

Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence.

A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed "replicative senescence." Alternatively, oxidative stress in the form of hydrogen peroxide (H(2)O(2)) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress-induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro-survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor-beta1 had a pronounced "rescue effect" in the aged, preventing entrance of HDFs into cellular senescence.

Transdermal sustained-delivery oxygen improves epithelial healing in a rabbit ear wound model.

HYPOTHESIS: Transdermal sustained-delivery oxygen therapy improves wound healing. DESIGN: Experimental study using a well-established rabbit ear model for acute wound healing. SETTING: Wound-healing research laboratory in a university center. METHODS: Four full-thickness 7-mm punch wounds were created on each ear of young, female New Zealand white rabbits. Treated ears received transdermal sustained delivery of oxygen via silicone tubing tunneled subcutaneously to a pocket under a semiocclusive dressing. Oxygen production (100% oxygen at 3 mL/h continuously) relied on a small, self-contained device connected to the silicone tubing and secured to the rabbit's back for the duration of the experiment using a body harness. Ears were harvested at each of 2 time points: day 5 and day 8. RESULTS: Histologic analysis of the wounds showed significantly greater healing at both day 5 and day 8 in response to oxygen therapy. Most significantly, epithelial wound coverage was almost doubled in treated ear wounds when compared with controls. CONCLUSION: Our results suggest that epithelial wound healing is improved by transdermal sustained-delivery treatment with 100% oxygen.

The future of wound healing: pursuing surgical models in transgenic and knockout mice.

BACKGROUND: Transgenic and knockout technologies have made determination of the molecular basis of wound healing possible. But there is no comprehensive or standardized approach to the investigation of wound healing in the mouse. A convention is proposed for assessing the multiple dimensions of wound healing. An approach to phenotyping a transgenic or knockout animal in a reproducible fashion is presented using this convention. STUDY DESIGN: Age- and gender-matched wildtype and knockout mice were characterized using six parameters of wound healing: epithelialization, granulation tissue formation, contraction, tensile strength, angiogenesis, and response to ischemia. Six surgical (four standard and two impaired) models were designed and used to quantitate these parameters. These models can be combined to efficiently maximize the data from any given subject. RESULTS: Each model leads to a rapid yield of results, with an average turnover of 4.9 days (range 3 to 7 days), and morbidity and mortality were minimal. A combinatorial approach elucidates the precise wound repair deficit of any subject. A case example is presented. CONCLUSIONS: Six surgical models investigating pertinent wound healing parameters are available. A factorial approach of quantitative wound healing assays maximizes data gathered from any one animal, minimizing the number of transgenic and knockout subjects needed; finely dissects molecular pathways of wound healing; and rapidly phenotypes a particular genetically altered mouse. We propose a standardized approach to wound healing assays that will elucidate critical cellular and molecular mechanisms and potential therapies.

Fibulin-5 promotes wound healing in vivo.

BACKGROUND: Fibulin-5 is a recently discovered multifunctional extracellular matrix protein that mediates endothelial cell adhesion through integrin ligation, regulates cell growth and motility in a context-specific manner, and prevents elastinopathy in vivo. Because fibulin-5 expression is induced dramatically in endothelial and smooth muscle cells in response to mechanical injury, my colleagues and I studied its role in dermal wound healing. STUDY DESIGN: We have used a gene therapy approach that used retroviral gene transfer to deliver fibulin-5 to the dermal wound milieu. Surgical generation of rabbit ear full-thickness dermal ulcers was performed in six female New Zealand white rabbits (6 months old). Wounds were injected with retrovirus containing either sense or antisense fibulin-5 or control vectors. Wounds were harvested on postwounding day 8 and analyzed by confocal microscopy, in situ hybridization, and histology. RESULTS: We report that fibulin-5 promotes wound healing in vivo. Wounds infected with fibulin-5 showed a considerable net increase (approximately 50%) in both newly formed granulation tissue volume and wound closure. Fibulin-5 expression stimulated substantial expression of collagen in dermal wounds. CONCLUSIONS: Taken together, our findings provide the first known example of overexpression of one extracellular matrix protein (fibulin-5) enhancing expression of another (type I collagen) in vivo. Our findings also demonstrate a novel role for fibulin-5 and suggest that altering extracellular matrix protein production through gene therapy may provide a novel means to promote wound healing.

Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing.

BACKGROUND: Tissue ischemia and aging are independent features associated with the healing impairment of cutaneous wounds. However, the pathophysiology of these processes as they relate to impaired-healing wounds is poorly understood. MATERIALS AND METHODS: A single full-thickness biopsy wound was made on both ears of young (3-6 month) and aged (>24 month) Fisher rats. One ear was rendered ischemic by transection of the vasculature at the ear base, while the other ear served as an internal nonischemic control. Wounds were harvested from 3 to 7 days and were evaluated histologically for either granulation tissue formation and epithelialization. Total RNA from wounds harvested at postoperative day 7 was probed using a nylon-based cDNA array to assess global genetic expression alterations. RESULTS: Healing in the rat ear model is impaired by both ischemia and advanced age as measured by granulation tissue formation and wound epithelialization. Granulation tissue formation was affected to a greater degree by ischemia than age (-58% versus -21%, respectively) while epithelialization displayed an opposite response (-17% versus -53%, respectively). Global analysis of gene expression suggests that ischemia engenders a marked increase in genes displaying altered expression in aged animals compared to young animals. Importantly, all possible alterations in gene expression are found in samples from aged ischemic wounds, indicating that gene regulation is not simply depressed by advanced age. CONCLUSIONS: Wound epithelialization appears to be affected to a greater degree by advanced age than by ischemia. The results demonstrate the distinctive phenotype presented by the clinically relevant combination of age and ischemia in an in vivo model of cutaneous wound healing.

Platelet-derived growth factor B, but not fibroblast growth factor 2, plasmid DNA improves survival of ischemic myocutaneous flaps.

HYPOTHESIS: Tissue flaps are commonly used for surgical reconstruction, especially to cover difficult wounds and in breast reconstruction following mastectomy. Complications due to inadequate flap perfusion are a source of morbidity and, in the lower extremity, can result in amputation. SETTING: Laboratory. INTERVENTIONS: We evaluated the ability of platelet-derived growth factor (PDGF) B and fibroblast growth factor 2 plasmid DNA, formulated in a type I collagen matrix, to promote tissue survival in a rat transverse rectus abdominis muscle flap model based on the inferior deep epigastric vascular supply. In the absence of any therapeutic agent, only about 24% of flap tissue survives in this model. The DNA/matrix formulations were delivered subcutaneously into the skin paddles 7 days before flap elevation, and tissues were harvested 7 days later. RESULTS: Our studies reveal dramatic increases in overall vascularity after treatment with PDGF-B and fibroblast growth factor 2 plasmid DNA; however, only PDGF-B increased flap survival (130% increase at 228 micro g/cm(2) of plasmid DNA vs controls; P<.01). Transdermal spectral imaging demonstrated an increase in patent vessels supporting blood flow in flaps treated with PDGF-B plasmid DNA vs the fibroblast growth factor 2 transgene. CONCLUSION: Matrix-enabled gene therapy may provide an effective nonsurgical approach for promoting flap survival and is well suited for surgical applications in which transient therapeutic transgene expression is desired.

A novel murine model of cyclical cutaneous ischemia-reperfusion injury.

BACKGROUND: Increasing evidence points to a principal role of ischemia-reperfusion in the pathogenesis of chronic skin ulceration, including pressure sores, diabetic ulcers, and venous ulcers. An incomplete understanding of this process and the limitations of current animal models of chronic wounds mandate a reproducible model in mice, in which transgenic and knockout technology are continually evolving. MATERIALS AND METHODS: A murine model of chronic skin ulceration based on cyclical magnetic compression is presented. Forty-three C57BL/6J mice underwent varying degrees of cyclical compression with defined periods of reperfusion. Injury was measured grossly as regional necrosis, and tissue was harvested for histology, DNA electrophoresis, and reverse transcription polymerase chain reaction. RESULTS: Skin necrosis became apparent only 12 h post cycling, and was cycle-responsive and quantitative in cycled subjects. Histopathologic analysis revealed a statistically significant doubling of the leukocyte count in sections from compressed skin versus sham controls. Moreover, apoptotic DNA laddering was evident in post ischemic skin and absent in controls. Real-time PCR analysis revealed a 300-fold higher expression in iNOS mRNA from cyclically compressed skin compared with normal skin: such expression was temporal in nature. CONCLUSIONS: A murine model of pressure necrosis, which bears all of the gross, histological, and molecular features of ischemia-reperfusion injury, has been established. Application of this model to the vast number of transgenic mice available will further our understanding of the mechanism of pressure sore development.

Use of hypoxia-inducible factor signal transduction pathway to measure O2 levels and modulate growth factor pathways.

Tissue PO2 levels are known to directly modulate numerous processes involved in the reparative response to cutaneous tissue injury, including cell differentiation and migration, extracellular matrix synthesis and maturation, and effectiveness of endogenous and exogenous growth factors. Oxygen is therefore likely the critical variable determining the healing capabilities of any tissue. Significant advances in the understanding of cutaneous wound healing progressed with advances in the measurement of tissue PO2, which has advanced over the past several decades from implantable probes to now include molecular tools such as the transcription factor hypoxia inducible factor-1 (HIF-1). HIF-1 modulates the expression of genes that drive the cellular adaptive response to hypoxia and possess the HIF-1 binding sequence named hypoxia response element within their promoter sequence. Molecular biology techniques are now allowing exploitation of the HIF-1/hypoxia response element pathway to drive the expression of potential vulnerary ectopic genes. Here we show the utility of the hypoxia response element for hypoxia-driven expression of the transforming growth factor-beta-signaling component Smad3 in vitro and the in vivo detection of ischemic hypoxia using luciferase. Smad3 is a positive effector of transforming growth factor-beta superfamily signal transduction. Such approaches are the latest evolution of work championed by Hunt and colleagues over the past 4 decades.

Inhibition of prolyl 4-hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring.

Hypertrophic scars result from excessive collagen deposition at sights of healing dermal wounds and can be functionally and cosmetically problematic. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. We tested the ability of the phenanthrolinone derivative FG-1648 (in 0.5% Carbopol 971 PNF gel, pH 6.5), a prolyl 4-hydroxylase inhibitor, to reduce hypertrophic scar formation in a rabbit ear hypertrophic scar model. New Zealand White rabbits were divided into two treatment groups (n = 12 wounds per group with an equal number of controls): low-dose group: 0.5% FG-1648; high-dose group: 1% FG-1648. Left ears were used for treatment and right ear for control. Four 7-mm dermal ulcer wounds were made on each ear. The inhibitor was topically applied to the wound at the time of wounding and once daily up to postoperative day 7. Wounds were harvested at postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. All wounds showed complete healing. Treatment of wounds with 1% prolyl 4-hydroxylase inhibitor decreased the scar elevation index by 26% compared to control wounds (p < 0.01). Wounds treated with 0.5% FG-1648 inhibitor showed no difference in scar elevation compared to control wounds. These results suggest that inhibition of prolyl 4-hydroxylase may be a suitable agent for topical treatment for the prevention of hypertrophic scar tissue.

Effect of Mederma on hypertrophic scarring in the rabbit ear model.

Currently accepted conservative treatments of hypertrophic scars are limited to steroid injections, radiation therapy, and silicone occlusive therapy. However, the use of Mederma for these problematic lesions has become quite prevalent in the clinical setting. Little scientific evidence exists to support the efficacy of this product in reducing hypertrophic scars. The aim of this study was to study the effects of Mederma on hypertrophic scars in the rabbit hypertrophic scar model, allowing the histologic quantification of scar elevation, dermal collagen organization, vascularity, and inflammation and the gross examination of scar erythema. Full-thickness wounds down to cartilage, four per ear, were created in four New Zealand White rabbits, for a total of 32 scars. Twenty-eight days after the initial wounding, the hypertrophic scars were photographed, and treatment of half of the scars on each ear was begun with Mederma three times per day for a total of 4 weeks. The untreated scars served as control scars and were left exposed to air. After 4 weeks of treatment, the scars were once again photographed. The rabbits were then killed, and the scars were analyzed histologically. The pretreatment and posttreatment photographs were compared by using computer quantification of magenta, yellow, and cyan expression within the scars. Histologic analysis demonstrated no significant reduction in scar hypertrophy or scar elevation index. However, a significant improvement in dermal collagen organization was noted on comparing Mederma-treated scars with untreated control scars (p < 0.05). No significant difference in dermal vascularity or inflammation was noted. Computer analysis of the scar photographs demonstrated no significant reduction in scar erythema with Mederma treatment. The active product in Mederma, allium cepa, has as its derivative quercetin, a bioflavonoid noted for its antiproliferative effects on both normal and malignant cells, and its antihistamine release effects. These properties could theoretically prove beneficial in reversing the inflammatory and proliferative responses noted in hypertrophic scars. Despite the authors' inability to demonstrate a reduction in scar hypertrophy, the improvement in collagen organization noted in the Mederma-treated scars suggests it may have an effect on the pathophysiology of hypertrophic scar formation.

Effect of age and hypoxia on TGFbeta1 receptor expression and signal transduction in human dermal fibroblasts: impact on cell migration.

Wound healing is critically affected by age, ischemia, and growth factors such as TGFbeta1. The combined effect of these factors on fibroblast migration, an essential component of wound healing, is poorly understood. To address this deficiency, we examined expression of TGFbeta receptor type I and II (TGFbetaRI and RII) under normoxia or hypoxia (1% O(2)) in cultured human dermal fibroblasts (HDFs) from young (ages 24-33) and aged (ages 61-73) adults. TGFbetaRI and RII expression was similar in both groups under normoxia. Hypoxia did not alter receptor levels in young HDFs but significantly decreased TGFbetaRI in aged cells (12 and 43%, respectively). Additionally, young cells displayed a 50% increase in activation of p42/p44 mitogen-activated kinase by TGFbeta1 (2-200 pg/ml) under hypoxia while aged cell levels of active p42/p44 decreased up to 24%. To determine functional outcomes of these findings, we measured the migratory capacity of the cells on type I collagen using a gold salt migration assay. Hypoxia increased the migratory index (MI) of young HDFs over normoxia by 30% but had no effect on aged cells. Under normoxia, TGFbeta1 (1-1000 pg/ml) increased young HDF migration in a concentration-dependent manner up to 109% over controls but minimally increased aged HDF migration (37%). Under hypoxia, TGFbeta1 significantly increased young cell MI at all concentrations but was without effect on the aged HDF response. These data demonstrate that aged fibroblasts have an impaired migratory capacity with complete loss of responsiveness to hypoxia and deficits in the migratory and signal transduction responsiveness to TGFbeta1 that may partly explain diminished healing capabilities often observed in aged patients.