Claire Linge

Differential gene expression in response to transforming growth factor-β1 by fetal and postnatal dermal fibroblasts

The multipotent growth factor transforming growth factor (TGF)‐β1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissues predominance of the TGF‐β3 isoform over the profibrotic TGF‐β1 and 2. Nevertheless, TGF‐β1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF‐β1stimulation. This study aimed to determine whether TGF‐β1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real‐time polymerase chain reaction. The regulation pattern of a number of TGF‐β response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF‐β1 autoinduction did not occur in fetal fibroblasts and genes that are normally up‐regulated, connective tissue growth factor and collagen type I were actually down‐regulated. Furthermore, other response genes responded in a delayed fashion (TGF‐β3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF‐β stimulation in postnatal cells, TGF‐β2 and collagen III, were up‐regulated in fetal cells. These developmentally related differences in fibroblast response to TGF‐β1 may influence wound‐healing outcome, i.e., perfect regeneration or fibrosis.

Dermal fibroblasts derived from fetal and postnatal humans exhibit distinct responses to insulin like growth factors

BackgroundIt has been well established that human fetuses will heal cutaneous wounds with perfect regeneration. Insulin-like growth factors are pro-fibrotic fibroblast mitogens that have important roles in both adult wound healing and during development, although their relative contribution towards fetal wound healing is currently unknown. We have compared responses to IGF-I and -II in human dermal fibroblast strains derived from early gestational age fetal (<14 weeks) and developmentally mature postnatal skin to identify any differences that might relate to their respective wound healing responses of regeneration or fibrosis.ResultsWe have established that the mitogenic response of fetal cells to both IGF-I and -II is much lower than that seen in postnatal dermal fibroblasts. Further, unlike postnatal cells, fetal cells fail to synthesise collagen in response to IGF-I, whereas they do increase synthesis in response to IGF-II. This apparent developmentally regulated difference in response to these related growth factors is also reflected in changes in the tyrosine phosphorylation pattern of a number of proteins. Postnatal cells exhibit a significant increase in phosphorylation of ERK 1 (p44) in response to IGF-I and conversely the p46 isoform of Shc on IGF-II stimulation. Fetal cells however only show a significant increase in an unidentified 100 kDa tyrosine-phosphorylated protein on stimulation with IGF-II.ConclusionDermal fibroblasts exhibit different responses to the two forms of IGF depending on their developmental maturity. This may relate to the developmental transition in cutaneous wound healing from regeneration to fibrosis.

Retrospective study of the association between hypertrophic burn scarring and bacterial colonization.

Although the association between hypertrophic burn scarring and infection is well described, an association with colonization has not been established. This retrospective study sought to determine whether a significant association between hypertrophic scarring and bacterial colonization exists. Details from the case notes of all patients seen in our institution’s burns unit over a two-year period were recorded and the incidence of hypertrophic scarring and burn bacterial colonization was noted. A total of 127 scars were recorded, and of these, 51 were hypertrophic and 76 nonhypertrophic. The incidence of bacterial colonization in the hypertrophic scar group was 88%, an association that achieved significance (P < .05) in comparison with nonhypertrophic scars (27%). This association holds true for individual organisms such as Staphylococcus aureus and Escherichia coli and for all burn depths and healing times. This study suggests that burn wound bacterial colonization may be more important than previously believed and perhaps suggests that striving toward an aseptic burn wound environment may reduce the incidence of hypertrophic scarring.

ruby Laser-assisted Hair Removal Success in Relation to Anatomic Factors and Melanin Content of Hair Follicles

Ruby laser-assisted hair removal is thought to work via selective photothermolysis, which relies on light reaching the deeper layers of skin, and the absorption of light by the target chromophore, melanin. It is therefore possible that efficacy of treatment is affected by anatomic factors that determine the amount of light reaching the hair bulbs (i.e., skin color, depth of intracutaneous hair, epidermal thickness and dermal density) and the melanin content of hair. To examine this hypothesis, a prospective study was performed. Forty-eight volunteers were treated with the Chromos 694 Depilation Ruby Laser at a single standard fluence of 11 J/cm2. Treatment efficacy was determined by measuring hair density at 3 and 7 months after treatment. Epidermal depth and dermal density were measured from 2-mm biopsies taken before treatment, and the intracutaneous hair length was determined from plucked hair. Skin color was assessed using a spectrophotometer, and melanin content of dissolved hair was assessed using spectrophotometry. Efficacy of treatment for each patient was compared with the patients age, intracutaneous hair length, epidermal depth, dermal density, skin color, and total melanin content and relative eumelanin content of hair. No correlation was found between the efficacy of treatment and age and the various anatomic factors. Patients with higher eumelanin content in their hair had better long-term results (Spearman rank test, p = 0.00219). The results suggested that the efficacy of treatment did not depend solely on the amount of laser light penetrating the skin but correlated well with the eumelanin content of hair. The clinical implication of this finding is discussed.

An investigation to optimize angiogenesis within potential dermal replacements.

Background: Acute and chronic wounds are costly and invariably expose significant structures. Surgical reconstruction causes donor-site morbidity, scarring, and the need for intensive care. Reconstruction using an artificial dermis avoids donor sites, but available collagen-based solutions are susceptible to poor take. Using an in vitro angiogenic assay, the authors investigated dermal matrices for potential inclusion in a second-generation proangiogenic synthetic dermal replacement. Methods: Human placental endothelial cells were cocultured on Cytodex beads (Pharmacia Biotech) and plated in eight different extracellular matrix gels (collagen, fibrin, four glycosaminoglycans, vitronectin, and fibronectin), with or without stimulation from two soluble angiogenic factors. Three different cell lines were used, with 30 beads per condition. Cellular invasion into gels was calculated using Sigma Scan computer software, and statistical comparisons were made. Results: The authors found that fibrin provided greatest stimulus for endothelial invasion, with invasion in fibrin inhibited by collagen in a concentration-dependent fashion. Invasion by alternative extracellular matrix components and soluble angiogenic factors was far less than that in fibrin. Conclusions: The authors identified that extracellular matrices can provide greater angiogenic potential than soluble angiogenic factors. Fibrin provided a better proangiogenic scaffold than collagen. This could well be used to encourage blood vessel ingress and eventual take of a second-generation proangiogenic synthetic dermal replacement.

Ruby laser irradiation (694 nm) of human skin biopsies: assessment by electron spin resonance spectroscopy of free radical production and oxidative stress during laser depilation.

Abstract Human skin biopsies (hair‐bearing scalp skin and non‐hair‐bearing breast skin) were treated with t‐butylhydro‐peroxide, irradiated with UV light (UVR) or irradiated with 694 nm ruby laser red light. Free‐radical production and oxidative stress were assessed with electron spin resonance spectroscopy (ESR) using the ascorbate radical as a marker. In comparison with both UVR and t‐butyl‐hydroperoxide (which readily induce the ascorbate radical in hair‐bearing and hairless skin), 694 nm red light does not result in the formation of the ascorbate radical in detectable concentrations. Spin‐trapping experiments with the spin trap 5,5‐dimethyl‐l‐pyrroline N‐oxide (DMPO) showed that while free radicals could be detected after treatment of skin with t‐butylhydroperoxide, radicals could not be trapped after laser treatment. Treatment of lasered skin (containing DMPO) with t‐bu‐tylhydroperoxide produced radical adducts as well as the ascorbate radical, demonstrating that the laser neither depletes endogenous ascorbate nor the preadministered spin trap. It is concluded that 694 nm red light does not induce oxidative stress in human skin in levels comparable either to t‐butyl hydroperoxide or UV light.

The effect of ruby laser light on ex vivo hair follicles: clinical implications.

Several clinical studies on the efficacy of ruby laser-assisted hair removal have reported that regrowth of hair after treatment is common. One of the reasons for the regrowth of hair is the incomplete destruction of germinative hair cells due to the insufficient penetration of the ruby laser in the skin. It was the aim of this study to estimate the extent of damage to the hair follicles after one ruby laser treatment and to determine whether the ruby laser destroyed the bulbs and the bulge regions of hair follicles. The extent of laser damage in hair shafts was determined by serial examination of six specimens of ex vivo scalp skin lasered with the Chromos 694 Depilation Ruby Laser at 14 J per square centimeter and 20 J per square centimeter. Another nine specimens of ex vivo scalp skin were similarly lasered, and monoclonal antibody LP2K was used to identify the bulge regions of the hair follicles using the immunoperoxidase technique. Damage to the bulge region was assessed from consecutive specimens, which were stained with hematoxylin-eosin stain. The mean depth of laser damage sustained by hair follicles was 1.34 mm (14 J per square centimeter) and 1.49 mm (20 J per square centimeter) underneath the skin surface. Most of the laser damage involved the bulge regions but fell short of the hair bulbs. The laser damage did not seem to extend far enough down the hair shafts to result in permanent hair destruction. The clinical implications of this finding are discussed.

In vitro optimisation of topical negative pressure regimens for angiogenesis into synthetic dermal replacements

BACKGROUND The use of synthetic dermal replacements (SDRs) in the treatment of large wounds, which have associated morbidity and mortality, has attracted great interest. However, because of poor outcome, SDRs have limited use. The addition of topical negative pressure (TNP) has increased their success, but little research has focused on the underlying mechanisms. This paper studies the in vitro effects of TNP on commonly used SDRs to identify the most effective TNP regimen and optimum SDR for encouraging endothelial cell ingress. METHODS Endothelial cells were co-cultured in vitro on four SDRs with or without TNP. Negative pressure (125mmHg) was applied intermittently, continuously, for 4h per day, or not at all. Endothelial ingress was measured for each condition. RESULTS In the collagen controls, cell migration was minimal. Integratrade mark gave the greatest endothelial cell migration (p<0.05, n=3). TNP increased endothelial cell migration, intermittent application being the optimum regimen. CONCLUSIONS Integratrade mark has an open sponge structure which may account for greater angiogenicity than Allodermtrade mark, Permacoltrade mark and Xenodermtrade mark. In vitro intermittent TNP stimulates the greatest angiogenic response. The majority of clinical studies investigating SDR success with TNP have used continuous regimens; this study suggests a change in clinical practice to intermittent application.

Does Low Penetration of Human Skin by the Normal Mode Ruby Laser Account for Poor Permanent Depilatory Success Rates

Abstract. Studies reported to date have shown a good depilatory response from patients treated with the normal mode ruby laser (NMRL) over 12 weeks, but a low response over a time period greater than this. Previous publications have suggested that this could be accounted for by the apparently poor skin penetration of laser light and so this study attempted to assess whether this was indeed the case. Skin samples of varying thicknesses were taken from six Caucasian patients and their depths measured. Each was laid individually on an energy meter before having pulses from an NMRL compatible with clinical doses (4.75 J/cm2, 9.24 J/cm2 and 13.41 J/cm2) fired on the epidermis. Several samples had the laser fired repetitively on the surface to assess whether this caused any change in laser/skin fluence depth profiles. Repetitive firing of the NMRL on the epidermis of skin samples did not alter the energy recorded by the meter beneath. The fluence/depth profiles were constructed showing the majority of energy was lost within the first 1 mm of the skin surface (50%) which then further reduced over distance but at a much slower rate. The maximum depth of penetration was 14.8 mm (SD±0.478) which appeared to be a function of wavelength and not fluence. The results suggest that laser penetration of skin should be adequate for generating enough heat at the hair bulge and bulb, potentially causing permanent damage. The implications of this study are that it is probably the presence of the correct chromophore in large enough amounts which is required for successful permanent depilation to occur.

Cutaneous Scarring: A Clinical Review

Cutaneous scarring can cause patients symptoms ranging from the psychological to physical pain. Although the process of normal scarring is well described the ultimate cause of pathological scarring remains unknown. Similarly, exactly how early gestation fetuses can heal scarlessly remains unsolved. These questions are crucial in the search for a preventative or curative antiscarring agent. Such a discovery would be of enormous medical and commercial importance, not least because it may have application in other tissues. In the clinical context the assessment of scars is becoming more sophisticated and new physical, medical and surgical therapies are being introduced. This review aims to summarise some of the recent developments in scarring research for non-specialists and specialists alike.