Infant skin maturation: Preliminary outcomes for color and biomechanical properties.

BACKGROUND: Newborn infant skin changes after birth but studies have focused on the epidermal barrier. Dermal properties are relevant for care, but literature on postnatal changes is sparse. We further characterized skin maturational changes in lightness, color and response to biomechanical stress. METHODS: Normal skin sites from subsets of participants in a trial on the progression and stage of infantile hemangiomas were retrospectively examined. Standardized photographs were analyzed as L*, a*, and b* images. Biomechanics were measured with the Cutometer® . RESULTS: Color changed significantly with increasing age. Skin was darker and redder at 2.0 vs. 5.4, 8.5 and 12.8 months. Yellow color increased, with higher values at 12.8 vs. 2.0, 3.5 and 5.4 months. Chest tissue was consistently more elastic than arm and face sites, with significantly higher elasticity for the youngest and oldest age groups. Biological elasticity, elastic recovery, and total recovery were significantly greater for the oldest subjects. Viscoelasticity and elastic deformation were lower at 5.5 vs. 8.8 and 17.6 months. Arm viscoelastic creep was highest at 2.8 months. CONCLUSION: Skin maturation continues into year two. Increasing elasticity and decreasing viscoelasticity may reflect increased collagen structure/function. The findings have implications for prevention of skin injury associated with mechanical forces.

Biomechanical properties of infantile hemangiomas: clinical stage and effect of age.

BACKGROUND: Infantile hemangiomas (IHs) are benign vascular neoplasms with rapid capillary proliferation shortly after birth and slow involution with diminishing capillary proliferative activity, fibrosis, and fatty replacement over 7-10 years. METHODS: Hemangiomas and contralateral control sites in 88 subjects were measured using a suction device, 6-mm probe and 200 mbar negative pressure. Mechanical properties were assessed vs. controls and effects of body site, depth, clinical stage, histology diagnosis, and time. RESULTS: Biological elasticity, overall elasticity, net elasticity, total recovery, and elastic recovery were lower for IH vs. controls (P < 0.001). IH total deformation, elastic deformation, viscoelastic creep, and residual deformation were higher than controls (P < 0.001). Involuting IHs had lower viscoelasticity than proliferating and stable lesions (P < 0.001) and lower viscoelastic creep than stable IHs (P = 0.04). IH viscoelasticity was higher at 2.3 than 12.9, 23.7, and 61.0 months and at 4.9 and 8.1 than 61.0 months. IH elastic recovery varied by body site with larger differences vs. control for abdomen and leg. Elastic recovery differences from control were smaller at younger vs. older ages. CONCLUSIONS: Measurement of biomechanical properties may be useful to characterize IH progression and treatment response in clinical settings.

The uptake of water hardness metals by human hair.

The objective of this work was to examine the variables that influence the interaction between water hardness metals and human hair. Hair extracts various constituents from the tap water used during daily hygiene practices and chemical treatments. Calcium and magnesium metal ions are the most prevalent and give water "hardness." Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to quantify the metal content of hair, which was studied as a function of the following variables: hair condition (oxidative damage), level of water hardness, and water pH. We have demonstrated that these variables impact water hardness metal uptake to varying extents, and the effects are driven primarily by the binding capacity (available anionic sites) of the hair. The condition of the hair, a key representation of the binding capacity, was most influential. Interestingly, water hardness levels had only a small effect on uptake; hair became saturated with notable amounts of water hardness metals even after repeated exposure to soft water. Water pH influenced metal uptake since side chains of hair proteins deprotonate with increasing alkalinity. These insights highlight the importance to the hair care industry of understanding the interaction between water hardness metals and hair.

The structural implications of water hardness metal uptake by human hair.

Human hair can extract significant levels of calcium and magnesium, water hardness metals, from tap water immediately following chemical treatments and during hygiene practices. We have previously shown that this uptake is primarily a function of the condition of the hair. Depending on the extent of chemical damage, the hair can extract notable amounts of water hardness metals even from soft water. As water hardness metals concentrate primarily in the cuticle layers of the hair fibre, it is hypothesized that their presence will affect the structural properties that are chiefly driven by the cuticle. We examined hair mechanics and styling by technical measures of single-fibre torsional and tensile properties, combability and style retention as a function of the calcium and magnesium content of virgin and bleached hair. Our work has indicated that water hardness can affect hair properties. Fibre stiffening was induced by the presence of water hardness metals inside the fibres of both virgin and bleached hair. A reduction in combing forces was also observed, and this effect is believed to be a result of the stiffening. The style retention of virgin hair was improved by water hardness metals, whereas that of bleached hair was slightly reduced.

Role of TNF-α polymorphism -308 in neurosensory irritation.

Neurosensory cutaneous discomfort in response to topical products is common, yet the relationship between symptoms such as stinging and visible irritation is currently unclear. The presence of a polymorphism at position -308 on the TNF-α gene has been associated with skin irritation, i.e., erythema, dryness. Individuals with a G to A transition (AA/GA genotypes) have a lower threshold to experimentally induced irritation than those with the wild type (G allele, GG genotype). We investigated the effect of this polymorphism on neurosensory irritation (NSI). DNA genotyping was used to determine the allele type amongst a population of health care workers. The neurosensory response to lactic acid and water on the nasolabial folds and hands was assessed using a quantitative lactic acid sting test. Both genotypes had a more intense response to lactic acid compared with water on the face. The AA/GA genotypes had directionally higher scores from lactic acid (P = 0.1) and significantly higher stinging intensities from water (P = 0.001) on the face. For the hands, stinging intensities were higher for lactic acid and water amongst the AA/GA genotypes (P = 0.03 and 0.006 respectively). NSI to lactic acid was significantly higher on the face than on the hands (P < 0.05). Our findings indicate that subjects with the A transition at position -308 on the TNF-α gene experience more intense NSI with common ingredients, i.e., lactic acid and water, than those with the wild type. TNF-α polymorphism -308 may account for some of the inter-individual variability in response to skin care practices.

Mechanism of tyrosinase inhibition by deoxyArbutin and its second-generation derivatives.

BACKGROUND: Disorders, such as age spots, melasma and hyperpigmentation at sites of actinic damage, emanate from the augmentation of an increased amount of epidermal melanin. OBJECTIVES: The ineptness of current therapies in treating these conditions, as well as high cytotoxicity, mutagenicity, poor skin penetration and low stability of skin-depigmenting formulations led us to investigate new compounds that meet the medical requirements for depigmentation agents. We have shown previously that the tyrosinase inhibitor deoxyArbutin (dA) is a more effective and less toxic skin lightener than hydroquinone (HQ). METHODS: The efficacy and reversibility of dA and its derivatives on inhibiting tyrosine hydroxylase and DOPAoxidase was assessed using standard assays. RESULTS: dA and its second-generation derivatives inhibit tyrosine hydroxylase and DOPAoxidase activities of tyrosinase dose dependently thereby inhibiting melanin synthesis in intact melanocytes, when used at concentrations that retain 95% cell viability in culture. This depigmenting effect was completely reversible when the compounds were removed. Tyrosinase inhibition was also observed in vitro when tested using human and purified mushroom tyrosinase, establishing that they are direct enzyme inhibitors. Lineweaver-Burk reciprocal plot analysis using mushroom tyrosinase illustrated that dA and its derivatives are more robust competitive inhibitors than HQ, when tyrosine is used as substrate. CONCLUSIONS: Thus, dA and its second-generation derivatives, which inhibit melanogenesis at safe concentrations by specifically acting on the tyrosinase enzyme at a post-translational level, are promising agents to ameliorate hyperpigmented lesions or lighten skin.

Effect of oral intake of choline-stabilized orthosilicic acid on hair tensile strength and morphology in women with fine hair.

The appearance of hair plays an important role in people's overall physical appearance and self-perception. Silicon (Si) has been suggested to have a role in the formation of connective tissue and is present at 1-10 ppm in hair. Choline-stabilized orthosilicic acid ("ch-OSA") is a bioavailable form of silicon which was found to improve skin microrelief and skin mechanical properties in women with photoaged skin. The effect of ch-OSA on hair was investigated in a randomized, double blind, placebo-controlled study. Forty-eight women with fine hair were given 10 mg Si/day in the form of ch-OSA beadlets (n = 24) or a placebo (n = 24), orally for 9 months. Hair morphology and tensile properties were evaluated before and after treatment. Urinary silicon concentration increased significantly in the ch-OSA supplemented group but not in the placebo group. The elastic gradient decreased in both groups but the change was significantly smaller in the ch-OSA group (-4.52%) compared to placebo group (-11.9%). Break load changed significantly in the placebo group (-10.8%) but not in the ch-OSA supplemented group (-2.20%). Break stress and elastic modulus decreased in both groups but the change was smaller in the ch-OSA group. The cross sectional area increased significantly after 9 months compared to baseline in ch-OSA supplemented subjects but not in the placebo group. The change in urinary silicon excretion was significantly correlated with the change in cross sectional area. Oral intake of ch-OSA had a positive effect on tensile strength including elasticity and break load and resulted in thicker hair.

Assessment of skin absorption and irritation potential of arachidonic acid and glyceryl arachidonate using in vitro diffusion cell techniques.

Arachidonic acid (AA), a precursor of pro-inflammatory mediators, and its glycerin ester, glyceryl arachidonate (GA), are reportedly used in cosmetic products. In vitro skin penetration of AA and GA and GA's ester hydrolysis was determined in flow-through diffusion cells. AA penetration with human and rat skin was 19.5% and 52.3% of the applied dose respectively, a substantial amount of which remained in the skin at 24h. Similar penetration results were obtained with GA in human skin. However, GA penetration through cultured skin (EpiDerm) was 51% of the applied dose, almost all of which appeared in the receptor fluid. At least 27.8% of GA penetrating skin was hydrolyzed to AA. In vitro methods were used to assess skin irritation in diffusion cells. Skin irritation of AA, sodium lauryl sulfate (SLS), and Tween 80 was determined by changes in transepidermal water loss (TEWL), skin viability (3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, formation), and cytokine release (IL-1alpha). SLS irritation was much less pronounced in an emulsion versus an aqueous vehicle. No significant irritation was observed in vitro from AA in an emulsion. This work predicts that AA would penetrate human skin in vivo and that it could be formed in skin from topically applied GA.

In vitro and in vivo percutaneous absorption of catechol.

The Cosmetic Ingredient Review Expert Panel found insufficient data to conclude that catechol could be used safely in permanent hair dye products. Information was lacking on the extent of oxidation and skin absorption of remaining catechol. In vitro percutaneous absorption studies were conducted in human and rat skin using a consumer permanent hair dye spiked with 0.6% catechol. A 30-min application demonstrated 0.4% of the applied dose was absorbed through human skin and 0.2% through rat skin. The minimal absorption observed was due to the short exposure time and to partial oxidation of catechol by the dye developer. The fate of catechol remaining in rat skin after exposure in vitro and in vivo was investigated with additional absorption studies using catechol in ethanol. At 72 h, 24-h application of 4% catechol resulted in skin absorption of 81% of the applied dose in vitro and 53% in vivo. Skin levels measured at 24 h remained unchanged after 72 h. Therefore the skin reservoir did not contribute to the estimated systemic absorption. A deconvolution technique employed to predict skin absorption using plasma levels from intravenous and dermal administration overestimated in vivo skin absorption due to volatility of catechol in an ethanolic vehicle.

Keratinocytes play a role in regulating distribution patterns of recipient melanosomes in vitro.

Melanosomes in keratinocytes of Black skin are larger and distributed individually whereas those within keratinocytes of Caucasian skin are smaller and distributed in clusters. This disparity contributes to differences in skin pigmentation and photoprotection, but the control of these innate distribution patterns is poorly understood. To investigate this process, cocultures were established using melanocytes and keratinocytes derived from different racial backgrounds and were examined by electron microscopy. Melanosomes transferred to keratinocytes were categorized as individual or in various clusters. Melanosome size was also determined for individual and clustered melanosomes. Results indicate that, in our model system, melanosomes in keratinocytes from different racial backgrounds show a combination of clustered and individual melanosomes. When keratinocytes from dark skin were cocultured with melanocytes from (i) dark skin or (ii) light skin, however, recipient melanosomes were individual versus clustered in (i) 77% vs 23% and (ii) 64% vs 36%, respectively. In contrast, when keratinocytes from light skin were cocultured with melanocytes from (iii) dark skin or (iv) light skin, recipient melanosomes were individual versus clustered in (iii) 34% vs 66% and (iv) 39% vs 61%, respectively. These results indicate that recipient melanosomes, regardless of origin, are predominantly distributed individually by keratinocytes from dark skin, and in membrane-bound clusters by those from light skin. There were also differences in melanosome size from dark or light donor melanocytes. Melanosome size was not related to whether the melanosomes were distributed individually or clustered, however, in cocultures. These results suggest that regulatory factor(s) within the keratinocyte determine recipient melanosome distribution patterns.

Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system.

We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co-cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co-cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co-culturing melanocytes with keratinocytes, and processing the co-cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co-culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co-cultures inhibited transfer of fluorochrome by approximately 15-44% as assessed by flow cytometry, and of melanosomes by 67-93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.

The ability of electrical measurements to predict skin moisturization. I. Effects of NaCl and glycerin on short-term measurements.

Non-invasive methods to evaluate skin hydration by measuring electrical properties are widely used in the cosmetic industry. However, there is still some controversy about factors that affect measurement. For example, concerns have often been expressed about the possible confounding effect of salts, either in the formulation or on the skin. Ionized salts on the skin may increase electrical conductivity and may lead to changes in electrical properties that are not related to increased water content. We have performed a systematic study of the effects of salt, i.e., sodium chloride, and glycerin on the electrical properties of skin as measured by the three most commonly used instruments, the Nova DPM 9003, the Corneometer CM 825, and the Skicon 200. Formulations containing salt from 0-3% and glycerin from 0-10% were tested for their effects at one and two hours after a single application. Salt lowered the readings in the absence of glycerin and increased the reading in the presence of glycerin. For all three instruments, there was a linear correlation between the measurement and the glycerin level in the presence or absence of salt.

The ability of electrical measurements to predict skin moisturization. II. Correlation between one-hour measurements and long-term results.

We investigated the ability of short-term (one-hour) electrical measurements with three different commonly used instruments to predict the effects of long-term treatment with glycerin-containing formulations on moderately dry leg skin. We report the moisturizing effects of glycerin on healthy female adult skin in a two-week study as measured by electrical conductance and capacitance, transepidermal water loss (TEWL), and clinical grading of skin dryness. The test formulations contained 1.5% NaCl and levels of glycerin from 0% to 15%. Results obtained with a smaller cohort of ten subjects, one hour after treatment, were predictive of moisturizing efficacy in the two-week period among twenty subjects with dry leg skin. Our results show that single application tests can be predictive of longer-term results with humectant-based moisturizers and that electrical measurements of skin conditions correlate well with skin grades.

Surface free energy characterization of vernix caseosa. Potential role in waterproofing the newborn infant.

BACKGROUND/AIMS: Vernix caseosa is a proteolipid biofilm synthesized by the human fetus, which progressively covers the fetal skin surface during the last trimester of pregnancy. The exact physiological functions of vernix are unclear. Hypothetically, it serves a role in "waterproofing" the fetus during the critical period of epidermal barrier development before birth. Vernix may also play a role in adaptation of the fetal skin surface to the dry, cool extrauterine environment after birth. Given the strategic position of vernix on the fetal skin surface and the rapidly changing environment encountered by the skin at birth, we proposed that investigation of vernix surface characteristics would facilitate understanding its putative physiological roles. METHODS: In this paper, we focused on the determination of the surface free energy (SFE) of vernix caseosa. Different approaches were used to calculate the SFE of vernix from contact angle (theta) measurements between vernix and various liquids (benzyl alcohol, diiodomethane, glycerol, and water). The critical surface tension (CST) of vernix was calculated using Zisman plots. The dispersive and the polar components of vernix SFE were calculated using the Owens-Wendt geometric mean method. Vernix was contrasted with petrolatum, a commonly used skin protectant. RESULTS: CST of fresh vernix was 40.5 dyne/cm while that of petrolatum was 35.8 dyne/cm. Fresh vernix polar SFE was 1.5 dyne/cm while petrolatum had almost no polar SFE component (0.03 dyne/cm). For all liquids (except the nonpolar diiodomethane) there was a significant decrease in contact angle with time. CONCLUSIONS: The CST and the total SFE values suggest that vernix has very low surface energy and is highly unwettable. These findings are significant insofar as the main component in vernix is water, which is highly energetic. Although vernix has a very high water content, the major part of its SFE is hydrophobic (dispersive). The limited interaction between vernix and hydrophilic liquids supports the hypothesis that vernix acts as a natural protectant cream to "waterproof" the fetus in utero while submerged in the amniotic fluid.

Effect of semipermeable membranes on skin barrier repair following tape stripping.

Reports in the literature suggest that the permeability of a wound dressing to water transport is an important variable in the healing of superficial wounds. Factors that influence skin hydration during barrier repair, therefore, are important in the optimization of wound treatments. In this study, the effects of semipermeable films on human skin following a standardized wound (tape stripping) were evaluated using measurements of transepidermal water loss (TEWL), skin hydration, rate of moisture accumulation, and erythema. Wounds treated with semipermeable films underwent more rapid barrier recovery than either unoccluded wounds or wounds under complete occlusion. Barrier films that produced intermediate levels of skin hydration during recovery produced the highest barrier repair rates. The results support the hypothesis; that semipermeable wound dressings augment barrier repair and skin quality by providing an optimized water vapor gradient during the wound healing process. The choice of wound dressing is discussed within the larger context of the design of vapor-permeable fabrics (smart materials) and the new fields of corneotherapy and comfort science.

Characterization of vernix caseosa as a natural biofilm: comparison to standard oil-based ointments.

The application of occlusive films and oil-in-oil ointments has been reported to improve epidermal barrier function in very low birthweight, preterm infants. Such infants have a structurally immature stratum corneum and lack a surface coating of vernix caseosa. In this study we examined the short-term effects of topical application of vernix caseosa to human skin and contrasted these effects with commonly used ointments and water-in-oil emulsions. Specifically, vernix, Eucerin(R), Aquaphor(R), and petrolatum were applied to the volar skin surface of adult volunteers. Surface electrical capacitance (SEC) and transepidermal water loss (TEWL) were measured as indices of surface hydration. Sorption-desorption profiles were performed to determine skin surface hydrophobicity. Particular attention was given to monitoring the acute (0-120 minutes) changes following vernix treatment in order to compare these effects with earlier reports on the rate of skin surface drying in newborn infants following birth. Immediately after vernix application there was an increase in the rate of water loss from the skin surface. Relative to control skin and skin treated with the ointments and water-in-oil emulsions, the application of vernix to freshly bathed human skin resulted in a unique profile of temporal change in baseline surface hydration, moisture accumulation, and water-holding capacity. These results demonstrate major differences between human vernix and standard oil-based topical ointments. The results provide a framework for discussing the various properties of topical barriers applied to the very low birthweight infant.

Interaction between pulmonary surfactant and vernix: a potential mechanism for induction of amniotic fluid turbidity.

The development of amniotic fluid turbidity during the third trimester is a known marker of fetal lung maturity. We hypothesized that this turbidity results from detachment of vernix caseosa from the fetal skin secondary to interaction with pulmonary-derived phospholipids in the amniotic fluid. To test this hypothesis, we exposed vernix to bovine-derived pulmonary surfactant over a physiologically relevant concentration range. Ten milligrams of vernix was evenly applied to the interior walls of 1.5-mL polypropylene microfuge tubes. Surfactant phospholipids were added to the tubes followed by slow rotation at 37 degrees C overnight. The liquid was decanted and spectrophotometrically analyzed at 650 nm to detect solution turbidity due to vernix detachment and/or emulsification. Increasing concentrations of surfactant phospholipids produced a dose-dependent increase in solution turbidity. A phospholipid mixture closely approximating natural pulmonary surfactant but devoid of surfactant-associated proteins yielded no increase. In other studies, the flow properties of vernix were studied in a Haake flow rheometer at 23 degrees C and 37 degrees C. There was a marked temperature-dependent effect with lower stress required to elicit flow at 37 degrees C compared with 23 degrees C. This temperature dependence was also demonstrated in the turbidity assay with a 124% increase in turbidity at body temperature compared with room temperature. We conclude that under in vitro conditions, pulmonary surfactant interacts with vernix resulting in detachment from a solid phase support. We speculate that in utero, this phenomenon contributes to the increase in amniotic fluid turbidity that is observed near term.

Assessment with the dermal torque meter of skin pliability after treatment of burns with cultured skin substitutes.

The assessment of visco-elastic (V-E) properties in cutaneous scars is critical to reduction of impairment and restoration of function after grafting of excised burns. Cultured skin substitutes (CSS) that consist of autologous keratinocytes and fibroblasts attached to biopolymer substrates are alternatives for permanent closure of excised, full-thickness burns, but assessment of scarring has been subjective. V-E properties of CSS were measured with a Dia-Stron Dermal Torque Meter (DTM 310, Dia-Stron, Ltd, Broomall, Pa), which applies a constant torque (10 mNm) for a fixed interval (10 seconds) and measures rotational deformation and recovery. Parameters of skin deformation were measured in patients (n = 10) after grafting of CSS or meshed skin autograft. Native human skin (NHS) of healthy volunteers (n = 13) served as the control. Skin healed after treatment with CSS or autograft was evaluated for 1 year or longer after grafting. Elastic stretch (Ue), viscous stretch (Uv), total extensibility (Uf), elastic recovery (Ur), total recovery (Ua), and residual plasticity (R) were measured as degrees of rotation, were tested for significance (P < .05) by Student t test comparisons between treatment groups and controls, and were subjected to regression analysis. Assessment of burn scar with the Dermal Torque Meter detected time-dependent increases of all individual parameters of V-E properties for both CSS and autograft during the first year after grafting. At 1 year or later, no statistical differences were found between CSS and autograft for individual parameters, but Ue and Ur for autograft were significantly lower than for NHS. At 1 year or longer, autograft was significantly different from CSS or NHS, with a greater ratio of Uv to Ue, and both graft types had a lower ratio of Ur to Uf than NHS had. These results suggest that instrumental measurement of scar pliability may increase objectivity in assessment of patient recovery and establish an absolute scale for quantitative analysis of V-E properties in skin after grafting of conventional or alternative skin substitutes.

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice.

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.