Validation of finite-element-simulated orthodontic forces produced by thermoplastic aligners: Effect of aligner geometry and creep.

PURPOSE: Finite element (FE) models for determining the orthodontic forces delivered by clear aligners often lack validation. The aim of this study was to develop and validate accurate FE models for clear aligners, considering the small but important geometrical variations from the thermoforming process and the creep behavior of the aligner material. METHODS AND MATERIALS: The tooth misalignment considered was a 2.4° torque aberration (rotation about the mesial-distal axis at the level of the center of resistance) of the maxillary left central incisor. FE models were created from Micro-CT scans of a model dental arch and five nominally identical aligners with the aforementioned misfit. Fitting of the aligners onto the dental arch was simulated using Abaqus's Interference Fit function, followed by surface-to-surface frictional interaction. Stress relaxation of the aligner material was measured using double-cantilever beam bending and modeled with a Prony series. The assembled FE models were validated by comparing the predicted forces and moments delivered to the maxillary left central incisor with experimental data, obtained with a custom-built but fully calibrated apparatus. RESULTS: Good agreement between prediction and measurement was obtained for both the short- and long-term forces and moments. In the short-term, i.e., after 30 s, the dominant force in the labial-lingual direction had a maximum difference of 2.9% between experiment and simulation, and the dominant moment about the mesial-distal axis had a maximum difference of 8.3%. In the long-term, i.e., after 4 h, the experimental and numerical forces had a maximum difference of 8.4%. There were statistically significant differences in the forces delivered among the nominally identical aligners, which were predicted by the geometrically accurate FE models and attributed to the variations in the points of contact between the aligners and the dental arch. The decay in force applied was affected by both the viscoelastic material behavior and friction between the aligner and arch. CONCLUSION: For accurate prediction of the forces and moments delivered by thermoplastic aligners, FE models that can accurately capture the point contacts between the aligners and the underlying teeth are essential. Stress relaxation of the aligners could be adequately modeled using the Prony series to represent the temporal changes of their elastic modulus.

Validation of finite element models for orthodontic aligners.

PURPOSE: Clear thermoplastic aligners have become popular in orthodontics, but the biomechanics of these devices is not well understood. Neither is the tooth movement induced by such devices. The aim of this study was to develop and validate finite element (FE) models for clear thermoplastic teeth aligners for orthodontic force prediction. METHODS AND MATERIALS: FE models were created from Micro-CT scans of an aligner and a model arch of teeth with one of the incisors tipped buccal-lingually by 2.4°. The models were uniformly meshed with 0.3-mm long elements. Linear-elastic mechanical properties provided by the material manufacturers were used. Fitting of the two components was simulated using Abaqus's interference fit, followed by frictional surface-to-surface interaction. The assembled FE model was validated by comparing its prediction for the teeth-aligner gaps and aligner surface strains with experimental data. The experimental teeth-aligner gaps were obtained from the Micro-CT scans whereas the aligner surface strains were measured using a 2-camera digital image correlation (DIC) system. RESULTS: Good agreement between prediction and measurement was obtained for both the teeth-aligner gaps and aligner surface strains. The linear regression between prediction and measurement for teeth-aligner gaps sampled at different positions had a R2 value of 0.99. The mean difference between prediction and measurement for the aligner surface strains (von Mises) over 1544 nodes on the labial side and 1929 nodes on the lingual side was 0.07% and 0.01%, respectively, both being lower than the mean background noise. CONCLUSION: A FE model for clear thermoplastic teeth aligners has been successfully developed and validated. The model can therefore be used with confidence to predict the forces and moments applied to teeth by the aligners, thus improving our understanding of the biomechanics of such devices and the tooth movement they induce.

Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol-digitonin structural complexes; (b) distinguishes cholesterol-rich and -poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.

Desmoglein isoform distribution affects stratum corneum structure and function.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Neuromuscular transmitter substance in insect visceral muscle.

Stimulation of the nerves innervating the proctodeum (hindgut) of the cockroach Periplaneta americana (L.) causes a slow-type, graded contraction of the longitudinal muscles. An unidentified substance, or substances, present in the foregut and hindgut, the specific activity of which is highest in the nerves innervating these organs, effects a similar contraction. This "gut-factor" is depleted from the hindgut after surgical section of the proctodeal nerves. None of Factors P(1) and P(2), 5-hydroxytryptamine, acetylcholine, adrenaline, noradrenaline, alpha-ami-nobutyric acid or glutamate duplicates the pharmacological behavior of this substance. The active factor is associated with subcellular particles that require centrifugal forces of approximately 1,000,000 g-min for sedimentation. The substance is inactivated in homogenates of gut tissue in the absence of suitable precautions. It is proposed that the "gut-factor" functions as an excitatory neuromuscular transmitter substance in insect visceral muscle.

Long-term impacts of rising sea temperature and sea level on shallow water coral communities over a ~40 year period.

Effects of combined rising sea temperature and increasing sea level on coral reefs, both factors associated with global warming, have rarely been addressed. In this ~40 y study of shallow reefs in the eastern Indian Ocean, we show that a rising relative sea level, currently estimated at ~11 mm y-1, has not only promoted coral cover but also has potential to limit damaging effects of thermally-induced bleaching. In 2010 the region experienced the most severe bleaching on record with corals subject to sea temperatures of >31 °C for 7 weeks. While the reef flats studied have a common aspect and are dominated by a similar suite of coral species, there was considerable spatial variation in their bleaching response which corresponded with reef-flat depth. Greatest loss of coral cover and community structure disruption occurred on the shallowest reef flats. Damage was less severe on the deepest reef flat where corals were subject to less aerial exposure, rapid flushing and longer submergence in turbid waters. Recovery of the most damaged sites took only ~8 y. While future trajectories of these resilient reefs will depend on sea-level anomalies, and frequency of extreme bleaching the positive role of rising sea level should not be under-estimated.

The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.

Aged epidermis displays altered drug permeability, increased susceptibility to irritant contact dermatitis, and often severe xerosis, suggesting compromise of the aged epidermal barrier. To delineate the functional, structural, and lipid biochemical basis of epidermal aging, we compared barrier function in young (20-30 yr) vs aged (> 80 yr) human subjects, and in a murine model. Baseline transepidermal water loss in both aged humans and senescent mice was subnormal. However, the aged barrier was perturbed more readily with either acetone or tape stripping (18 +/- 2 strippings vs 31 +/- 5 strippings in aged vs young human subjects, respectively). Moreover, after either acetone treatment or tape stripping, the barrier recovered more slowly in aged than in young human subjects (50 and 80% recovery at 24 and 72 h, respectively, in young subjects vs 15% recovery at 24 h in aged subjects), followed by a further delay over the next 6 d. Similar differences in barrier recovery were seen in senescent vs young mice. Although the total lipid content was decreased in the stratum corneum of aged mice (approximately 30%), the distribution of ceramides (including ceramide 1), cholesterol, and free fatty acids was unchanged. Moreover, a normal complement of esterified, very long-chain fatty acids was present. Finally, stratum corneum lamellar bilayers displayed normal substructure and dimensions, but were focally decreased in number, with decreased secretion of lamellar body contents. Thus, assessment of barrier function in aged epidermis under basal conditions is misleading, since both barrier integrity and barrier repair are markedly abnormal. These functional changes can be attributed to a global deficiency in all key stratum corneum lipids, resulting in decreased lamellar bilayers in the stratum corneum interstices. This constellation of findings may explain the increased susceptibility of intrinsically aged skin to exogenous and environmental insults.

Cholesterol synthesis is required for cutaneous barrier function in mice.

Previous studies have shown that topical acetone treatment results in the removal of stratum corneum lipids and disruption of the permeability barrier. This disruption stimulates epidermal lipid synthesis which is associated with the rapid restoration of stratum corneum lipids and barrier function. The aim of this study was to determine the role of cutaneous cholesterol synthesis in the barrier recovery. Here we show that topical lovastatin, a competitive inhibitor of HMG CoA reductase, inhibits cholesterol synthesis. After acetone disruption of the barrier, the normal rapid return of cholesterol to the stratum corneum and recovery of barrier function is impaired in animals treated topically with lovastatin. When lovastatin animals are simultaneously treated topically with either mevalonate, the immediate product of HMG CoA reductase, or cholesterol, the final end product of the pathway, the recovery of the barrier is normalized. Lovastatin resulted in the delayed secretion and abnormal appearance of lamellar bodies. These results provide the first evidence demonstrating that cholesterol synthesis is required for the maintenance of barrier structure and function and suggests a crucial role for cholesterol synthesis in allowing for terrestrial existence.

Stratum corneum lipids in disorders of cornification. Steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis.

The pathological scaling in recessive x-linked ichthyosis is associated with accumulation of abnormal quantities of cholesterol sulfate in stratum corneum (J. Clin. Invest. 68:1404-1410, 1981). To determine whether or not cholesterol sulfate accumulates in recessive x-linked ichthyosis as a direct result of the missing enzyme, steroid sulfatase, we quantitated both steroid sulfatase and its substrate, we quantitated both steroid sulfatase and its substrate, cholesterol sulfate, in different epidermal strata, as well as within stratum corneum subcellular fractions obtained from normal human and neonatal mouse epidermis and from patients with recessive x-linked ichthyosis. In normal human and mouse epidermis, steroid sulfatase activity peaked in the stratum granulosum and stratum corneum, and negligible activity was detectable in lower epidermal layers. In contrast, in recessive x-linked ichthyosis epidermis, enzyme levels were virtually undetectable at all levels. In normal human stratum corneum, up to 10 times more steroid sulfatase activity was present in purified peripheral membrane preparations than in the whole tissue. Whereas in normal human epidermis cholesterol sulfate levels were lowest in the basal/spinous layer, and highest in the stratum granulosum, in recessive x-linked ichthyosis the levels were only slightly higher in the lower epidermis, but continued to climb in the stratum corneum. In both normal and in recessive x-linked ichthyosis stratum corneum, cholesterol sulfate appeared primarily within membrane domains, paralleling the pattern of steroid sulfatase localization. Finally, the role of excess cholesterol sulfate in the pathogenesis of recessive x-linked ichthyosis was directly tested by topical applications of this substance, which produced visible scaling in hairless mice in parallel to an increased cholesterol sulfate content of the stratum corneum. These results demonstrate an intimate relationship between steroid sulfatase and cholesterol sulfate in normal epidermis: both are concentrated in the outer epidermis (stratum corneum and stratum granulosum), and both are localized to membrane domains. Presumably, as a result of this distribution pattern, continued enzymatic degradation of substrate occurs in normal epidermis, thereby preventing excessive accumulation of cholesterol sulfate. In contrast, in recessive x-linked ichthyosis, degradation of cholesterol sulfate does not occur and cholesterol sulfate accumulates specifically in the stratum corneum, where it produces visible scale.

The permeability barrier in essential fatty acid deficiency: evidence for a direct role for linoleic acid in barrier function.

Essential fatty acid (EFA) deficient rodents demonstrate abnormal epidermal permeability barrier function and differentiation, defects which can be corrected by either topical or systemic administration of linoleic acid. Since linoleic acid is a precursor of prostaglandins, correction of the defect in barrier function may either reflect a prostaglandin-mediated return toward normal epidermal differentiation, or, instead, a direct effect of linoleic acid. To test these possibilities severely EFA-deficient mice were pretreated daily with indomethacin and/or 5,8,11,14-eicosatetrayeonic acid, and then placed on normal (lineolic acid-supplemented) diets. Endogenous formation of prostaglandin E2 was determined by thin-layer chromatography after transformation into prostaglandin B2 with ethanolic-hydrochloric acid. Animals treated with both indomethacin and TYA DEMONSTRATED SUBSTANTIAL REDUCTIONS IN PROSTAGLANDIN E2 levels in liver and skin. Animals replenished with linoleic acid invariably demonstrated a rapid return of barrier function toward normal whether or not they were blockaded, while nonreplenished animals, with or without inhibition of prostaglandin biosynthesis, demonstrated continued deterioration in barrier function. In other experiments, topically applied linoleic acid rapidly reversed the defect in barrier function at the sites of application prior to systemic correction of the EFA deficient state. These results suggest that: (1) defective cutaneous barrier function in EFA deficiency can be corrected locally without prior systemic reversal of the deficiency state; and (2) that linoleic acid may play a direct role in the epidermal permeability barrier independent of its role in prostaglandin metabolism.

Percutaneous transport in relation to stratum corneum structure and lipid composition.

Despite the acknowledged importance of the stratum corneum in limiting water loss and in controlling skin permeability, the basis for these functions remains unknown. To pinpoint those factor(s) of importance for cutaneous barrier function, we correlated the thickness, number of cell layers, and lipid composition of leg vs. abdominal stratum corneum samples with penetration of 3H-water and 14C-salicylic acid across the same tissue sample. Viable upper epidermal sheets were obtained by incubating fresh autopsy or amputation full-thickness skin with staphylococcal exfoliatin. Each sheet was divided into 3 portions. The first piece was mounted in a diffusion cell for penetration studies. The second stratum corneum sample was frozen sectioned, stained with the fluorochrome, ANS, and measured with a micrometer eyepiece. The 3rd piece was pooled with other leg (n = 6) and abdomen (n = 15) specimens for determination of lipid weight percent. In all cases, leg stratum corneum was congruent to 2 times more permeable than abdominal stratum corneum to water and slightly more permeable to salicylic acid, as well. Penetration of both substances correlated inversely with lipid weight % of leg (mean = 3.0%) vs. abdomen (mean = 6.8%), but neither the penetration of water nor of salicylic acid was influenced by the number of cell layers or the thickness of the stratum corneum. We conclude that: differences in the thickness and the number of cell layers in the stratum corneum are insufficient to account for differences in percutaneous transport across leg and abdomen, and that total lipid concentration may be the critical factor governing skin permeability.

Membrane structural alterations in murine stratum corneum: relationship to the localization of polar lipids and phospholipases.

During the formation of the mammalian epidermal permeability barrier, lipids are sequestered in the stratum corneum intercellular spaces, transforming from a relatively polar lipid mixture to predominantly nonpolar species. Certain lipid catabolic enzymes, which co-localize with these lipids, may regulate this process. In order to localize the sites within the outer epidermis where polar lipids are catabolized, and their relationship to the alterations in membrane structure that occur in these layers, we compared the biochemical localization of polar lipids, the ultrastructure, and freeze-fracture morphology, as well as the localization of phospholipases within the outer epidermis. Both histochemical staining of frozen sections and biochemical studies of protease- and tape-stripped whole stratum corneum demonstrated small amounts of polar lipids in the stratum compactum, while in contrast, the stratum disjunctum was devoid of both phospholipids and glycosphingolipids. Phospholipase activity was present within lamellar bodies, among secreted lamellar body disks at the granular-cornified layer interface, and within the intercellular spaces of the stratum compactum. Both the depletion of polar lipids from the stratum compactum and deletion of these substances from the stratum disjunctum correlated with sequential changes in membrane structure observed by transmission electron microscopy and freeze-fracture. Thus, a phospholipase-mediated attack on phospholipids (with a parallel assault by other lipid catabolic enzymes on other polar species), may induce both the initial fusion and elongation of lamellar body disks and the subsequent formation of the hydrophobic membrane bilayers found in the mid-to-outer stratum corneum. These studies also may require modification of traditional views of the stratum corneum as a metabolically inert tissue, revealing its intercellular lipid domains to be partially in an active state of flux.

Effect of essential fatty acid deficiency on cutaneous sterol synthesis.

The fact that the skin is a major site of total body sterologenesis, coupled both with the apparent absence of low density lipoprotein receptors on keratinocytes and with the lack of influence of serum cholesterol on epidermal sterologenesis, has created the impression that epidermal lipid synthesis might be autonomous, i.e., nonregulatable. Recent studies have shown, however, that disruption of cutaneous barrier function with acetone or detergents stimulates epidermal sterologenesis (J Lipid Res 26:418-427, 1985). To correlate further sterologenesis with barrier function, we measured de novo synthesis of cholesterol and total nonsaponifiable lipids in essential fatty acid-deficient (EFAD) hairless mice. Animals with defective barrier function, manifested by abnormal transepidermal water loss, demonstrated a 2-fold increase in epidermal cholesterol and total nonsaponifiable lipid synthesis over controls while synthesis in the dermis was unchanged. Epidermal sterologenesis in EFAD animals, repleted with linoleic acid either systematically or topically, returned toward normal as barrier function improved. Moreover, plastic occlusion of EFAD mouse skin normalized epidermal sterologenesis at 1 and 3 days. These results provide further evidence that epidermal sterologenesis is not entirely autonomous, and can be regulated by water barrier requirements.

Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice.

The epidermis of aged mice displays decreased stratum corneum (SC) lipid content and decreased extracellular bilayers, which result in impaired barrier recovery following the solvent treatment or tape stripping. We assessed the role of altered lipid synthesis as the cause of the abnormal barrier and lipid content in aged epidermis, both under basal conditions and in response to acute barrier perturbations. In aged epidermis ( > or = 18 months), synthesis of one of the three key lipid classes (cholesterol) is decreased under basal conditions, and sterologenesis fails to attain the levels reached in young epidermis following comparable acute perturbations. In contrast, fatty acid and sphingolipid synthesis in aged epidermis increase sufficiently to approach the levels attained in stimulated young epidermis. The abnormalities in sterologenesis in aged epidermis are paralleled by a decrease in activity of its rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, under basal conditions, and enzyme activity also fails to increase as much as in young epidermis after barrier disruption. That defective lipid generation contributes to the barrier defect is shown directly by the ability of either a cholesterol-containing mixture of SC lipids or cholesterol alone to enhance barrier recovery. Finally, lipid-induced acceleration of barrier recovery in aged epidermis correlates with repletion of the extracellular spaces with normal lamellar structures. Thus, a deficiency in lipid synthesis, particularly in cholesterologenesis, accounts for the barrier abnormality in aged epidermis.

Glucosylceramides stimulate mitogenesis in aged murine epidermis.

Glucosylceramides (GlcCer) and ceramides (Cer) appear to have opposite effects on epidermal growth and differentiation. Whereas Cer inhibit mitosis and induce terminal differentiation and apoptosis in cultured keratinocytes, GlcCer is mitogenic in young murine epidermis. Using a recently described murine model of chronologic senescence we explored whether GlcCer is mitogenic in aged epidermis. Epidermal GlcCer content increases following topical applications of either conduritol-B epoxide (CBE), an inhibitor of GlcCer hydrolysis, or exogenous GlcCer in a penetration-enhancing vehicle. During chronologic aging in the hairless mouse, baseline epidermal DNA synthesis rates remain normal until 18 mo, but decline significantly at 24 mo. Topical CBE stimulates a 1.5- to 1.9-fold increase in epidermal DNA synthesis in all age groups (i.e., 1-2, 18, and 24 mo). Although the CBE induced increase in [3H]thymidine incorporation in 24 mo old animals is significant (p < 0.01), it is not sufficient to reach the absolute levels reached in similarly treated, younger mouse epidermis. Moreover, topical GlcCer induced mitogenesis is both dose dependent and hexose specific in young (1-2 mo old) animals, and remains effective in aged (< or = 24 mo old) animals. Furthermore, the CBE induced increase in DNA synthesis in aged epidermis is sufficient to produce epidermal hyperplasia. Finally, although an increased GlcCer:Cer ratio can alter stratum corneum barrier function and membrane structure, neither stratum corneum function nor extracellular membrane structure change under these experimental conditions, and therefore the mitogenic effects of increased epidermal GlcCer cannot be attributed to effects on the stratum corneum. These results show that: (i) elevations in endogenous GlcCer are mitogenic for aged as well as young murine epidermis; (ii) topical GlcCer is also mitogenic when delivered in an enhancing vehicle; and (iii) despite the putative importance of epidermal DNA synthesis for barrier homeostasis, these mitogenic alterations do not alter stratum corneum function.

Localization of de novo sterologenesis in mammalian skin.

Previous studies have demonstrated that the skin is an important site of de novo sterol synthesis and that there is a sex difference in cutaneous sterologenesis with male animals synthesizing more sterols than females. The aim of the present study was to localize the major sites of sterol synthesis within the skin and to determine which of these sites accounted for the sex differences in sterologenesis. In male and female rats whose dermal and epidermal layers are separated by dithiothreitol treatment, the dermis is the major site of skin sterologenesis (males 86% of total, females 82% of total). Moreover, the sex difference in skin sterol synthesis is quantitatively localized primarily within the dermal layer (approximately 2.5-fold greater in the dermis of males). Sterol synthesis is also increased in the epidermis of males. To rule out the possibility that sebaceous gland production accounted for our observations, we treated animals with isotretinoin (13-cis-retinoic acid), a drug that suppresses sebaceous gland sebum production. Sterol synthesis in the skin of both male and female rats is not significantly altered by isotretinoin administration and the sex difference in skin sterologenesis is unaffected. To further localize the sites of sterol synthesis within the skin, studies of hairless mice whose skin was split by DTT were initiated. In hairless mice, DTT separates the epidermis into upper (stratum corneum and granulosum) and lower (basal and spinous cells) strata. The basal layer was separated from the dermis by gentle scraping. As in rats, the dermis is the chief site of sterol synthesis in the skin. In addition, the lower layer of the epidermis (basal and spinous cells) is also a very active site of sterologenesis, accounting for 20% of total skin nonsaponifiable lipid synthesis. The upper epidermis accounted for only a small portion of total skin synthesis. It is highly likely that the bulk of cutaneous sterol synthesis occurs in the pilosebaceous epithelium.

The lovastatin-treated rodent: a new model of barrier disruption and epidermal hyperplasia.

Recent studies have linked epidermal cholesterol synthesis with maintenance of the permeability barrier. To assess directly the importance of cholesterol synthesis, we applied lovastatin, a potent inhibitor of cholesterol synthesis, to hairless mouse skin. Transepidermal water loss (TEWL) began to increase after four to six daily applications. Co-application of cholesterol blocked the expected increase in TEWL, demonstrating the importance of cholesterol for development of the lesion. The histology of lovastatin-treated skin revealed epidermal hyperplasia, accompanied by accelerated DNA synthesis. Whereas cholesterol synthesis initially was reduced in lovastatin-treated epidermis, with further treatment cholesterol synthesis normalized, while fatty acid synthesis accelerated greatly. Although the total free sterol content of lovastatin-treated epidermis remained normal, the fatty acid content increased coincident with barrier disruption. Finally, morphologic abnormalities of both lamellar body structure and their deposited, intercellular contents occurred coincident with the emerging biochemical abnormalities. Thus, the abnormal barrier function in this model can be ascribed to an initial inhibition of epidermal sterol synthesis followed by an alteration in cholesterol and fatty acid synthesis, leading to an imbalance in stratum corneum lipid composition and abnormal membrane bilayer structure.

Antimicrobial activity of stratum corneum lipids from normal and essential fatty acid-deficient mice.

Among the cutaneous effects of an essential fatty acid deficient (EFAD) diet are hyperdesquamation, increased transepidermal water loss (TEWL), and altered lipid profiles, characteristics also common to inflammatory dermatoses. Because fatty acids are antimicrobial, we examined the indigenous skin flora of normal and EFAD hairless mice, and compared the antimicrobial efficacy of lipids extracted from their stratum corneum. EFAD mice supported 100-fold more bacteria than normal mice, and were the only group from which Staphylococcus aureus were routinely isolated. Despite this greater carriage, in vitro experiments demonstrated that EFAD lipids are more lethal than normal lipids against Streptococcus pyogenes, S. aureus, S. epidermidis, Micrococcus sp., and a coryneform. Skin fungi were equally susceptible to both extracts. After thin layer chromatography, the most active fractions were found to be glycosphingolipids and phospholipids. EFAD extracts had 35% more free fatty acids and 75% more glycosphingolipids; normal extracts had more triglycerides and phospholipids. S. aureus strain 502A survived equally well on EFAD as on normal mice. Normal lipids applied on EFAD mice had no additional effect, but EFAD lipids on normal mice brought about a 35% reduction of the inoculated bacteria. If the mice were pretreated with alcohol, carriage of strain 502A was reduced by 71%. If instead the mice were previously washed with acetone to increase TEWL, a 97% reduction of the staphylococcus occurred. The application of normal flora to such acetone-washed mice decreased the efficacy to 76%. EFAD and normal lipids on human subjects were equally ineffective in eliminating strain 502A.(ABSTRACT TRUNCATED AT 250 WORDS)

Stratum corneum lipid abnormalities in ichthyosis. Detection by a new lipid microanalytical method.

Although the biochemical diagnosis of the ichthyoses is still in its infancy, the two recessively inherited types, recessive X-linked ichthyosis (RXLI) and nonbullous congenital ichthyosiform erythroderma (CIE), are accompanied by stratum corneum lipid abnormalities. However, in RXLI, cholesterol sulfate accumulates; in CIE, massive quantities of n-alkanes accumulate. The diagnosis of these disorders has required large quantities of scale for sequential, quantitative thin-layer chromatography (TLC). In this study, we sought to confirm the previously described lipid abnormalities with the use of a rapid, recently developed microchromatographic technique that employs silica gel-coated quartz rods and flame ionization detection (Iatroscan). The cholesterol sulfate content of RXLI (n = 5) scale and the n-alkane content of CIE (n = 8) scale were determined by both TLC and the microchromatographic technique. Less than 10 mg of scale and even single punch biopsy specimens sufficed for the microchromatographic technique, whereas more than 50 mg of scale were required for TLC. Since the microchromatographic technique can rapidly detect diagnostic biochemical abnormalities from readily obtainable, small tissue samples, this method could eventually supplant or supplement standard lipid biochemical techniques for the diagnosis of cutaneous lipidoses.

Exogenous nonphysiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction.

BACKGROUND AND DESIGN: Although barrier function requires cholesterol, free fatty acids, and ceramides, applications of one or two of these lipids to damaged skin impedes barrier recovery, while equimolar mixtures allow normal recovery. Both incomplete and complete mixtures appear to be internalized within the epidermal nucleated layers, followed by the secretion of abnormal vs normal lamellar body contents, respectively. We compared the ability of complete physiologic lipid mixtures vs a nonmetabolized hydrophobic lipid, petrolatum, to repair the barrier and the requirement for intracellular processing of these lipids within the epidermis. RESULTS: Neat petrolatum, which remains restricted to the stratum corneum, produces more rapid improvement in barrier function than the solvent-dispersed physiologic lipids, and its effects are not altered by coapplication of either monensin or brefeldin A (both from Sigma Chemical Co, St Louis, Mo), known inhibitors of exocytosis and organellogenesis, respectively. In contrast, the physiologic lipids enter the nucleated layers in substantial amounts and require longer to produce barrier recovery. Whereas monensin blocks their ability to facilitate barrier recovery, the physiologic lipids overcome brefeldin A-induced delays in barrier recovery, bypassing the subcellular site of brefeldin A blockade, normalizing both lamellar body contents and intercellular bilayers. CONCLUSIONS: While petrolatum remains restricted to the stratum corneum, physiologic lipid mixtures influence barrier recovery after transport to subjacent, nucleated layers, followed by internalization, apparent transport to the distal Golgi apparatus, and incorporation into nascent lamellar bodies.