strategy to improve skin barrier function and repair
Rißmann, R.
Citation
Rißmann, R. (2009, March 17). Development of a vernix caseosa substitute : a novel strategy to improve skin barrier function and repair. Retrieved from https://hdl.handle.net/1887/13664
Version: Not Applicable (or Unknown)
License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/13664
Note: To cite this publication please use the final published version (if applicable).
General introduction
Introduction
Birth is a milestone in human adaptation to terrestrial life. The body undergoes a substantial change from an aqueous, warm and sterile surrounding into a gaseous, cold and xenobiotic-containing environment. Along with other organs of the body, the skin needs to adapt to this major transition [1]. The skin plays a key role in protecting the body; it separates the internal, vital organs from the external environment and comprises the main barrier. During birth, the dramatic change in the environment proceeds with the maturation and development of the skin-surface material vernix caseosa (VC), a creamy-white and viscous biofilm (Fig. 1). VC is produced during the last trimester of gestation on the foetal skin surface [2].
The exact role of VC in the adaptation process of the skin is still discussed but several, unique properties of VC have been addressed in literature [3-5].
Therefore, it has been suggested that this natural biofilm has great beneficial potential for underdeveloped skin of preterm infants, barrier-deficient or diseased skin of the adult population [5-7]. However, its clinical application is restricted due to the limited availability and the risk of transmission of diseases [8]. Therefore, the design and generation of synthetic biofilms which closely mimic the composition and properties of natural VC has been proposed [5, 6, 8].
These innovative biofilms may promote barrier repair processes of burned or injured skin in a similar way as suggested for native VC [5]. Furthermore, the novel biofilms contain lipids similar to the uppermost layer of the skin, the stratum corneum (SC), which might be of benefit for the treatment of dry or diseased skin.
This thesis describes the rational design of biofilms aiming to mimic closely the properties of VC. In the first part of the introduction, brief descriptions of the skin structure and the development of the epidermal barrier are provided. The second main part of the introduction portrays VC, its composition, ultrastructure and biological role. Then the characterization of the epidermal barrier at birth and possible applications of biofilms are described. Finally, the objectives and the outline of this thesis are presented.
Figure 1. The creamy white biofilm vernix caseosa covers the skin of the newborn (photo of Tristan Le Dévédec). During the last trimester of gestation it covers the skin of the human fetus to various degrees and after delivery it dries spontaneously.
The skin barrier
The skin is the largest organ of the human body, it is between 1.5 and 2 m2 in size and constitutes approximately 10% of the body mass [9]. It comprises the main physical barrier between the internal organs and the external environment.
Despite the barrier function, it also plays a crucial role in thermoregulation, perception of heat and pain and has an important sensory and appearance function [10]. It is a multilayered structure and consists of three main layers from inside to outside: the hypodermis, the dermis and the epidermis. The hypodermis, also known as subcutaneous fat tissue, consists mainly of adipocytes (fat cells) and has an important role in energy storage and metabolism, thermal insulation and support of the other skin layers [11]. Above the hypodermis lies the dermis, a layer formed by fibrous, filamentous and amorphous connective tissue that serves as supporting tissue. In this layer also hair roots, sweat glands, nerve fibres and blood vessels are present, the latter supplying the skin with oxygen and nutrients [11]. The uppermost layer of the skin is the epidermis and consists of a viable and a dead part. The most abundant cells are keratinocytes. From the stratum basale, the deepest layer of the epidermis, these cells migrate towards the surface of the skin. During this process, differentiation of the cells takes place which results finally in a complete cornification of the keratinocytes. At this stage, the cells that are called corneocytes, are devoid of all cell organelles (e.g. nucleus and cytoplasm) and form together with the intercellular lipid matrix the uppermost layer of the epidermis, the SC. The structure of SC is also compared to a wall-like structure,
since regularly arranged corneocytes represent the bricks and the lipids stand for the mortar [12]. The corneocytes are linked together by the corneodesmosomes, protein regions spanning the intercellular regions. The hydrophilic corneocytes have a characteristic hexagonal, flat shape and are typically surrounded by a tight, cross-linked structure, the cornified envelope [13]. To the surface of the envelope proteins, -hydroxyceramides are covalently bound and a lipid–
protein complex is formed [14, 15]. The major constituents of the intercellular, free lipids are the barrier lipids: cholesterol (CHOL), free fatty acids (FFA) and ceramides (CER), typically present at approximately equimolar ratios [16]. The CER are sphingolipids consisting of a non-hydroxy (N), -hydroxy (A) or - hydroxy (O) fatty acid linked to a base of either sphingosine (S), phytosphingosine (P) or 6-hydroxy sphingosine (H) (Fig. 2).
Figure 2. Molecular structures of free ceramides (CER) present in human stratum corneum (adapted from [17]) with the number based system according to their chromatographic behaviour.
Furthermore, the letter-based terminology by Motta et al. [18] is presented: the structures are classified according to the sphingoid base (S - sphingosine, P – phytosphingosine, H - 6- hydroxysphingosine) and the N-acyl fatty acid (A - -hydroxy group, O - -hydroxy group, N – non- hydroxy group, E - esterified in -hydroxy position).
Remarkably, the acyl-CER (EOS, EOH, EOP) consist of unusual chains comprising an unsaturated linoleic acid moiety which is esterified (E) to a long
–hydroxy fatty acid (O). The free fatty acids of SC consist mainly of long and saturated chains whereas low levels of oleic and linoleic acid represent the only unsaturated fatty acids [19].
It is generally accepted that the lipid organization plays a key role for the barrier function of the skin [20]. The intercellular lipids of SC from human, pig and mouse are highly organized and form crystalline lamellae with a repeat distance of approximately 6 and 13 nm referred to as short periodicity phase and long periodicity phase (LPP), respectively [21-23]. This lipid organization is very characteristic for the SC [24-26]. The lipid composition and organization are key factors for the barrier properties of the SC and various studies indicated that lipid abnormalities in SC are also observed in diseased skin, e.g. ichthyosis, psoriasis and atopic dermatitis [27-29].
Development of the epidermal barrier in utero
In terrestrial life, the skin is mainly exposed to air whereas during the foetal stage, the skin is developing completely surrounded by the amniotic fluid. 7-8 days after conception, the first single-layered, primitive epidermis is identified whereas two distinct layers of epithelium – the periderm and a layer of basal keratinocytes – are developed in week 5 of gestation [30]. The periderm serves as protective barrier for the epithelial maturation underneath. At the end of the second month ‘intermediate cells’ are formed on the foetal skin surface. The epidermal differentiation is completed (i.e. all epidermal layers are present) at the end of the second trimester and simultaneously the periderm is sloughed into the amniotic fluid [30]. At the same time SC development and thereby the barrier formation in utero commences. This coincides also with the formation of VC [31].
At first only thin SC is noticed but during the last trimester the thickness increases gradually [32]. Based on transepidermal water loss (TEWL) and percutaneous absorption studies, almost complete activity of the skin barrier has been reported between gestation week 30 [33], week 34 [34] and week 37 [35]. In contrast, other literature suggests that infants born at term still need time to develop the full functionality of the skin barrier [36]. However, the development of the epidermal barrier is patterned and typically occurs at first around the hair follicles, i.e. pilosebaceous apparatus [4, 7]. Under aqueous conditions, the skin development is biologically challenging, since adult skin exhibits damage of the skin barrier function under these conditions [37]. Similarly, human skin cultures show incomplete stratification of the keratinocytes under submerged conditions [38].
Vernix caseosa
To our knowledge, VC is unique for the human fetus. VC consists of approximately 80% water, 10% proteins and 10% lipids [2, 6]. The amount of VC present on the skin before and after birth correlates with the gestational age [39].
VC is a semi-solid material and is relatively intractable after birth [8]. The term vernix caseosa derives from “veronix” which means fragrant resin (Latin) [40].
Furthermore, the word ”varnish” signifies to cover with a coating, whereas caseosa derives from “caseous” meaning “cheesy” or “cheese-like”.
Ultrastructure
VC consists of hydrophilic, dead corneocytes which probably originate from the fetus hair follicle i.e. the pilosebaceous apparatus. VC has structurally similarities to SC as the corneocytes are embedded in a lipid matrix [41]. A schematic drawing of VC’s structure is presented in figure 3. Structurally, the corneocytes are of ovoid, polygonal shape and are typically larger than corneocytes of SC and vary in size between 5 and 50 μm in diameter [41]. Further ultrastructural investigation of VC by transmission and cryo-scanning electron microscopy revealed additional features: I) intercellular connections (desmosomes) are lacking, II) the water is mainly localized in the corneocytes and III) a layered orientation of the lipids was occasionally observed [2]. However, VC lacks desmosomes and the more viscous appearance suggested the view of VC as a “fluid or mobile phase” SC [2].
Figure 3. The schematic drawing of the ultrastructure of vernix caseosa depicts corneocytes (dead cells) which are embedded in a lipid matrix (grey). The hydrophilic, water containing corneocytes are of longitudinal, polygonal shape and are surrounded by a tight, cross-linked protein structure – the cornified envelope (thick black line).
intercellular lipids cornified
envelope hydrated
corneocyte
Composition of VC a. Lipids
VC consists of a wide range of lipids with different polarity. One can distinguish the sebum-derived nonpolar lipids and the more polar barrier lipids. Haahti et al.
[42] and Kaerkkaeinen et al. [43] provided the first information on the free lipid composition in VC. Major fractions could be identified containing sterol esters (SE) and triglycerides (TG). In addition, it was shown that the fatty acids of individual lipid classes, such as SE, wax esters and TG, consist of straight and methyl branched fatty acids [43]. Subsequent studies confirmed these results [44] and provided information on the double bond positions and more detailed information on the fatty acid composition [45]. Hoeger et al. [6] found that all barrier lipids of SC, i.e. CHOL, FFA and CER, are present in VC but in lower levels than in SC. In VC also all the CER, that are also found in SC, are present.
However, in addition to the SC CER, two new acyl-CER were identified [46].
Because of the presence of the common sebum marker squalene [47] VC lipids were first thought to be exclusively originating from sebaceous glands. However, the presence of barrier lipids clearly indicates that VC lipids also derive from epidermal lipids.
b. Peptides and proteins
The foetal corneocytes of VC are mainly composed of keratin, the protein that forms the scaffold of the corneocytes. Hydrolysis of the proteins revealed an abundant presence of asparagine and glutamine in VC [48]. This latter is of particular interest, because when VC is swallowed [49], glutamine is suggested to be involved in the foetal gut development [50]. Furthermore, several reports on the antimicrobial activity of VC have been published showing that the main families of mammalian antimicrobial peptides, the cathelicidins and the defensins are present in VC [51-53]. The human antibacterial peptide LL-37 and lysozym have also been demonstrated to be present [19]. Moreover, the collectin associated surfactant proteins A and D were found [49]. Complete proteomic analysis of VC was reported by Tollin et al. [54]. This study confirmed the presence of antibiotics and showed also the presence of peptides which control the innate immune function.
Role and functions of VC
Ample biological functions have been described for VC. Already in the 1940s studies showed that addition of VC to the food of tadpoles is followed by a marked acceleration of growth and metamorphosis suggesting that VC has beneficial effects as the first nutrition of the fetus [55]. More recently, multiple biological functions before, during and after birth were reported. The
hydrophobic nature of VC, that has been reported based on measurements of the surface free energy [56], suggests that in utero VC exhibits waterproofing properties which presumably facilitate the formation of the skin under submerged conditions. VC’s function as a protectant was underlined by showing that it prevents excessive penetration of the chymotrypsin from the amniotic fluid into the skin [57]. During delivery VC acts as a lubricant and reduces the friction, while it exhibits anti-infective properties postnatally. The latter might be due to I) mechanical obstruction of bacterial passage [58] and II) the presence of antimicrobial peptides, proteins and branched fatty acids [51, 59]. The anti- oxidant -tocopherol is also known to be present [60] which indicates that VC might also be able to reduce and compensate for oxidative stress of the skin at birth. Skin cleansing properties were reported [3] as well as the importance of VC for hydration and acid mantle development of the newborn’s skin [4]. The high level of water present in VC makes it also an excellent natural moisturizing cream [2]. Upon application of VC on healthy skin of human volunteers, an increased water loss and a temporal change in the skin surface hydration was observed which underlines that VC modifies the moisture gradient within the skin [61].
Application of VC on trophic ulcers of the lower extremities in humans gave evidence that it also possesses wound healing properties [62]. These multiple biological functions clearly suggest that VC has a very fundamental role in adaptation for the rapidly changing environment of the skin during birth.
Moreover, these unique properties of VC point out that the generation of biofilms which mimic these properties could have beneficial potential for different skin disorders.
Barrier of the (pre)term infant
In very low birth weight infants, born before 32 weeks of gestation, an increased frequency of complications in fluid balance, temperature control, infections and trauma have been reported [63]. All these symptoms can be explained by a poorly developed skin barrier due to an underdeveloped SC which leads to a reduced barrier function [64]. Preterm infants are also lacking VC [63] and nosocomial and community acquired infections occur more often [5] attributable to the inadequate developed epidermal barrier. These infants showed significantly higher TEWL which indicates a reduced barrier of the epidermis compared to term infants [65].
The thinner epidermis allows penetration of drugs or xenobiotica at higher rates than observed for fully developed adult skin [66, 67]. In contrast, infants born at full term have a fully developed skin barrier as TEWL and permeability are even lower than in adult skin [65]. Furthermore, their epidermis is characterized by similar thickness and appearance as adult skin [64]. In summary, babies born at
term are characterized by a fully developed and competent SC barrier. This is surprising since the fetus (skin) is exposed to amniotic fluid during its entire prenatal development and adult skin exhibits quickly signs of barrier damage after exposure to water for an extended period of time [37, 68]. However, for the underdeveloped skin barrier of preterm infants natural VC and the synthetic biofilms might be of great benefit [6], since the high water loss from the body could be prevented while the moisture is retained within the skin.
Functionality of biofilms for skin treatment
The optimal water level of the SC is known to be of importance for skin (barrier) function [69]. An imbalance of moisturization can be caused by several environmental factors (chemicals, microbes or weather conditions), diseases or premature birth. The activity of enzymes within the SC is disturbed which can lead to abnormal desquamation or disturbed SC maturation [70]. To prevent or cure pathological skin conditions with reduced water levels in the skin, topical application of formulations is a standard treatment [71]. One distinguishes (water- free) ointments and (water-containing) creams or so called emollients or barrier creams. Typically, besides water also moisturizers, i.e. substances that increase SC moisture level by penetrating into the skin, are present in the formulation. One classical example of a moisturizer is the hygroscopic molecule glycerol which is a standard hydrophilic moisturizer [72]. Another treatment strategy of increasing SC hydration is the application of occluding ointments such as the oil-based ointment Vaseline [73]. Due to long-term occlusion of the skin, Vaseline treatment can lead to SC over-hydration. This might cause SC perturbation [37] or prolonged skin barrier damage [74]. However, also the non-occluding ointments or barrier creams suffer from an uncontrolled water release and might also cause too high water levels in the SC. The suboptimal enzyme activity will cause typical dry skin symptoms, i.e. itching, scaling and irritation [69] which is a major pitfall of current therapies.
With the development of synthetic biofilms which closely mimic structure and properties of natural VC, a novel generation of barrier creams will be created. The structure of highly hydrated (synthetic) corneocytes embedded in a lipid matrix will enable our biofilms to achieve a well defined water release. This may be of great advantage for dry or diseased skin as it is aiming to compensate for the low water levels in the SC of these skin disorders. Furthermore, the presence of barrier lipids in the synthetic biofilms will be of advantage because these lipids have been shown to promote the formation of SC in barrier disrupted skin [75, 76]. If this indeed will be observed, the synthetic biofilms might be applicable as additional layer on barrier-deficient skin e.g. for extremely low birth weight infants where
VC is absent [6, 63]. Furthermore, beneficial effects can be expected for applications in order to enhance wound healing in adult skin [5]. Skin disorders where imbalances occur either in lipid composition or moisturization level, might be also be targeted by the newly developed biofilms.
Aim of this thesis
The goal of this thesis is the rational design of synthetic biofilms mimicking the properties and structure of VC. In order to do so, the main objectives of the thesis can be defined as follows:
I) Thorough characterization of VC concerning (lipid) composition, ultrastructure and physicochemical properties as bases for the development of synthetic biofilms in a later stage of the project.
II) Design of lipid mixtures that mimic composition and organization of VC lipids.
III) Generation of synthetic corneocytes which closely resemble the hydrophilic natural counterpart. This particular research is conducted by M.H.M. Oudshoorn (Utrecht University) and resulted in a dissertation [77].
IV) Preparation of synthetic biofilms which are composed of the synthetic corneocytes and the selected lipid mixtures and subsequent characterization regarding homogeneity, stability, (ultra-) structure, rheological and water-handling properties in respect to natural VC.
V) Evaluation of the synthetic biofilms in vivo to prove their efficacy and potential applications.
Outline of this thesis
The thesis is organized in three experimental parts.
Part I – Detailed characterization of vernix caseosa (lipids)
The thorough characterization of VC and its lipids concerning lipid composition and organization, ultrastructure and temperature dependent features are described (Chapters 2-4).
Part II –Mimicking vernix caseosa (lipids)
The second main part of the thesis describes the development of synthetic biofilms which mimic the composition and properties of VC (Chapters 5 and 6).
To resemble the structure of VC as closely as possible, both, the VC lipids and the highly hydrated corneocytes, are imitated. The lipid mixtures are selected on the basis of similarities in lipid composition, lipid organization and thermotropic behaviour compared to the natural VC lipids. The synthetic corneocytes are prepared from derivatized hyperbranched polyglycerol and mimic closely the highly hydrated natural corneocytes. The synthesis of these polymers and the preparation of the particles are described in the thesis of M.H.M. Oudshoorn [77].
The synthetic biofilms are prepared by embedding the synthetic corneocytes into
the lipids. Characterization studies and comparison to natural VC properties are then performed.
Part III – In vivo studies to test the biological efficacy of natural and synthetic biofilm(s) All in vivo studies are described in the third part of this thesis (Chapters 7-9). A reliable model to study the biological effect of natural VC and its synthetic counterpart is developed with which VC is tested. Finally, the different synthetic biofilms, standard pharmaceutical ointments are being evaluated and compared to native VC using the optimized in vivo model.
The obtained results are summarized, discussed and put in perspective (Chapter 10).
A more detailed outline of the thesis is given below:
Although VC lipid composition has been extensively studied [6, 42-44], no integral data on the overall free and bound lipid composition are currently available. All studies performed until now focused either on the nonpolar free lipids sterol esters, wax esters and triglycerides or on the barrier lipids of VC.
Concerning the fatty acid distribution, very long chain fatty acids were often neglected as most of these studies were carried out focusing on acyl chain length distribution up to a length of 20 carbon atoms. Hence, a very detailed analysis of VC lipids is initially performed (Chapter 2) and secondly, the lipid organization of VC is investigated in more detail by small-angle X-ray diffraction (SAXD) and freeze-fracture electron microscopy. To gain more insights into the interior of the corneocytes, especially focusing on the water distribution, cryo-scanning electron microscopy is employed.
Changes in physicochemical properties of VC during birth are of great interest in order to better understand its role after birth and are important for the synthetic counterparts. We investigate the structural and physicochemical features in VC which accompany physiologically relevant variations in environment parameters such as temperature and humidity in Chapter 3. Temperature-dependent water- handling properties, rheological properties as well as the thermotropic phase behaviour of VC are reported.
As the main classes of SC barrier lipids are also present in VC, we explore whether VC lipids are also able to form the lipid lamellar phases present in SC (Chapter 4). In order to investigate this, the phase behaviour of VC lipid mixtures prepared with various methods is studied by SAXD. To mimic the physiological situation as closely as possible, no equilibration step at elevated temperatures is included.
Based on the knowledge of chapter 2 and 3, in the second part of the thesis, biofilms mimicking the structure and properties of VC are prepared. In a first step, suitable lipid candidates are selected and an optimization of lipid mixtures mimicking VC lipids in composition, organization and thermotropic phase behaviour is performed in Chapter 5. A series of semi-synthetic lipid mixtures comprising lanolin (wool wax) as primary compound are assessed and the most appropriate lipid mixture is selected.
In Chapter 6, the preparation of synthetic biofilms is described. Synthetic corneocytes [77] are embedded in the selected lipid mixture. The preparation and optimization of the biofilms concerning different particle/lipid ratios as well as different water contents of the synthetic corneocytes are evaluated.
Characterization of these biofilms is performed regarding homogeneity, stability, water-handling and rheological properties as well as thermotropic behaviour and compared to natural VC.
In the third part of this thesis, in vivo studies are described to generate a reliable in vivo model for skin barrier disruption and barrier recovery. Different levels of barrier disruption by tape stripping are assessed (Chapter 7). The most severe barrier disruption was selected as the best performing model to study barrier repair. This model was used to study the effect of VC.
In Chapter 8, the acetone-induced barrier disruption is described. We employed this model as it is often used as a barrier repair model. We focus on the effect of acetone treatment on lipid composition and organization as well as morphological changes in the SC.
Various selected biofilms, standard oil-based ointments and lipid formulations are investigated regarding their in vivo efficacy on the obtained mouse model (Chapter 9). The performance of the individual formulations is evaluated by scoring the redness of the disrupted skin, monitoring the TEWL and assessing the histological features of biopsies during barrier recovery.
Chapter 10 provides a summary on the obtained results and some future perspectives.
References
[1] Mauro T, Behne M. Acid Mantle. In: Neonatal Skin: Structure and Function (SB Hoath, HI Maibach and EK Boisits, eds), Marcel Dekker, New York 2003:pp. 47-58.
[2] Pickens WL, Warner RR, Boissy YL, Boissy RE, Hoath SB. Characterization of vernix caseosa: water content, morphology, and elemental analysis. J Invest Dermatol 2000 Nov;115(5):875-81.
[3] Moraille R, Pickens WL, Visscher MO, Hoath SB. A novel role for vernix caseosa as a skin cleanser. Biology of the neonate 2005;87(1):8-14.
[4] Visscher MO, Narendran V, Pickens WL, LaRuffa AA, Meinzen-Derr J, Allen K, et al. Vernix caseosa in neonatal adaptation. J Perinatol 2005 Jul;25(7):440-6.
[5] Haubrich KA. Role of Vernix caseosa in the neonate: potential application in the adult population. AACN Clin Issues 2003 Nov;14(4):457-64.
[6] Hoeger PH, Schreiner V, Klaassen IA, Enzmann CC, Friedrichs K, Bleck O. Epidermal barrier lipids in human vernix caseosa: corresponding ceramide pattern in vernix and fetal skin. Br J Dermatol 2002 Feb;146(2):194-201.
[7] Hoath SB, Pickens WL, Visscher MO. The biology of vernix caseosa. Int J Cosmet Sci 2006 Oct;28(5):319-33.
[8] Youssef W. Physical characterization of vernix caseosa: Implication for biological function.
Dissertation, University of Cincinnati, Cincinnati, OHIO, USA 2001.
[9] Moore L, Chien YW. Transdermal drug delivery: a review of pharmaceutics, pharmacokinetics, and pharmacodynamics. Crit Rev Ther Drug Carrier Syst 1988;4(4):285- 349.
[10] Williams AC, Barry BW. Skin absorption enhancers. Crit Rev Ther Drug Carrier Syst 1992;9(3-4):305-53.
[11] Schaefer H, Redelmeier T. In: Skin barrier Principles of percutaneous absorption, Karger AG, Basel 1996.
[12] Elias PM. Epidermal Lipids, Barrier Function, and Desquamation. Journal of Investigative Dermatology 1983;80:S44-S9.
[13] Odland GF. Structure of the skin. In: Physiology, Biochemistry and Molecular Biology of the Skin, ed Lowell A Goldsmith, Oxford University Press 1991:pp.3-62.
[14] Swartzendruber DC, Wertz PW, Madison KC, Downing DT. Evidence that the corneocyte has a chemically bound lipid envelope. J Invest Dermatol 1987 Jun;88(6):709-13.
[15] Wertz PW, Madison KC, Downing DT. Covalently bound lipids of human stratum corneum. J Invest Dermatol 1989 Jan;92(1):109-11.
[16] Coderch L, Lopez O, de la Maza A, Parra JL. Ceramides and skin function. Am J Clin Dermatol 2003;4(2):107-29.
[17] de Jager MW, Gooris GS, Dolbnya IP, Ponec M, Bouwstra JA. Modelling the stratum corneum lipid organisation with synthetic lipid mixtures: the importance of synthetic ceramide composition. Biochim Biophys Acta 2004 Aug 30;1664(2):132-40.
[18] Motta S, Monti M, Sesana S, Caputo R, Carelli S, Ghidoni R. Ceramide composition of psoriatic scale. Biochim Biophysic Acta 1993;1182:147-51.
[19] Ponec M, Gibbs S, Pilgram G, Boelsma E, Koerten H, Bouwstra J, et al. Barrier function in reconstructed epidermis and its resemblance to native human skin. Skin Pharmacol Appl Skin Physiol 2001;14 Suppl 1:63-71.
[20] Bouwstra JA, Ponec M. The skin barrier in healthy and diseased state. Biochim Biophys Acta 2006 Dec;1758(12):2080-95.
[21] Bouwstra JA, Gooris GS, Bras W, Downing DT. Lipid organization in pig stratum corneum.
J Lipid Res 1995 Apr;36(4):685-95.
[22] Bouwstra JA, Gooris GS, van der Spek JA, Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering. J Invest Dermatol 1991 Dec;97(6):1005-12.
[23] White SH, Mirejovsky D, King GI. Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study. Biochemistry 1988 May 17;27(10):3725-32.
[24] Bouwstra JA, Gooris GS, Dubbelaar FE, Weerheim AM, Ijzerman AP, Ponec M. Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J Lipid Res 1998 Jan;39(1):186-96.
[25] de Jager M, Groenink W, Bielsa i Guivernau R, Andersson E, Angelova N, Ponec M, et Bouwstra JA. A novel in vitro percutaneous penetration model: evaluation of barrier properties with p-aminobenzoic acid and two of its derivatives. Pharm Res 2006 May;23(5):951-60.
[26] Swartzendruber DC. Studies of epidermal lipids using electron microscopy. Semin Dermatol 1992 Jun;11(2):157-61.
[27] Elias PM, Williams ML, Maloney ME, Bonifas JA, Brown BE, Grayson S, et al. Stratum corneum lipids in disorders of cornification. Steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis. The Journal of clinical investigation 1984 Oct;74(4):1414-21.
[28] Fartasch M. Epidermal barrier in disorders of the skin. Microscopy research and technique 1997 Aug 15;38(4):361-72.
[29] Yamamoto A, Serizawa S, Ito M, Sato Y. Stratum corneum lipid abnormalities in atopic dermatitis. Archives of dermatological research 1991;283(4):219-23.
[30] Holbrook KA. Structure and Function of the Developing Human Skin. In: Physiology, Biochemistry and Molecular Biology of the Skin, ed Lowell A Goldsmith, Oxford University Press 1991:pp. 63-110.
[31] Hardman MJ, Moore L, Ferguson MW, Byrne C. Barrier formation in the human fetus is patterned. J Invest Dermatol 1999 Dec;113(6):1106-13.
[32] Chiou YB, Blume-Peytavi U. Stratum corneum maturation. A review of neonatal skin function. Skin Pharmacol Physiol 2004 Mar-Apr;17(2):57-66.
[33] Kalia YN, Nonato LB, Lund CH, Guy RH. Development of skin barrier function in premature infants. J Invest Dermatol 1998 Aug;111(2):320-6.
[34] Evans NJ, Rutter N. Development of the epidermis in the newborn. Biology of the neonate 1986;49(2):74-80.
[35] Visscher MO, Chatterjee R, Munson KA, Pickens WL, Hoath SB. Changes in diapered and nondiapered infant skin over the first month of life. Pediatr Dermatol 2000 Jan-Feb;17(1):45- 51.
[36] Harpin VA, Rutter N. Barrier properties of the newborn infant's skin. J Pediatr 1983 Mar;102(3):419-25.
[37] Warner RR, Stone KJ, Boissy YL. Hydration disrupts human stratum corneum ultrastructure. J Invest Dermatol 2003 Feb;120(2):275-84.
[38] Ponec M, Weerheim A, Kempenaar J, Mommaas AM, Nugteren DH. Lipid composition of cultured human keratinocytes in relation to their differentiation. J Lipid Res 1988 Jul;29(7):949-61.
[39] Behrman RE, Kliegman RM, Jensen He. In: Nelson’s Text book of Pediatrics, 16th ed Philadelphia, Penna: Saunders 2000:pp.1965-74.
[40] Soanes C, Hawker S, Elliott Je. In: Oxford Dictionary of Current English,, 2006.
[41] Agorastos T, Hollweg G, Grussendorf EI, Papaloucas A. Features of vernix caseosa cells.
Am J Perinatol 1988 Jul;5(3):253-9.
[42] Haahti E, Nikkari T, Salmi AM, Laaksonen AL. Fatty acids of vernix caseosa. Scand J Clin Lab Invest 1961;13:70-3.
[43] Kaerkkaeinen J, Nikkari T, Ruponen S, Haahti E. Lipids Of Vernix Caseosa. J Invest Dermatol 1965 May;44:333-8.
[44] Fu HC, Nicolaides N. The structure of alkane diols of diesters in vernix caseosa lipids.
Lipids 1969 Mar;4(2):170-5.
[45] Nicolaides N, Fu HC, Ansari MN, Rice GR. The fatty acids of wax esters and sterol esters from vernix caseosa and from human skin surface lipid. Lipids 1972 Aug;7(8):506-17.
[46] Oku H, Mimura K, Tokitsu Y, Onaga K, Iwasaki H, Chinen I. Biased distribution of the branched-chain fatty acids in ceramides of vernix caseosa. Lipids 2000 Apr;35(4):373-81.
[47] Schaefer H, TE. R. Composition and structure of the stratum corneum. In: Skin barrier, Principles of percutaneous absorption, Karger AG, Basel, Switzerland 1996:pp 43-86.
[48] Baker SM, Balo NN, Abdel Aziz FT. Is vernix caseosa a protective material to the newborn?
A biochemical approach. Indian J Pediatr 1995 Mar-Apr;62(2):237-9.
[49] Narendran V, Hull WM, Akinbi HT, Whitsett JA, Pickens WL, Lambers DS, et al. Vernix caseosa contains surfactant proteins: Potential role in innate immune function in the fetus.
Pediatric Research 2000 Apr;47(4):420a-a.
[50] Buchman AL. Glutamine: is it a conditionally required nutrient for the human gastrointestinal system? J Am Coll Nutr 1996 Jun;15(3):199-205.
[51] Akinbi HT, Narendran V, Pass AK, Markart P, Hoath SB. Host defense proteins in vernix caseosa and amniotic fluid. Am J Obstet Gynecol 2004 Dec;191(6):2090-6.
[52] Marchini G, Lindow S, Brismar H, Stabi B, Berggren V, Ulfgren AK, et al. The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa. Br J Dermatol 2002 Dec;147(6):1127-34.
[53] Yoshio H, Tollin M, Gudmundsson GH, Lagercrantz H, Jornvall H, Marchini G, et al.
Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense. Pediatr Res 2003 Feb;53(2):211-6.
[54] Tollin M, Jagerbrink T, Haraldsson A, Agerberth B, Jornvall H. Proteome analysis of vernix caseosa. Pediatr Res 2006 Oct;60(4):430-4.
[55] Lajos L, Szontagh F. Evolution and Growth of Tadpoles by Feeding Vernix Caseosa, Progesterone and Folliculin. J Obst and Gynaecol Brit Emp 1948;55(3):281-&.
[56] Youssef W, Wickett RR, Hoath SB. Surface free energy characterization of vernix caseosa.
Potential role in waterproofing the newborn infant. Skin Res Technol 2001 Feb;7(1):10-7.
[57] Tansirikongkol A, Wickett RR, Visscher MO, Hoath SB. Effect of vernix caseosa on the penetration of chymotryptic enzyme: potential role in epidermal barrier development.
Pediatr Res 2007 Jul;62(1):49-53.
[58] Joglekar VM. Barrier Properties of Vernix Caseosa. Arch of Dis in Child 1980;55(10):817-9.
[59] Tollin M, Bergsson G, Kai-Larsen Y, Lengqvist J, Sjovall J, Griffiths W, et al. Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.
Cell Mol Life Sci 2005 Oct;62(19-20):2390-9.
[60] Pickens WL, Zhou Y, Wickett RR, Visscher MO, Hoath SB. Antioxidant defense mechanisms in vernix caseosa: potential role of endogenous vitamin E. Pediatr Res 2000;47:425A.
[61] Bautista MI, Wickett RR, Visscher MO, Pickens WL, Hoath SB. Characterization of vernix caseosa as a natural biofilm: comparison to standard oil-based ointments. Pediatr Dermatol 2000 Jul-Aug;17(4):253-60.
[62] Zhukov BN, Neverova EI, Nikitin KE, Kostiaev VE, Myshentsev PN. [A comparative evaluation of the use of vernix caseosa and solcoseryl in treating patients with trophic ulcers of the lower extremities]. Vestn Khir Im I I Grek 1992 Jun;148(6):339-41.
[63] Rutter N. The immature skin. Eur J Pediatr 1996 Aug;155 Suppl 2:S18-20.
[64] Holbrook KA. A histological comparison of infant and adult skin. . In: Neonatal Skin:
Structure and Function, Marcel Dekker, New York 1982:pp.3-31.
[65] Hammarlund K, Sedin G. Transepidermal water loss in newborn infants. III. Relation to gestational age. Acta Paediatr Scand 1979 Nov;68(6):795-801.
[66] McCormack JJ, Boisits EK, Fisher LB. An in vitro comparison of the permeability of adult versus neonatal skin. In: Neonatal Skin: Structure and Function; New York 1982:pp.149-64 [67] Wester RC, Maibach HI. Comparative percutaneous absorption. In: Neonatal Skin:
Structure and Function, Marcel Dekker, New York 1982: pp.137-47.
[68] Warner RR, Boissy YL, Lilly NA, Spears MJ, McKillop K, Marshall JL, et al. Water disrupts stratum corneum lipid lamellae: damage is similar to surfactants. J Invest Dermatol 1999 Dec;113(6):960-6.
[69] Caussin J, Gooris GS, Groenink HW, Wiechers JW, Bouwstra JA. Interaction of lipophilic moisturizers on stratum corneum lipid domains in vitro and in vivo. Skin Pharmacol Physiol 2007;20(4):175-86.
[70] Harding CR, Watkinson A, Rawlings AV, Scott IR. Dry skin, moisturization and corneodesmolysis. Int J Cosmet Sci 2000 Feb;22(1):21-52.
[71] Proksch E, Lachapelle JM. The management of dry skin with topical emollients--recent perspectives. J Dtsch Dermatol Ges 2005 Oct;3(10):768-74.
[72] Fluhr JW, Darlenski R, Surber C. Glycerol and the skin: holistic approach to its origin and functions. Br J Dermatol 2008 Jul;159(1):23-34.
[73] Rawlings AV, Scott IR, Harding CR, Bowser PA. Stratum corneum moisturization at the molecular level. J Invest Dermatol 1994 Nov;103(5):731-41.
[74] Fluhr JW, Lazzerini S, Distante F, Gloor M, Berardesca E. Effects of prolonged occlusion on stratum corneum barrier function and water holding capacity. Skin Pharmacol Appl Skin Physiol 1999 Jul-Aug;12(4):193-8.
[75] Mao-Qiang M, Brown BE, Wu-Pong S, Feingold KR, Elias PM. Exogenous nonphysiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction. Arch Dermatol 1995 Jul;131(7):809-16.
[76] Yang L, Mao-Qiang M, Taljebini M, Elias PM, Feingold KR. Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. Br J Dermatol 1995 Nov;133(5):679-85.
[77] Oudshoorn MHM. Composite of microgels and lipids as biofilm to restore skin barrier function. Dissertation, University of Utrecht, The Netherlands 2008.
