Depth‐dependent hydration dynamics in human skin: Vehicle‐controlled efficacy assessment of a functional 10% urea plus NMF moisturizer by near‐infrared confocal spectroscopic imaging (KOSIM IR) and capacitance method complemented by volunteer perception

Stratum corneum (SC) hydration is vital for the optimal maintenance and appearance of healthy skin. In this context, we evaluated the efficacy of an NMF‐enriched moisturizer containing 10% urea on different aspects of SC hydration of dry skin.


INTRODUCTION
The stratum corneum (SC) represents the outermost layer of the epidermis establishing a protective barrier against the external environment. For most body areas, SC thickness is reported to be around 20 μm. [1][2][3] Although it constitutes a nonviable tissue, the SC plays an essential role in regulating skin moisturization and water retention. Its water content decreases from a constant level of about 70% in the stratum granulosum to about 15-25% at the skin surface. [4][5][6][7][8] Hydration of the SC is a key factor for desquamation, generation of natural moisturizing factor (NMF), the optimal function of physiological and mechanical skin properties, as well as cutaneous metabolism and enzyme function. [9][10][11] An adequate moisturization of the SC is also essential for the healthy appearance of the skin. Maintenance of SC hydration is dependent on factors such as relative atmospheric humidity, the capability to balance evaporative water loss, the presence of extracellular lipids and the ability of corneocytes to bind water. 12 This water-binding capacity is dependent on NMF, a complex mixture of low-molecular-weight, highly hygroscopic, water-soluble compounds that are exclusively present in the SC. 9 NMF mainly consist of free amino acids, pyrrolidone carboxylic acid, lactates, citrate, sugars, and urea, forming a depth profile that peaks in the upper third of the SC. 5,13 Reductions or a deficiency in NMF have been associated with various skin conditions such as xerosis cutis. 13 To increase the diminished SC water content, moisturizers containing glycerol or urea are well established. 13,14 Glycerol enhances SC hydration, prevents epidermal water loss as well as SC thickening, and improves dry, scaly skin. [15][16][17] However, urea is currently considered the gold standard in the treatment of dry skin 18,19 since it has been demonstrated to improve hydration mechanisms and skin barrier function and its safety and efficacy have extensively been documented over the last century. [18][19][20][21] The efficacy of urea can be further enhanced by the addition of other NMF components, and glyceryl glucoside and glycerol. 19,22,23 The aim of this study was to investigate the objective and subjective hydrating efficacy of a moisturizer formulation containing these humectants compared to its emulsion base containing glycerol (referred to as the vehicle). The effects of formulations containing 10% urea on skin hydration have repeatedly been confirmed by us and others in vehicle-controlled studies using capacitance-based corneometry. 20,[23][24][25][26][27] Several investigations applying confocal Raman microspectroscopy in vitro and in vivo have elucidated the depth profile of SC water content in detail. 7,8,28,29 Here, we applied the recently developed near-infrared confocal spectroscopic imaging (KOSIM IR) method that allows a noninvasive measurement of skin hydration in vivo as a function of skin depth 6 to detect depth-dependent alterations in water content and related the results to Corneometer ® values.

Test formulations
The  Five days prior to the study start, subjects were asked to refrain from using skin care products. Measurements were carried out by trained and experienced personnel after acclimatization for 30 min under standard atmospheric conditions (21.0 • C ± 1.0 • C and 50% ± 5% relative humidity).

In vivo studies I and II
Skin moisturization of the lower legs was assessed using a Corneometer ® CM 825 (MDD4 device, Courage and Khazaka, Cologne, Germany). Ten measurements were performed per test site according to the European Group for Efficacy Measurements on Cosmetics and Other Topical Products (EEMCO) guidelines. 31 Also, subjects assessed their skin condition by using a self-grading questionnaire. Volunteers were requested to observe their skin and grade skin moisturization by means of a scale ranging from 1 (extremely dry and scaly) to 10 (extremely moisturized, no signs of dryness). Under supervision of the investigator subjects then treated one of their lower legs with the moisturizer and the other one with the vehicle, applying approximately 2 g each. Corneometer ® measurements and self-gradings were repeated 30 min and 24 h after application.
Twenty-four hours after application, volunteers additionally evaluated product performance using a two-tiered rating scale: 1 = no and 2 = yes. If a volunteer skipped a question, the answer was saved as 0 = I do not know.  All statistical tests were two-sided at significance level alpha = 0.05.

Study I: Corneometer measurements and skin moisturization assessments
Data were tested for conformity with a normal distribution using Shapiro-Wilk's test. In the case when the conformity was absent, a logarithm transformation of the data was performed. Statistical analysis utilized pairwise Wilcoxon's signed rank test (when conformity of data with a normal distribution was absent even after transformation).

3.1.1
In vivo determination of skin moisturization using corneometry As illustrated in Figure 3    Additionally, according to the self-assessment questionnaire 64% of subjects rated the moisturizer as more effective against signs of dry skin than the vehicle (22% preference), significantly favoring the moisturizer (p < 0.01).

3.2.1
In vivo determination of skin moisturization using KOSIM IR Figure 5 depicts the hydration profile of SC and viable epidermis to a depth of 50 μm. Compared to the untreated area, the application of the vehicle resulted in a significantly higher water content in the SC at depths of 5, 7.5, and 10 μm ( Table 1). The moisturizer induced a significantly higher water content in the SC at all depths compared to both the untreated area and the vehicle. Moreover, the AUC revealed a significantly higher water content after treatment with the moisturizer compared to the vehicle. AUC values of sites treated with the moisturizer or the vehicle were significantly higher in comparison to control sites (Table 1).

DISCUSSION
In the studies reported here, we assessed the hydrating capacity of a moisturizer containing 10% urea, supplementary NMF components, glyceryl glucoside, and glycerol in comparison to its vehicle containing glycerol in dry skin by applying two different methods. Corneometry is a well-established capacitance method that provides a single dielectric value (arbitrary units) of skin hydration per measurement, integrating skin hydration values to a depth of 45 μm. 31,32 F I G U R E 4 Self-assessment of skin hydrating product properties 24 h after a single application of the moisturizer and the vehicle (n = 42). Results are depicted as confirmation rates. Significant agreement rates are marked with an asterisk (*p < 0.05).

F I G U R E 5
Water concentration profiles in the SC of dry forearm skin in m% versus depth determined using near-infrared confocal spectroscopic imaging (KOSIM IR) after 14 days of regular treatment with the moisturizer (n = 27) or the vehicle (n = 26). Significant differences are marked with an asterisk (*p < 0.05 comparison to vehicle; # p < 0.05 comparison to untreated control).
The Corneometer ® assessments revealed statistically significant higher values after application of the moisturizer, compared to the vehicle, 30 min and 24 h after treatment, indicating an instant and sustainable effect on skin hydration. These findings were reflected by the volunteers' ratings of the skin hydrating properties of the moisturizer, resulting in significant confirmation rates. Accordingly, the moisturizer was significantly more frequently rated as effective against signs of dry skin than the vehicle. A high level of acceptance is critical for treatment adherence that depends on patient preference. 33 Moreover, these findings corroborate an earlier vehicle-controlled study of the moisturizer in subjects with very dry skin that evidenced the effect of the applied humectants on skin hydration as paralleled by improvements in clinical severity grading. 23 Accordingly, NMF components are regarded crucial for the maintenance of adequate skin hydration in the treatment of xerosis and other skin conditions. 13,14,19 To investigate influences of the moisturizer and its vehicle on SC hydration dynamics in more detail, we furthermore assessed the intra-

CONCLUSION
Taken together, our results demonstrate that the moisturizer containing 10% urea, supplementary NMF components, glyceryl glucoside, and glycerol improved SC hydration in total and, moreover, as a func-