In vivo examination of healthy human skin after short‐time treatment with moisturizers using confocal Raman spectroscopy and optical coherence tomography: Preliminary observations

Skin is our barrier against environmental damage. Moisturizers are widely used to increase hydration and barrier integrity of the skin; however, there are contrasting observations on their in vivo effects in real‐life settings. In cosmetic studies, corneometers and tewameters are traditionally used to assess skin hydration. In this study, two novel noninvasive diagnostic techniques, optical coherence tomography (OCT) and confocal Raman spectroscopy, were used to analyze stratum corneum and epidermal thickness (ET), water content, blood flow in function of depth, skin roughness, attenuation coefficient, natural moisturizing factor, ceramides and free fatty acids, cholesterol, urea, and lactates in 20 female subjects aged between 30 and 45 before and after 2 weeks application of a commercially available moisturizing lotion on one forearm. The untreated forearm served as control. A third measurement was conducted 1 week after cessation of moisturizing to verify whether the changes in the analyzed parameters persisted. We noticed a reduction in skin roughness, an increase in ceramides and free fatty acids and a not statistically significant increase in ET. As a conclusion, short time moisturizing appears insufficient to provide significant changes in skin morphology and composition, as assessed by OCT and RS. Novel noninvasive imaging methods are suitable for the evaluation of skin response to topical moisturizers. Further studies on larger sample size and longer treatment schedules are needed to analyze changes under treatment with moisturizers and to standardize the use of novel noninvasive diagnostic techniques.


INTRODUCTION
Skin has a crucial role as a barrier against environmental factors and must be regularly hydrated for protecting its integrity and function.
The outermost layer of the epidermis, the stratum corneum (SC), is primarily responsible for preventing dehydration of underlying tissues.
Its complex structure is composed of corneocytes, their hygroscopic material, collectively referred to as natural moisturizing factor (NMF), and an intercellular lipid bilayer matrix. NMF is the degradation product of filaggrin, acting as an efficient humectant and containing amino acids, ceramides, cholesterol, and fatty acids. If SC water content reduces to a critical level, normal desquamation is impaired, leading to the accumulation of corneocytes on the skin surface and causing the appearance of dryness and scaling. 1 Moisturizers increase skin hydration and re-establish skin lipid film after cleansing; they can also function as vehicles to deliver active ingredients into the skin. 1 The growing interest for skin barrier function together with the flourishing cosmetic industry led to an increasing number of studies trying to analyze the short-and long-term effects of moisturizers on dry skin. However, many studies lack standardized methods and devices that can be perfectly reproducible; on the other hand, difficulty in performing invasive biopsies for such studies limits the accuracy of the results.
Noninvasive imaging techniques can be used for a painless, in vivo investigation of skin morphology and physiology, thus avoiding biopsies. Optical coherence tomography (OCT), mainly employed for skin cancer diagnosis, offers two-dimensional vertical imaging of the skin up to a depth of 1.5-2 mm 2-8 ; it can assess skin thickness with a good correlation with histopathology. 9 Confocal Raman spectroscopy (CRS), on the other hand, provides information on the molecular composition of the skin including water content, NMF, ceramide, and cholesterol levels. CRS has been successfully used to assess pathophysiological skin conditions and physiological skin parameters. [10][11][12][13][14][15][16][17][18] In addition, when combined with OCT, it showed a positive correlation in measuring SC thickness. 19 Even though various studies have been conducted on the effects of moisturizer application on skin, there are little data available assessing the posttreatment effects of moisturizers using novel noninvasive imaging methods. 2,3,[19][20][21][22] The aim of this study was to noninvasively investigate skin changes following the application of a hygroscopic moisturizer over 2 weeks and to follow up its short-term efficacy 1 week after cessation of application, using OCT and CRS.

Optical coherence tomography
The OCT device used in our study (VivoSight; Michelson Diagnostics, Kent, UK) scans an area of 6 × 6 mm, reaching a penetration depth of

Confocal Raman spectroscopy
CRS analyses were performed using a confocal Raman spectrometer

Data and statistical analysis
Data extrapolation of Raman spectroscopy was performed semiautomatically using the Skintools 3 software (RiverD International B.V.).
The two-way analysis of variance (ANOVA) test was used to assess the variables in three measurement timepoints. Paired t-tests were used for pairwise comparisons. Shapiro-Wilk's tests were applied for confirming the assumption of normal distribution in the data.
Outliers located higher than 1.5* interquartile range above the upper quartile or lower than 1.5* interquartile range below the lower quartile were capped.

Epidermis thickness manually measured by OCT
The control arm had a mean ± SD ET of 0.094 ± 0.014 mm, and the treated arm of 0.094 ± 0.013 mm at baseline. In the treated arm, ET F I G U R E 1 Epidermis thickness manually measured by optical coherence tomography (OCT) (A) and automatically measured by OCT VivoTools software (B) in the treated arm (yellow) and in the control arm (blue) measured by OCT significantly increased from baseline to T1 (0.108 ± 0.016 mm), remaining stable at T2 (0.108 ± 0.012 mm). The control arm did not show a statistically significant change (T1 0.093 ± 0.015 mm, T2 0.096 ± 0.014 mm) ( Figure 1A, Figure S1).

Ceramides and free fatty acids assessed by CRS
Mean ± SD ceramide and free fatty acids concentration was 0.040 ± 0.005 at baseline in the treatment group, which slightly increased to 0.043 ± 0.007 at T1, and decreased after stopping treatment to 0.038 ± 0.007 at T2.
On the other hand, mean ± SD ceramide and free fatty acids concentration in controls was 0.042 ± 0.006 at baseline, reduced to 0.039 ± 0.006 at T1, and remained stable at T2.

NMF assessed by CRS
Mean ± SD NMF of the treatment group was 0.000481 ± 0.000138 at baseline, decreased to 0.000459 ± 0.000135 at T1, and increased to 0.000499 ± 0.000133 at T2. The changes were not statistically significant (p = 0.68). Mean ± SD NMF of the control group was 0.000480 ± 0.000132 at baseline, decreased to 0.000470 ± 0.000124 at T1, and increased to 0.000484 ± 0.000129 at T2. The changes were not statistically significant (p = 0.8) (Figure 4). were not statistically significant ( Figure 5).

Urea assessed by CRS
Mean ± SD urea was 0.007 ± 0.002 at baseline in the treatment group and increased to 0.008 ± 0.002 at T1 and 0.009 ± 0.003 at T2. The changes were not statistically significant (p = 0.32). Mean ± SD urea was 0.008 ± 0.002 at baseline in the control group, remained stable to 0.008 ± 0.002 at T1, and increased to 0.009 ± 0.003 at T2. The changes were not statistically significant ( Figure 6).

Lactate assessed by CRS
Mean ± SD lactate was 0.009 ± 0.002 at baseline in the treatment group and increased to 0.012 ± 0.004 at T1 and 0.010 ± 0.005 at T2.
Mean ± SD lactate was 0.010 ± 0.002 at baseline in controls and remained stable to 0.010 ± 0.005 at T1 and 0.010 ± 0.004 at T2.
The two-way repeated measures ANOVA showed a statistically significant two-way interaction between treatment and time (p = 0.026).
Pairwise comparisons between time points showed a statistically significant difference in the treatment group between T0 and T1 (p = 0.0008), but not between T1 and T2 (p = 0.079) (Figure 7).  Figure 8).

Blood flow at 0.1 mm depth measured by OCT VivoTools software
Mean ± SD baseline BF in the treatment and control groups was 0.006 ± 0.01 and 0.006 ± 0.005, respectively. At T1, it was 0.006 ± 0.005 in the control group and 0.008 ± 0.007 in the treatment group. At T2, it was 0.008 ± 0.007 in both treated and untreated arms, and F I G U R E 5 Cholesterol assessed by confocal Raman spectroscopy (CRS) in the treated arm (yellow) and in the control arm (blue) F I G U R E 6 Urea assessed by confocal Raman spectroscopy (CRS) in the treated arm (yellow) and in the control arm (blue) F I G U R E 7 Lactate assessed by confocal Raman spectroscopy (CRS) in the treated arm (yellow) and in the control arm (blue)

F I G U R E 8 Water content assessed by confocal
Raman spectroscopy (CRS) in the treated arm (yellow) and in the control arm (blue) F I G U R E 9 Blood flow at 0.1 mm depth measured by OCT VivoTools software in the treated arm (yellow) and in the control arm (blue) the difference was statistically significant only in the treated arm (p = 0.004) (Figure 9).

Blood flow at 0.3 mm depth measured by OCT VivoTools software
Mean ± SD baseline BF of the control and treatment groups was 0.053 ± 0.026 and 0.04 ± 0.019. respectively. At T1, it was 0.059 ± 0.024 in the control group and 0.054 ± 0.022 in the treatment group. At T2, it was 0.067 ± 0.022 in the control group and 0.068 ± 0.018 in the treatment group; the differences between timepoints were statistically significant only in the treated arm (p < 0.05) ( Figure 10).

Skin roughness (Rz) measured by OCT VivoTools software
Mean ± SD Rz of the treatment group in T0, T1, and T2 was 98.641 in the treated arm (p = 0.01) (Figure 12, Figure S2).

Attenuation coefficient measured by OCT VivoTools software
Mean ± SD AC in the untreated arm was 1.996 ± 0.291, in the moisturized arm of 1.942 ± 0.303 at baseline. AC increased, however, in both untreated and moisturized arms from baseline to T1 (2.24 ± 0.506 and 2.262 ± 0.514, respectively) and from T1 to T2 (2.566 ± 0.43 and 2.721± 0.544) so that there was no statistical significance in the group comparison ( Figure 13).

DISCUSSION
The integrity of the skin is needed to preserve water in the epidermis, which is essential for hydrolytic enzymatic reactions in keratinocytes and maintaining the defence function against microorganisms, chemicals, and mechanical stress.
Moisturizers can be used to support the natural skin barrier. They act through two basic mechanisms: occlusion and humectancy. Occlusive moisturizers function as a water-impermeable barrier over the skin surface, thereby preventing Transepidermal water loss (TEWL) and creating an optimal environment for restoration of the SC barrier.
Rehydration of SC occurs by water that is attracted from the deeper Recently, a new user-friendly and semi-automated CRS was able to determine in vivo real-time skin water profiles in the function of depth, estimate SC thickness, and the effect of moisturizers on the skin by combining the principle of confocal microscopy with Raman spectroscopy. 18 A study that measured the effects of moisturizers and compared CRS and OCT obtained a positive correlation between these two methods in measuring SC thickness. 19 More recently, CRS alone has been used to assess the efficacy of moisturizers on skin hydration in 12 patients; the authors of this pilot study compared water and NMF contents of skin after the application of commercial moisturizer products and stated that different products do not hydrate the skin to the same level, and that some of them might even dehydrate the skin, depending on the formulations. 18 If we focus on OCT, the device was reported to be an efficient tool for the characterization of skin morphology, also compared to fluorescence laser scanning microscopy and conventional light microscopy. 15 (Figure 2). F I G U R E 1 3 Attenuation coefficient measured by OCT VivoTools software in the treated arm (yellow) and in the control arm (blue) In our previous study, 2 we also observed an increase in the ET by OCT by manual measurements following 2-week application schedule, and hypothesized a deeper and enhanced penetration of hygroscopic lotions. In this study, we aimed to verify the above-mentioned hypothesis and detect the distribution of volume increase among skin compartments through the standardized and objective measures of CRS.
In fact, we demonstrated that the thickness of SC did not change significantly at the different time points analyzed, but showed a trend toward reduction at T1 in the treatment arm, which is against the common belief the moisturizers accumulate in and causes the swelling of SC. [36][37] Such a reduction of SC thickness after short-term treatment was also observed in another study with CRS. 19 This might be due to the increased exfoliation of the superficial SC produced by the mechanical action of the regular moisturizer application.
On the other hand, we found an increase in the ET following mois- NMF showed a negative trend from baseline to T1, and we observed a positive trend from T1 to T2 (after suspending treatment). This is in line with previous findings showing a reduction of NMF after treatment with moisturizers, following the hypothesis that lotions can temporar-ily interfere with the functional organization of SC and composition of the skin lipid film. 18 The ceramide and free fatty acids content in the skin showed a statistically significant improvement from baseline to T1 (during daily application of the moisturizer), which decreased after stopping treatment. Ceramides are produced in stratum granulosum and transported to SC and constitute almost 50% of the total SC volume. 38 Although we showed a diminished SC following moisturizing, an induction of ceramide production by the ingredients of the moisturizer could be speculated. This result also supports the hypothesis that regular moisturizer application improves the skin barrier function.
Concerning skin morphology and blood vessels analysis, we reported an increase in BF at a depth of 0.3 mm between timelines T1 and T2 in the treated arm, probably due to mechanical stimulation causing vasodilatation. Accordingly, we saw a significant decrease in the skin roughness parameters (Ra and Rz) between baseline and T1 only in the moisturized arm, indicating a smoothening of the skin surface, in line with our previous study. 33 We found no statistically significant changes in the AC; in particular, we could not observe the expected decrease of AC observed in other studies after treatment with glycerol, probably because our measurements were not taken immediately after moisturizing. 39 In conclusion, we observed an increase in ceramides and free fatty acids and a decrease in skin roughness indicating a temporary swelling of lower epidermal compartments (thus, smoothening of the skin surface) under treatment with a moisturizing lotion; we observed a tendency to increased ET which could not reach statistical significance.
However, novel noninvasive diagnostic methods such as OCT and CRS are suitable for monitoring the effects of moisturizers in vivo, and they can be combined to observe morphological and molecular perspectives. Further studies on larger sample size and longer treatment schedule could be helpful to better standardize the use of the abovementioned tools in cosmetic studies and better characterize the skin changes.