The study was approved by the institutional ethics board of the Ethical Committee of the Euroderma Clinic in Sofia from 9.07.2020 with protocol number 2/9.7.2020. All volunteers gave their written informed consent prior to the start of the study. 25 healthy volunteers (mean age, gender?) were included and completed the study. The statistical analysis was performed with Prism 6. The male-female ratio was 10:15; mean age 30.7 years (SEM 2.1), BMI 21.7 (SEM 0.8), smoker: non-smoker ratio 12:13.
Design of the study
Initial values on stratum corneum hydration, surface pH, skin color and barrier function as well as microbiome analysis were performed at baseline. Tape stripping was carried out with 5 sequential strippings with Corneofix® applied with standardized pressure of 225gr/cm² for 5 seconds each [27, 28]. Three different areas on the volar forearms were tape-stripped, one remained the untreated control area. Definition of the sites was achieved by using a template adjusted to the inner fold of both elbows for measurements and microbiome swabs during the study. Skin color, barrier function and microbiome swabs were obtained immediately after tape stripping as well as 2 h later. Further measurements of the skin physiology parameters and microbiome swab sampling were performed after 2 days (48 hrs.) and 7 days (168 hrs.).
Skin Physiology Parameters
Epidermal barrier function was evaluated with the open-chamber system Tewameter TM300. The values are given in g/m²hr. Stratum corneum hydration was measured with Corneometer CM 825. The values are given in arbitrary units (AU). Surface pH was quantified with the pH-Meter including a flat-glass electrode from Mettler-Toledo. The values are given in pH-units. Skin color was measured with the Mexameter MX 18 with the erythema index and the melanin index; both in arbitrary units. All instruments are manufactured by Courage&Khazaka electronics (Cologne, Germany). All probes were attached to a central unit (multiprobe adaptor MPA-9) and subsequently to a PC. The published guidelines were respected [29, 30] . The acclimatization time at constant temperature (21 oC) and air humidity (55%) prior to the measurements of skin parameters was between 20 and 30 minutes.
Lotion A with a pH of 5.5 (range 5.2 – 5.8) (named lotion A) and a product of the same composition with a pH of 9.3 (range 9.1 – 9.5; adjusted and buffered by the addition of Bicarbonate and Sodium Hydrogencarbonate) (named lotion B) were compared. The INCI list of lotion ingredients was as following: Aqua, Glycerin, Cetearyl Alcohol, Sorbitol, Hexyldecanol, Hexyldecyl Laurate, Chamomilla recutita flower extract, Allantoin, Sodium Cetearyl Sulfate, Citric Acid, Sodium Hydroxide, Sodium Acetate, Sodium Ascorbate, Xanthan Gum, Dimethicone, Parfum, Alcohol, Phenoxyethanol, Sodium Benzoate, Benzyl Alcohol.
Lotion A and B treatment areas as well as TS-untreated and non-TS untreated areas were assigned with a randomization list. A fingertip unit of lotion A and B, respectively, was applied to the assigned test areas twice daily and rubbed in until they were completely absorbed for 6 consecutive days.
Washing of the forearms, showering or bathing with water was allowed only 24 h before and not at all 3 hours prior to the baseline examination. No skin care products were to be applied on the forearms 24 h prior to the baseline examination. 12 h before the analysis on day 2 and day 7 no washing of the forearms, showering or bathing with water or use of any cosmetic products including the test products, was allowed.
DNA was extracted and purified into 50µl elution buffer using the DNeasy PowerSoil Pro-Kit (Qiagen). 2.6 pg of DNA from Salinibacter ruber (DSM 13855) was added as an internal spike-in control. Amplification of the V3-V4 region of the 16S rRNA gene was done with the UCP multiplex PCR mastermix (Qiagen) from 5µl DNA using the primers CCTACGGGNGGCWGCAG (forward) and GACTACHVGGGTATCTAATCC (reverse) following the Illumina 16S library preparation protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Sequencing was done on an Illumina MiSeq (v2 reagents) with 2x250bp paired-end reads.
After sequencing, the UPARSE 16S-protocol was used to merge the paired reads by their 3’-ends, filter reads, and cluster unique reads into OTUs using a 97% identity threshold, and quantify OTU abundances by mapping reads to the OTU sequences. OTUs with a relative abundance of less than 0.01% were discarded. Each OTU sequence was searched in the NCBI Targeted Loci 16S database using NCBI BLAST with an identity cut-off of 97% and E-value cut-off of 0.01, and assigned to the annotated species. If no match was found, the sequence was also searched in the NCBI nt database using the same cut-offs but also excluding unclassified species or environmental sequences. If no match was found in either database, the OTU was labelled as unclassified. OTUs with multiple best matches were assigned to the least common ancestor of all alignments with the same BLAST bit score. OTUs assigned to the Salinibacter ruber spike-in were discarded.
Alpha diversity is measured as the number of OTUs in each sample. Relative abundances for species were calculated from classified OTUs and summarized on taxonomic ranks from species to phylum. Visualisation of microbiome data was done using ggplot2.
Biophysical analysis: One way ANOVA analysis within time groups was calculated followed by Sidak's multiple comparisons test. The significance level was set at p<0.05.
Microbiome: Comparison of diversity between treatment groups was performed with the Durbin-Conover test and Holm multiple testing correction using ggstatsplot.