A preliminary 1-month study, subsequently extended to 3 months, in Portugal, and a second 6-month study in Italy, included volunteers with primary and secondary weak and brittle nails. Globally, a cohort of 60 patients was initially included in the study and evaluated at 1 month. Seven subjects from the Portuguese cohort were not invited to continue the study after the first visit and 1 subject withdrew before completing the 3-month study. Therefore, 52 subjects were evaluated at 6 months. For all patients, the diagnosis was established on clinical grounds and from their medical history. When suspected, onychomycosis was excluded through clinical examination, direct microscopy and/or culture. None of the patients were receiving systemic or other topical treatments.
Portuguese study: 30 female patients (mean age 46.28, from 23 to 63 years) with primary fingernail fragility were evaluated for 1 month, of which 23 continued to follow-up at 3 months of treatment; however, one subject was lost to follow-up at 3 months. Therefore, 22 subjects were evaluated at 3 months.
Italian study: 30 patients, 9 males and 21 females (mean age 52.37, from 18 to 82 years). In three patients nail fragility was due to trachyonychia associated with alopecia areata, in two patients it was due to onychotillomania and in three patients it was due to mild nail lichen planus confirmed by histopathology. In one patient nail fragility was a result of chronic paronychia. The other 21 patients were diagnosed as having idiopathic nail fragility, involving the fingernails in 14 patients and the toenails in seven patients.
The product tested is a new water-based nail-strengthening solution specially formulated with a combination of active ingredients (silanediol salicylate and Pistacia lentiscus gum) that increase the quantity and quality of silicon and keratin in the nails. The gum from the tree Pistacia lenticus contains numerous biologically active molecules including essential oils of which α-pinene, b-myrcene, b-pinene, linalool, limonene and caryophyllen are the main constituents. The polymer polymyrene represents about 25% of the gum. The biologically active substances are in part triterpenic acids (C29–C31), including masticadienonic acid, isomasticadienonic, oleanonic acid and moronic acid. Pistacia lentiscus gum has demonstrated in vitro that it increased the synthesis of hard keratins (K31, K83, K85) and in vivo it improved nail strength and thickness (data on file). The product also contains cationic hyaluronic acid that adheres to the surface and deeply moisturizes the nails and cuticles. This combination of ingredients (patent pending) helps the natural process of nail repair and growth. The product was applied to the entire nail surface and the periungual tissues once daily for 3 months (Portugal) or 6 months (Italy).
In both studies, the evaluation of treatment efficacy and tolerability was performed after 2 weeks (V2), 1 month (V3), and 3 months (V4); in the Italian study an additional 6-month evaluation (V5) was performed (Figs. 1, Fig. 2). Both studies evaluated the fragility of target nails according to the investigator’s assessment using a 4-point global assessment scale (GAS): 0 (clear of any sign of splitting, ridges or grooves; normal nail plate thickness), 1 (mild tendency to breakage; very few distal horizontal splits and longitudinal grooves), 2 (moderate tendency to breakage with horizontal splitting of the nail; few deep ridges and longitudinal grooves; distal longitudinal fissures of the nail plate free edge; mild thinning of the nail) and 3 (severe lamellar or transversal splitting; more than 70% of the nail showing deep ridges and grooves; extended longitudinal fissures of the nail plate margin; a dramatic reduction of the nail plate thickness).
In addition, in both studies, patient assessment scales (PAS) of treatment efficacy were performed with a specific questionnaire with a scale from 0 to 3, where 0 indicated no improvement; 1, some improvement; 2, clear improvement; and 3, very good improvement.
For both studies, each follow-up visit (week 2, months 1, 3 and 6) included standardized photos of the target nails, expert investigator grading and subject-reported outcome measures.
During and at the end of treatment, patients were asked to give their opinion about the efficacy and the cosmetic properties of the product (e.g., ease of application).
Finally, the tolerability of the product was evaluated through examination of investigational areas (nails and skin around the nails) and evaluation of other symptoms (pain, stinging/burning) manifested by subjects at each visit.
A Vivascope 1500 (Mavig GmbH, Munich, Germany) in vivo confocal microscopy system was used to obtain uniform imaging of the nails. From these 2-dimensional images, the total nail thickness, thickness of the superficial layer of the nail, and the density of the nail were assessed [6, 7].
PRIMOS (phaseshift rapid in vivo measurement of skin) optical 3D is an in vivo optical measurement device that uses a fringe projection technique based on micromirror display devices. A fringe standard is projected on the nail and detected by the system’s CCD camera. The 3D effect is calculated from the deflection in the fringes which provides quantitative information on the nail profile [8, 9].
The standard roughness was calculated from the full aligned image. The parameters evaluated were Ra (arithmetic mean of nail surface) and Rz (mean of the 5 highest “peaks” and 5 lowest “valleys” in the image area).
Onychoscopy evaluation was supported and documented by standardized dry videodermoscopy (Fotofinder Thricoscan®) of the target nail. The evaluation used a 4-point onychoscopy assessment scale (OAS), from 0 (clear of any sign of splitting, ridging or grooves) to +3 (severe lamellar o transversal splitting, more than 70% of the nail showing deep ridging and grooves; extended longitudinal fissures of the nail plate margin).
Dynamic Optical Coherence Tomography (D-OCT)
D-OCT was performed on the top of the nail plate through the proximal nail fold and from the end of the nail plate through the distal margin of the considered nail, by means of a commercially available D-OCT (VivoSight®: Michelson Diagnostics Ltd, Maidstone, UK) and 3D image reconstruction software. The VivoSight® OCT scanner uses eye-safe infrared laser light to obtain a 3D block of image data at a high resolution (< 10 μm). OCT has a central wavelength of 1305 nm and scans an area of 6 mm by 6 mm up to a depth of 2 mm. D-OCT allows the description of blood flow in vivo and visualization of the tissue microvasculature. In order to minimize normal anatomical variation, the same depth was chosen for all images using the software tool “OCTFITTER” (University of Modena and Reggio Emilia, Modena, Italy) that provide horizontal D-OCT images at three selected depths (150, 300 and 500 μm) [10,11,12].
Reflectance Confocal Microscopy (RCM)
RCM (VivaScope® 3000: Mavig GmbH, Munich, Germany) was used to visualize the nail plate. It uses a diode laser at 830 nm and < 22 mW power at the tissue level, for the in vivo imaging of nails with cellular resolution. Each individual RCM image corresponds to a horizontal section of approximately 1 × 1 mm, at a selected tissue depth, with a lateral resolution of 1 μm. Crodamol oil was employed as a coupling medium between the probe and the nail surface. The images were taken from the top of the nail plate through the nail bed and at the distal margin of the nail. RCM and D-OCT (cross-sectional and en face) images were obtained from each patient before, during, and after the application of the nail gel for qualitative visual comparison . Reflectance confocal microscopy (RCM) (VivaScope® 1500) assessed the total nail thickness, the thickness of the superficial layer of the nail and the density of the nail. The parameters were measured according to a routine that included a two-dimensional evaluation of the image of the nail obtained. The results were presented in pixels.
Due to the cosmetic nature of the intended use of the product studied, ethics committee approval was not required. Both studies were performed in line with the Declaration of Helsinki (1964) and its subsequent amendments. In both studies, the subjects were informed about the expected benefits of the tested products and the constraints of study participation and signed an informed consent form, including consent for the use of photographs taken during the studies, prior to the start of the studies.
Portuguese study: values are expressed as the mean and standard deviation (SD). A Willcoxon’s test for paired samples was performed to evaluate changes between the data obtained at baseline and at each time-point. Differences were considered significant at p < 0.05. Statistical analysis was performed using SPSS software (version 20.0; SPSS Inc, Chicago, IL, USA).
Pool analysis: the analysis has been carried out on the set of data from the different individual databases of each of the aggregate studies, taking into account that exactly the same information has been collected with the same schedule. Continuous variables have been described by the number of valid cases, mean and standard deviation. Comparisons of categorical variables have been made using the chi-square test. In the case of continuous variables, the Student t-test was used for independent data. For all comparisons a level of statistical significance of 0.05 bilateral was considered. All analyses were performed on the data set using all available information with intention to treat (ITT) criteria. Statistical analysis was performed using the SAS (Statistical Analysis System) program, version 9.2.