|Original Article, Biomed Biopharm Res., 2022; 19(2):410-423
doi: 10.19277/bbr.19.2.299; PDF version here [+] ; Portuguese html version [PT]
Evaluation of the influence of the application of a cosmetic formulation on the skin morphological characteristics by Reflectance Confocal Microscopy
Patrícia Maria Berardo Gonçalves Maia Campos* & Gabriela Maria D’Angelo Costa
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. Av do Café, s/nº, Monte Alegre, Ribeirão Preto, SP, Brazil 14040-903
Reflectance Confocal Microscopy (RCM) is a non-invasive imaging technique with near-histological resolution that allows evaluation of the morphological and structural characteristics of the skin. In this context, the aim of the study was to evaluate the influence of the application of a cosmetic formulation on the skin morphological characteristics by RCM. The study included six female subjects, aged 40 to 50 years. Measurements in terms of epidermis thickness, dermal papillae depth (DPD), interkeratinocytes brightness (IB), furrows morphology, skin surface homogeneity (SSH), honeycomb pattern, skin hyperpigmentation, dermal papillae and dermis morphology were performed using RCM before and after a 90-day period of formulation application on the face. The studied formulation showed a significance increase of DPD after the 90 day period, showing an improvement on the dermal-epidermal junction (DEJ). In addition, an increase of IB in the granular layer and an improvement of dermis morphology were observed after the treatment. In conclusion, the obtained results by RCM showed an improvement of morphological characteristics of the skin after application of the studied cosmetic formulation for 90 day period when compared to the initial baseline, as it improved epidermis and dermoepidermal junction morphology.
Keywords: Clinical efficacy, Reflectance Confocal Microscopy, cosmetic formulation
Received: 13/11/2022; Accepted: 31/12/2022
The search for active ingredients to improve the characteristics of skin aging is important for innovation in the cosmetic area. Thus, clinical efficacy studies are necessary to prove the effectiveness of cosmetic formulations containing these active ingredients (1,2,3).
Skin aging is a process induced by intrinsic and extrinsic factors (4). The changes of intrinsic aging are the presence of a thinner epidermis, decreased depth of the dermoepidermal junctions that compromise skin nutrition, and decrease basal cell proliferation (5,6). Solar radiation significantly contributes to accelerated skin aging by promoting photoaging, which results in skin changes such as an increased of collagen degradation, presence of solar elastosis and hyperpigmentations regardless of chronological age or sex (7,8).
There are active ingredients that can improve aged skin conditions. Peptides have been used in cosmetics since they have presented potential for the skin care (9). In addition, di- and tripeptides extracted from rice (Orysza sativa) are compatible with the skin and present potential for application in cosmetic formulation for the treatment of aging skin (10).
A previous study of our research group (Maia Campos et al., 2019) showed that a cosmetic formulation with di- e tripeptides from rice was effective in the improvement of skin elasticity and dermis density for women aged 40 to 50 years (10). Another study from our research group, Shirata et al (2021), showed that cosmetic formulations containing rice di- and tripeptides were effective for the improvement of skin hydration and the prevention of skin photodamage in young age groups (11). Thus, the evaluation of skin morphological and structural characteristics is very important to elucidate the efficacy of cosmetics in dermis epidermal junction, dermal papillae and collagen morphology in the papillary dermis.
Clinical studies using instrumental measurements such as non-invasive imaging techniques are very important to evaluate the efficacy of cosmetic products in real conditions of use (3,7,10,12). Reflectance Confocal Microscopy (RCM) is a non-invasive advanced imaging technique with near to histological resolution that allows evaluation of morphological and structural skin characteristics in real time. It is a technique widely used in dermatology and can be applied in the cosmetic area to characterize different types of skin and evaluate the effectiveness of cosmetics without the need for biopsies (12, 13).
In this context, the application of RCM to evaluate the clinical efficacy of cosmetic formulations can bring an important contribution to elucidate the benefits of application of these formulations in the treatment of photoaged skin.
Thus, the aim of the study was to evaluate the influence of the application of a cosmetic formulation containing di-and tripeptides of hydrolyzed rice protein on the skin morphological characteristics by Reflectance Confocal Microscopy.
Materials and methods
After approval by Ethnics Committee of Faculty of Pharmaceutical Sciences of Ribeirão Preto – University of Sao Paulo (CEP/FCFRP no339), six female subjects, aged 40-55 years (mean age 48 ± SD: 4.95 years), skin Fitzpatrick Phototypes II and III, Caucasian descent were enrolled. (mean age: 48.09 ± SD: 4.85 years)
A cosmetic formulation based on hydroxyethylcellulose (1.8%), methylphenyl polysiloxane (2.0%), cyclomethicone (1.5%), cyclomethicone and crosspolymer dimethicone (1.5%), hydrosoluble filter UVA/UVB (7.5%), propylene glycol (2.5%), glycerin, 2‐Phenoxyethanol (And) Methylisothiazolinone (0.8%), (And) Aqua/Water was added to 4% of the di-and tripeptides of hydrolyzed rice protein and applied on the face twice a day by subjects for a 90 day period. Instrumental measurements by RCM were performed before (baseline) and after 90 days of the application of studied formulation in the periorbital region of the face. The subjects were required to remain in the laboratory [located in the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil (21°100 S, 47°480 W)] with controlled climatic conditions of 21.5 ± 1oC and 50 ± 5% of relative humidity for 20 minutes before the beginning of the measurements.
Skin imaging technique - RCM
Reflectance Confocal Microscopy (VivaScope® 1500, Lucid, New York, USA) emits a low-powered laser (22 mW) with a wavelength close to the infrared (830 nm). This equipment obtains several microscopic images captured as horizontal sections with a field of view of 500 x 500 µm and a high-resolution of 1000 x 1000 pixels. The VivaStack® program allows the capture of images in depth from the stratum corneum to the papillary dermis. The depth of the layers, dermal papillae depth and epidermis thickness is calculated with the “z-depth” (Table 1). Analyzes of interkeratinocyte brightness, furrows morphology and skin surface homogeneity were performed using a standardized score in our NEATEC (Center of Advanced Studies in Cosmetic Technology) research group (14).
In addition, other scores adapted from Longo et al. (2013) were assessed as honeycomb pattern, skin hyperpigmentation, dermal papillae morphology, dermis morphology (15). The score definitions are shown in Table 2.
The normal distribution of the data was evaluated by the Shapiro-Wilk test in the OriginPro® 8 software. After defining whether the data were parametric or non-parametric, the paired Student's t or Wilcoxon tests were performed, respectively. GraphPad Prism® 5 software was used to perform the statistical analyses. The value of p <0.05 was considered statistically significant.
The results showed a significant increase (p=0.0328) of dermal papillae depth after the use of studied formulation for 90 days. In addition, an increase in the total epidermis thickness was observed after the 90 day period, which is according to improvement of dermal papillae depth (Table 3).
The results of RCM score analyses are shown in Figures 1 to 7.
The skin surface homogeneity score analysis showed an improvement of skin regularity of stratum corneum since the participants with Score 1 increased after 90 days of treatment (Figure 1A). This improvement can be seen in representative RCM images (Figure 1B).
The furrows morphology score improved after the 90 day formulation application period as the Score 0 (regular) increased (Figure 2A). Representative RCM images showed an improvement in the furrow morphology noted as straighter edges of the furrows relative to the baseline (Figure 2B).
The interkeratinocyte brightness score analysis showed an increase after 90 days of treatment with the studied formulation. The score represents the low interkeratinocyte brightness (Figure 3A). An increase of interkeratinocyte brightness in the granular layer is demonstrated in representative RCM images after 90 days of treatment (Figure 3B).
The score analysis of the honeycomb pattern showed better homogeneity of keratinocytes with a more defined honeycomb pattern, as a Score 1 was found in 100% of the subjects after 90 days of treatment with the formulation (Figure 4A). The improvement in the honeycomb pattern is observed in representative RCM images (Figure 4B) in which the keratinocytes are homogeneous in size and shape and with defined cell borders after the treatment period.
The score analysis of the skin hyperpigmentation showed a reduction after the use of the formulation under study for 90 days, with an increase in participants with Score 1 (Figure 5A). The basal layer observed in the representative RCM images (Figure 5B) shows a reduction of basal layer brightness after 90 days of treatment.
The score analysis of the dermal papillae morphology showed an improvement after the use of the formulation as Score 2 was observed in 100% of participants after 90 days of treatment (Figure 6A). Representative RCM images showed an improvement in the dermal papillae morphology after 90 days of treatment, in which the dermal papillae were rounded and partially outlined rings contours instead of polycyclic papillae (Figure 6B).
The score analysis of the dermis morphology showed an improvement of collagen after the use of the studied formulation for 90 days. The presence of coarse collagen (Score 3) was presented in 50% of participants after 90 days of treatment (Figure 7A). The dermis morphology was represented as coarse and huddled collagen at the initial time and was observed as coarse collagen after the treatment period (Figure 7B).
In order to prove the effectiveness of the topical peptides, clinical study is important for the development of effective and innovative cosmetic formulations (16). For that, the RCM imaging analysis is essential to evaluate the effects of the active ingredients in skin depth as it evaluates the morphological and structural characteristics of the epidermis in real time and in a non-invasive way (12).
According to the results obtained, improvements of regularity of stratum corneum, furrow morphology and increase of interkeratinocyte brightness were oberved after the use of the formulation under study for 90 days. These findings corroborated with the study of Manfredini et al. (2013) that suggested an increase of skin hydration due to a decrease in the irregularity of the skin surface, an improvement in the morphology of furrows and an increase in the interkeratinocytes brightness after application of the cosmetic formulations (17). In addition, Shirata and Maia Campos (2021) showed a significant increase in the granular layer thickness in photoaged skin after a 90 day application period of a formulation containing di- and tri peptides, which suggest an increase of skin hydration (11).
Moreover, the improvement in the honeycomb pattern suggests an improvement in the skin conditions, since the irregularity of the honeycomb pattern is present in skin photoaging (7,18).
A significant increase of dermal papillae depth and the improvement of dermal papillae morphology after 90 days of treatment with the formulation under study suggests that the formulation was effective for the treatment of alterations in the aged skin since the improvement in the dermo-epidermal junction can increase the skin nutrition and basal cell proliferation that are compromised with skin aging (5).
Another finding that suggests the efficacy of the studied formulation on aged skin was the improvement of the collagen morphology, since there was a decrease of huddled collagen and increase of coarse collagen after the use of the proposed formulation for 90 days (15).
These findings are very important since the aging process cause changes in the layers of dermis, superficial papillary layer and deeper reticular layer, which have distinct collagen fibrils and fibroblast properties (19). However, the papillary layer shows more remarkable changes with aging than the reticular layer (20).
In addition, there was a reduction of skin hyperpigmentation after the use of the proposed formulation for 90 days, which can suggest that there was an improvement in the photoaged skin, since hyperpigmentation is present on skin exposed to higher incidence of UV radiation (18). This result corroborate with the findings reported by Shirata e Maia Campos (2021), who observed a significant hyperpigmentation reduction after a 90 day period of use of the cosmetic formulations based on rice peptides in combination with ascorbyl tetraisopalmitate (11).
In addition, the oral and topical use of peptides is commonly used in dermatology for skin care (21). Oral collagen supplements are known to increase skin elasticity, hydration and dermal collagen density (22, 23). In addition, previous studies by our research group have shown a significant increase in dermis echogenicity, hydration and viscoelastic properties of the skin after using formulations containing di- and tripeptides extracted from rice for 90 days of topical or oral treatment (10).
In summary, the present study complements the previous study of our research group since it showed that the proposed cosmetic formulation was effective in the improvement of honeycomb pattern of epidermis, dermoepidermal junction and dermis morphology due to an improvement of collagen morphology after a 90 day period of application.
Finally, the study has as limitations the lack of a control group and the small number of participants in the clinical study.
The results obtained in RCM imaging analyses showed an improvement of morphological characteristics of the skin after 90 days of twice-daily application of the studied cosmetic formulation containing di- and tripeptides extracted from rice when compared to baseline - initial time, before the formulation application, as it improved epidermis and dermoepidermal junction (DEJ) morphology.
Reflectance Confocal Microscopy can be suggested as a valuable tool for evaluating the effectiveness of cosmetic formulations on the skin, in an objective and non-invasive way under the real conditions of use.
The authors would like to express their thanks for the financial support of the Fundação de Amparo à do Estado de Sao Paulo - Brasil – FAPESP (grant number 2017/19278-0) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
The contributions of Patrícia M. B. G. Maia Campos were conceptualization of the study, writing, revision, and editing. The contributions of Gabriela M. D’Angelo Costa were data and image analysis, statistical analysis and writing.
Conflicts of Interest
The Editor involved in the authorship of this manuscript had no participation in the review or decision process. All authors have stated that there are no financial and/or personal relationships that could represent a potential conflict of interest.
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