Evaluation of the Anti-Ageing Potential of a New Dermocosmetic Containing Retinoids Mix Through In Vitro Models

Gasperina DD and Guida S

Published on: 2021-07-27

Abstract

Physiological ageing due to the passing of time and prolonged exposure to harmful sun rays generate wrinkles and reduce skin elasticity. These visible and clinical signs can be prevented or reversed rely on known strategies, as the daily use of cosmetic products with antioxidant combinations or retinoids. A new dermocosmetic formulation enriched with a complex of retinoids, called RETINOIDS SERUM, was investigated through in vitro assays using human skin cells. The experiments were carried out to assess the anti-ageing activity in normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT). After the preliminary MTT assay, the proliferation together with the synthesis of collagen and elastin fibers was performed on NHDF cells after 24 h treatment with the two non-cytotoxic concentrations. Using UVB-irradiated HaCaT cells, the measurement of matrix metalloproteinase-1 (MMP-1) levels was also investigated. Potential irritating and hazard risk was evaluated on Reconstructed Human Epidermis (RHE) and Human Reconstructed Corneal Epithelium (HCE) 3D models, close to human skin and eye tissues. In vitro studies show the safety of the dermocosmetic product and the biological effectiveness, improving collagen and elastin synthesis together with a renewal of dermal fibroblasts. In addition, a reduction in the MMP-1 secretion was also highlighted in UVB-irradiated HaCaT cells. These results demonstrate that a panel of in vitro assays can be helpful to support the safety and the anti-ageing effect of the cosmetic formulation containing functional compounds as retinoids to prevent the natural sign of ageing.

Keywords

Evaluation of the Anti-Ageing Potential of a New Dermocosmetic Containing Retinoids Mix Through In Vitro Models

Introduction

The skin, whose main function is to defend the body from external agents, presents also an important role that cannot be underestimated: feeling young and healthy helps in the social context [1]. However, this protection can be compromised with ageing due to reduced epidermal thickness and increased in wrinkles and dryness. Skin ageing is a complex biological process influenced by a combination of many factors related to intrinsic events as genetics, hormonal changes and metabolic processes, whereas ultraviolet (UV) radiation, pollution, or chemical exposure are more associated with environmental stressors (extrinsic events). All these factors lead to gradual but cumulative alterations where the formation of wrinkles, loss of elasticity, thinning of the skin, and roughness are some examples. Almost all of them are correlated with reduced extracellular matrix (ECM) proteins, especially collagen and elastin in the dermis [2,3]. Collagen fibers represent the main component of ECM and they are responsible for the structural integrity and tensile strength of the body. Quantitative biochemical analysis showed that people with advanced age (80 years) had a quantity of fragmented collagen 4.3 times greater than in younger people aged between 21 and 30 years (in the human dermis) in vivo (4). Elastin has the main function of providing elasticity to the tissues, being ten thousand times more flexible than collagen itself [5]. With ageing, the number of these two fibers described above is reduced. In addition to the physiological decay, chronic exposure to sunlight, in particular UV radiation, can cause a further worsening of the normal functions and characteristics of the skin. Among solar radiations, UVB are the most damaging rays, capable to induce DNA damage and the generation of excessive reactive oxygen species (ROS) production. ROS induce the activation of the mitogen-activated protein kinase (MAPK) cascade and the transcription factor named activator protein-1 (AP-1). The latter, in turn, regulates the transcription of metalloproteinases (MMPs), a large and complex family of zinc-dependent endopeptidases, able to break down almost all of the ECM components [6]. The MMPs are classified based on their domain structure into collagenases, gelatinases, stromelysins, and matrilysins. Among all MMPs, the interstitial collagenase metalloproteinase-1 (MMP-1) is the most investigated for its ability to cleave primarily the collagen type I, the dominant form with 80-90% secreted in human skin [7]. On the other side, many of these features are targets of the majority anti-ageing application or procedures for research strategies pertaining to the skin, from “anti-wrinkle creams” to various filling agents. It is noteworthy that the topical application of cosmetic products supplemented with functional compounds as antioxidant combinations or retinoids represents a valid way to contrast the signs of ageing. In this study, the effects of RETINOIDS SERUM (Matex Lab Spa, Brindisi, Italy) were taken under investigation with in vitro studies for its innovative formulation enriched with vitamin A derivatives characterized by a 1% of a retinoids mix including 0.1% of retinol, 0.1% retinaldehyde, and 1% of bioretinol. The retinoid class includes vitamin A (retinol), a 20-carbon molecule characterized by a cyclohexenyl ring and an alcohol end group, and its synthetic and natural derivatives (retinaldehyde, retinoic acid, and retinyl esters). Retinoids are required in many cellular processes, such as cell growth and differentiation, apoptosis, inflammation and immune modulation. Many of their molecular mechanisms are mediated by the interaction with specific cellular and nucleic acid receptors as the Cellular Retinoic Acid Binding Protein (CRABP) types I and II [8]. In the skin, retinoids play a key role in inducing keratinocyte cell differentiation with a reduction in epidermis cell adhesion and fibroblast proliferation enhancing also collagen and elastin fibrils synthesis [9]. Cosmetic products are daily used by people of any age or sex and ensure their safety is very important as well as prove their effectiveness. From March 2013 animal experiments for all cosmetics were replaced by in vitro test based on the 3R principle (Reduction, Refinement and Replacement) proposed by William and Rex in 1945 [10]. Organization for Economic Co-operation and Development (OECD) edited test guidelines for the safety assessment of cosmetic chemicals (T-439) using 3D reconstructed skin models. Therefore, to evaluate the safety of the serum, Reconstructed Human Epidermis (RHE) and Human Reconstructed Corneal Epithelium (HCE) were selected as ideal models close to human skin and eye tissues. To demonstrate and sustain the anti-ageing efficacy of the product containing retinoids mix, in vitro experiments were carried out in two of the most representative cell lines of the human skin: normal human dermal fibroblasts to evaluate the proliferation and the production of collagen and elastin fibers and keratinocytes to investigate the MMP-1 secretion after UVB-radiation.

Materials and Methods

Sample Preparation and Reagents

The tested functional product contains 1% of a retinoids mix (0.1% of retinol, 0.1% retinaldehyde, and 1% of bioretinol). To perform the experimental panel, the product was solubilized at a concentration of 10 mg/mL directly in culture medium. All the chemical reagents used were provided from Merck (Darmstadt, Germany) unless otherwise specified.

Cell Cultures and UVB Irradiation

Primary normal human dermal fibroblasts (NHDF-Ad-Der Fibroblasts FGM-2, code CC-2511) were purchased from Lonza (Basel, Switzerland), while human immortalized keratinocytes (HaCaT, code BS CL 168) were provided by I.Z.L.E.R (Istituto Zooprofilattico della Lombardia ed Emilia Romagna, Italy). Both cell lines were grown in cell culture medium constituted by High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM - Lonza, Basel, Switzerland) with 10% Fetal Bovine Serum (FBS - Thermo Fisher Scientific, Monza, Italy) and 1% of L-glutamine and antibiotics (penicillin G sodium and streptomycin sulfate - Lonza, Basel, Switzerland), and maintained in incubation at 37 °C with 5% carbon dioxide (CO2) atmosphere. Before use, NHDF and HaCaT cells were tested to verify the absence of mycoplasma contaminations with the MycoAlertTM Mycoplasma Detection Kit (Lonza, Basel, Switzerland). For the irradiation protocol, cells were seeded in a 6-well plate for 24 h and then the treatment with the product for further 24 h was performed. The day after, cells were washed with phosphate buffer saline (PBS) and exposed to UVB lamp (CAMAG® UV Lamp 4, wavelength 302 nm - Muttenz, CH) with a dose equal to 5 mJ/cm2. After irradiation, cells were maintained in a complete medium for a recovery time of 24 h at 37 °C before cell supernatant recovery. Untreated cells were used as control in all experimental sets.

Cytotoxicity Assay (MTT Test)

The preliminary cytotoxicity assay was performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagent [11]. NHDF and HaCaT cells were homogeneously seeded in 96-well plates with a density equal to 2 x 104 cells per well. After 24 h, cells were treated with different concentrations of product, starting from 10 mg/mL and following dilution (1:2 ratio), prepared directly in a complete culture medium. After subsequent 24 h of treatment, plates were processed with 50 µL of MTT solution (1 mg/mL) at 37 °C for 2 h. Afterwards, the supernatant was discarded and 100 µL of isopropanol was added to each well. Absorbance was read at 560 nm using a microplate reader (Glomax®, Promega Corporation, USA). Cell survival was obtained by measuring the difference in optical density (OD) of the tested product at all concentrations with respect to control (untreated cells).

Cell viability (%) = [OD570nm test product/OD570nm control] x 100

Brdu Incorporation Assay

To investigate the proliferation rate, a synthetic analogue of the pyrimidine deoxynucleoside thymidine, the 5-bromo-2’-deoxyuridine (BrdU), was used. NHDF cells were seeded on a coverslip into Petri dishes. After 24 h, the two non-cytotoxic concentrations of the product were chosen to treat cells for other 24 h. Subsequently, cells were incubated with BrdU during the last hour of culture and then fixed with 70% ethanol for 2 h at -20 °C. After a fixation step, samples were washed for 30 min with blocking solution, composed of 0.4% Triton-X, 1% bovine serum albumin (BSA) in PBS, incubated first with anti-BrdU antibody (clone B44 - Becton Dickinson, Sunnyvale, CA, USA) for 1 h and then with a FITC-conjugated antibody (Alexa Fluor 488 - Thermo Fisher Scientific, Monza, Italy) for 30 min. At the end of the assay, coverslips were washed, counterstained for DNA with Hoechst 33258, and mounted on microscope slides. Visualization was performed with a Nikon Eclipse E400 fluorescence microscopy, equipped with a Canon A590 IS camera.

Collagen Quantitative Analysis

The amount of the collagen secreted by dermal fibroblasts was measured using a colourimetric kit (SircolTM, Soluble Collagen Assay Kit - Biocolor Life Science, Carrickfergus, United Kingdom). NHDF cells were homogeneously seeded with 8 x 104 cells/well density in a 24-well plate. After 48 h, two of the tested product concentrations proved to be non-cytotoxic and presenting the best solubility in culture medium were chosen. The measurement of collagen synthesis was performed following the manufacturer's instructions. Briefly, at the end of incubation, 200 μL of Tris-HCl pH 7.4 containing polyethylene glycol were added to the recovered supernatant for the isolation and concentration of collagen, then stored overnight at 4 °C. The day after, samples were centrifuged at 12000 rpm for 10 min and incubated for 30 min with a Sircol dye reagent able to bind collagen. During this phase, the coloured complex that is formed tends to precipitate. After centrifugation (12000 rpm for 10 min), a reagent containing acetic acid, sodium chloride and surfactants was added to remove the unbound bye. After last centrifugation, a 0.5 M of sodium hydroxide solution was added to solubilize the collagen precipitated. 200 μL of the sample were collected and placed into a 96 well-plate for spectrophotometric OD reading at a wavelength of 555 nm (Multiskan, Thermo Scientific, USA). The final concentration of collagen (µg/mL) was calculated using a standard curve (0-10 µg).

Elastin Quantitative Analysis

Total elastin, as α-elastin (soluble tropoelastins, lathyrogenic elastins and insoluble elastins), was evaluated in NHDF cells after 24 h treatment, using a colourimetric kit (FastinTM, Elastin Assay kit - Biocolor Life Science Assays, Carrickfergus, United Kingdom). For the preparation of the assay, cells were homogeneously seeded in a 12-well plate at 1.8 x 105 and incubated at 37 °C. After 48 h, the same concentrations chosen for the collagen test were used. At the end of treatment, cells were detached with a cell dissociation solution and diluted 3:1 with 1 M oxalic acid (final concentration 0.25 M). Subsequently, samples were incubated at 95 ± 5 °C for 1 h, with intermittent mixing, and then the measurement of elastin produced by each sample was performed following the manufacturer's instructions.

Analysis of MMP-1 Expression

For the measurement of MMP-1 production, an ELISA kit (MMP-1 Human, Biotrak ELISA System - Amersham, GE Healthcare, United Kingdom) was used. HaCaT cells were homogeneously seeded in 6-well plates and incubated at 37 °C. After 24 h, the two highest concentrations of the product (0.313 and 0.625 mg/mL), demonstrated to be non-cytotoxic after a preliminary MTT assay (data not shown), were chosen to be tested in this assay. After treatment and a rapid wash with PBS, cells were irradiated with a single sub-toxic dose UVB (5 mJ/cm2) as a good MMPs stimulator and incubated at 37 °C with 5% CO2 for a further 24 h. UVB-irradiated cells were used as a positive control. Subsequently, cell culture supernatants were collected and the concentration of MMP-1 in each sample was determined by interpolation from a 4PL standard curve (6.25-100 ng/mL).

Skin Irritation on Reconstructed Human Epidermis (RHE)

The potential irritation risk of the formulation was evaluated on RHE 3D inserts purchased by Episkin® Laboratories (Lyon, France) according to the standard method DB-ALM Protocol n°135. The reconstructed tissue is represented by a multi-lamellar architecture of keratinocytes grown for 17 days on a 0.5 cm2 inert polycarbonate filter. Upon receipt, RHE inserts were placed in a Growth medium (Episkin® Laboratories, Lyon, France) overnight in standard and sterile incubation conditions (37 °C, 5% CO2 and 95% humidified atmosphere). After this equilibration period, 32 µL/cm2 of the formulation was applied upon the insert surface for 42 min. The efficacy of the test method was evaluated by testing in parallel negative controls (RHE inserts treated with Dulbecco's phosphate-buffered saline, DPBS) and positive controls (RHE inserts treated with Sodium Dodecyl Sulphate, SDS); all conditions were evaluated in triplicate. After exposure, the RHE inserts were gently rinsed twenty-five times with DPBS and placed in a 6 well-plate containing Growth medium (Episkin® Laboratories, Lyon, France) for 42 h in standard and sterile incubation conditions. After recovery time, RHE viability was assessed by MTT (1 mg/mL) and incubated for 3 h at 37 °C, 5% CO2 and 95% humidified atmosphere. The extraction of formazan crystals was performed with isopropanol and the absorbance of the samples was read at 570 nm with a microplate reader. Cell viability was calculated as a ratio of the OD of the test-inserts versus the negative controls (RHE inserts treated only with DPBS). Furthermore, in association with the inserts viability, the interleukin (IL)-1α release in each tested condition was also evaluated by an ELISA kit (Diaclone, Besan?on cedex, France) following the manufacturer’s instruction. The absorbance of each sample was measured at 450 nm with a microplate reader and the IL-1α amount was quantified plotting the mean absorbance value with a linear regression standard curve (3.9 – 250 pg/mL).

Eye Irritation on Human Reconstructed Corneal Epithelium (HCE)

The potential ocular irritation risk was screened on HCE inserts provided by Episkin® Laboratories (Lyon, France), a reconstructed 3D model human cornea-like epithelium. The test protocol was performed in agreement with the reference standard method DB-ALM Protocol n°190. After arrival, the inserts were placed in a 6 well-plate filled with the maintenance medium (Episkin® Laboratories, Lyon, France) and incubated overnight in sterile condition at 37 °C, 5% CO2 and 95% humidified atmosphere. After equilibration, 30 µL of the serum in toto and 10 µL of DPBS were applied on the insert's surface (test-inserts), 30 µL of DPBS and 30 µL methyl acetate added with 10 µL DPBS were applied on the negative and positive insert's surface, respectively. Each condition was tested in triplicate. After 30 min of exposure, the inserts were rinsed twice with DPBS, placed in a 24 well-plate containing the maintenance medium and incubated at 37 °C, 5% CO2 and 95% humidified atmosphere for other 30 min. After recovery time, inserts were transferred in a 24 well-plate prefilled with MTT solution (1 mg/mL) and incubated for 3 h. The isopropanol extraction was performed for 4 h and then the absorbance was read at 570 nm with a microplate reader. The viability of tissue inserts was calculated as a ratio of the mean of each sample OD versus the mean control (inserts treated only with DPBS) OD.

Statistical Analysis

Data are presented as mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was calculated in all experimental sets using the One-way ANOVA followed by Fisher’s LSD multiple comparisons post-test by the GraphPad Prism version 9.0.0 software (GraphPad Software, Inc) and differences with p values < 0.05 were considered statistically significant compared to the relative controls.

Results

Evaluation of MTT Assay

MTT test was performed to study the cell viability of NHDF cells and to select the concentrations that do not cause a decrease in cell respiration exceeding 20%. NHDF cells were treated with scalar concentrations of the product (0.625 - 10 mg/mL) for 24 h. Figure 1 shows that increasing concentrations of the product induce a dose-dependently reduction of cell viability. Concentrations of 2.5, 5, and 10 mg/mL induced a cytotoxic effect, with cell viability of about 77.7, 64.2, and 53%, respectively. The first two non-cytotoxic concentrations having a viability > 80% were identified in 0.625 and 1.25 mg/mL and used for the subsequent tests.

Figure 1: Cell viability expressed as a percentage after 24 h of treatment on NHDF cells with the product compared to the control (untreated cells) (n=3, replicates=3).

Evaluation of Cell Proliferation

After cytotoxicity evaluation, the product was tested on dermal fibroblasts to investigate its proliferative capacity to promote cell renewal by the BrdU assay. NHDF cells were treated with the two highest non-cytotoxic concentrations (0.625 and 1.25 mg/mL) for 24 h. In Figure 2, treatment with the product showed a greater proliferative rate compared to untreated cells (CTRL). After 24 h treatment with 0.625 mg/mL, the percentage of BrdU positive cells is equal to 55.12% versus 38.46% of control cells, indicating a significant increase of 43% (* p values < 0.05). A similar result was confirmed after treatment with the highest concentration used of 1.25 mg/mL (+32% compared to untreated cells, * p values < 0.05).

Figure 2: Representative images of immunofluorescence BrdU assay (A) DNA is marked in blue with Hoechst 33258 while BrdU is visible in green. (B) Quantitative assessment of BrdU positive cells expressed as a percentage compared to control after 24 h of treatment with the product (0.625 and 1.25 mg/mL). Values of * p < 0.05 were considered statistically significant compared to untreated cells (CTRL).

Evaluation of Collagen Synthesis

Collagen is the main structural protein in connective tissue and fibroblasts are the cell population most responsible for its synthesis. To evaluate the capability of the product to modulate collagen production and therefore to perform its function as an anti-ageing product, NHDF cells were treated with 0.625 and 1.25 mg/mL and a colorimetric assay was performed. Figure 3 shows collagen levels produced by the cells after 24 h treatment. The obtained results demonstrate that the product determines a significant increase in the level of newly synthesized collagen equal to 355% (* p values < 0.05) and 425% (** p values < 0.01) after treatment with 0.625 and 1.25 mg/mL, respectively, compared to control (untreated cells).

Figure 3: Collagen levels expressed as µg/mL (A) and percentage (B) after treatment with the product compared to control (CTRL, untreated cells). Values of * p < 0.05 ** p < 0.01 were considered statistically significant compared to CTRL (n=3, replicates=2).

Evaluation of Elastin Synthesis

Together with collagen, elastin is the second principal component of ECM representing 2% of the dermis weight. Its role is closely linked to collagen fibers and it is mainly responsible for the elasticity of the skin. After evaluating the production of new collagen in normal human dermal fibroblasts, the synthesis of elastin was also investigated after 24 h treatment with 0.625 and 1.25 mg/mL. As shown in Figure 4, treatment with the product determines a significant increase in elastin levels. In particular, the amount of elastin measured results to be statistically significant with an increase equal to 63% after treatment with 1.25 mg/mL of the tested product (* p values < 0.05).

Figure 4: Elastin levels expressed as µg (A) and percentage (B) after treatment with the product compared to control (CTRL, untreated cells) in NHDF cells. Values of * p < 0.05 were considered statistically significant compared to CTRL (n=3, replicates=2).

Evaluation of MMP-1 Levels

Alterations in the expression of MMP-1 is correlated predominantly to skin photo-ageing. The possible protection from MMP-1 production was investigated in HaCaT cells using UVB as a source of irradiation. After 24 h treatment, HaCaT cells were treated with the highest non-cytotoxic concentrations selected by a preliminary MTT test, equal to 0.313 and 0.625 mg/mL and UVB irradiation. As shown in Figure 5, the results demonstrated that UVB irradiation markedly increases the MMP-1 production by 25% (** p values < 0.01), compared to non-irradiated control cells (CTRL). In the UVB condition, treatment with the product at the two non-cytotoxic concentrations induced a modest reduction of MMP-1 levels after 24 h, compared to the positive control cells (UVB). In particular, it is possible to observe a reduction of 14% after treatment with 0.625 mg/mL of the product (* p values < 0.05). With the lower concentration tested (0.313 mg/mL), there was a tendency to decrease (about 9%) but without statistical significance.

Figure 5: MMP-1 levels measured in ng/mL (A) and percentage (B) after treatment with the product at the concentrations of 0.313 and 0.625 mg/mL in HaCaT cells. CTRL: untreated cells; UVB: irradiated cells. Values of * p < 0.05 and ** p < 0.01 were considered statistically significant (n=3, replicates=2).

Skin Irritation on RHE

Skin irritation was investigated on 3D Reconstructed Human Epidermis (RHE) evaluating tissue viability by MTT test and the IL-1α amount. The data obtained show that the serum does not present irritant activity after 42 min of exposure upon tissue surface with a viability greater than 50% (equal to 103%), and an interleukin (IL)-1α amount lower than 9 International Unit (IU)/mL (equal to 2.22 IU/mL), thresholds used to identify a substance as not irritant, according to DB-ALM protocol n°135 (*** p values < 0.001) Figure 6.

Figure 6: RHE viability (%) after 42 min of exposure with the product and 42 h of recovery time (A) and IL-1α (IU/mL) amount in the tissue medium after a recovery time of 42 h post-exposure (B). CTRL -: inserts treated with DPBS; CTRL +: inserts treated with SDS; RETINOIDS SERUM: inserts treated with the product in toto. Values of *** p < 0.001 were considered statistically significant (n=1, replicates=3).

Eye Irritation on HCE

The potential eye irritation of the serum was investigated on 3D Human Reconstructed Corneal Epithelium (HCE) evaluating tissue viability by MTT test after 30 min of direct exposure with the tissue. The results show a viability of 75%, thus indicating the absence of irritant or dangerous risk in the periocular zone (threshold ≥ 60%) Figure 7.

Figure 7: HCE viability (%) after 30 min of exposure with the product and 30 min of recovery time. CTRL -: inserts treated with DPBS; CTRL +: inserts treated with methyl acetate; RETINOIDS SERUM: inserts treated with the product in toto (n=1, replicates=3).

Discussion

The aesthetic condition of the skin is gaining importance over the years because of its social impact. The appearance of expression lines that become with age deep wrinkles, loss of tone and elasticity of the skin, irregular pigmentation together with a reduction in the thickness of the epidermis and dermis, represent the typical signs of skin-ageing. All of these changes are induced by chronological ageing or can be accelerated by external factors [12]. Several studies prove that a daily and constant use of cosmetic products enriched with functional compounds, such as vitamins, can help to counteract the signs of ageing and photo-ageing. In particular, in the last two decades, retinoids, like vitamin A and its derivates, have been frequently added to cosmetic formulations. All of them have important effects on skin cells: they can stimulate or maintain the skin keratinocyte’s differentiation from the basal layer to the most superficial one, they are fundamental in epidermal renewal both caused by normal skin turnover or in case of injury, and they also influence the ECM composition. Being lipophilic molecules, retinoids diffuse well across the cell membrane and they can modulate the expression of genes involved in cell proliferation and differentiation by binding specific nuclear receptors, such as retinoid X receptors (RXRs) and retinoic acid receptors (RARs). The retinoids receptor signaling increases the production of procollagen, thus leading to the synthesis of new collagen and, at the same time, inhibits MMPs [13-16]. In cosmetics, the most commonly used retinoids are retinol and retinyl esters. Several articles have been published highlighting the beneficial role of retinoids in the treatment of skin ageing. Kligman reported the positive effects of topical application of retinoids on photodamaged skin, highlighting that the UV damage was partially repaired after constant application of a 0.05% tretinoin cream thanks to the production of new collagen in the dermis [17]. A similar positive effect was observed after treatment with formulations composed of 0.1% retinaldehyde [8, 18, 19]. Moreover, retinaldehyde is well tolerated on human skin with fewer irritative side effects than other vitamin A derivatives, such as retinoic acid [20-23]. Rouvrais and colleagues demonstrated the potential effect of a new dermocosmetic containing retinaldehyde (RAL), delta-tocopherol glucoside, and glycylglycine oleamide with in vitro and clinical studies. Using UV-irradiated fibroblasts, it has been demonstrated a restoration and improvement of the ECM complex with new elastin fibers formation and cell-cell interactions [24]. In this study, we investigated the in vitro effects of a new formulation produced by MatexLab Spa, containing vitamin A derivatives in a mix that included 0.1% of retinol, 0.1% retinaldehyde, and 1% of bioretinol, focusing on the induction of proliferation and ECM synthesis in human dermal fibroblasts (NHDF). The proliferation process was evaluated through a 5-bromo-2'-deoxyuridine (BrdU) assay, showing a statistical increase of the proliferative rate of NHDF cells after 24 h treatment with the two non-cytotoxic concentrations identified by MTT assay. Since the proliferative capacity of fibroblasts is frequently associated with the production of ECM, we then studied the synthesis of collagen and elastin. Our results showed that the product increased the secretion of these ECM components in a significant way in dermal fibroblasts. At the same time, we focused on assessing the effect of the dermocosmetic product on human keratinocytes. The photo-ageing is induced by exposure to UV rays, especially in a chronic manner, and can further affect the normal functions of the skin, especially of its outermost layer, mainly composed of keratinocytes. At this level, UVB induces the generation of high levels of ROS, which mediates the expression of MMPs, as confirmed by in vitro studies [6]. Among the MMPs, MMP-1 cleaves fibrillary collagen type I which is the most present protein in the connective tissue of the skin. Therefore, the photoprotective capacity of the product was evaluated through the measurement of MMP-1 levels, showing a markedly decreased UVB-mediated MMP-1 production in HaCaT cells. To complete our investigation the potential skin irritant risk of the serum was screened in a model of Reconstructed Human Epidermis (RHE) composed by a multi-lamellar architecture of keratinocytes and representing an ideal model closer to human skin. In particular, the tendency to decrease the tissue viability as well as the ability to modulate interleukin (IL)-1α release, represents a valid way to identify potential irritant chemicals. The eye irritant potential risk of the serum was evaluated on Human Reconstructed Corneal Epithelium (HCE), useful for the safety evaluation of the cosmetics application in periocular zone. Data show a good biocompatibility of the serum after exposure of both type of tissues demonstrating its safety and efficacy profile for topical application.

Conclusion

Taken all together, these results show a panel of in vitro assays helpful to support firstly the safety and then the anti-ageing effect of the formulation RETINOIDS SERUM, characterized by a complex of retinoids, suggesting an interesting use of this product to prevent the natural signs of skin ageing.

Acknowledgements

The research was supported and funded by MatexLab S.p.A., Via C. Urbani 2, Ang. via E. Fermi, Brindisi, Italy.

Declaration of Interest Statement

Zerbinati N. is the Scientific Director of Matex Lab. The authors declare no other conflicts of interest.

References

  1. Zhang S, Duan E. Fighting against Skin Aging: The Way from Bench to Bedside. Cell Transplant. 2018; 27: 729-738.
  2. Shin JW, Kwon SH, Choi JY, Na JI, Huh CH, Choi HR, et al. Molecular Mechanisms of Dermal Aging and Antiaging Approaches. Int J Mol Sci. 2019; 20.
  3. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008; 30: 87-95.
  4. Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, et al. Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin. Am J Pathol. 2009; 174: 101-114.
  5. Weihermann AC, Lorencini M, Brohem CA, de Carvalho CM. Elastin structure and its involvement in skin photoageing. Int J Cosmet Sci. 2017; 39: 241-247.
  6. Xuan SH, Park YM, Ha JH, Jeong YJ, Park SN. The effect of dehydroglyasperin C on UVB-mediated MMPs expression in human HaCaT cells. Pharmacol Rep. 2017; 69: 1224-1231.
  7. Docherty AJ, O'Connell J, Crabbe T, Angal S, Murphy G. The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases. Trends Biotechnol. 1992; 10: 200-207.
  8. Mukherjee S, Date A, Patravale V, Korting HC, Roeder A, Weindl G. Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Interv Aging. 2006; 1: 327-348.
  9. Jurzak M, Latocha M, Gojniczek K, Kapral M, Garncarczyk A, Pierzcha?a E. Influence of retinoids on skin fibroblasts metabolism in vitro. Acta Pol Pharm. 2008; 65: 85-91.
  10. Flecknell P. Replacement, reduction and refinement. Altex. 2002; 19: 73-78.
  11. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55-63.
  12. Ganceviciene R, Liakou AI, Theodoridis A, Makrantonaki E, Zouboulis CC. Skin anti-aging strategies. Dermatoendocrinol. 2012; 4: 308-319.
  13. Katsambas AD, Katoulis AC. Topical retinoids in the treatment of aging of the skin. Adv Exp Med Biol. 1999; 455: 477-482.
  14. Griffiths CEM. The role of retinoids in the prevention and repair of aged and photoaged skin. Clin Exp Dermatol. 2001; 26: 613-618.
  15. Zasada M, Budzisz E. Retinoids: active molecules influencing skin structure formation in cosmetic and dermatological treatments. Postepy Dermatol Alergol. 2019; 36: 392-397.
  16. Lee ES, Ahn Y, Bae IH, Min D, Park NH, Jung W, et al. Synthetic Retinoid Seletinoid G Improves Skin Barrier Function through Wound Healing and Collagen Realignment in Human Skin Equivalents. Int J Mol Sci. 2020; 21.
  17. Kligman AM, Leyden JJ. Treatment of photoaged skin with topical tretinoin. Skin Pharmacol. 1993; 6: 78-82.
  18. Boisnic S, Branchet-Gumila MC, Nocera T. Comparative study of the anti-aging effect of retinaldehyde alone or associated with pretocopheryl in a surviving human skin model submitted to ultraviolet A and B irradiation. Int J Tissue React. 2005; 27: 91-99.
  19. Rouvrais C, Baspeyras M, Mengeaud V, Rossi AB. Antiaging efficacy of a retinaldehyde-based cream compared with glycolic acid peel sessions: A randomized controlled study. J Cosmet Dermatol. 2018; 17: 1136-1143.
  20. Creidi P, Vienne MP, Ochonisky S, Lauze C, Turlier V, Lagarde JM, et al. Profilometric evaluation of photodamage after topical retinaldehyde and retinoic acid treatment. J Am Acad Dermatol. 1998; 39: 960-965.
  21. Sorg O, Saurat JH. Topical retinoids in skin ageing: a focused update with reference to sun-induced epidermal vitamin A deficiency. Dermatology. 2014; 228: 314-325.
  22. Saurat JH, Didierjean L, Masgrau E, Piletta PA, Jaconi S, Chatellard-Gruaz D, et al. Topical retinaldehyde on human skin: biologic effects and tolerance. J Invest Dermatol. 1994; 103: 770-774.
  23. Stratigos AJ, Katsambas AD. The role of topical retinoids in the treatment of photoaging. Drugs. 2005; 65: 1061-1072.
  24. Rouvrais C, Bacqueville D, Bogdanowicz P, Haure MJ, Duprat L, Coutanceau C, et al. A new dermocosmetic containing retinaldehyde, delta-tocopherol glucoside and glycylglycine oleamide for managing naturally aged skin: results from in vitro to clinical studies. Clin Cosmet Investig Dermatol. 2017; 10: 35-42.