Research Article

Reorganizing Dermis Macromolecular Structure with Gigartina Stellata Extract to Counteract the Damaging Effect of Earth’s Natural Gravity on Skin

Meunier M¹, De Tollenaere M¹*, Marine Bracq¹, Chapuis E¹, Lapierre L¹, Tiguemounine² J, De Bizemont A¹, Humeau A¹, Scandolera A¹ and Reynaud R¹

¹Sciences & Technologies, Givaudan France SAS, Argenteuil, France

²Plastic Surgery Service, Polyclinique Courlancy, Reims, France

Submission: September 30, 2024; Published: October 16, 2024

*Corresponding author: Morgane De Tollenaere, Sciences & Technologies, Givaudan France SAS, Argenteuil, France, Email: morgane.de_tollenaere@givaudan.com

How to cite this article: Meunier M, De Tollenaere M*, Marine Bracq, Chapuis E, Lapierre L, et al. HIFU with Oral Collagen Vs HIFU Alone in Vaginal Rejuvenation. JOJ Dermatol & Cosmet. 2024; 6(2): 555687.DOI: 10.19080/JOJDC.2024.06.555687

Abstract

Background: Earth’s natural gravity is known to exacerbate skin ageing by causing visible face sagging, especially at the lower part of the face. We hypothesized that reorganization of the dermis scaffold is essential for developing skin resistance against sagging.

Methods: Skin explants from young and mature donors were compared, proteomics and atomic force microscopy were used to examine the impact of ageing on dermis structure in skin explants. A smart extract from Gigartina stellata was developed to reorganize the dermis scaffold and its benefits were evaluated on skin explants and on volunteers.

Results: Age-related differences were found in proteins involved with elasticity and collagen structure. These were associated with reduced skin suppleness and a unidirectional orientation of collagen in mature skin, making it more susceptible to the effects of gravity. Gigartina stellata extract restored the dermis 3-D organization in skin explants and improved lower face sagging.

Conclusions: Ageing disrupts the structural organization of the dermis, leading to sagging. Reorganization of the dermis scaffold can enhance skin resilience, prevent the dermis scaffold from collapsing, and hinder gravity-induced face sagging.

Keywords: Gravity; Dermis; Macromolecules; Sagging; Resistance

Abbreviation: UV: Ultraviolet; VOC: Volatile Organic Compounds; FBLN5: Fibulin-5; MAGP: Micro-fibril-Associated Glycoproteins; LOXL1: Lysyl Oxidase Like 1; MFAP: Microfibrillar-Associated Protein; NHDFs: Normal Human Dermal Fibroblasts; GADD45A: Growth Arrest and DNA Damage Inducible Alpha; RQ: Relative Quantification; MS: Mass Spectrometer; QNM: Quantitative Nanomechanical Mapping; 3-D: 3 Dimensional; AFM: Atomic Force Microscopy

Introduction

Over the past few decades, many skin ageing mechanisms have been described, including natural chronological ageing of the body. Skin ageing can be aggravated by various factors in the exposome [1]. Amongst these are ultraviolet (UV) radiation, outdoor pollution from hydrocarbons and heavy metals, indoor pollution from cigarette smoke, volatile organic compounds (VOC), and artificial light. Such external factors are well known to accelerate the natural skin ageing process [2]. Despite extensive investigation of the exposome, an additional external factor that has so far received little attention is the Earth’s natural gravity. Indeed, gravity is often neglected because all individuals are exposed to it daily and equally, in contrast to UV radiation, pollution, artificial light, etc.

In their study on the impact of gravity on skin, Flament et al postulated that the direction of gravitational force on the body varies according to the angle of the body relative to the Earth’s horizontal plane [3]. Moreover, the upright position of volunteers exacerbates the appearance of vertical wrinkles (e.g., nasogenian wrinkles). These wrinkles were much smoother in the supine position, especially in people aged over 40 years. The work by Flament et al confirmed the hypothesis that gravity has an effect on the clinical signs of facial skin ageing, especially on the lower part of the face. An internal survey of 4100 consumers in 10 countries confirmed this result, since 54% spontaneously declared they were affected by sagging skin, lack of skin tonicity and firmness, loss of well-defined face contour, or the development of a doublechin [4].

To explain the effect of gravity on sagging of the lower face, and more especially to understand why this loss of resistance with age, we hypothesised that it may be due to alteration of the skin macromolecular structure. The structure and function of the dermis have been well studied. It is composed of a tangle of macromolecules that form a scaffold. This confers the skin with biomechanical properties such as firmness, elasticity and resistance to deformation. Among the macromolecules, type I and type III collagen are the major components of collagen fibers. These play a structural role in providing firmness to the skin and conferring tissues with mechanical resistance against traction [5]. Collagen fibers are intertwined with elastic fibers. Functional elastic fibers are composed of a complex of fibulin-5 (FBLN5) that interacts with fibrillin-1 (FBN1), micro-fibrilassociated glycoproteins (MAGP), e.g. microfibrillar-associated protein (MFAP)-2, -4 and -5), lysyl oxidase like 1 (LOXL1), and tropoelastin [6]. Fibulin-5 functions as a scaffold for elastic fibers [7], but its expression in the skin declines with age [8]. MFAP-4 and MFAP-5 interact and colocalize with fibrillin-1, tropoelastin, and the cross-linking enzyme desmosine. They also promote tropoelastin self-assembly on top of fibrillin microfibrils, while MFAP-2 binds strongly to fibrillin microfibrils [9]. Finally, LOXL1 is responsible for the crosslinking between collagen and elastin fibrils [10]. These elastic fibers provide the skin with its ability to undergo repetitive extension or deformation, and then to return to its original size [11]. The recoiling force of elastic fibers is believed to be the force that leads to the contraction of extended collagen fibers [12].

During skin ageing, type I and type III collagen (e.g. COL1A1 and COL3A1) are progressively degraded by matrix metalloproteinases due to loss of expression of metallopeptidase inhibitor 1 (TIMP1) [13,14]. This leads to the loss of 75% of collagen density between 30 and 70 years of age [15]. Elastic fibers progressively degrade and lose their capacity, resulting in generalized atrophy of the extracellular matrix [16]. With this background knowledge, we hypothesized that this loss of extracellular matrix component synthesis and organization we are facing with age could be an aggravating factor that contributes to the gravity-induced face sagging [17].

To address this issue, we compared the clinical features of skin from of different ages and analyzed the macromolecular organization and biomechanical properties of their skin. In a second step, we investigated a natural product to reorganize the skin macromolecular organization, and thereby, reduce clinical features such as face sagging and loss of resistance to deformation.

Materials and Methods

Preparation of Gigartina stellata Extract

An aqueous suspension of Gigartina stellata starting with 4.8% dry matter was prepared. The algae material was first cut with scissors into small pieces of 15-20 mm length and then macerated under stirring for 15 minutes at room temperature. The biomass (first solid phase) and supernatant (first liquid phase) were then separated by filtration (Whatman filter, 40 μm). Subsequently, 1,3-propanediol was added to the supernatant at a final concentration of 20% to obtain the first part of the extract. The insoluble part of the previous extraction (first solid phase) was suspended again to 4.8% of dry matter in osmosed water, stirred, acidified to pH=2 with 96% sulphuric acid solution, and then heated to 80°C for 2 h. Next, the suspension was cooled and neutralized to pH=5 with 10 M sodium hydroxide solution. The extract was centrifuged (4500 rpm, 20°C, 20 min), the supernatant was separated from the solid, and 1,3-propanediol was added to the supernatant (second liquid phase) at a final concentration of 20% to obtain the second part of the extract. The first and second extracts were combined, stirred and heated at 60°C for 30 minutes. The mixture was then filtered first with a 2.5 μm pore size and then with a 0.3 μm pore size to obtain the final Gigartina stellata extract.

In vitro / ex vivo Biological Evaluation

Cells and Skin Source

All experiments were performed on primary cells and skin explants obtained from skin surgical residues following plastic surgery. Skin explants were obtained from donors who underwent abdominoplasty and lifting at the Polyclinique Courlancy, Reims. All donors had read, understood and signed an Information and No Objection Form for the Use of Tissues, Cells, and Products of the Human Body Collected During Surgery (Surgical Residues) for Dermocosmetic Research Purposes, as outlined in articles L. 1211-2 alinéa 2 and L. 1245-2 Code de la santé publique.

Transcriptome Analysis of Fibroblasts

Normal human dermal fibroblasts (NHDFs) were seeded at 300,000 cells per well in 6-well plates in the presence of Dulbecco’s Modified Eagle Medium (DMEM, Gibco®, Life Technologies, California, USA) supplemented with 10% fetal calf serum (FCS, Hyclone, Utah, USA) and 1% antibiotic (Sigma-Aldrich, Missouri, USA). After 48 h of culture, the cells were washed twice with Phosphate-Buffered Saline solution (PBS, Gibco®) and allowed to rest overnight in basal medium before being stimulated with 0.5% (v/v) Gigartina stellata extract in basal DMEM. Control cells were left untreated. After 6 and 18 h of treatment, RNA was extracted using Trizol (Thermo Fisher Scientific, Massachusetts, USA) and the RNA quality was evaluated. Reverse transcription to obtain cDNA was performed using the Verso cDNA kit (Thermo Fisher Scientific). RT-qPCR was performed on specific pre-coated plates (Biorad, California, USA) designed to study the transcriptomic expression of various genes involved in dermal function. 10 ng of cDNA per well was used with CFX96 Touch (Biorad) and an iTaq Universal Sybr Green supermix (Biorad). The relative quantification (RQ) of gene expression was calculated relative to the expression of PES1 (Pescadillo Ribosomal Biogenesis Factor 1), GADD45A (Growth Arrest and DNA Damage Inducible Alpha) and HMBS (hydroxymethylbilane synthase) housekeeping genes.

Proteome Analysis of Skin Explants

Sample preparation: Skin explants obtained from young donors (average age 28 years) and mature donors (average age 59 years) were maintained in an air-liquid interface. The skin explants from young donors were kept untreated, while those from mature donors were topically treated with a 1% extract of Gigartina stellata. Treatment and medium (MIL217C from Biopredic International, Saint-Grégoire, France) were renewed every day for 5 days. After 5 days of stimulation, skin explants were washed twice with PBS (Gibco®) and frozen at -80°C for later analysis.

Samples treatment: Sheared tissue was added to a zirconia oxide bead mix in 700 μl of iST LYSE buffer (Deoxycholate, TCEP and Chloroacetamide). Samples were lysed by two rounds of a bead-beating cycle. The homogenate was transferred into Diagenode protein extraction tubes and any co-extracted nucleic acid and organelles were sheared by micro-cavitation (Bioruptor Pico, Diagenode, Liège, Belgium). Proteins were solubilized, reduced, and alkylated by boiling in iST LYSE buffer. The protein concentration was determined with the BCA method. Peptide extracts were prepared ac-cording to the iST (in-Stage Tip) method. 50μg of protein was digested with a mixture of LysC and trypsin. The resulting peptides were purified by mixed-mode reverse-phase cation exchanger SPE (Solid Phase Extraction; PreOmics GmbH, Martinsried, Germany), dried and solubilized in 100 μl of 3% acetonitrile / 0.1% formic acid aqueous solution. The peptide concentration was then determined using the BCA method.

LC-MS/MS: Peptides (300 ng) were injected in triplicate for each sample. Chromatography was performed using an Ultimate 3000 (Dionex, California, USA) instrument with C18 (75 μm x 50 cm, 2 μm material) column. Following a 3-minute trapping step on a precolumn, a 2.5% to 35% acetonitrile gradient was applied over 120 minutes at a flow rate of 300 nl/min. Data were acquired using a Q-Exactive (Thermo Fisher Scientific, Massachusetts, USA) mass spectrometer (MS). The MS scan was performed at a resolution of 70000 and an accumulation time of 60 ms. MS/MS scan was performed with a resolution of 17500 on the 10 most intense ions of each cycle, with an accumulation time of 60 ms. A total of 6545 cycles were performed, with an average of 17 cycles per chromatographic peak.

Protein identification: Proteins were identified using the SEQUEST-HT algorithm against a database containing the human reference proteome and enzymes used for digestion mined from UNIPROT. The search parameters were: enzyme = trypsin (full); allowed miscleavage = 2; precursor error tolerance = 10 ppm; fragment error tolerance = 0.02 Da; dynamic modification = oxidation (M), deamidation (N/Q); protein terminus modification = acetylation; static modification = carbamidomethyl (C). Determination of the False Discovery Rate (FDR) was made using the Percolator algorithm.

All spectra reported with a confidence of less than high by SEQUEST-HT was considered as not identified. These were reprocessed with the MS Amanda 2.0 algorithm against the same database as above. Search parameters were: enzyme = trypsin (full); allowed miscleavage = 2; precursor error tolerance = 10 ppm; fragment error tolerance = 0.02 Da; dynamic modification = oxidation (M)(P), deamidation (N/Q); protein terminus modification = acetylation, Met-Loss, Met-Loss+Acetyl; static modification = carbamidomethyl (C). The determination of FDR was made using the Percolator algorithm.

Protein quantification: Data were processed using Minora and feature mapper for Proteome Discoverer 2.3 software (Thermo Fisher Scientific). Peak integration parameters were: post-acquisition recalibration = true (fine parameters); minimum trace length = 5; minimum number of isotopes = 2; max Delta RT for isotope = 0.2 min PSM (Peptide-Spectrum match); confidence level for integration = high.

Chromatographic alignment parameters were: RT alignment= true; parameter tuning = fine; max RT shift= 5 min; mass tolerance = 10 ppm. Feature mapping parameters were: RT tolerance = automatic; mass tolerance = automatic; S/N threshold = 2. Statistical analyses were performed using the software Precursors Ions quantifier node for Proteome Discoverer 2.4 (Thermo Fisher Scientific).

General Quantification Settings were: Peptide to use = Unique + RAZOR (Unique = peptides that are not shared by different proteins or protein groups; RAZOR = peptides shared by multiple protein groups but only used to quantify protein with the largest number of unique peptides and the longest amino acid sequence); Consider Proteins Groups for Peptide Uniqueness = True; Reject Quan Results with Missing Channels = False. Precursor Quantification Settings were: Precursor Abundance Based on Area; Min number replicate feature = 50% (to be used in quantification, peptides must be detected in at least 50% of samples from one group). Normalization settings: total peptide amount (calculates the total sum of abundance values for each injection over all peptides identified. The injection with the highest total abundance is used as a reference to correct abundance values for all other injections by a constant factor per injection, so that at the end the total abundance is the same for all injections).

Data selection: A first selection was performed by selecting only those proteins that were significantly affected by ageing (mature donor vs young donor) and that were reversed by Gigartina stellata extract (treated mature donor vs untreated mature donor).

Evaluation of the Biomechanical Properties of Skin

Sample preparation: Skin explants from young (average age 19 years) and mature (average age 49 years) donors were maintained in an air-liquid interface. Skin explants from young donors were left untreated, while those from mature donors were topically treated with a 1% Gigartina stellata extract. Treatments and medium were renewed every day for 3 days. After 3 days of stimulation, skin explants were washed twice with PBS and cryopreserved in OCT™ compound mounting medium (VWR, Pennsylvania, USA) and cryosectioned at 20 μm thickness.

Analysis of elastic modulus by Atomic Force Microscopy: The Atomic Force Microscope (AFM) used in this study was a Bioscope Resolve (Bruker, Massachusetts, USA) instrument, onto which was added an epifluorescence microscope (DMi8, Leica, Wetzlar, Germany). This configuration allows precise positioning of the AFM probe on the sample, and allows the acquisition of correlative images from mechanical to fluorescent. The Quantitative Nanomechanical Mapping (QNM) Peakforce® mode was used in this study. The AFM probe harbors a 0.4 N/m theoretical spring constant and has a curvature radius of < 10 nm. Before each use, probe deflexion sensitivity was measured on a sapphire and the spring constant was also calibrated using the thermal noise method. Force measurements were performed in air (PBS 1X, Batch number: CP20-3404). AFM measurement involves acquisition of the Force-volume on the dermis, as illustrated in (Figure 1).

Study of Collagen Network Organization using Bi-Photonic Microscopy: A ZEISS LSM880 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) was used for imaging Second Harmonic Generation. The laser was a coherent, biphoton-pulsed Cameleon laser, and the ob-jective was a “C-Apochromat” water x40 objective. To image collagen, the samples are excited at 900 nm wavelength and light is collected with a filter set at 445 nm. Three large images (600 μm x 600 μm) were taken per condition. Data analysis was performed with image J software and the mean grey value of three surface images (50 μm x 50 μm) was analysed. Collagen fiber orientation was analysed with Rozeta software on three 50 μm x 50 μm size images.

First Clinical Evaluation of Gigartina stellata Extract

Clinical studies were carried out in compliance with the most recent recommendations of the World Medical Association ethical principles for medical research involving human subjects (Helsinki Declaration, 64th WMA General Assembly, Fortaleza, Brazil, October 2013). Participation in the study was only permitted after providing informed consent.

INCI formula

The formula for INCI is: AQUA/WATER, ISODECYL NEOPENTANOATE, CETYL ALCOHOL, GLYCERYL STEARATE, GLYCERIN, ± GIGARTINA STELLATA, PEG-75 STEARATE, CETETH-20, STEARETH-20, PHENOXYETHANOL, DIMETHICONE, METHYL PARABEN, PROPYLPARABEN, ETHYL PARABEN, FRAGRANCE.

Study description

A placebo-controlled, single blind clinical study was carried out on 44 volunteers with dry skin (corneometry measurement <70 a.u.) who presented with wrinkles in the crow’s feet area, as well as chin ptosis. The subjects were randomly divided in two equal groups: one applied a placebo cream (22 women; average age 49±5 years), while the other applied a cream containing a 1% extract of Gigartina stellata (22 women; average age 49±6 years). Each group of volunteers applied the cream every day in the morning and evening for 28 days. During the study, skin biomechanical properties were analysed using Cutometer® (Courage+Khazaka, Köln, Germany) analysis and by fringe protection with AEVA-HE® (EOTech, Michigan, USA).

Skin Biomechanical Properties Analyzed by Cutometry

Assessment of the biomechanical properties of skin allows the functional state of the following tissue structures to be evaluated: i. Elastic structures (elastic fibers, curvature of the connective bundles, wrinkles of the stratum corneum) ii. Viscous-behaving structures (interstitial fluids, internal adherences).

The study was performed using the Cutometer® MPA 580 instrument (Courage and Khazaka), with the measuring principle based on the suction method. Negative pressure is created in the device and the skin is drawn into the cylindrical aperture (2 mm diameter) of the probe. The depth of penetration inside the probe was determined by an optical measuring system. Each suction phase is followed by a relaxation phase.

The program used for this study was: length of cycle = 4 seconds (suction: 2 seconds / relaxation: 2 seconds); negative pressure = 450 millibars; diameter of the chamber = 2 mm; area measured = crow’s feet.

The resistance of skin to being sucked up by the negative pressure, and its ability to return to the original position, were displayed as curves at the end of each measurement. The parameters are then calculated from these curves. During the suction phase, deformation of the skin by negative pressure first measures the elastic resistance, followed by the viscous component. Together, these measurements represent skin firmness. During the relaxation phase, immediate recovery of the skin measures shear cutaneous elasticity, whereas the delayed return of skin to its initial position measures the viscoelastic component. Details of these measurements are shown in (Figure 2).

This study focused on the following parameters:

i. R0 (or Uf), which represents the amplitude of the skin during the suction phase. At equal pressure, the more flexible the skin, the greater the amplitude. R0 therefore evaluates the viscoelastic dispensability, or in other words the firmness of the skin.

ii. R5 represents net elasticity. This is the elastic portion of the relaxation region divided by the elastic portion of the suction region (Ur/Ue).

These parameters were measured at D0 and D28.

Analysis of double chin volume by AEVA-HE®

The AEVA-HE system is based on a patented (US 7,821,649) fringe projection unit combined with stereo imaging techniques. This method allows various measurements ranging from the reduction of wrinkles to body reshaping. It is designed to quantify the efficacy of cosmetics, aesthetic and dermatology products, and treatments.

AEVA-HE is particularly well suited to the following applications:

i. Face (wrinkles, volume, fine lines and pores, glabella, eye bags, nasogenian fold, lips, sagging)

ii. Body reshaping and firmness (circumference of waist, legs, and breasts)

iii. Automatic or interactive areas of extraction

iv. Amplitude, roughness, volume, areas, circumference evaluation.

Second Clinical Evaluation of Gigartina stellata Extract

INCI formula:

AQUA/WATER, ISODECYL NEOPENTANOATE, CETYL ALCOHOL, GLYCERYL STEARATE, ± GIGARTINA STELLATA, PEG- 75 STEARATE, CETETH-20, STEARETH-20, PHENOXYETHANOL, DIMETHICONE, 1.2 HEXANEDIOL, CAPRYLYL GLYCOL, FRAGRANCE

Panel Description

A placebo-controlled, single blind clinical study was carried out on 40 volunteers with wrinkles and a visibly sagging lower face (V-shaped re-shaping). The subjects were randomly divided into two equal groups: one applied a placebo cream (20 women; average age 63±5 years), while the other applied a cream containing a 1% extract of Gigartina stellata (19 women; average age 65±5 years). Each group of volunteers applied the cream every day in the morning and evening for 56 days. The collagen level on the oval of the face was measured using SIAscope® prior to, and after 56 days application of cream with or without the Gigartina stellata extract.

Measurement of collagen density by SIAscope®

The SIAscope® method of spectrophotometric intracutaneous analysis provides insight into how light interacts with the skin structure. This makes it possible to visualize the distribution of chromophores such as melanin, hemoglobin, and collagen up to 2 mm below the skin surface. A SIAscope® portable scanning device connected to Siametrics® software was placed in contact with the skin and used to illuminate. Some of the light is reflected and scatters away from the surface, while the remaining light is transmitted through the top layers of the skin. Varying fractions of the incoming light are absorbed by the melanin in the epidermis before penetrating the dermis where they are absorbed by hemoglobin in the blood vessels. Scattering also occurs in the dermis when the light interacts with collagen fibers, resulting in a portion of the light being redirected back to the surface. SIAscope® interprets the combination of wavelengths that are returned to the surface and subsequently produces SIAscans®. These are generated using inbuilt, proprietary mathematical models of skin optics. The concentration and distribution of collagen in this study was analysed at D0, D28 and D56.

Illustrative pictures obtained by LC-OCT®

Line-Field Optical Coherence Tomography (LC-OCT®) is an innovative, non-invasive skin imaging technique. It combines the high resolution of confocal microscopy with the significant tissue penetration of Optical Coherence Tomography (OCT) in one device. LC-OCT® was used to visualize the dermal fibrous network at the level of the facial oval at D0, D28 and D56.

Elasticity Test

The elasticity test consists of applying a 20 g mass suspended by a thread and attached by a strip to the oval of the face. This test was carried out at D0, D28 and D56 and pictures taken at each time to record the evolution of skin elasticity and resistance to deformation by the suspended mass.

Statistical Analysis

Data normality for in vitro, ex vivo and clinical data was first examined by Gaussian law using the Shapiro-Wilk test. Depending on the result, either parametric or non-parametric tests were used to examine the effect of Gigartina stellata extract compared to the untreated condition or placebo. Statistical significance was denoted as: #p<0.1, *p<0.05, **p<0.01, and ***p<0.001.

Results

Expression to Dermis Macromolecules Organization

We first tried to understand the molecular basis for the skin alteration with age. Comparison of the two age groups (26 years old of average age versus 54 years old) by proteomic analysis revealed a significant negative impact of ageing on the expression of proteins involved in the organization of elastic fibers (MFAP4, -10%; TIMP1, -60%; FBLN5, -76%) and on other structural proteins of the dermis (COL1A1, -75%; COL2A1, -75%) (Figure 3). These results confirmed the negative impact of ageing on the composition of skin extracellular matrix and on skin elasticity. Moreover, they highlight the need to develop active ingredients that could restore the expression of these proteins.

The loss of protein expression in mature skin was associated with loss of the dermal 3 dimensional (3-D) scaffold. Moreover, by using bi-photonic microscopy, a single orientation of collagen fibers was found in mature skin instead of the multiple orientations observed in younger skin (Figure 4). As shown by atomic force microscopy, this collapse of the dermis scaffold led to a 10-fold increase in elastic modulus, translating into a loss of skin suppleness (Figure 5).

Effect of Gigartina stellata Macroalgae on Dermis Macromolecules Reorganization

We next studied the link between perturbation of the dermis scaffold and the loss of skin biomechanical properties. The aim was to reorient dermis macromolecules in order to reorganize the dermis scaffold, thereby improving skin biomechanical properties at the clinical level. After screening several molecules, interesting results were obtained with an extract from Gigartina stellata macroalgae.

Gene regulation

We first evaluated the potential benefits of Gigartina stellata extract on the elastic properties of skin by a transcriptomic study. Results for the fold-changes and standard error of measurement (SEM) are shown in (Table 1).

After 6 h of treatment, Gigartina stellata extract showed significant bioactivity in the dermis by inducing the expression of genes that control elastic fiber organization (FBLN5, +34%; LOXL1, +32%; MFAP2, +10%), while inhibiting the expression of a gene involved in elastic fiber degradation (heparanase (HPSE), -53%). These pathways were altered in a similar manner after 18 h of treatment with the Gigartina stellata extract, as shown by the upregulation of genes involved in elastic fiber organization (FBLN5, +24%; MFAP2, +24%; MFAP5, +6%), and downregulation of HPSE (-40%). Gigartina stellata extract also significantly upregulated the expression of genes involved in organization of the extracellular matrix (COL3A1, +33%; CD44, +58%). These results highlight the strong potential of this active ingredient to improve skin elasticity.

Protein Expression

Transcriptomic analysis indicated that Gigartina stellata extract could improve the organization of skin elastic fibers. Next, we evaluated the beneficial effect of this extract by examining the protein level in skin explants from mature donors. The expression of five proteins that decreased during ageing was significantly increased by topical application of Gigartina stellata extract for 5 days. Indeed, the expression of MFAP4, TIMP1 and FBLN5 involved in elastic fiber organization increased by 64%, 23% and 95%, respectively, while the expression of COL1A1 and COL2A1 increased by 17% and 14%, respectively (Figure 6). The above results on protein expression confirm that Gigartina stellata extract has biologically relevant anti-ageing properties with regard to dermis structure, and in particular with regard to the organization of elastic fibers.

Skin Biomechanical Properties

After studying the effect of Gigartina stellata extract on dermis and elastic fiber structure at the level of gene and protein expression, we next evaluated its effect on the biomechanical properties of skin. Topical application of Gigartina stellata extract to the mature skin surface significantly reduced elastic modulus in the dermis by 88%, thus providing evidence of improved skin suppleness (Figure 7). This result was consistent with the improved elastic fiber organization, as demonstrated by the upregulated expression of relevant proteins

Analysis of Dermis Scaffold

The properties of skin suppleness and rigidity are linked to organization of the dermis scaffold. Interestingly, the topical application of Gigartina stellata extract on mature skin explants led to reorganization of the collagen fibers in multiple orientations (Figure 8). This redistribution favors reorganization of the dermis scaffold, which could explain the improvement in skin biomechanical properties revealed by atomic force microscopy (AFM).

Clinical Improvement in Skin Biomechanical Properties and Double-Chin Reduction

A clinical study was performed with a cream formula containing a 1% Gigartina stellata extract versus placebo to evaluated the effect of this extract on volunteers presenting dry and mature skin, crow’s feet wrinkles, and facial ptosis (V-shaped reshaping). The results showed that Gigartina stellata extract significantly increased skin firmness after 28 days of application, as shown by a 7-fold reduction in the R0 parameter versus placebo, and a 2.7-fold increase in skin elasticity (R5 parameter) versus placebo (Figure 9). These results are consistent with the earlier in vitro / ex vivo results showing that reorganization of dermis components such as collagen and elastin fibers correlated with improved skin elasticity (Figures 4 & 5).

Next, the effect of the extract on double chin volume was evaluated in the same volunteers. AEVA-HE® revealed that application of Gigartina stellata extract for 1 month reduced the double chin volume by 18 mm3. Interestingly, the placebo cream appeared to have a deleterious effect, with a slight increase in the double chin volume (Figure 10). The effect of the extract was also clearly visible, as shown in the case of a representative volunteer (Figure 11).

Clinical Improvement in Dermis Macromolecules Organisation

A second clinical study was performed on 40 volunteers who presented with wrinkles and visible sagging of the lower face. Measurement with Siascope® revealed a significant improvement in skin collagen density following application of the extract, as shown by a 3.4-fold increase versus placebo after 28 days, and a 2.8-fold increase after 56 days (Figure 12). These results were consistent with earlier in vitro and ex vivo results that showed increased gene and protein expression of collagen after treatment with Gigartina stellata extract (Table 1 and Figure 6).

We further evaluated collagen synthesis using Line-Field Optical Coherence Tomography (LC-OCT®). This tool was used here to visualize the skin internal structure via light penetration to several defined depths. The recorded images clearly show the appearance of collagen fibers in the skin of volunteers who applied the Gigartina stellata extract for 2 months, but not in the skin of volunteers who applied placebo cream (Figure 13). These images complement the measurements made with Siascope® and suggest the active ingredient not only boosts collagen synthesis in the skin but also favors the formation of functional collagen fibers. Finally, we investigated whether neo-formation of collagen fibers in the dermis and increased collagen density following application of the extract would also improve skin resistance to deformation, as observed by an amelioration of skin elasticity and firmness during the first clinical evaluation (Figure 9). To address this, a skin deformation test was performed by hanging a 20 g weight to the cheek. Following application of the active cream, the skin in the V-shape of the face was found to be firmer and more tonic with a better resistance to gravity, while no effect was observed with the placebo cream (Figure 14).

Discussion

Many factors from the exposome have been extensively studied over the years in relation to their effect on human skin. Some studies have reported an effect of gravity on facial appearance, especially sagging of the lower part of the face [3]. To complete this knowledge, a past internal study on the evaluation of a cosmetic ingredient on 80 volunteers of various ages evidenced that volunteers aged 60-69 years presented 3-times more wrinkles, a 4.4-fold higher total wrinkles area and a 1.3-fold higher count of pores than volunteers aged 20-29 years (data not shown).

To understand the difference with younger volunteers, we compared macromolecular organization in the dermis between young and mature skins. Confocal microscopy was used to study collagen autofluorescence through second harmonic generation, revealing a loss of scaffold structure with age. Indeed, the collagen fibers in young skin were organized in multiple orientations, giving rise to a 3-D scaffold organization. In mature skin, the collagen fibers were organized mainly in a single direction, indicating collapse of the dermis scaffold. These results correlated with changes in the skin’s biomechanical properties. For example, the elastic modulus was increased in older skin, implying a decrease in skin suppleness due to loss of the dermis scaffold. The loss of skin macromolecular organization may underlie a loss of resistance to gravity and hence to subsequent face sagging.

To confirm this hypothesis, we specifically developed an active ingredient to improve the macromolecular organization of the dermis. For this purpose, a Gigartina stellata extract was found to induce the expression of genes involved in the structure of elastic fibers. It also protected against the breakdown of extracellular matrix organization, thus potentially improving skin organization and elasticity. These results were confirmed by measuring proteins in skin explants from mature donors treated with the extract. Treatment with the active ingredient significantly increased the expression of proteins involved in elastic fiber organization and dermis structure. Our results confirm that Gigartina stellata extract has interesting dermis restructuring properties that are related to effects on collagen fibers and also to effects on the organization of elastic fibers.

The apparent benefits of Gigartina stellata extract with regard to dermis organization and biomechanical properties were confirmed with skin explants from mature donors. These showed evidence for the reorganization of collagen fibers in multiple orientations, which could be explained by an improvement in the structure of elastin fibers induced by Gigartina stellata extract. Indeed, Ueda et al. reported that the recoil force of elastic fibers may lead to the contraction of extended collagen fibers [12]. This redistribution favors reorganization of the dermis scaffold, which could explain the improved biomechanical properties of skin revealed by AFM.

To further investigate our hypothesis, we conducted two clinical studies specifically on the lower part of the face, as this area is highly concerned by face sagging. Cutometry revealed a significant improve-ment in skin firmness and elasticity following treatment with Gigartina stellata extract. This was consistent with earlier in vitro / ex vivo results that showed reorganization of the dermis macromolecular structure. The beneficial effects of the extract on skin biomechanical properties were also evaluated by studying double chin volume. Double chin formation can have multiple causes, including natural skin sagging due to ageing. Since we showed the Gigartina stellata extract caused reorganization of the dermis macromolecules to improve the biomechanical properties of skin, we hypothesized this would lead to a tensing effect on the double chin and thus to a reduced volume. Reconstructed images from AEVA-HE® revealed a significant reduction in double chin volume after 1 month of treatment with Gigartina stellata extract. This product improved skin collagen density at the lower part of the face, as shown with the Siascope®, and also induced the appearance of newly formed collagen fibers in the skin. The complete reorganization of dermis scaffold shown in vitro led to improved skin resistance to deformation, as shown by images from the skin deformation test. This last clinical result evidenced that Gigartina stellata extract is able to give the skin resistance against gravity-induced face sagging.

Conclusion

This work showed that while ageing, skin progressively lose its macromolecular or-ganization responsible of skin 3-D scaffold, leading to a progressive loss of resistance against Earth’s gravity, resulting in an exacerbation of face sagging during ageing. Our research based on the results obtained with the application of Gigartina stellata extract, indicates that reorganization of the dermis macromolecular structure is a promising approach to counteract the damaging effects of gravity on human skin. Conflicts of Interest: The authors declare no conflicts of interest.

Institutional Review Board Statement: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Acknowledgments

The authors would like to thanks all team members for their participation on the conducting of the studies. Thank you also to Phylogène and Biomeca for their involvement and their help in the completion of this study.

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