{"id":3946,"date":"2023-05-30T11:23:49","date_gmt":"2023-05-30T10:23:49","guid":{"rendered":"https:\/\/site.suisselle.ch\/?p=3946"},"modified":"2023-11-21T09:34:03","modified_gmt":"2023-11-21T09:34:03","slug":"issue-12-cellbooster-glow-anti-aging-and-depigmentation-effect-of-a-hyaluronic-acid-mechanically-stabilized-complex-on-human-skin-explants","status":"publish","type":"post","link":"https:\/\/suisselle.com\/de\/articles\/issue-12-cellbooster-glow-anti-aging-and-depigmentation-effect-of-a-hyaluronic-acid-mechanically-stabilized-complex-on-human-skin-explants\/","title":{"rendered":"Issue 11: CELLBOOSTER\u00ae GLOW: Anti-Aging and Depigmentation Effect of a Hyaluronic Acid Mechanically Stabilized Complex on Human Skin Explants"},"content":{"rendered":"

Gabriel Siquier-Dameto 1,2,\u2020, Sylvie Boisnic 3,\u2020, Pere Boadas-Vaello 2 and Enrique Verd\u00fa 2,*<\/strong>
\n1 Dameto Clinics International, 1171 VC Badhoevedorp, The Netherlands; info@dametoclinics.com
\n2 Research Group of Clinical Anatomy, Embryology and Neuroscience (NEOMA), Department of Medical
\nSciences, University of Girona, E-17003 Girona, Catalonia, Spain; pere.boadas@udg.edu
\n3 Groupe de Recherche et d\u2019Evaluation en Dermatologie et Cosm\u00e9tologie (GREDECO), 75116 Paris, France;
\ngredeco@orange.fr
\n* Correspondence: enric.verdu@udg.edu
\n\u2020 These authors contributed equally to this work.<\/p>\n

Abstract:<\/strong> Solar radiation and environmental pollutants are factors that cause changes in the skin
\nthat trigger skin aging. The objective of the study is to evaluate the rejuvenating effects of a complex
\nformed by hyaluronic acid supplemented with vitamins, amino acids and oligopeptides in explants
\nof human skin. For this, surplus skin samples have been obtained from donors that have been resected
\nand cultivated on slides with membrane inserts. The complex was administered to some skin
\nexplants and the percentage of cells with low, medium and high levels of melanin was evaluated as
\nan indicator of the degree of pigmentation. Other skin segments were irradiated with UVA\/UVB,
\nthen the product was administered on several slides and the levels of collagen, elastin, sulfated GAG
\nand MMP1 were evaluated. The results show that the administration of the complex significantly
\nreduces the percentage of skin cells with a high melanin content by 16%, and that in skin irradiated
\nwith UVA\/UVB, there is a decrease in the content of collagen, elastin and sulfate GAGs, and the
\ncomplex reverses this reduction without changing MMP1 levels. This suggests that the compound
\nhas anti-aging and depigmentation effects on the skin, giving a skin rejuvenation appearance.<\/p>\n

Keywords<\/strong>: skin explants; melanin; extracellular matrix; hyaluronic acid; aging; depigmentation
\n————————-
\n1. Introduction<\/strong>
\nHyaluronic acid (HA) is one of the most widely used biopolymers of extracellular
\nmatrix components in dermo-aesthetic medicine, with the aim of rejuvenating the skin [1\u2013
\n4]. In human skin, hyaluronic acid is located in the epidermis, except in the upper granular
\nlayer and the stratum corneum, and in the first layers of the dermis (upper and lower
\ndermis) [5,6]. Hyaluronic acid content decreases with aging, both in men\u2019s and women\u2019s
\nskin [7], so its injection improves the aged appearance of the skin, especially to smooth
\nwrinkles. In this context, it has been observed that dermal injection of chemically crosslinked
\nhyaluronic acid (CL-HA) fills spaces, smooths wrinkles and favors collagen formation
\nby skin fibroblasts [8]. CL-HA injections revealed increased collagen formation
\naround the filler [9]. Injection of chemically cross-linked high-molecular-weight HA supplemented
\nwith 0.3% lidocaine hydrochloride increases skin volume, improving the aesthetic
\nappearance of the skin [10]. Intradermal injections of cross-linked HA cause a reduction
\nin wrinkles [11]. Injections of non-cross-linked HA also improve skin hydration
\nand elasticity. In the periorbital area, they cause an improvement of between 25% and 50%
\nin skin brightness, texture and turgor [12,13]. The facial application of Viscoderm\u00ae Hydrobooster
\n(IBSA SA, Collina d\u02b9Oro, Switzerland), slightly cross-linked HA, induces a significant
\nimprovement in facial wrinkles and static facial lines [14].<\/p>\n

Non-animal stabilized hyaluronic acid (NASHA) injection improves skin texture [15]
\nand skin elasticity [16]. The subcutaneous injection of low-molecular-weight hyaluronic
\nacid fragments mixed with amino acid (HAAM) in aged skin improves the aesthetic appearance
\nof the skin, since it induces the production of collagen III by skin fibroblasts [17].
\nHA and hydroxyapatite complexes have also been used as skin fillers [18\u201322]. Combined
\nHA with glycerol improves skin quality by altering viscoelastic skin properties and skin
\ndensity [23], and HA plus mannitol also is effective for skin hydration [24].
\nUsing CD1 mice, the effect of various HAs has been studied, such as Viscoderm 0.8 a
\nlinear HA with a molecular weight of about 1 \u00d7 106 Da, Profhilo a hybrid cooperative complex-
\nbased compound with low-molecular-weight HA (\u224865\u2013110 kDa) and high-molecular-
\nweight HA (\u22481.4\u20132.1 \u00d7 106 Da), Profhilo Structura a hybrid cooperative complex-based
\ncompound containing low- and high-molecular-weight HA and Aliaxin GP a cross-linked
\nHA (molecular weight 1000 kDa\u20132000 kDa). This study shows that the degradation kinetics
\nof subcutaneous implants of these four types of HA are 4, 10, 29 and greater than 33
\nweeks post-injection for Viscoderm, Profhilo, Profhilo Structura and Aliaxin GP, respectively.
\nThis suggests that HA fillers with high molecular weight or a mixture of high and
\nlow molecular weight have a greater capacity for skin integration [25]. Likewise, in guinea
\npigs, the intradermal administration of Aliaxin GP, composed of cross-linked HA, and
\nViscoderm\u00ae Skink\u00f2 E (SE), composed of non-cross-linked HA supplemented with ions,
\namino acids and vitamins, shows that the injection of Aliaxin GP has slow reabsorption,
\ninducing scratching of the skin. In the skin of these animals treated with Aliaxin GP, no
\nincrease in infiltrates was observed compared to the control group treated with saline solution.
\nIn animals treated with Viscoderm\u00ae SE, reabsorption is rapid, with slight post-injection
\nerythema being observed. At the histological level, an increase in the deposit of
\ncollagen, elastic fibers and proliferation of fibroblasts in the area of administration is observed,
\nwith no increase in infiltrates compared to the control group [26].
\nAll these studies are an example of the multiple types of HA used in aesthetic medicine
\nto rejuvenate the skin, ranging from non-cross-linked HA, cross-linked HA, autocross-
\nlinked HA, as well as low- and high-molecular-weight HA, and supplemented HA
\nwith amino acids, ions and vitamins, and preclinical studies provide valuable information
\non the skin behavior of these HA-based compounds. In summary, this diversity of HAbased
\nproducts with different molecular weights and rheological characteristics constitutes
\nvery useful tools in aesthetic medicine to reduce, delay and partially repair age-related
\nskin changes [27]. In the present study, the effect of treatment with CELLBOOSTER\u00ae
\nGlow (Suiselle SA, Yverdon-les-Bains, Switzerland), a revitalizing complex of hyaluronic
\nacid, made up of HA, amino acids (cysteine, glycine, lysine, proline and valine) oligopeptides
\n(glutathione) and vitamins (C, biotin), has been tested. CELLBOOSTER\u00ae Glow (CG)
\nis made up of high-molecular-weight hyaluronic acid, not cross-linked and mechanically
\nstabilized by shear deformation and simultaneous pressure. The objective of the study
\nwas to demonstrate the efficacy of this revitalizing HA complex in skin depigmentation
\nand anti-aging when injected into the skin of human donors. The results show that in
\nhuman skin explants, treatment with CG significantly reduces the percentage of cells with
\na high melanin content, which suggests a depigmentation effect, and that in explants irradiated
\nwith UVA\/UVB, treatment with CG increases the levels of components of the dermal
\nextracellular matrix, such as collagen type 1, elastin and sulfated GAGs, components
\nthat had decreased in irradiated explants. Taken together, these results suggest that CG
\nhas a depigmentation and rejuvenation and\/or revitalization effect on the skin.<\/p>\n

2. Materials and Methods<\/strong>
\n2.1. Human Skin Samples Collection and In Vitro Maintenance of Skin Explants<\/em><\/p>\n

In the present study, skin samples from four female donors between 29 and 57 years
\nof age have been used. The human skin used is excess skin from abdominoplasty (skin #1;
\n50 years old), from tight lift (skin #2; 57 years old) and breast reduction (skin #3, 30 years old, and skin #4, 29 years old).
\nThe donor women signed an informed consent in which they accepted that the resected skin from their interventions
\ncould be used for scientific purposes. All procedures performed in this study were in accordance with the ethical
\nstandards of the Helsinki Declaration.<\/p>\n

In the first hour after excision of the skin, under a laminar flow hood and under sterile
\nconditions, the skin explants were washed with a PBS\u2013antibiotic solution, and the subcutaneous
\nfat and lower dermis were mechanically removed using a surgical scarpel. Under
\nthese conditions, the skin was cut into small fragments of approximately 1 cm2, which
\nwere placed inside plate inserts with a 3 \u03bcm pore membrane (# 10769-210; VWR, Rosnysous-
\nBois, France) in 12-well culture plates (Costar, VWR, France). The culture medium
\nwas placed at the bottom of the wells, ensuring skin survival by slow diffusion between
\nthe two compartments through the porous membrane. Medium changes were made 3
\ntimes per week. The culture medium used was Dulbecco\u2019s Modified Minimal Essential
\nMedium (DMEM; Life Technologies, Saint-Aubin, France) enriched with antibiotics (penicillin
\n100 \u03bcg\/mL, streptomycin 100 \u03bcg\/mL and amphotericin B 250 \u03bcg\/mL; Life Technologies),
\nbovine pituitary extract (Life Technologies), L-glutamine (200 \u03bcg\/mL; Sigma-Aldrich-
\nMerck, Saint-Quentin-Fallavier, France) and fetal calf serum (Sigma-Aldrich-
\nMerck). The culture plates were placed in a humidified incubator in a 5% CO2 atmosphere
\nat 37 \u00b0C. In each experimental series, the control and test conditions were compared between
\nskin fragments from the same donors [28,29].<\/p>\n

2.2. Experimental Series and Experimental Design<\/em><\/p>\n

The effect of CELLBOOSTER\u00ae Glow has been tested in two experimental series of
\nhuman skin explants. In the first experimental series, the depigmentation effect has been
\nanalyzed, for which 3 administrations of 10 \u03bcL per cm2 of the product were injected in the
\nsuperficial dermis and in the upper part of the middle dermis, of the skin samples. For
\neach donor, duplicates of skins treated with CG and control skins non-treated were made.
\nThe day of the injection was considered as day 0 (D0), and the depigmentation effect was
\nevaluated during the following 12 days post-injection. Subsequently, skin samples were
\nformalin-fixed and histologically processed to detect melanin pigment.<\/p>\n

In the second experimental series, the anti-aging effect of CG was studied, using a
\nmodel of aging by ultraviolet radiation. For this, the skin samples were irradiated with
\nultraviolet A light (UVA; 8 J\/cm2) and ultraviolet B light (UVB; 1 J\/cm2) for 60 min by UVA
\nand 2 min by UVB. The source of ultraviolet irradiation was a Vilber Lourmat simulator
\n(Vilber Lourmat, Marnes-la-Vall\u00e9e, France) fitted out with a UVA irradiation source (365
\nnm) composed of Vilber Lourmat tubes T-20.L-365 mercury vapor tubes, low pressure.
\nThe UVB irradiation source (312 nm) is composed of Vilber Lourmat tubes T-20.L-312
\nmercury vapor tubes. A radiometer was associated with a microprocessor programmable
\nin energy (J\/cm2). The skin samples have been irradiated with UVA and UVB because they
\nare the ultraviolet radiations that have the greatest penetrating capacity in the skin, ranging
\nfrom the epidermis to the dermis, and even the hypodermis. These ultraviolet radiations
\ngenerate free oxygen radicals, and this oxidative stress is responsible for skin aging
\n[30\u201332]. The day of radiation of the skin samples was considered day 0 (D0). After the
\nultraviolet radiation session, the skin samples received 3 injections of 10 \u03bcL\/cm2 in the
\nsuperficial dermis and in the upper part of the mid-dermis of CG. Irradiated non-treated
\nskin and non-irradiated skin also were used as controls. The anti-aging effect of the skin
\nhas been studied during the 12 days following irradiation. Between days 1 and 4 (D1-D4),
\nthe culture media were collected, mixed and frozen. These media were used for the analysis
\nof metalloproteinase type 1 (MMP1) activity. On day 4 (D4), skin fragments were frozen,
\nwhich were used for the analysis of glycosaminoglycans (GAGs). Finally, on day 12
\n(D12), the skin fragments were frozen and used for pro-collagen-I and elastin analysis.<\/p>\n

2.3. Histological Evaluation of the Melanin Pigment of the Skin<\/em><\/p>\n

Skin samples treated with CG and control skin samples were fixed in formalin and
\nsubsequently embedded in paraffin. The paraffin blocks with the skin inside were cut to
\na thickness of 4 \u03bcm and collected on pregelatinized slides. Histological sections of skin
\nwere stained with the method of Fontana-Masson silver (FMS) stain. FMS stain is a histochemical
\ntechnique that oxidizes melanin and melanin-like pigments as it reduces silver
\n[33]. This histochemical method has been used to determine the degree of depigmentation
\ninduced by CG. For this, the histological sections have been deparaffinized and rehydrated
\nwith distilled water. Next, the Fontana-Masson stain kit (#HT200-1KT; Sigma-Aldrich-
\nMerck) was used, and the procedure described by the manufacturer was followed.
\nFinally, the histological sections were dehydrated in increasing solutions of ethanol,
\nbathed for 5 min in xylene, and coverslips were mounted with DPX (#06522; Sigma-Aldrich-
\nMerck).<\/p>\n

A quantitative numeration of cells containing melanin pigments (black stain) was
\nmade under an optical microscope at x400 magnification on about 300 basal cells of the
\nepidermis. Three types of cells were counted: unpigmented cells or cells presenting rare
\nmelanin pigments isolated in the cytoplasm (score 1); cells presenting moderate melanin
\npigment in all over the cytoplasm (score 2) and cells presenting important melanin pigment
\nin all over the cytoplasm (score 3). Then the epidermal melanin content was calculated
\nas the percentage of the epidermal cells in each cell type counted.<\/p>\n

2.4. Biochemical Evaluation of Skin Samples<\/em>
\n2.4.1. Biochemical Assay of Type 1 Pro-Collagen<\/p>\n

During the aging process, the metabolism of fibroblasts is reduced. The objective of
\nan anti-aging product such as CG is to stimulate this metabolism in order to achieve collagen
\nand other extracellular matrix molecules synthesis. Thus, the analysis of the excretion
\nof the single-stranded molecule of type I alpha 1 pro-collagen, before the formation
\nof the triple helix, constitutes an interesting approach to evaluate an anti-aging effect. For
\nit, the skin fragments collected at D12 were weighed and then put in PBS (0.1 M, pH = 7.4)
\nAfter grinding with a potter, the amount of type I pro-collagen (\u03bcg\/mL) was evaluated by
\nan ELISA method (# DY6220-05; Human Pro-Collagen I alpha 1, BioTechne), following the
\nprocedure described by the manufacturer. The final result was expressed in pg of type 1
\npro-collagen\/mg of biopsy.<\/p>\n

2.4.2. Biochemical Assay of Elastin
\nElastin, the main component of elastic fibers, provides stretch and elasticity to the
\nskin. During the aging process, the synthesis of elastin decreases which impacts skin structure,
\nfunction and youthful appearance. The objective of an anti-aging product such as
\nCELLBOOSTER\u00ae Glow is also to stimulate the synthesis of elastin. Therefore, the assessment
\nof the elastin content in skin samples constitutes an interesting contribution to assess
\nthe anti-aging effect.
\nAfter the 12-day survival period, insoluble elastin was extracted from skin samples
\nwith 0.25 M oxalic acid at 100 \u00b0C as soluble alpha-elastin polypeptide fragments. After
\ncentrifugation to remove undigested tissue, the Fastin Interchim Elastin Assay Kit (#F200;
\nBiocolor, Montlu\u00e7on Cedex, France) was used, following the manufacturer\u2019s procedure.
\nFinally, the amount of soluble elastin was measured by a spectrocolorimetric assay
\nmethod at 513 nm. To compare the different results, the quantity of elastin was related to
\nthe quantity of total proteins in the sample. The protein concentration (\u03bcg\/mL) was determined
\nspectrophotometrically at 562 nm, using the Pierce BCA protein assay kit (# 23225;
\nThermoFisher, Waltham, MA, USA), and following the procedure indicated by the manufacturer.
\nFinally, results were expressed in \u03bcg elastin\/mg protein.<\/p>\n

2.4.3. Biochemical Assay of Sulfated Glycosaminoglycans
\nIn this assay, frozen skin fragments from day 4 (D4) have been used. These fragments
\nwere enzymatically digested with papain at 50 \u00b0C, overnight. And then, the activity of the
\nfibroblasts to synthesize sulfated GAGs was evaluated by a spectrocolorimetric method at
\n656 nm, using the Sircoll blyscan GAGs kit (#AA4881; Bicolor, Montlu\u00e7on Cedex, France),
\nand following the procedure indicated by the manufacturer. To compare the different results,
\nthe amount of GAGs was related to the amount of total protein in the sample. The
\nassay of the protein concentration was carried out spectrophotometrically at 562 nm, using
\nthe Pierce BCA protein assay kit (# 23225; ThermoFisher, Waltham, MA, USA), and
\nfollowing the procedure indicated by the manufacturer. The results were expressed in \u03bcg
\nof sulfated GAGs\/mg protein.<\/p>\n

2.4.4. Biochemical Assay for the Analysis of Metalloproteinase Type 1 (MMP1) Activity
\nMetalloproteinases are enzymes involved in the degradation of macromolecules of
\nthe extracellular matrix. The interstitial collagenase of fibroblasts MMP1 is specific to type
\nI, II and III fibrillar collagens. MMP1 is first secreted into the culture medium by the fibroblast
\nin its zymogenic form, pro-MMP1, which is then activated into MMP1 by the action
\nof other proteinases.
\nIn this assay, the culture media collected between days 1 and 4 (D1-D4) have been
\nused. This culture medium has been thawed and centrifuged at 900 rpm to remove potential
\ncell debris and other impurities. The amount of MMP1 (pg\/mL) contained in the supernatant
\nhas then been analyzed by the ELISA technique with a spectrophotometric
\nreader at 450 nm, and using the Human MMP-1 (Matrix Metalloproteinase 1) ELISA Kit
\n(# E-EL-H6073; Elabscience Biotechnology Inc, Houston, TX, USA), and following the procedure
\ndescribed by the manufacturer. The final result was expressed in pg of MMP1\/mg
\nof biopsy.<\/p>\n

2.5. Statistical Analysis<\/em>
\nThe mean and standard deviation (SD) were calculated in tests performed in doublet
\nin 4 donors in duplicate (8 values). After checking the normality of the groups using the
\nShapiro\u2013Wilk test, the comparison of the parameter was made with the Student\u2019s T test
\n(alpha risk of 5%). If the groups did not follow a normal distribution, a Wilcoxon test (5%
\nalpha risk) was performed. The IBM SPSS 25.0 statistical package for Windows (IBM Corp.
\nReleased 2017; Armonk, NY, USA) was used for the statistical analysis.<\/p>\n

3. Results<\/strong>
\n3.1. Treatment with CELLBOOSTER\u00ae Glow Significantly Reduces the Percentage of Skin Cells<\/em>
\nwith High Melanin Content<\/em><\/p>\n

In human skin explants, three injections of CG were administered. The percentage of
\ncells with a melanin score of 1 was 18.30% and 21.93% in the control and treated groups,
\nrespectively. No significant differences were observed between both groups (p > 0.05). Regarding
\nthe percentage of pigmented cells with a score of 2, it was 42.83% and 45.52% in
\nthe control and treated groups, respectively. Neither were significant differences observed
\nbetween both experimental groups (p > 0.05). Finally, the percentage of cells with a score
\nof 3 was 38.87% and 32.55% in the control and treated groups, respectively. For this score,
\nsignificant differences were observed between both experimental groups (p < 0.05) (Figure
\n1). CG treatment does not reduce the percentage of skin cells with low and medium melanin
\ncontent (scores 1 and 2); however, it does significantly reduce the percentage of skin
\ncells with significant melanin content (score 3). This reduction is approximately 16%.
\nTaken together, these results indicate that CG treatment slightly but significantly reduces
\nskin pigmentation. This indication may favor a less-aged cosmetic appearance.<\/p>\n

\"\"<\/p>\n

Figure 1.<\/strong> Histological results of skin pigmentation: (A) Histogram of the percentage of cells with
\nlow (score 1), medium (score 2) and high (score 3) melanin content in both experimental groups
\n(Control, CBG). Values are mean \u00b1 standard deviation (n = 8 values). * p < 0.05 compared to the
\ncontrol group. (B,C) Light microscope images of skin donor #1, and (D,E) images of skin donor #2.
\nImages (B,D) correspond to the control, and images (C,E) after CG treatment. Comparatively, a
\nslight decrease in black marking (cells with melanin) can be observed in images (C,E) compared to
\nimages (B,D). All images were captured at \u00d7400 magnification.<\/p>\n

3.2. In Skin Treated with Ultraviolet Radiation, Treatment with CELLBOOSTER Glow Significantly<\/em>
\nIncreases the Content of Pro-Collagen Type I, Elastin and Sulphated GAGs, but without<\/em>
\nCausing Changes in MMP1 Levels<\/em>
\nUltraviolet irradiation causes a significant decrease in the levels of type I pro-collagen,
\nelastin and sulfate GAGs in human skin explants compared to the control, which
\ncorresponds to non-irradiated skin (Figure 2A\u2013C), while it significantly increases the levels
\nof MMP1 compared to the control (Figure 2D).<\/p>\n

\"\"<\/p>\n

Figure 2.<\/strong> Biochemical results of skin irradiated with UVA\/UVB and treated with CELLBOOSTER\u00ae
\nGlow (CG): (A) Histogram of the results of the content of pro-collagen type I in control, irradiated
\nwith ultraviolet, and in skin irradiated with ultraviolet and treated with CG; (B) histogram of the
\nelastin content and (C) of sulfated GAGs, in the skin in the same three experimental groups; (D)
\nhistogram of MMP1 activity and\/or content in the skin of the three experimental groups. * p < 0.05;
\n** p < 0.01; *** p < 0.001. Values are mean \u00b1 standard deviation (n = 8 values).<\/p>\n

When UV-irradiated skin is treated with CG, the levels of pro-collagen type 1, elastin
\nand sulfated GAGs increase compared to irradiated skin without treatment, to levels similar
\nto those observed in the control group (Figure 2A\u2013C). However, MMP1 levels remained
\nsimilar between the groups of skin irradiated with and without CG treatment
\n(Figure 2D). Ultraviolet irradiation of the skin caused a reduction of 28.9%, 13.2% and
\n19.2% in the content, respectively, pro-collagen type 1, elastin and GAGs, compared to
\nwhat was observed in the control group. Likewise, in ultraviolet-irradiated skin, treatment
\nwith CG caused a 47.9%, 25.3% and 22.4% increase in the content of pro-collagen type 1,
\nelastin and GAGs, respectively, compared to what was observed in untreated irradiated
\nskin (UV group). In skin irradiated with ultraviolet, a significant increase of 40.9% in the
\nlevel of MMP1 was observed, compared to what was observed in the control, while in the
\nUV + CBG group, a non-significant reduction of 6.7% was observed compared to the UV
\ngroup. Taken together, these biochemical results suggest that ultraviolet irradiation of the
\nskin causes changes compatible with aging that manifest as a reduction in the content of
\npro-collagen type 1, elastin and sulfated GAGs, as well as an increase in MMP1 activity,
\nand the CG treatment reverses these changes by increasing the levels of these extracellular
\nmatrix components, except for MMP1 activity, which can be attributed to its rejuvenating
\neffect. In summary, treatment with CG in skin irradiated with ultraviolet radiation increases
\nthe levels of pro-collagen type 1, elastin and sulfate GAGs, without modifying the
\nactivity of MMP1.<\/p>\n

4. Discussion<\/strong>
\nIn the present study, it has been observed that in human skin explants, treatment
\nwith CG significantly reduces skin pigmentation, especially a significant reduction of 16%
\nof cells with high melanin content has been observed. And in skin treated with ultraviolet
\nA and B radiation, CG treatment significantly increases the content of components of the
\nextracellular matrix of the dermis\u2013epidermis, especially collagen type 1, elastin and sulfated
\nGAGs. All these changes observed in the skin after treatment with CG suggest that
\nthis treatment promotes skin rejuvenation.<\/p>\n

CELLBOOSTER\u00ae Glow (CG) consists of non-cross-linked and mechanically stabilized
\nHA supplemented with amino acids and vitamins, specifically cysteine, glycine, lysine,
\nproline and valine amino acids, glutathione as an oligopeptide, and as vitamins it
\ncontains biotin and vitamin C. The observed effects in the present study may be due to
\nthe components of CG. In this context, it is known that ultraviolet radiation causes an
\nincrease in the levels of enzymes that degrade the extracellular matrix of the skin, such as
\ncollagenases, gelatinases and metalloproteinase type I, which leads to a decrease in collagen
\ncontent and unstructured resynthesis of this collagen [34]. Ultraviolet radiation also
\ncauses a reduction in the release of elastase by skin fibroblasts, so the structural changes
\nthat this radiation induces on the elastic fibers of the skin are not easily degraded, which
\nbecome curly and tortuous, losing skin elasticity and maintaining wrinkles [35]. Ultraviolet
\nradiation triggers the aggregation of elastin. Elastin aggregates are not eliminated by
\nthe set of extracellular chaperones present in the skin, because ultraviolet radiation also
\ninduces the inactivation of these chaperones. Consequently, these elastin aggregates remain
\nin the skin and this decreases its elasticity [36]. Taken together, all these findings
\nsuggest that the skin\u2019s elasticity is compromised by ultraviolet radiation from the sun,
\ngiving it a more aged appearance, with the formation of wrinkles. Likewise, ultraviolet
\nradiation also induces a reduction in the expression of hyaluronic synthase enzymes (type
\n1, 2 and 3), transforming growth factor beta-1, and its type II receptor. All this leads to a
\nlower proliferation of fibroblasts, with a lower synthesis and release of components of the
\nextracellular matrix (e.g., HA and collagen) [37\u201339]. Finally, an in vitro study demonstrated
\nthat ultraviolet radiation also causes a reduction in elastin content [40] and a
\nreduction of GAGs in cultured human fibroblasts [41]. In the present study, it has been
\nobserved that in human skin explants ultraviolet radiation reduces the content of elastin,
\ntype I collagen and sulfated GAGs. In turn, it is known that sulfated GAGs play an important
\nrole in establishing interactions with other components of the extracellular matrix,
\nallowing them to retain water, thus maintaining the degree of dermal hydration; retain
\ngrowth factors and act as co-receptors of these growth factors, responsible for the survival
\nof skin cells; and modulate the inflammatory\u2013immune response of the skin by interacting
\nwith cytokines\u2013chemokines [42]. Consequently, the reduction of these sulfated GAGs in
\nskin samples subjected to ultraviolet radiation can compromise all these functions, favoring
\nthe appearance of skin signs associated with aging.<\/p>\n

As in the present study, other previous studies have also observed an increase in
\nMMP1 levels in UV-irradiated skin samples [43,44] and in cultured human fibroblasts [45].
\nMatrix metalloproteinase type 1 (MMP1) belongs to the collagenase subgroup and its
\nfunction is to degrade type I and type III collagen [46]. Type I collagen is synthesized and
\nsecreted by dermal fibroblasts and represents 80\u201390% of skin collagen. Fibroblasts also
\nsynthesize and release type III collagen, which represents 15% of the collagen in the skin.
\nBoth are of the fibril-forming types. Collagen fibers form extensive and robust networks
\nproviding the dermis with strength, firmness and elasticity. A collagen fiber is essentially
\ncomposed of bundles of smaller fibrils. Collagen fibrils are approximately 10 to 300 nm in
\ndiameter and several micrometers in length. A collagen fibril is a bundle of triple-stranded
\ncollagen molecules (about 1.5 nm in diameter and approximately 300 nm long). This triple-
\nhelix, coiled structure is stereo-dynamically favorable to allow strands to be interwoven
\ntogether and this incredibly robust structure can persist in tissues for many years. The
\nformation of fibers is dependent on interaction with other ECM components including
\nelastin proteins and GAGs [47]. The increase in MMP1 after ultraviolet irradiation of the
\nskin suggests a degradation of these collagen fibers, a loss of mechanical firmness of the
\nskin and the ability to stretch the skin, as well as a decrease in skin elasticity, and all these
\ncause the appearance of signs of skin aging.<\/p>\n

Treatment with CG reverses the observed effects of ultraviolet radiation to levels
\ncomparable to control. Specifically, the levels of type I collagen, elastin and sulfated GAGs
\nare restored, although it does not cause a decrease in MMP1. The increased levels of biopolymers
\nin the ECM can be explained by a reactivation of skin fibroblasts by the CG
\ncomponents. In vitro studies show that HA promotes the proliferation of fibroblasts [48\u2013
\n52] that secrete a greater amount of type I collagen [52]. HA binds to the CD44 receptor,
\nexpressed by dermal fibroblasts [53], and attenuates MMP1 overexpression [54]. HA can
\nalso bind the CD44 receptor on keratinocytes [55] inducing the growth, survival and migration
\nof these epidermis cells [56], which implies an acceleration of the epidermal rejuvenation
\nprocess. Likewise, the interaction of HA with the CD44 receptor of the keratinocyte
\nalso favors the formation and release of lamellar bodies into the extracellular space
\nof the epidermis, which favors the maintenance of the degree of hydration and the rejuvenation
\nof the epidermis [57]. Epidermal melanocytes also express CD44 receptors [58],
\nand sulfate GAGs enhance the melanotic phenotype of these cells through binding to
\nCD44 receptors [59]. In a physiological situation, melanocytes have a layer of hyaluronic
\nacid that surrounds them and that prevents them from synthesizing and secreting cytokines
\nand chemokines. Ultraviolet radiation favors hyaluronidases to degrade HA and
\nmelanocytes to secrete these pro-inflammatory factors [60]. It is well known that pro-inflammatory
\nfactors, especially cytokines, play a crucial role in the manifestation of decreased
\nskin collagen content, decreased skin thickness and dry and wrinkled skin [61].
\nTherefore, the application of CG, which provides hyaluronic acid, can potentially restore
\nthis HA layer in the melanocytes, preventing these epidermal cells from secreting the inflammatory
\ncytokines that trigger the signs of skin aging. Preclinical evidence indicates
\nthat the suppression of the synthesis and release of pro-inflammatory cytokines by melanocytes
\nwould be mediated by the binding of HA to CD44 receptors [60]. In addition, in
\nvitro studies show that HA prevents the secretion of inflammatory cytokines by
\nPolymers 2023, 15, 2438 9 of 13 keratinocytes induced by ultraviolet radiation [62] and ionizing radiation [63].
\nTaken together, all these evidences suggest that the HA biopolymer present in CG has a skin rejuvenation
\neffect through its interaction with CD44 receptors, favoring the proliferation of
\ndermal fibroblasts and the release of extracellular matrix proteins (type I and III collagen,
\nsulfated GAGs, elastin) and preventing the release of inflammatory cytokines by epidermal
\nmelanocytes and keratinocytes.<\/p>\n

CELLBOOSTER\u00ae Glow also contains biotin and vitamin C. Biotin promotes protein
\nbiosynthesis in the liver, intestinal wall and skin [64]. Fibroblasts require biotin for their
\nsurvival and proliferation [65]. In animals subjected to a diet deficient in biotin, alterations
\nin the composition of fatty acids of the skin are observed, such as accumulation of oddchain
\nfatty acids and abnormal metabolism of long-chain polyunsaturated fatty acids
\n[66,67]. In these animals, a loss of Langerhans cells in the epidermis is also observed,
\nwhich compromises the innate immune system of the skin [68]. On the other hand, vitamin
\nC plays different biological functions in the skin, such as promoting collagen synthesis
\nand structural stabilization of collagen fibers; it is a powerful antioxidant that neutralizes
\nand removes oxidant molecules; reduces the synthesis of melanin in melanocytes and
\nfacilitates the proliferation and differentiation of keratinocytes and the release of lipids
\nthat surround the corneocytes from the stratum corneum of the epidermis, thus facilitating
\nthe renewal of the epidermis layers [69]. Vitamin C inhibits the hyaluronidase enzymes
\nresponsible for the breakdown of hyaluronic acid in the epidermis [70]. Furthermore,
\nskin pigmentation requires multiple steps, namely, the activation of melanocytes,
\nthe synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites
\nand the transfer of melanosomes from melanocytes to surrounding keratinocytes. It has
\nbeen shown that vitamin C inhibits melanin synthesis through downregulation of tyrosinase
\nenzyme activity [71]. Vitamin C also interferes with the transport of melanosomes to
\nthe tips of the dendrites of melanocytes, since this vitamin reduces the expression of
\ntransport proteins of these melanosomes (e.g., kinesin) [72]. All these findings suggest that
\nthese two vitamins present in CG promote the activation of dermal fibroblasts and with it
\nthe synthesis of new extracellular matrix proteins; they maintain the proliferation of
\nkeratinocytes allowing adequate renewal of the epidermis, as well as the lipid content of
\nthe stratum corneum of the epidermis, which contributes both to epidermal renewal and
\nto maintaining the degree of skin hydration; they prevent the degradation of the HA that
\nsurrounds the melanocytes, preventing the release of inflammatory cytokines and they
\ndecrease the synthesis of melanin and the transport of melanosomes to the dendritic ends
\nof the melanocytes, which has a depigmentation effect. All of these changes can contribute
\nto a more rejuvenated appearance of the skin. Regarding skin pigmentation, in the present
\nstudy, it has been verified that CG reduces the percentage of epidermal cells with a high
\nmelanin content and limits the percentage of cells with a low and intermediate content.<\/p>\n

Cysteine, glycine, lysine, proline and valine are the amino acids present in CG. Cysteine
\nis an important amino acid in the synthesis of keratins by the keratinocytes of the
\nepidermis. Lysine is an amino acid that is incorporated into skin proteins and post-translational
\nchanges of this residue allow the maturation of these skin proteins. The oxidation
\nof lysine allows cross-linking of the collagen fibers, which gives the stretching strength
\nand insolubility of these protein fibers. Glycine and proline are the two most abundant
\namino acids in collagen fibers. Proline also undergoes post-translational changes allowing
\nthe maturation of collagen fibers. Proline is also an amino acid that promotes skin elasticity
\n[73]. Additionally, glutathione that is present in CG is a tripeptide made up of the
\namino acid glutamate, cysteine and glycine. It has been shown that glutathione (GSH) not
\nonly acts as an antioxidant by scavenging free radicals, but it is also involved in
\npheomelanin formation and regulating melanogenesis [74]. Oral administration of GSH
\nresulted in the lightening of skin color in humans [75]. Moreover, the oxidized form of
\nglutathione (GSSG) was also found to have anti-melanogenic effects in humans [76,77].
\nAll these evidences suggest that GSH present in CG has anti-aging effects on the skin,
\nsequestering free radicals that cause cell damage that accelerate skin aging, as well as
\nreducing the degree of skin pigmentation by having anti-melanogenic effects, all these
\ncontributing to skin rejuvenation. GSH may also contribute to the depigmentation results
\nobserved in the present study.<\/p>\n

The evidence described above suggests that CG components have skin rejuvenating
\neffects, at least on human skin explants. A good part of the changes observed may be due
\nto the effect of hyaluronic acid on the different cellular elements of skin explants and, to a
\nlesser extent, to vitamins, amino acids and oligopeptides. Recently, newer technologies in
\ntransdermal delivery systems had become effective [78] for skin rejuvenation. Nevertheless,
\nclinical studies are necessary to corroborate these effects of CG.<\/p>\n

5. Conclusions<\/strong><\/p>\n

The objective of this study is to evaluate the rejuvenating effects of a stabilized compound
\nformed by HA supplemented with vitamins, amino acids and oligopeptide in human
\nskin explants. Transformations in the skin, which activate skin aging, can be caused
\nby elements such as the sun\u2019s ultraviolet light and atmospheric contaminants. HA is one
\nof the most widely used biopolymers of extracellular matrix components in dermo-aesthetic
\nmedicine, with the aim of rejuvenating the skin. The effect of various HAs has been
\nstudied in vivo and in vitro. In the present study, the effect of treatment with CELLBOOSTER
\n\u00ae Glow (CG) has been tested when injected into the skin of human donors. CG
\nis made up of high-molecular-weight hyaluronic acid, non-cross-linked and mechanically
\nstabilized. The results show that in human skin explants, treatment with CG significantly
\nreduces the percentage of cells with a high melanin content, which confirms a depigmentation
\neffect of the formula. In explants irradiated with UVA\/UVB, treatment with CG increases
\nthe levels of components of the dermal extracellular matrix, such as collagen type
\n1, elastin and sulfated GAGs, components that had decreased in irradiated explants. Taken
\ntogether, these outcomes suggest that CG has a depigmentation and rejuvenation and\/or
\nrevitalization effect on the skin. This indication of a slight reduction of skin pigmentation
\nand an increase of ECM components may favor a less-aged cosmetic appearance.<\/p>\n

Author Contributions:<\/strong> All authors have contributed sufficiently to be included as authors. Conceptualization,
\nmethodology and validation, S.B. and G.S.-D.; formal analysis and investigation, G.S.-
\nD., S.B., P.B.-V. and E.V.; resources, S.B. and E.V.; data curation, G.S.-D., S.B., P.B.-V. and E.V.; writing\u2014
\noriginal draft preparation, E.V. and P.B.-V.; writing\u2014review and editing, G.S.-D., S.B., P.B.-V.
\nand E.V.; visualization, G.S.-D., P.B.-V. and E.V.; supervision, G.S.-D., S.B., P.B.-V. and E.V.; project
\nadministration, S.B. and E.V.; funding acquisition, G.S.-D., S.B., E.V. and P.B.-V. All authors have
\nread and agreed to the published version of the manuscript.<\/p>\n

Funding:<\/strong> This research was funded by Suiselle SA, Rue Galil\u00e9e 6, 1400 Yverdon-les-Bains, Switzerland
\n(grant number 88.7.22 and grant number 045\/22).<\/p>\n

Institutional Review Board Statement:<\/strong> The study was conducted in accordance with the Declaration
\nof Helsinki. The patients who participated in this study signed an informed consent in which
\nthey gave up the excess skin from the operations they underwent for scientific studies. As the present
\nstudy has been carried out with these human skin explants, the experimental procedure has
\nnot been required to be approved by an Ethics Committee.<\/p>\n

Informed Consent Statement:<\/strong> Informed consent was obtained from all subjects involved in the
\nstudy.<\/p>\n

Data Availability Statement:<\/strong> All data generated or analyzed during this study are included in this
\npublished article.<\/p>\n

Acknowledgments:<\/strong> The authors thank the entire GREDECO technical team (France) for technical
\nassistance in the realization of the present study.<\/p>\n

Conflicts of Interest:<\/strong> The authors declare no conflict of interest.<\/p>\n

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——————————
\nCitation: Siquier-Dameto, G.;
\nBoisnic, S.; Boadas-Vaello, P.; Verd\u00fa,
\nE. Anti-Aging and Depigmentation
\nEffect of a Hyaluronic Acid
\nMechanically Stabilized Complex on
\nHuman Skin Explants. Polymers
\n2023, 15, 2438. https:\/\/doi.org\/
\n10.3390\/polym15112438
\nAcademic Editor: Alina Sionkowska
\nReceived: 3 May 2023
\nRevised: 22 May 2023
\nAccepted: 23 May 2023
\nPublished: 24 May 2023
\nCopyright: \u00a9 2023 by the authors. Licensee
\nMDPI, Basel, Switzerland.
\nThis article is an open access article
\ndistributed under the terms and conditions
\nof the Creative Commons Attribution
\n(CC BY) license (https:\/\/creativecommons.
\norg\/licenses\/by\/4.0\/).<\/p>\n","protected":false},"excerpt":{"rendered":"

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