Native collagen sheet mask improves skin health and appearance: A comprehensive clinical evaluation

Collagen, a critical structural protein found abundantly in animal skin and bones, has become increasingly recognized for its potential therapeutic role in skincare. Despite growing interest, the scientific evidence for the efficacy of collagen sheet masks remains limited. The principal objective of our study was to provide insights into the multifaceted role of collagen in skin health, with a specific focus on its application in collagen sheet masks.


| INTRODUC TI ON
Collagen, an abundant structural protein, is found in animal skin and bones.[3][4] Comprising primarily of amino acids such as glycine, proline, and hydroxyproline, the collagen structure involves these amino acid chains shaping into a helix.The physical attributes of collagen are fundamentally linked to their primary sequence, affecting conformation and fibril assembly. 5,6Its unique native triple helical structure is critical for the desired properties of collagen-based biomaterials. 7rrently, 29 different types of collagen have been identified, with each type exhibiting variations in chain composition, particle isoforms, and supramolecular structures. 8,9Collagen's biocompatibility, non-toxicity, and biodegradability have contributed to its wide range of applications in various industries such as cosmetics, pharmaceuticals, medical, and food. 1,8llagen tissue formation begins with the entwining of three collagen polypeptide chains.These chains comprise over 1050 amino acids, forming a collagen molecule that adapts a rigid triple helix structure.Water plays a crucial role in this molecular behavior, with collagen molecules aggregating to form an insoluble collagen fibril.[12] The skin is subjected to degeneration under internal aging factors and external environmental stressors. 4,13,14Photoaging is characterized by reduced skin elasticity, moisture, and softness, and is accompanied by the appearance of brown spots and wrinkles. 15llagen plays a vital role in maintaining the health and appearance of the skin by acting as a structural protein, promoting cell adhesion, regulating cell behavior, and aiding in the wound healing process.
Supplementing with type I collagen may help to attenuate skin photodamage.However, collagen has a stable structure and a large molecular weight of over 300 kDa, making it difficult for the body to absorb and use collagen directly. 3,16when collagen is exposed to acid, alkali, heat, or enzymes, it can be broken down into smaller peptides with lower molecular weights.These peptides are more easily digested and absorbed by the human body. 17In recent years, more and more studies have shown that low-molecular weight collagen peptides have a beneficial effect on preventing skin aging caused by sunlight exposure. 4llagen is a primary component in many cosmetic formulations due to the fact that it is a natural humectant and moisturizer. 1,6corporating collagen in skincare products can replenish and maintain the skin's collagen levels, improving its texture, elasticity, and overall appearance. 4[20][21][22] Yet, the impacts of temporary skin treatment like collagen masks are not thoroughly documented.
Collagen sheet masks have gained popularity in recent years and are projected to grow at a compound annual growth rate (CAGR) of 8.3%. 23Collagen sheet masks, 23 offer a convenient, hygienic, singleuse method to deliver moisturization and active ingredients to the skin.These masks are typically made from a hydrogel or biocellulose material that is infused with hydrolyzed or denatured collagen and other nourishing ingredients.
However, despite growing popularity, the scientific evidence for the efficacy of collagen sheet masks is limited.Current research primarily focuses on the potential of collagen as a bioactive ingredient in these products, but studies examining the practical skin benefits of these masks are still emerging.Questions remain concerning the bioavailability of collagen when delivered as a topical formulation and its ability to penetrate the multilayer structure of the epidermis to render its skin rejuvenating effects.
In contrast to most available collagen sheet masks, which are inert materials infused with ingredients, we developed a collagen sheet mask that consists of >92% native bovine collagen type I, II and V. To explore the biochemical and physical attributes of this sheet mask and ascertain its effects on skin tolerance, appearance, hydration, and facial wrinkles, we undertook a suite of pre-clinical and clinical tests.Despite the diversity of these testing methods, we chose to integrate them in our study due to their synergistic contribution in comprehending the potential of our masks from both an essential scientific viewpoint and a practical, user-oriented approach.

| Collagen sheet mask
Bovine split skins, sourced as by-products from other bovine industries, are processed in accordance with Good Manufacturing Practice according to Cosmetic Regulation (EG) 1223/2009.The split skins are washed, decellularized, and subsequently freeze dried and lyophilized according to a proprietary method.The result is a waterless, preservative free collagen dry sheet mask, containing >92% type I, II, and V native collagen (Native Collagen Mask-MedSkin Solutions Dr. Suwelack, Billerbeck, Germany).

| Analysis of soluble protein components
We conducted a quantitative protein determination of the collagen mask using the biuret method.The collagen mask, with a dry weight of 3.36 g, was extracted using water and then lyophilized.The resulting lyophilized extract was dissolved in 2 mL HPLC water.To determine the protein concentration, 250 μL of this solution was used with the biuret method.A calibration curve was constructed using gelatin and standard solutions ranging from 1 g/L to 8 g/L.
In addition, we performed an amino acid analysis of the collagen mask using quantitative RP-HPLC.The redissolved freezedried extract from the collagen mask was hydrolyzed with 6N hydrochloric acid.Afterward, the hydrolysate was derivatized and analyzed for its amino acid composition using RP-HPLC.The percentages of individual amino acids in relation to the total peak area were calculated.
The size of the soluble collagen components was determined by gel permeation chromatography/Size Exclusion Chromatography (SEC) of the freeze-dried collagen sheet.
The collagen biomatrix extract underwent chromatographic separation using a FPLC 12-column and detection was performed through flow measurement at a wavelength of 214 nm.The resulting elution profile was then compared to the profile of a pre-determined amount of collagen peptide standard mixture, which was obtained using the same experimental conditions.
The size of the low-molecular soluble collagen components was determined with low-molecular weight fraction of the extract from freeze-dried collagen sheet with MALDI-TOF (mass spectrometry).
To extract the soluble fraction of the collagen biomatrix from its accompanying salts, micro RP columns were utilized for separation and concentration.Subsequently, the concentrated solution was co-crystallized with cinnamic acid.The co-crystallized specimen was then subjected to analysis using laser-desorption ionization in a mass spectrometer (MALDI) integrated with a time of flight (TOF) detector.

| Influence of soluble collagen components on fibroblast regulation
This in-vitro study was set up to investigate the influence of the collagen matrix on the regulation of (wound healing-related) proteins in fibroblasts.Primary fibroblasts were grown in the presence of the collagen matrix and exposed to soluble collagen components without physical contact to matrix.Treated cells and controls were collected at several time-points (12 h, 24 h, and 48 h) and mRNA of cells were obtained for further analysis.Regulation of 87 proteins related to wound healing was analyzed and changes in expression patterns were quantified.Changes were considered as relevant when showing values higher than twofold or lower than half of control signal.

| Scanning electron microscope analysis of the collagen mask
Scanning electron microscope (SEM) images were made (magnification 200×) of the collagen mask, and a biocellulose mask (SkinCeuticals, Germany).
To evaluate the efficacy of the collagen mask in calming and caring for irritated skin an in-vitro skin model system was used.Human skin explants were obtained, and slightly irritated using a standard cytokine protocol.The irritated skin explants were then treated with the collagen mask, and skin samples were obtained before and after treatment.The skin samples were analyzed using scanning electron microscopy and was used to obtain skin side view images to visually analyze the effects of the treatment.

| Clinical evaluations
The test product was assessed for its safety of use before the clinical evaluations took place.In compliance with EU cosmetic Regulation no.1223/2009, a cosmetic product should not induce harm to human health when used under normal or reasonably predictable conditions.Prior to exposing human subjects to the product, it is necessary to evaluate its safety of use, thereby eliminating the need for additional ethical approval.
We conducted a thorough safety assessment of our collagen masks by evaluating the ingredients, packaging, and labeling.As part of our safety review, we applied a traditional skin patch test to identify any potential adverse reactions.Additionally, each raw material in our product passed safety testing and conformed with required cosmetic regulation guidelines.Even though our collagen mask does not have a specific certification at present, it meets all necessary safety standards, and strictly adheres to the legislative requirements for cosmetic products.
The studies were performed in accordance with ethical principles registered in the Declaration of Helsinki and subjects were informed and consented to study participation.
No sample size computations were performed for the clinical evaluations in this study.Taking into consideration factors such as anticipated effect sizes, practical constraints related to the study population, and considerations regarding the conditions under examination, we accordingly determined the sizes of various study groups.
All study procedures were carried out in the same conditions, with the room temperature at 22.5 ± 1.5°C and relative humidity at 50.0% ± 10.0%.

| Clinical microbiome evaluation
This clinical study aimed to compare the diversity of the skin microbiome before and after 28 days of using the collagen mask with an activator liquid.
The secondary aims of this study were to compare the differences in skin microbiome diversity, skin pH, and transepidermal water loss (TEWL) between the treated and untreated skin sites, as well as to compare the changes in skin pH and TEWL after 28 days of consecutive product applications with the baseline values.
Twenty-eight female healthy subjects aged between 50 and 60 years old, with a baseline pH ≥5.5, were enrolled in this study.The main inclusion criteria were: Being in the menopause, with no menstrual period for at least 1 year prior to the beginning of the study, and not changing any of the daily habits during the study period (habits of exercise, skin hygiene and alimentary habits, etc.).Main exclusion criteria were presence of skin diseases, immunosuppression, using pharmacological treatment or antibiotics.Subjects were instructed not apply any topical product (cosmetic, pharmaceutical or otherwise) in the evaluation area (forearms), in the 24 h that precede the first measurement visit and during the 28 days of the study performance, including make-up products and other cosmetic products such as hydrating products, body lotions, etc., excluding cleansing products and the investigational products.and cleaning their forearms only with water.Subjects were instructed not to go swimming during the study and avoid using hot tubs, steam baths and sauna in the 48 h preceding the measurement visits.
The collagen masks were applied every other day on the forearm, in the evenings, for a period of 28 days.The investigational product and the untreated skin site were randomly assigned to an outlined site of 3 × 3 cm on the volar part forearm with the help of a plastic marker.
Premoistened swabs were collected from each skin site of each subject before products' application (Day 0) and again 28 days after consecutive product applications (Day 28).Swabs for each subject were collected at the same time of day for each time-point of analysis.After collection, DNA was extracted to preserve the integrity of the sample, and Metagenomics NGS was used to assess the DNA samples.The MiSeq platform (by Illumina) or the Oxford Nanopore platform was used.After amplification, the quantity and integrity of the DNA were verified and normalized by agarose gel electrophoresis.The total number of copies of 16S rRNA was quantified and the results were expressed as the percentage of each species relative to the total number of all species present in the skin.The analysis included the global state of the skin microbiome, the individual analysis of the species P. aeruginosa and C. acnes, and the calculation of the Shannon's Diversity index.
Skin pH was measured in triplicate on each site using the equipment HALO pH/TEMP PROBE HI14142 (Hanna Instruments, Romania) before product application (Day 0) and 28 days after consecutive product applications (Day 28).TEWL was also measured on each site using a Tewameter® TM 300 (Courage + Khazaka electronic GmbH, Germany) before product application and 28 days after consecutive products' applications (Day 28).

| Clinical evaluation of tolerability and local irritating reactions in atopic dermatitis
This study investigated the efficacy and safety of a collagen mask for treating atopic dermatitis.It involved 15 male and female subjects aged 18 to 65 years with manifest atopic dermatitis from a single center.All subjects received all treatments, which were randomly assigned to the test fields.The treatments included a collagen mask, sodium chloride (negative control), and sodium dodecyl sulfate (positive control).The treatments were applied to the test fields using special test chambers (Finn Chambers®, Epitest Ltd.Oy, Finland, 18 mm inside in diameter).The test chambers were filled with the individual treatments in accordance with the randomization list.
Test product and controls were occlusively applied once daily for a 5 day period, with visual assessments performed on days 2 to 5.
The adhesive patches were removed by the subjects after 22 ± 1 h of occlusion.
The subjects were required to have skin types I and II according to Fitzpatrick and lesional skin sufficient for at least two test fields.
They were excluded if they had certain conditions, such as known allergies to ingredients of the test product or adhesive patches, or were taking systemic glucocorticoids or immune modulators within 3 months before the study.The use of skin care products in the test fields, bathing, sauna, sunbathing, or solarium was prohibited throughout the study.The dermal reactions in the test fields were graded according to erythema and scaling scales, ranging from 0 to 4, where 0 is no reaction and 4 is severe erythema with induration and/or epidermal defect.

| Clinical evaluation of skin redness after UV radiation
The test was carried out based on the specifications provided by Ljunggren and Moller (1973)  24 and modified according to the Din 67 501 regulations and findings by Farr and Diffey (1984). 25 Next, seven square fields (side length of 1.5 cm each) were marked on both sides of the back.Subsequently, all test fields were irradiated with 1.5 MED.
Immediately after irradiation, an after-sun lotion (L'Oreal Ambre Solair) and a gel containing dimenthindene maleate (Fenistil gel) were applied, and a piece of the collagen mask and a piece of fabric were placed on the moistened test areas, blotted with a damp sponge, and lightly massaged.The exposure time was 30 min.One area was left untreated as a control.The treatment was repeated 6 h after irradiation.
The degree of erythema was measured 24 h after irradiation and two test product applications.The measurement was carried out using a Chromameter (Konica Minolta).

| Clinical evaluation of hydration and skin roughness
The study was conducted on 10 adult, female participants who met the inclusion criteria of having healthy skin in the test area and being 30 years of age or older.Exclusion criteria included severe or chronic skin inflammation, serious inner or chronic diseases, intake of drugs that could interfere with skin reactions, application of pharmaceutical or skin care products with active ingredients until 7-10 days before testing, severe allergies, sunbathing or usage of tanning beds during the study period, known cancer, and pregnancy or lactation The first half of the collagen mask was placed on one side of the face and rehydrated with water (Vittel).The other half of the mask was placed on the other side of the face and rehydrated with an activator liquid.The mask was left in place for 20 min.

| Clinical evaluation of long-term effect on wrinkles in the periorbital area
The purpose of this study was to determine the product's tolerance according to clinical-dermatological test criteria and to examine the depth of wrinkles before and after the application period by means of PRIMOS optical 3D measurement.
The study included five female test participants who met the inclusion and exclusion criteria.The inclusion criteria included subjects aged 30 years and older with normal to dry skin and healthy skin in the test area.Exclusion criteria included severe or chronic skin inflammation, serious inner or chronic diseases, intake of drugs that possibly can interfere with skin reactions, application of pharmaceutical products and skin care products with active ingredients until 7-10 days before testing, severe allergies or occurred severe side effects after the usage of cosmetic products, sunbath or usage of the tanning bed during the study period, known cancer, pregnancy, or lactation period.
The test started with a dermatological examination for all the test subjects.Then, all test subjects applied a kidney-shaped collagen mask with instant rehydration with an activator liquid (INCI: Aqua (Water), Butylene Glycol, Glycerin, 1,5-Pentanediol, Leuconostoc/ Radish Root Ferment Filtrate, Sodium Hyaluronate) twice a week for 15 min for a period of 4 weeks on the test area according to the instructions in the eye area.Skin roughness parameters were assessed at baseline, after 2 and 4 weeks.

| Clinical evaluation of skin hydration and wrinkles in comparison to a pre-soaked sheet mask
The objective of this dermatological study was to determine the influence of the collagen mask in comparison to a pre-soaked cellulose sheet mask with respect to skin smoothness (eye wrinkles)/ skin roughness (micro relief of the skin) and skin hydration at 20, 40, and 120 min after a single application for 20 min.The collagen mask was rehydrated with an activator liquid containing Aqua, Butylene Glycol, Glycerin, Pentylene Glycol, Leuconostoc/Radish Root Ferment Filtrate, Sodium Hyaluronate.
Twenty qualified female volunteers of Fitzpatrick phototype I-IV and aged between 35 and 65 years (mean age 56.1 ± 4.9 years) were included in the study.All subjects had good health and mental condition.Subjects were excluded if they had acute skin diseases, warts, scabs, tattoo, many hairs, or skin irritations/diseases that would interfere with the evaluation of the product under test, known allergies to cosmetics or ingredients of cosmetic test products, topical medication or systemic medication with anti-inflammatory agents, antibiotics or corticoids, pregnancy, period of breastfeeding, immunological disorders, severe disorders within the last 6 months, disorders within the last 8 days before starting the study with a fever period of more than 24 h, participating in a study with pharmaceutical within a period of at least 4 weeks prior to this study, consumption of alcohol or drugs before study examination, alcohol and drug abuse, intellectual or mental inability to follow study instructions, sauna or swimming on the study day, and lack of compliance.
Before the start of measurements and treatments, a preconditioning period of 7 days was provided, during which subjects were not allowed to use skin care products or dermatological therapeutics on the forearms and periorbital areas or use shower oils.In the morning of the measurements, subjects were not allowed to use decorative cosmetics except for the lips.The test areas were localized on the inner sides of both forearms and the periorbital areas of both eyes.Each test area on the forearm (one test area per volar forearm) was located about 7 cm away from the crook of the arm and measured 6 × 6 cm.Within this test area, three sub-test sites were marked for measurement of skin roughness.The kidney-shaped periorbital test area included the outer corner of the eye and a part of the lower palpebra.Within the periorbital test area, a sub-test site was marked at the section of the outer corner of the eye for determination of skin roughness.

| Analysis of soluble protein components
The lyophilized collagen biomatrix that was analyzed has soluble protein components that are extractable in water.The amount of soluble components in the extracted material accounts for 0.25% of the dry mass.For a collagen face mask with a mass of 2.0 g, this corresponds to 5 mg of extractable protein.The composition of amino acids in the extract is consistent with that of collagen.The composition of the analyzed extract closely resembles that of other collagen materials, such as bovine skin.Table 1 provides a comparison of the amino acid distribution between the extract solution and bovine skin.
Based on these findings, it can be concluded that the extracted protein components from the collagen mask are indeed collagen.
The slight variations in glycine, proline, and arginine values are within the allowable margin of error for the RP-HPLC method used in this analysis and are therefore not significant.As a result, there is no evidence of foreign proteins in the extract solution.
The lyophilized collagen biomatrix also contains smaller collagen peptides that are soluble and water-extractable.The main components of these peptides have molecular weights below 13 kDa.When compared to a 5-component molecular weight standard, the elution profile of the collagen extract exhibits a continuous, bell-shaped curve.In comparing the molecular weights of the individual signals from the standards, it is evident that the peak of the size distribution is approximately 13 kDa.However, the primary component of the collagen peptides is significantly smaller than 13 kDa (Figure 1,B).
Further analysis of the low-molecular fraction of the collagen extract reveals the presence of collagen peptides that are smaller than 2 kDa.The mass/charge values in this range demonstrate consistent and reproducible small collagen peptides ranging from 500 to 2000 Da.Considering the average molecular weight of amino acids, with glycine at 75 Da, hydroxyproline at 131 Da, and proline at 115 Da, these peptide lengths correspond to approximately 4-18 amino acids.

| Influence of soluble collagen components on fibroblast regulation
Our study observed relevant changes in the levels of 87 proteins related to wound healing.Of these, 10 proteins were found to have significant changes.Stimulation of growth factor (TNFa) and collagen type IV was noted while the levels of degrading enzymes (MMP7, MMP9) and precursor of MMP-activating enzymes (Plasminogen) were reduced.Additionally, there was a reduction in levels of inflammation mediating cytokines such as Interleukine 1b, 2, 4, 6, and 10.
These findings suggest a potential mechanism of action for promoting fibroblast activity, even without physical contact to the collagen matrix (Figure 2A-C).

| Scanning electron microscope analysis of the collagen mask
SEM analysis of the collagen mask shows a native structure of collagen highly similar to the fiber and fibril structure of human dermis.
(Figure 3A,B) When compared to other sheet masks, the collagen mask shows high interconnected porous structure with the presence of a multitude of pores, in contrast to the biocellulose mask which shows very limited porosity Figure 3C,D.
TA B L E 1 Amino acid composition of collagen mask compared to bovine skin.The SEM results of the skin irritation samples indicate that the collagen mask tested was effective in providing visible calming of the irritated skin explants, as well as in moisturizing and plumping the skin.Additionally, the treatment resulted in a significant reduction in the gap of the dermal-epidermal junction (DEJ), suggesting an improvement in overall skin health and integrity (Figure 3E).

| Clinical microbiome evaluation
Regarding the global state of the skin microbiome at genus level obtained after 28 days (t1) of application of the investigational product Collagen Mask the most common genus was Staphylococcus followed by Corynebacterium, Streptococcus, Cutibacterium, Enhydrobacter, and Acinetobacter.These genera were also the most Regarding the relative abundance of the species S. epidermidis, a statistically significant (p ≤ 0.05) decrease was observed between the two time-points.However, comparing with the results obtained for the untreated area, non-statistically significant differences (p > 0.05) were observed.
Furthermore, non-statistically significant (p > 0.05) differences were observed among the relative abundance of the species C. acnes between the two time-points.As well, comparing with the results obtained for the untreated area, non-statistically significant differences (p > 0.05) were observed (Table 2).
These results show that this investigational product maintained the skin microbiome diversity, not having an impact in the global skin microbiome.
A statistically significant (p ≤ 0.05) mean difference of −1.79% in skin pH values was observed when comparing the results obtained after 28 days of product's application every other day (t1) to the ones obtained at baseline (t0).Comparing with the untreated area, statistically significant differences (p ≤ 0.05) variations were observed.These results show that the collagen mask had a statistically significant impact in skin pH, decreasing the skin pH in 77.27% of the subjects, with a maximum decrease of −6.78%.(Table 3).
After 28 days of study performance, statistically significant (p ≤ 0.05) differences were obtained for the untreated area when compared to the TEWL results at the baseline.The collagen mask also significantly increased the TEWL, in the same way that was observed in the untreated area, so no differences were found between the skin sites where the product was applied and the untreated skin sites.(Table 4) In fact, the temperatures registered during the day 28 visits were a little higher than the ones observed at the baseline; nevertheless, the range between the values obtained at each timepoint was more or less the same, meaning that the results of every subject were affected equally.

| Clinical evaluation of tolerability and local irritating reactions in atopic dermatitis
On intact skin treated with collagen mask, only a few slight erythematous reactions and a few slight scaling reactions were noted, similar to the scores given for the test fields treated with the negative control (NaCl 0.9%).The positive control (SOS 0.2%) led to the MedSkin Solutions' Native Collagen Human Dermis expected distinctive scaling reactions and cumulative erythemal reactions (Figure 4).
On lesional skin, scores for erythema and scaling for both the negative control and the test product decreased slightly from baseline during the study.This positive effect might be caused by occlusion and the good weather period with high UV-levels during the study period.
Under the conditions of this study, the dermal tolerance of the collagen mask on intact and lesional skin in subjects with atopic dermatitis was very good.
There were no adverse events related to the test product during the study.

| Clinical Evaluation of skin redness after UV radiation
The study aimed to determine the efficacy of various skin care products in reducing erythema, or skin redness, caused by exposure to UV radiation.The erythema threshold of the participants was first established through irradiation of five fields of varying exposure durations.Seven test fields were then irradiated with 1.5 MED and treated with either an after-sun lotion, dimenthindene maleate gel, collagen mask, or fabric (wet towel).Erythema measurements after 24 h after UV exposure, show that all products used, including the wet cloth, result in statistically significant decreases in redness compared to the untreated area (p < 0.05).The collagen mask has the most significant effects.The after-sun lotion and dimenthindene maleate gel are both equally effective and result in a statistically significant reduction in redness compared to treatment with the wet cloth (Figure 5).

| Clinical evaluation of hydration and skin roughness
The participants tolerated the product very well with none of study participants reporting issues during the course of the application test under dermatological and clinical criteria.There were no pathological skin reactions in the test area.
The average R z value for skin roughness in the test area before application of the collagen mask, after 15 min after mask removal, and 120 min after mask removal are provided in Table 5.The average percentual decrease in skin roughness is displayed in Figure 6.These results indicate that after 20 min of application, the collagen mask itself has strong smoothing effect which can be improved by taking another activator with additional ingredients than water.
The average values for skin moisture measured with a corneometer in the test areas before application of the collagen mask in the control area, after 15 min and after 120 min are listed in Table 5.The average percentual increase in skin hydration is displayed in Figure 7.
These results indicate that the collagen mask itself has strong hydrating effect which can be improved by taking another activator with additional ingredients than water.

| Clinical evaluation of long-term effect on wrinkles in the periorbital area
All of the five study participants tolerated the kidney-shaped collagen mask very well during the 4-week application test under dermatological and clinical criteria.There were no pathological skin reactions in the test area.
After 2 weeks, in the treated area an improvement in skin roughness of about 16.56% was measured and after 4 weeks an improvement in skin roughness of 27% was measured (Figure 8).

TA B L E 4
Transepidermal water loss.

| Clinical evaluation of skin hydration and wrinkles in comparison to a pre-soaked cellulose sheet mask
In this study with 20 participants, the collagen mask demonstrated a statistically significant increase in skin hydration in the periorbital test area at 20, 40, and 120 min after a single treatment when compared to baseline.Specifically, the skin hydration increased by about 59% after 20 min, 53% after 40 min, and 37% after 120 min(p < 0.001).The volume of eye wrinkles also decreased by about 15% after 20 min, 12% after 40 min, and 11% after 120 min after a single application of the collagen mask when compared to baseline.These results were statistically significant when compared to the test site treated with the pre-soaked cellulose sheet mask (p < 0.05), which showed a lower decrease in the volume of eye wrinkles by about 6% after 20 min, 5% after 40 min, and 1% after 120 min after a single application.The pre-soaked cellulose sheet mask also demonstrated a statistically significant increase in skin hydration in the periorbital test area after 20 min, but a decrease of 7% after 40 and a decrease of 19% after 120 min (Figure 9 and Figure 10).

| DISCUSS ION
Numerous scientific papers have been dedicated to collagen and its applications; however, limited records in common databases pose a significant challenge when attempting to compare and consolidate existing knowledge on collagen's use in cosmetics.Although collagen is widely incorporated into cosmetic products, not all research studies are published, as cosmetic companies often conduct proprietary research that is not made publicly available through scientific publications. 6r work helps to broaden the knowledge on the application and potential benefits of collagen in skin care and cosmetics.
Skin consists of mainly type I, III, and V collagen.The most prevalent collagen in the skin is the type I. 26 Bovine collagen contains mainly in this type of collagen, and native non-denatured collagen is highly desired in cosmetic and biomedical applications. 6,27e collagen mask investigated demonstrates several skin benefits.It significantly increases hydration, reduces erythema, minimizes wrinkles, and maintains a healthy skin microbiome and skin barrier.
Moisturization of skin, by skin care products, is predominantly produced by the water content of the product. 28,29[34][35] The collagen mask produced has a high similarity to human dermis, as demonstrated by the SEM images.The visual characteristics and structural organization of collagen fibrils closely resemble those of intact collagen observed in the dermis.Collagen matrices for tissue regeneration that are produced according to the same proprietary method as the collagen mask, have been described to have a native structure similar to human dermis and a close to human biological behavior. 36,37The native collagen scaffolds showed a significant increase in cell attachment and proliferation compared to other scaffolds.Furthermore, the cell morphology observed on these scaffolds exhibited a more asymmetrical shape, closely resembling the structures found in the native in vivo environment. 36e to its moisturizing, regenerating, and film-forming properties, collagen is a key component in many cosmetic formulations.Its superior ability to bind water helps to maintain optimal moisture levels in the skin, ensuring it remains supple and moisturized throughout the day.In addition to its natural humectant properties, collagen's film-forming properties also help to minimize transepidermal water loss (TEWL), thereby helping to protect the skin against damage caused by mechanical factors.Furthermore, the occlusive effect of collagen helps to create a more radiant, illuminated, and smooth complexion. 38,39Our collagen mask consists of collagen fibers and fibrils as well as a high amount of collagen peptides and molecules.
In contrast to other sheet masks, it is highly porous and the mask application results in an occlusive effect with slowly releasing moisture to the skin.The released peptides and molecules form a moisture film for further effect.
Our work shows that the collagen mask supports the skin barrier function and maintains a healthy skin microbiome.The collagen mask with the activation liquid has a low pH and resulted in a significant lowering of skin pH.These findings align with prior research demonstrating that skin care products formulated with a pH ≤4.5 can effectively acidify the skin, preserving its physiological pH and skin barrier function.In addition, collagen peptides are rich in NMF, such as Ser, Asp, Hyl, Hyp, and various hydrophilic groups including hydroxyl and carboxyl. 43These components improve the skin's ability to bind and retain water, effectively enhancing the water content of photoaging skin. 44tural moisturizing factor (NMF) is derived from the filaggrin protein and includes high-efficiency humectants like urea and uric acid, which help absorb water from the environment and maintain the water content in the skin. 4,45,46NMF also helps increase the elasticity of the skin's cuticle by reducing the molecular force between fibers.The decrease in NMF content is mainly caused by UV radiation, which impairs the hydrolysis of filaggrin protein and leads to decreased skin hydration ability. 47e amino acid composition of the collagen mask contributes to the hydration mode of action.
The demand for type I collagen in the healthcare, food, and cosmetics industries is substantial, leading to its frequent use as a biomaterial.To obtain collagen for industrial purposes, animal tissues are typically purified, rather than utilizing recombinant production systems. 48The challenge of reproducing the complete collagen molecule with its natural post-translational modifications, such as hydroxylation, has led to a decline in attempts to synthesize it using various types of hosts including prokaryotic and eukaryotic organisms such as yeast, bacteria, mammalian cells, insects, or plants. 49,6vine collagen type I has a 97% sequence homology to human type I collagen, making it a preferred source. 27The preservation of fiber packing to a certain extent, is a crucial element as it not only impacts bioengineering parameters but also the way cells interact with the biomaterial. 36,37,50,51Cells recognize and utilize the nanometric fibrillar organization as a guide for their growth and migration during the remodeling phase.The collagen molecule, in its triple helical conformation, plays a significant role in cellular signaling and contains multiple integrin binding sites.These binding sites, commonly known as the "GxOGER" sequence, comprise specific amino acids, including glycine (G), hydroxyproline (O), glutamate (E), arginine (R), and a hydrophobic residue (x).They are essential for cell adhesion and interaction. 20,21,6erefore, the non-structural functions of collagen are crucial for cellular communication, proliferation, and differentiation. 6,27ditionally, it has been observed that short polypeptides and small peptides can effectively penetrate deeper layers of the skin, facilitating the regeneration of skin properties. 52llagen peptides have been shown to have a positive impact on skin health by increasing the abundance of collagen, reducing the These effects contribute to the improvement of UV-induced skin damage. 31,53e collagen mask is rich in the amino acids associated with cell signaling, as is demonstrated by the amino acids extraction test.Our findings further suggest a potential mechanism of action for promoting fibroblast activity, even without physical contact to the collagen matrix.Clinically, a significant increase in skin hydration was observed, which lasted longer than a cellulose mask.
Furthermore, the application of collagen masks demonstrated a substantial and sustained reduction in wrinkles by 27%, resulting in an average Rz value of 110 μm, thereby reducing the appearance of primary wrinkles.
According to research, 54,55 wrinkles are the most visible when they measure between 100 μm and several millimeters in depth, which is influenced by environmental factors and an individual's age.Our result highlights the potential of collagen as a promising biomaterial and its application in the cosmetic industry.Moreover, other studies have indicated the significant correlation between the organization of collagen fibers in the papillary dermis and cutaneous relief at the primary and secondary lines. 56,57The destruction of the elastic network in the papillary dermis can also lead to changes in cutaneous relief, such as the flattening of the papillae due to age and exposure to UV radiation. 55Therefore, the ability of the collagen mask to reduce wrinkles may be attributed to its interaction with the skin.This finding adds to the understanding of how collagen can combat signs of skin aging.
While our research provides valuable insights, it does come with limitations.Population sizes in our studies were determined based on practical factors, without pre-set sample size calculations.We used relatively small groups, but the results were statistically significant, indicating a consistent trend.Recognizing that larger samples would be ideal for studies on visual conditions like wrinkles, we remain cautious while extrapolating results to broader populations.Notwithstanding these limitations, our findings contribute to the research field and lay the foundation for future, larger studies.
In conclusion, collagen is a valuable and versatile biomaterial that shows promise in the field of cosmetics and skincare.The collagen mask described in this manuscript demonstrated several skin benefits, such as increasing hydration, reducing erythema, minimizing wrinkles, and maintaining a healthy skin microbiome and skin barrier.
Collagen's natural humectant and film-forming properties make it an ideal component in cosmetic formulations aimed at moisturizing and protecting the skin against damage caused by mechanical factors.
Collagen's ability to combat signs of skin aging is an area of significant interest and research.Furthermore, collagen's potential as a biomaterial in tissue regeneration is well documented, with studies showing its ability to promote cell signaling, proliferation, and differentiation.Further research is necessary to explore the full potential of collagen in cosmetics and other applications, but the available evidence suggests that it holds great promise for enhancing skincare and promoting healthy aging.
Test areas were established on the back skin on both sides of the spine below the shoulder blades.Ultra-Vitalux lamps (Osram) were used as the source of radiation, installed 40 cm away from the skin.The exposure time was 10 min, and non-exposed areas were covered with opaque covers.Initially, the individual light sensitivity of the participants (erythema threshold, MED) was determined by irradiating five fields of approximately 0.5 cm 2 .The exposure duration of each field was 60 s, 84 s, 120 s, 168 s, and 240 s.The determination of the individual MED value based on these five irradiated fields was done after 24 h.The average exposure duration for erythema threshold was between 1 and 3 min.
period.The subjects were acclimatized for 45 min at a temperature of 22 degrees centigrade and 60% relative humidity before skin moisture values were measured at three different places within the testing area by using a Corneometer.Skin roughness and wrinkle depth was measured with the PRIMOS optical 3D measuring device.The R z value was measured (Rz (DIN) from the greatest individual roughness depth measurements of five adjacent individual measurements of equal length of the (digitally) filtered profile).The control measurement area was the untreated skin close to the test area.Skin measurement values were taken before the application, 10-15 min, and 120 min after a single application.
The skin roughness was measured at four different time-points: immediately before the start of the first product application (t0), 20 min (t1), 40 min (t2), and 120 min (t3) after the treatment.The measurements were carried out on the forearms (PRIMOS® compact, in vivo, SQ-value, 3 repeated measurements) and on the periorbital areas (PRIMOS®, in vivo, volume of one eye wrinkle, 3 repeated measurements).Statistical analysis was performed, and the distribution of the data was analyzed.For analysis of the significance of differences the two-sided t-test for dependent samples or Wilcoxon test for paired samples was used.Statistical hypotheses were tested two-sided at a significance level of 0.05.Regarding the outcome measurements, the original data and the relative data (relative to t0 measurement) were analyzed.
common at the baseline (t0) and for the untreated area after 28 days (t1) after product's application.After 28 days of product's application every other day (t1), a statistically significant (p ≤ 0.05) increase of the Shannon's Diversity index mean values was observed when compared to the baseline (t0).However, non-statistically significant (p > 0.05) differences were obtained when compared to the Shannon's Diversity index results obtained for the untreated area (code: E).A statistically significant (p ≤ 0.05) decrease of the relative abundance of the genus Staphylococcus was observed between the two time-points.However, comparing with the results obtained for the untreated area, non-statistically significant differences (p > 0.05) were observed.Regarding the relative abundance of the genus Corynebacteriumand Pseudomonas, non-statistically significant (p > 0.05) differences were observed between the two time-points, and also F I G U R E 1 (A) Elution profile of the collagen extract.(B) Representative diagram of an intensity to mass/charge spectrum of the MALDI-TOF-analysis.F I G U R E 2 (A) Change in expression (fold change)-reduction in degrading enzymes (MMP7, MMP9).(B) Change in expression pattern (fold change)-downregulation of Interleukines 1b, 2, 4, 6, 10. (C) Change in expression pattern (fold change)-up-regulation of Collagen Type IV, TGFa. the results obtained for the untreated area (code: E).
U R E 3 (A) Mask scanning electron microscope (SEM) image of the collagen fiber and fibril structure of human dermis and collagen mask.(B) Collagen mask scanning electron microscope (SEM) image (200× magnification).(C) Biocellulose mask scanning electron microscope (SEM) image (200× magnification).(D) Hydrogel mask scanning electron microscope (SEM) image (200× magnification).(E) SEM skin side view before and after application of collagen mask on irritated skin explants.
with moisturization of the skin, as is demonstrated in our studies.The addition of an activator liquid containing hyaluronic acid and butylene glycol further increases the hydration potential of the collagen mask.The film-forming properties of collagen are enhanced F I G U R E 4 Sums of scores of erythematous and scaling reactions on intact skin and lesioned skin.

5
Reduction in skin redness (in %) caused by exposure to UV radiation compared to control area.*p < 0.05 ***p < 0.001.
may lead to enhanced skin barrier function.Studies have shown that extended use of skincare products with an adjusted pH of 4.0 significantly improved the function of the outermost layer of the skin, known as the epidermal barrier, compared to identical products with a pH of 6.0.42

F I G U R E 8 9 1 0
Long-term skin roughness effects in periorbital area after twice a week 15-min application of collagen mask.*p ≤ 0.05.Average increase of skin hydration for collagen mask and pre-soaked cellulose mask.***p ≤ 0.001.Average volume of eye wrinkles collagen mask and pre-soaked cellulose mask.*p ≤ 0.05. of type I collagen, maintaining a balanced ratio of type III and type I collagen, and decreasing the thickness of the dermis.

Amino acid Percentage (%) collagen extract Percentage (%) cattle skin
Shannon's diversity index, relative abundance of the genus Corynebacterium, Staphylococcus, and Pseudomonas and relative abundance of the species Cutibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa obtained before (t0) and after 28 days (t1) of application of the investigational product Native Collagen Mask and on the untreated area.Skin pH results.
TA B L E 2 Microbiome results.*ANOVA,**Not calculated.Note: *Paired t-test; **Wilcoxon test.Skin pH results obtained before (t0) and after 28 days (t1) of application of the investigational product Native Collagen Mask and on the untreated area.TA B L E 3Note: *Paired t-test; **Wilcoxon test.TEWL mean results obtained before (t0) and after 28 days (t1) of application of the collagen mask and on the untreated area.

z value-before mask application (μm) R z value-15 min after mask removal (μm) R z value-120 min after mask removal (μm)
40,41Regular application of the collagen mask TA B L E 5 Skin roughness and skin hydration outcomes.R