The use of animal testing in the cosmetic industry is already prohibited in more than 40 countries, including those of the EU. The pressure for it to be banned worldwide in the future is increasing, so the need for animal alternatives is of great interest today. In addition, using animals and humans in scientific research is ethically reprehensible. This study aimed to prove some of the anti-aging properties of elastin (EL), hydrolyzed collagen (HC), and two vegan collagen-like products (Veg Col) in a tri-layered chitosan membrane that was ionically crosslinked with sodium tripolyphosphate (TPP). In the first approach, as a way of representing different layers of a biological system, such as the epidermis and the two dermis sublayers, EL, HC, or Veg Col were independently introduced into the two inner layers (2L (i+b) ). Their effects were compared with those of their introduction into three layers (3L). Different experiments were performed on the membrane to test its elasticity, hydration, moisture retention, and pore reduction at different concentrations of EL, HC, and Veg Col, and the results were normalized vs. a blank membrane. This new alternative to animal or human testing can be suitable for proving certain efficacy claims for active ingredients or products in the pharmaceutical, nutritional, and cosmetic fields.
The effects of the active ingredients and Veg Col products on the previous properties varied depending on the layer in which they were introduced.
It was demonstrated that the effects of some active ingredients, namely EL, HC, and two Veg Col products, were evidenced in terms of pore reduction, water permeation, elasticity tests, swelling, and moisture retention. Hence, anti-aging claims could be proven.
From the results of this study, it can be stated that these membranes are sensitive to specific properties.
Hydration is a complex process composed of two mechanisms&#;the barrier effect or moisture retention&#;and is the ability of a system to avoid water loss. In the skin, this is known as transepidermal water loss (TEWL). On the other hand, humidity absorption is the capacity to absorb water from the environment [ 36 38 ].
With an excess of Ch, a hydrogen-bonding-type complex can be formed in addition to an electrostatic complex, whereas a self-crosslinking phenomenon is induced with an excess of Col [ 16 , 34 ]. However, the ionic interaction of TPP with Col chains is small since the number of exposed amino groups in Col chains is very small.
In Col, different hydrogen bonds can be formed, such as those between chains by hydroxyproline &#;OH groups, those between other side groups, those involved in the formation of fibrils, and those with the &#;OH and NH 2 groups from Ch. Additionally, hydrogen bonds can be formed between the end groups of &#;COOH and NH 2 of Col and the &#;OH and &#;NH 2 groups of Ch, as Ch contains large numbers of &#;OH groups [ 33 ].
By combining Col with Ch, two kinds of interactions take place&#;hydrogen bonding and electrostatic interactions&#;and these reinforce the mechanical strength [ 31 ]. According to Taravel et al. [ 20 , 32 ], there is a weak interaction between Ch and Col that forms polyanion-polycation complexes (&#;NH 3 + from Ch with the &#;COO &#; group from Col) in slightly acidic solutions, although this is obstructed by Col gel formation.
Col, especially in its hydrolyzed form, is a well-known anti-aging ingredient that is used in cosmetics, pharmaceuticals, healthcare, and the beverage and food industries [ 29 ]. It is also a widely used biomaterial scaffold, and chemical crosslinking is the most suitable method for type I Col. However, most crosslinkers are expensive, difficult to manage, and cytotoxic, as with glutaraldehyde; in most cases, they cannot be applied alone, as in the case of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylamino-propyl) (EDC) [ 30 ]. Col is used as a skin substitute in different commercial products, such as Integra ® (Princetown, NJ, USA), OrCel ® (Forticell Bioscience, New York, NY, USA), Promogran Prisma ® (3M, Two Harbors, MN, USA), and PuraPly ® (Organogenesis, Inc., Canton, MA, USA), to name a few [ 12 ].
It was already reported that Ch was used in combination with Col due to its antibacterial properties and because it reduces the biodegradation of the scaffold [ 12 ]. Col can improve the tensile strength and elasticity of chitosan scaffolds depending on the dosage used [ 10 , 16 , 28 ].
Col, with a molecular weight around &#;300 KDa, is produced by fibroblasts. It provides tensile strength to the skin [ 15 ]. It is located in the dermis, provides physical support to tissues, plays an important role in the structural and biological integrity of the ECM, and represents 75% of the dry weight of the skin. It is also an amphoteric macromolecule [ 27 ].
The addition of EL to a Ch membrane can increase its elasticity and mechanical strength, improve hydrophilicity, and increase degradation rates [ 25 ]. Hydration of EL is necessary for obtaining elasticity. EL monomers are disordered and flexible in water. They can act as plasticizers in water by interacting with the water molecules that are bound to the main chain, favoring its movement. A hydrated elastin chain can form transient hydrogen-bonded turns. These turns are not random but, rather, dynamic conformation structures that confer flexibility and contribute to high disorder. Furthermore, the hydrophobic segments prevent the formation of large secondary structures that could block chain motion [ 26 ].
In contrast to HC, EL, whether or not it is in its hydrolyzed form, has not been extensively employed in cosmetics or as a nutraceutical, despite some of its beneficial effects that have been found in skin, such as its improvement of skin elasticity in combination with Col and with some other biomaterials [ 21 ]. EL is rarely used in bioscaffolds, and it is mainly used for blending with other polymers, such as Col or GAGs [ 22 , 23 , 24 ]. It is mainly used as an additive for other scaffold materials, such as Col, due to its poor mechanical strength and availability. The MatriDerm ® matrix (Billerberck, Germany) is an example of this, where EL is used to modify Col to mimic elastic fibers in the native dermis [ 12 ].
EL, with a molecular weight of around &#;70 KDa, is the main component of elastic fibers. Its role is closely linked with that of Col; it provides stretching, recoil, and elasticity to the skin. It is also located in the dermal layer of the skin and makes up approximately 2&#;4% of the dry weight of the dermis in the skin of adults [ 17 ]. It is composed of alternating hydrophilic and hydrophobic segments [ 18 , 19 , 20 ].
EL and HC were introduced into the 3L of the membrane to test their efficacy in the entire membrane. The three layers of the membrane were intended to represent the epidermis and the two dermis sublayers, namely, the papillary dermis and the reticular dermis. For this reason, EL and HC were also independently introduced into the two inner layers, where they are naturally found in human skin [ 6 ].
In this research, EL and HC were selected with the aim of testing their efficacy in a Ch-based membrane [ 13 , 14 ]. EL and Col, together with GAGs, are some of the key components of the ECM. The ECM is a non-cellular, three-dimensional macromolecular network in which cells reside, and it is found in all tissues and organs [ 15 , 16 ].
This kind of membrane based on chitosan could have potential for use in biomedical applications. Thanks to its hemostatic, antimicrobial, and antifungal properties, chitosan could be a suitable option for burn and wound treatments [ 11 12 ].
Chitosan is affordable compared to other biomaterials that can be naturally found in skin, such as collagen (Col), EL, and GAGs, such as hyaluronic acid.
Chitosan is characterized by the presence of primary amino groups along its polymer backbone, which causes its structure to interact with different proteins, cells, and living organisms.
Chitosan is derived from chitin, and it is probably the second most abundant polymer in nature, behind only cellulose.
Ionic crosslinking was chosen, among other reasons, because this kind of crosslink is created between collagen fibers and proteoglycans in the extracellular matrix (ECM); thus, the components of our membranes had a similar structure [ 8 ]. These crosslinks confer structure to the skin. In contrast to ionic crosslinking, covalent crosslinking is formed in the skin between the amino acids that form proteins located in the major part of the ECM, which is mainly to give strength and elasticity to the skin.
In our previous work [ 7 ], a tri-layered chitosan membrane that was ionically crosslinked with sodium tripolyphosphate (TPP) was created as a physical model in an attempt to simulate different skin layers with the aim of providing similar results to those obtained with human skin, but with strong simplifications. The use of living cells was avoided, as there was no goal of reproducing the conditions and complexity of human skin. The materials employed were cost-effective, and they could mimic several skin characteristics and properties, such as the different layers, skin pore size, elasticity, hydration, and moisture retention. In this work, two kinds of membranes were tested: a base membrane and an activated membrane. In the second case, pores were mechanically created, as was already reported [ 7 ].
Some synthetic or natural biomaterial-based scaffold skin models that mimic the complex and stratified structure of human skin have been developed to replace the use of animals. Some skin models represent the epidermis, including EpiSkin ® (L&#;Oreal, Île-de-France, France), Epi-Derm ® (MatTek Corporation, Ashland, MA, USA), SkinEthic ® (SkinEthics, Lyon, France), epiCS ® (CellSystems, Troisdorf, Germany), Holoderm ® , and Kaloderm ® (Seoul, Republic of Korea). Some other models represent the structure of the full thickness of human skin. Skin-on-a-chip models can also bring some other important aspects to light, such as the use of a vascular system, resulting in longer survival of the tissue [ 4 ]. However, these alternatives represent only some aspects of real human skin. Some other aspects, such as the skin barrier function, are poorly represented. Some major drawbacks of these models are that they can be time-consuming, dependent on expertise, and costly [ 5 ]. Hence, there is still much research with the aim of finding an alternative system in which the complexity of human skin can be achieved [ 6 ].
In March , the European Union started the first full ban on animal testing for cosmetics. For this reason, there is a need for the replacement of animal skin models, since they are forbidden in more than 40 countries for cosmetic purposes and are ethically regulated for medical purposes [ 2 , 3 ].
Animals have been widely used for biomedical research as an alternative to human beings, although it is important to exercise caution when extrapolating findings to human outcomes. Apart from these aspects, ethical concerns regarding the use of animals, together with the high costs, time-consuming protocols, lack of effective extrapolations, and lack of reproducibility in results, are important drawbacks in all basic research.
2. Results and Discussion
2.1. Pore Quantification
Pores have different sizes depending on age, sex, ethnicity, and body area. Facial skin pores are a great concern for the beauty market, and their size may vary for different reasons, such as high sebum excretion, decreased elasticity around pores, an increase in hair follicle volume, and dehydration [39,40,41].
shows the pore areas that were measured for activated membranes with extreme concentrations in order to see their differences with respect to blank activated membranes. It can be seen the different mean pore areas for some EL and HC studied concentrations and the percentage of pore reduction versus blank containing the active ingredient in 3L or M2L(i+b) according to Equation (4). All concentrations appearing in this work are expressed as w/w. Other expressions of concentrations will be specifically detailed.
Table 1
ID MNº Pores AreaSDPore Reduction M3L&#;Ch (blank),&#;M3L&#;0.085EL,&#;4.9M3L&#;0.28EL,&#;18M2L(i+b)&#;0.085EL ,&#;0.48M2L(i+b)&#;0.28EL ,&#;18M3L&#;2.6HC,&#;15M3L&#;10HC,&#;22M2L(i+b)&#;2.6HC ,&#;2.1M2L(i+b)&#;10HC ,&#;8.5Open in a separate window
As previously reported by Flament et al. [39], facial pore size varies depending on ethnicity, from 15,000 μm2 for the Chinese ethnicity to 92,500 μm2 for the Brazilian ethnicity. In the case of Asiatic facial pores, the pores are the smallest. For Caucasians, the skin values are in the middle, ranging between 40,000 and 55,000 μm2 (calculated as the area of a circle) [42]. All of these pore sizes can be studied with these kinds of membranes; the mean pore size was 39,045 µm2 for the blank activated membrane, which had even lower values than those of Caucasian skin at some concentrations of EL and HC.
When EL was included in the activated membrane, the effect on pore size reduction was most evident at the highest concentration, 0.28%, with the highest effect in both 3L and 2L(i+b); there was a pore reduction of &#;18%. A possible explanation for this having the same effect in both kinds of membranes could be that EL was able to diffuse from the inner to the top layer in 2L(i+b), thus producing the same effect as that in 3L. When the EL content was low (0.085%), the effect was a bit more pronounced when EL was included in 3L (&#;4.9%), but its effect at this low concentration was hardly perceived when it was included in 2L (&#;0.48%). In any case, the pore size reduction was directly proportional for both 3L and 2L(i+b).
In the case of HC, the effect on pore size reduction was also evidenced the most with the highest concentration (10%); again, the greatest effect on pore reduction was in 3L (&#;22%), which was in contrast to when it was included in 2L(i+b) (&#;8.5%). The pore size reduction was also directly proportional to the concentration for both 3L and 2L(i+b).
Nowadays, noticeable pore size is a great concern for consumers [43]. Hence, these results can be helpful for claiming the beneficial effects of these active ingredients or products on reducing pore size. The effect of HC on pore size reduction was also evidenced in previous scientific reports [44].
In , there is visual evidence of the most extreme cases of pore reduction for both the EL- and HC-activated membranes in comparison with the activated blank.
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When looking at the images obtained with the microscope, the pore size reduction was visually evident, and it can also be stated that the shapes of the pores varied arbitrarily.
A possible reason for these two phenomena could be the kinds of interactions between Ch, HC, or EL and TPP. When layers were composed of only Ch, well-shaped and firm channels were formed. Nevertheless, when HC or EL were included, some interactions could occur, as they could be located between adjacent molecules of Ch and TPP to produce arbitrary links. As a consequence, different effects could be promoted, such as increasing plasticity or disrupting the Ch structure. In addition, an increase in swelling could be achieved because of the nature of Col and EL, as the membranes were in contact with water during their formation. Consequently, water molecules could diffuse into the membrane and into the pores&#; surroundings. This fact could promote the disruption of the intermolecular forces that tightly held the polymer chains. Therefore, as a possible consequence of these two phenomena, the formation of irregularities in the channels and reductions in pore size could take place.
2.2. Permeation Tests
As a means of studying the permeability, 30 g of water was permeated through an activated membrane in a Franz diffusion cell. During the permeation tests, the blank activated membranes reached the highest permeation of water in the first minute, and the 30 g of water was completely permeated in 2.9 min on average. For this reason, the quantity of water permeated within the first minute was used to compare the pore sizes. The reference value for the experimental results was the mean water permeation through the blank activated membranes for one minute (15 g). The mean water permeation (%) of the different experiments was calculated according to Equation (5).
2.2.1. Permeability of the EL-Activated Membranes
Three different concentrations of EL were studied for the water permeation tests. The lowest one, 0.085%, fell within the margins of the EL content in dermal skin.
In and , the differences that were observed when EL was included in 3L and 2L(i+b), respectively, can be seen.
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As shown in , a 65% reduction in permeation was observed with 0.283% EL, which was a reduction in permeation by more than half with respect to the blank.
As shown in , when EL was included in 2L(i+b), a 60% water permeation reduction was observed at the highest concentration of EL (0.283%), showing the high power of EL in water permeation reduction, despite only being included in 2L(i+b).
These results show that EL had a very strong effect on the reduction in water permeation. It worked in a concentration-dependent manner for both kinds of membranes (3L and 2L(i+b)). The effect of water permeation reduction was slightly clearer when EL was introduced in 3L than in 2L(i+b), as there was no EL in the first layer. Thus, the water permeation reduction was less evident.
2.2.2. Mean Permeability of the HC- and Veg-Col-Activated Membranes
In the case of HC, eight different concentrations were studied to see their effects on water permeation. In addition, two different Veg Col products were also studied at the concentration recommended by the supplier (2%).
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In and , the differences observed when HC was just studied in 3L or 2L(i+b) at some concentrations can be seen.
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As shown in , the reduction in water permeation was, again, not as strong as when HC was introduced in 3L, but the effect was also concentration dependent.
Well-arranged values were observed for the eight different concentrations that were studied, as the water permeation decreased from &#;9.2% to &#;39%. It should be noted that HC is a small molecule compared to EL. Hence, although the concentration of HC was higher than that of EL, it was foreseeable that the water permeation reduction would not have as much of an impact as that of EL (from &#;23% to &#;65%), as the latter has longer hydrophobic segments.
In the case of Veg Col, the effect of water permeation reduction was barely noticeable in comparison with the HC of animal origin.
2.3. Relationship between the Mean Water Permeability and the Mean Pore Reduction
In , the relationship between the mean percentage of water permeation reduction and the mean percentage of pore reduction is shown for both active ingredients (EL and HC) that were studied.
Table 2
ID MWater Permeation ± SDWater Permeation ReductionPore ReductionM3L&#;Ch (blank)15 ± 2.6&#;&#;M3L&#;0.085EL12 ± 2.8&#;23&#;4.9M3L&#;0.28EL5.3 ± 0.07&#;65&#;18M2L(i+b)&#;0.085EL 114 ± 1.7&#;10&#;0.48M2L(i+b)&#;0.28EL 16.1 ± 1.1&#;60&#;18M3L&#;2.6HC11 ± 3.1&#;25&#;15M3L&#;10HC9.3 ± 1.7&#;39&#;22M2L(i+b)&#;2.6HC 114 ± 5.5&#;9.3&#;2.1M2L(i+b)&#;10HC 112 ± 1.4&#;21&#;8.5Open in a separate window
When comparing both active ingredients, it can be seen that in the case of EL, the water permeation reduction was greater than that with HC for similar values of pore reduction. The difference increased at high concentrations of both active ingredients. An explanation for this divergence could be because of the nature of EL. EL contains more hydrophobic segments as it has a longer chain compared to that of Col, which is hydrolyzed. This issue may impact the water permeability by slowing down the permeation process, despite the fact that the effective pore size obtained with EL was not much smaller than that obtained with HC.
From the results obtained, it can be seen that the greatest pore reduction was achieved when 10% HC was added to 3L, and the greatest water permeation reduction was obtained when 0.28% EL was added to 3L.
2.4. Rheological Tests
All elastic modulus results are mean values of G&#; (Pa) that were obtained within 2 min. For blank rheological tests, it was evident that the elasticity results were very sensitive to the room temperature during membrane formation. All membranes studied for the rheological tests were base membranes.
As can be observed in , the elasticity of the blank membranes followed a good model of linear regression (R2 = 0.) with respect to temperature in the range of room temperatures studied (from 17.5 to 25 °C).
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All elastic modulus results, which are shown in %, of the membranes containing the different active ingredients or products were normalized (Equation (7)) with respect to their blank membrane interpolation in the linear regression model (Equation (6)), as shown in , , and .
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In , it can be observed that, when EL was added to 3L, the elasticity decreased as the EL concentration increased.
In , it is shown that, when studying these three concentrations of EL in 2L(i+b), the tendency was the same, causing the percentage of elasticity to decrease as the EL concentration increased.
However, when comparing the same concentration of EL in 3L and 2L(i+b), the effect on elasticity was increased in 2L(i+b); the greatest difference was at the lowest concentration in the solution (0.085%), and this was from 18% (3L) to 71% (2L(i+b)). This concentration (0.085% EL) was equal to 2.6% EL in dry weight. This concentration fell within the margins of the content of EL in human dermal skin (2&#;4% in dry weight). This fact suggests that the EL content in human skin has the optimal concentration for achieving the highest elasticity capacity. For the last concentration that was studied (0.28%), there was almost no difference between the two kinds of membranes (3L or 2L(i+b)).
From the results obtained for EL, it can be seen that for the three concentrations studied, EL always produced an increase in elasticity, although it was inversely proportional to the concentration. An elastic booster effect was obtained when EL was included in 2L(i+b) at all concentrations studied.
A possible explanation for the increase in elasticity could be that when EL is in contact with Ch, a unique hydrogen binding site between EL and Ch is achieved. Thus, it could break down the inter- and intramolecular hydrogen bonding networks of Ch. So, hydrophobic segments of EL that can form disordered assemblies&#; structures may hinder the formation of intermolecular hydrogen bonding networks. Thus, this would favor the motion of Ch chains. This effect reached its maximum at the lowest concentration of EL, 0.085%. When the EL content was increased, what could have happened is that fewer hydrogen bonding sites could be achieved between Ch and EL, as there could be saturation of EL, so a decrease in elasticity was observed.
As shown in , at 0.1% and 0.2% HC, according to literature, when there is an excess of Ch, a hydrogen-bonding-type complex and an electrostatic complex could form between Ch and HC, thus producing some elasticity in the membrane. Apart from these two kinds of complexes, a small interaction between TPP and HC could be produced, which may have also helped to increase the elasticity.
For higher concentrations, we have considered the following hypothesis. When the concentration of HC began to increase from 0.4% to 2%, a more disorganized network was produced, resulting in a reduction in elasticity. When the concentration of Ch was almost equal to that of HC, it seemed that a more ordered network was formed, and this was able to create more bonds between Ch, TPP, and HC. Hence, an increase in elasticity was observed. At higher concentrations, from 4 to 10% HC, a more disorganized network was achieved, as the excess of HC disturbed the links between Ch and TPP. Hence, a disruption in molecules&#; alignment was caused, leading to a decrease in elasticity. At 7.5% HC, the greatest effect on elasticity reduction (&#;47%) was obtained.
An inversely proportional relationship between Ch and HC concentrations was also observed when the peak of elasticity found when both concentrations were almost equal was excluded.
For both kinds of Veg Col that were studied, a noticeable effect on elasticity was found at the concentration of 2% recommended by the supplier. In the case of Veg Col-A, the &#;collagen-like&#; active ingredient was combined with glycerin. Glycerin is known to have good plastic properties in the skin [45]. When it is combined with Ch, its single hydrogen bonding site can break down the inter- and intramolecular hydrogen bonding networks of, thus leaving hydrophobic C-H ending groups to limit intermolecular hydrogen bond formation and allowing free motion of the chitosan chains [46]. According to the supplier of Veg Col-A, glycerin was found in the product at 1.4% when using 2% Veg Col-A. Some elasticity tests were performed by adding 1.4% glycerin to the blank solution in order to check its influence on elasticity. There was a 16% increase in elasticity with respect to the blank. Hence, it seems that almost all increases in elasticity with Veg Col-A (17%) could be attributed to the plastifying effect of glycerin. In the case of Veg Col-B, the elasticity value of 15% could only be attributed to the active ingredient &#;vegan Col&#;.
As shown in , an even greater increase in elasticity was observed at 0.1% HC (40%) in 2L(i+b) than with the same concentration in 3L (19%). Both hydrogen bonding and electrostatic complexes could also be formed here. Our hypothesis in this case was that, apparently, when HC was included in 2L(i+b) at the lowest concentration (0.1%), it did not disturb the interaction of Ch and TPP. At the same time, HC could also form links with Ch from the same layer and from the top layer. A possible explanation could be that, in this case, the molecules could be organized in a way that favored linkages, thus acting as a dominant force for some other kinds of links in opposite directions, which could disturb the interaction. It seems that the HC located in the intermediate layer may still have had carboxylic groups that were able to react with free amino groups and &#;OH from the Ch stock solution in the last layer by forming more hydrogen bonds. It seems that in 3L at 0.1% HC, the molecules were a bit more disorganized than those in 2L(i+b), which had lower elasticity values (19%).
However, when the concentration of HC was 0.2 or 0.4%, the effect on elasticity was reduced to almost negligible. At 2% HC, a peak in elasticity was observed. A possible explanation of this effect could be that the diffusion of HC to the top layer may have had a similar effect on elasticity to that seen for the concentrations of 0.1 or 0.2% HC in 3L, suggesting that a similar concentration could diffuse to the top layer. Hence, the elasticity had a similar value to that obtained with those two concentrations (17%). For cases in which there was an excess of HC, the same effect on 3L was also visualized in 2L(i+b), but with slightly lower values of elasticity, as the concentration of HC in the whole membrane was not as high as in 3L. In the case of Veg Col-A, the elasticity was found to be similar for both kinds of membranes, and the positive effect of elasticity due to glycerin was observed in both cases (17 and 19%, respectively). For Veg Col-B, based only on the effect of the &#;collagen-like&#; active ingredient, the elasticity decreased when it was included in the two inner layers, and it was even lower than in the blank membrane (&#;23% in 2L(i+b) in contrast to 15% in 3L).
In the 2L(i+b) membranes, as shown in , an inversely proportional relationship between the Ch and HC concentrations was found, except for the peak of elasticity that was found when Ch was at 2.5% and HC was at 2%.
These elasticity results clearly demonstrate that EL, HC, and the two Veg Col products had an influence on the elasticity of the membrane, as they increased or decreased its value depending on the concentration used. It was also evidenced that when the active ingredient or Veg Col product was included in 2L(i+b), the elasticity values for the lowest concentration were boosted in comparison with the membrane in which the active ingredient or Veg Col product was included in 3L.
Some similarities with our results were found in the elasticity results obtained by Hydalgo-Vicelis et al. [28], where membranes were prepared in the form of films with the solvent evaporation technique and were composed of Ch and Col, which were crosslinked by a covalent crosslinker (EDC). In their case, different proportions of both Ch and Col were combined, unlike in our study, where the concentration of Ch was almost constant, but only the HC concentration varied. When a low concentration of Col was used, the elasticity obtained had the highest value, but, as the Col concentration increased, the elasticity values decreased. In this case, although they did not keep the Ch concentration constant, a tendency similar to that in our study was found despite the use of different types of crosslinkers.
According to Martínez et al. [16], they also tested EDC and TPP as crosslinkers for Ch membranes. In their case, the membranes were prepared by freeze-drying and combining three different proportions of Ch and Col. For TPP-crosslinked membranes with Col in the minority, the highest elasticity value was obtained. When the concentration of Col was equal to that of Ch, they found a reduction in the elasticity value, which was not in concordance with the values that we obtained. When the quantity of Col was in the majority, the elasticity increased, but not as much as when it was in the minority. For EDC-crosslinked membranes and when using a combination of both TTP and EDC, they showed a directly proportional concentration-dependent behavior as the Col concentration increased with the three concentrations that were studied.
These results show that, despite the use of the same crosslinker, depending on the technique of preparation of the membrane and the combination of concentrations of both Ch and Col, the tendencies of the elasticity results can substantially vary.
2.6. Moisture Retention
Moisture retention (MR) was measured in the base membranes containing the active ingredient or Veg Col products in 3L due to the conditions of this test. The water loss (in %) for the blank membranes was 60% at 15 min and 80% at 30 min. The moisture retention percentage for each membrane under study, normalized versus blank membrane, was calculated according to Equation (10).
As shown in , strong moisture retention abilities were observed for both concentrations of EL at 15 min; the value was higher (21%) at the highest concentration (0.27%). A lower moisture retention was observed at 30 min than at 15 min. These results suggest that EL has good short-term water retention abilities but a poor ability to retain moisture in the long term.
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As shown in , there was a negligible or negative effect on moisture retention for the studied concentrations of 2, 2.6, and 4% HC. This could mean that HC was able to disrupt the Ch structure, thus favoring water release.
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However, some moisture retention was observed for 10% HC at 15 min (6.3%). This could mean that this kind of membrane was more compact and closed than that in which Ch was in the majority. Water could diffuse easily because a more porous structure could be formed at 10% HC, and it was kept inside, so water retention was favored.
For the two types of vegan Col studied, Veg Col-B presented a strong moisture retention power in the short term (19%), unlike Veg Col-A, which had no positive effects (&#;1.2%). The moisture retention power of Veg Col-B (19%) was similar to that of the highest concentration of EL studied (21%).
As was shown, HC had a short-term capacity for water retention but almost no capacity for retaining water in the long term.
2.7. Comparison of the Studied Properties with Results in Human Skin Reported in the Scientific Literature
Some scientific papers have already stated claims based on efficacy tests that were performed with volunteers for HC and EL ingestion or topical application of cosmetic products containing these two active ingredients. Different clinical trials demonstrated that properties such as hydration, elasticity, transepidermal water loss (TEWL), reductions in wrinkles, and some other aspects of skin aging can be addressed by using these active ingredients. HC was used as both a nutraceutical and a cosmetic ingredient. The oral intake of a hydrolysate of EL showed beneficial effects on human skin, such as improvements in skin elasticity and wrinkles [21,53].
In most of the reported studies of oral ingestion of HC at different dosages, it was shown that improvements in skin elasticity and hydration were more evident than reductions in transepidermal water loss (TEWL) [44,54,55,56,57,58,59,60,61]. However, no effects on increased skin elasticity with the use of topical products with HC were reported. According to Berardesca et al. [62], topical application of HC produced significant improvements in skin hydration, surface smoothness, and luminosity. Ohara et al. [63] reported a dose-dependent effect of oral ingestion of two and a half or ten grams of HC on stratum corneum hydration, but it must be noted that only two dosages were studied. They also highlighted that stratum corneum hydration is the unique beneficial aspect of skin, but they found no significant differences in elasticity or TEWL improvement. In our study, hydration could be related to two properties: pore reduction and moisture retention. A directly proportional relationship was found between pore reduction and the concentrations of HC and EL. Although EL had positive effects on moisture retention in the short term, not enough concentrations were studied to see a clear tendency. In the case of HC, no tendencies were found, but a positive result was obtained for only the highest concentration: 10% HC.
As reported by Maia Campos et al. [64], the combination of topical products and oral supplementation of Col peptides can bring the best benefits for keeping skin in good condition, as their effects can be complementary. Topical products produce a short-term effect, and nutraceuticals have a long-term effect. They also demonstrated the pore miniaturization effect of HC.
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