Restoration of the Ultrastructural Integrity of the Dermal Collagen Network by 12-Week Ingestion of Special Collagen Peptides

Abstract

Introduction

This pilot study investigated the effects of a 12-week administration of a nutritional supplement containing special collagen peptides on the structural and molecular properties of the collagen fiber network in the human skin. For the assessments, the suction blister method and electron microscopical comparisons were used.

Methods

Three suction blisters were generated on the inner forearm of each test subject before and after the 12-week administration of the nutritional supplement. High-resolution scanning electron microscopy (SEM) was employed to meticulously investigate the structural characteristics of the skin’s collagen network, including the length and diameter of collagen fibers within the suction blister roof. Furthermore, the analysis included immunohistochemistry and fluorescence light microscopy to study hyaluronic acid within the extracellular matrix. Additional assessments encompassed changes in various epidermal parameters. Nine female participants within the age range of 43.7–61.8 years (mean: 52.5 ± 5.9 years) completed the study in accordance with the study protocol.

Results

Compared with baseline, the 12-week supplementation regimen led to a statistically significant average increase in the collagen fiber network size of 34.56% (p < 0.0001). Additionally, collagen fiber cross-linking and fiber length were substantially increased. The ingestion of the supplement also resulted in an 18.08% elevation in epidermal hyaluronic acid concentration (p < 0.0001). No adverse events were recorded during the study.

Conclusion

Using an innovative approach, this study demonstrated the ability of a targeted nutritional supplement to effectively restore the ultrastructural integrity of the dermal collagen network, which is typically disrupted by the natural aging process of the skin. These findings not only corroborate existing data regarding the positive effects of oral collagen peptides on skin structure and function but also contribute to our understanding of ultrastructural morphological aspects of changes in the skin’s collagen network. Supplementation can induce regeneration of the collagen fiber network in the human skin.

Trial Registration Number

German Clinical Trials Register, DRKS-ID DRKS00034161- Date of registration: 06.05.2024, retrospectively registered.

Plain Language Summary

Is it possible to improve the structure and molecular properties of the collagen fiber network in human skin by taking collagen peptides orally? To find out, suction blisters were created on the inner forearm of nine women before and after taking a nutritional supplement containing special collagen peptides for 12 weeks. To detect changes in the collagen fiber network, the inner surfaces of the roofs of the isolated suction blisters were examined using scanning electron microscopy. Additionally, hyaluronic acid levels were measured with fluorescence light microscopy. As a result, it was shown for the first time that a 12-weeks supplement regimen can lead to a statistically significant increase in the size of the collagen fiber network. Moreover, collagen fiber cross-linking and fiber length were substantially increased. The supplement also led to a statistically significant rise in epidermal hyaluronic acid levels. These findings not only support existing data on the positive effects of oral collagen peptides on skin structure and function but also enhance our understanding of the detailed morphological changes in the skin’s collagen network.

Key Summary Points

This pilot study investigated the effects of a 12-week administration of a nutritional supplement on the structural and molecular properties of the collagen fiber network in the human skin.

For the assessments, the suction blister method and electron microscopical comparisons were used.

Compared with baseline, the supplementation regimen led to a statistically significant average increase in the size of the collagen fiber network and the epidermal hyaluronic acid concentration, as well as a substantial increase in collagen fiber cross-linking and fiber length.

This study demonstrated the ability of a targeted nutritional supplement to effectively restore the ultrastructural integrity of the dermal collagen network.

Introduction

The collagen fiber network in the human skin and its age- and environment-related changes have been studied for over 30 years [1–5]. Biochemical and ultrastructural parameters were used to assess collagen changes in aging and photodamaged skin [5–12]. Extensive collagen fragmentation, clumping of the fragmented collagen, and a reduced quantity and quality of the collagen fiber network were observed in aged and photodamaged skin [2–4, 6, 8]. Sun-protected skin from young individuals exhibited little change or damage with an intact and large collagen fiber network [2, 4]. A uniform distribution of collagen fibrils was seen, and the extensive skin collagen network was intact and dense [2–4, 6]. Enhanced degradation of collagen fibers and the reduction of collagen synthesis in photodamaged skin leads to premature aging of the collagen fiber network [2–4]. The effect of supplementation on the collagen fiber network of the aging human skin has been investigated previously with different outcomes [5, 7, 9–15]. The majority of seven studies demonstrated strong effects of collagen supplementation on the ultrastructure of the fiber network in the human skin [5, 7, 9–11, 13, 15], whereas one study has not found significant effects [12].

The nutritional supplement containing special collagen peptides (ELASTEN^®) has been demonstrated to improve skin structure and function and to positively affect collagen ultrastructure [11, 16, 17]. Thus, the results of a blinded study using confocal laser scanning microscopy have demonstrated a significant improvement in the collagen structure of facial skin after the application of specific collagen peptides compared with placebo [11]. Furthermore, numerous studies have shown the positive effects of supplementation with collagen peptides, acerola extract, vitamin C and E, biotin, and zinc on skin parameters like hydration, elasticity and roughness [9–12, 16–21]. However, to explore the effects on skin structure and function, more studies regarding the impact of supplementation on the ultrastructure of the human skin collagen fiber network are needed [7, 22–32]. Studies have shown that collagen peptides can act synergistically with vitamin C, other vitamins, antioxidants, and zinc in facilitating the repair and regeneration of the endogenous collagen network in the skin [9–12, 16–21]. Currently, 14 studies demonstrate the age- and environment-related effects on the ultrastructure of the collagen fiber network [2, 4, 6, 33–43]. The damage to the collagen fiber network by endogenous and exogenous factors can be profound and the necessity for the development of new approaches to protection and regeneration is obvious [2, 4, 6, 33–43]. The unique potency of the nutritional supplement in improving skin appearance and physiology has been reviewed and confirmed in recent systematic reviews and meta-analysis on the efficacy of collagen supplements [29, 32]. Although highest level of evidence has been provided that the collagen supplement can improve skin structure and function, additional specific studies on how this supplementation affects the collagen fiber network of the skin and the extracellular matrix compound hyaluronic acid are necessary.

This exploratory pilot study investigated the effects of a 12-week administration of a nutritional supplement containing collagen peptides on the structural and molecular properties of the collagen fiber network in the human skin of nine female volunteers within the age range of 43.7–61.8 years (mean age: 52.5 ± 5.9 years). The aim of this pilot study was to investigate the question if the collagen supplement could and would affect the ultrastructure of the human skin collagen fiber network. The results indicate that a regeneration of the collagen fiber network in the human skin is possible by supplementation that is accompanied by an increase in skin hyaluronic acid content. These findings demonstrate that the ultrastructural appearance of the collagen fiber network as well as the hyaluronic acid concentrations in aged skin can be improved. The positive effects of supplementation on the endogenous collagen biomatrix in the skin indicates that such approaches are promising [5, 7, 9–11, 13, 44]. This study confirms and extends these previous findings.

Methods

Study Procedures

Study Design and Ethical Considerations

This clinical study, designed as a pilot study with a nutritional supplement, adhered to the principles of Good Clinical Practice, and followed a single-center, non-blinded, non-controlled, and non-randomized approach. Ethical approval was granted by the International Medical and Dental Ethics Commission GmbH (general approval issued on 17 January 2023; SGS ref. 307-01-0001, IRB Ref.-Nr: 2023/101), and all research activities adhered to the Declaration of Helsinki.

Suction Blister Generation

Suction blisters were induced on the inner forearm of each test subject using a plexiglass suction chamber with three circular openings of 7 mm diameter. Post-treatment blister generation was performed at the same forearm at a different site to exclude any influence of the natural wound healing process on the results. No control measurements on the potential effects of the blister generation have been performed. The blisters were generated through the application of vacuum pressure ranging from approximately 550–850 mbar within a period of 2.0–2.5 h. In the diameter of the plexiglass chamber openings, the blisters were formed after approximately 2 h, whereat the epidermis was slowly detached from the underlying dermis. After disinfection of the skin with Kodan^® Tinktur Forte skin disinfectant, the blister roofs were removed using sterile syringes, scissors, and forceps. The small wounds induced by this procedure were treated with Hansaplast “Schnelle Heilung” Strips and healed completely, scar-free, within 6–10 days. The blister roofs were collected (one roof per tube) in a buffer-solution (provided by Microscopy Service Dähnhardt (MSD)), cooled and forwarded to MSD on the day of generation for further analyses.

Study Schedule

After the initial generation of suction blisters on day 1, participants commenced a 12-week regimen by consuming the first one of 84 drinking ampoules of the test product. They were instructed to consume one ampoule daily at home. After the 12-week period, suction blisters were generated again using the same methodology for the final examination. Compliance checks were conducted at the 6 and 12-week marks.

Study Participants

A total of 14 healthy female subjects in good mental health ranging from 35 to 65 years of age were recruited, with a minimum of nine subjects completing the study. Key exclusion criteria included pre-existing skin damage or excessive hair in the test area, acute and/or chronic skin diseases, consumption of nutritional supplements in the 12 weeks prior to the study and within the study period, systemic therapy with immunosuppressive drugs and/or retinoids within 4 weeks of the study’s commencement, the use of anti-inflammatory agents within 2 weeks prior to the study, and the application of topical medications or any physical and/or cosmetic treatments at the measurement site within the month leading up to the study. Participants were also instructed not to expose the test area to natural sunlight or artificial ultraviolet irradiation, which could result in sunburn or tanning. They were instructed not to take any nutritional supplements other than the test product, not to use any skin care products, oily or moisturizing skin cleansing products on the forearms, to maintain their normal lifestyle habits, and not to change their diet and initiate or alter oral hormonal treatment or oral contraception. The consumption of any other products that could influence the skin during the study was strictly prohibited. Prior to study commencement, written informed consent was obtained from all study participants.

Test Materials

The test product, a nutritional supplement containing collagen peptides, was provided as 25 ml drinking ampoules. Each ampoule contained 2.5 g of special collagen peptides ([HC]-collagen-complex (10%), 666 mg aqueous acerola fruit extract 4:1 (2.7%), 80 mg l-ascorbic acid, 3 mg zinc citrate, 2.3 mg mixed natural tocopherols, and 50 µg biotin. Subjects were instructed to consume the contents of one ampoule daily, ideally in the morning and with or after a meal, for 12 weeks. The chosen dosage and duration of the supplementation were based on the experiences and results from previous studies with this supplement mentioned in the introduction [11, 17].

Measurements

Examination of Collagen Fiber Network

To investigate the collagen fiber network on the dermal side of the basement membrane, a new in vivo method was applied, which allows the underside of suction blister roofs to be analyzed using scanning electron microscopy [4]. After the generation of suction blisters, their roofs were re-fixed with osmium tetroxide, dehydrated using a graded ethanol series, and critically point-dried to avoid drying artifacts and optimally preserve fine structures. Subsequently, the samples underwent gold sputtering and were analyzed by SEM (DSM 940, Zeiss) at 15 kV accelerating voltage with various magnifications. Image acquisition was performed using the Image capture system DISS 5.0 (Point Electronic GmbH).

Detection of Hyaluronic Acid in the Epidermis

To discern any changes in hyaluronic acid levels induced by oral administration of collagen peptides, as described in the literature, [45] hyaluronic acid as part of the extracellular matrix was studied by immunohistochemistry using fluorescence light microscopy. Suction blister samples were fixed with modified Karnovsky fixative at 4 °C, dehydrated in ethanol, and embedded in LR White medium through low-temperature embedding. Sections measuring 200 nm were prepared, mounted on round glass coverslips, and treated with a washing buffer for 60 min to prevent non-specific binding. An anti-hyaluronic acid antibody (Abbexa), directly conjugated with fluorescein isothiocyanate, was applied for an additional 180 min. Following five washes, samples were stained using 4′,6-diamidino-2-phenylindole (DAPI) and embedded to visualize the nuclei using Roti^®-Mount Fluor Care DAPI (Roth). The samples were examined using a Leica fluorescence microscope (Leica DMLS), and images were captured with a coupled CCD Camera (ISH500 Tucsen) employing the ISIListen software from Tucsen Photonics. Measurement of fluorescence intensities was carried out using ImageJ software (www.nih.gov).

Analysis of the Quality of Collagen Fibers

The mean length and mean diameter of visible collagen fibers before and after the 12-week application of the test product were determined from the SEM images. To measure the change in fiber quality, the quotient of mean length and mean diameter was calculated for each subject. The difference between the quotient before and after the 12-week application was expressed as a mean percentage.

Statistical Analyses

The analysis of the study objective was performed by SGS INSTITUT FRESENIUS GmbH using the computer software Microsoft EXCEL (Office 365) and STATISTICA (version 13.5). Microsoft EXCEL was used for the calculation of the relative data, sample sizes, arithmetic means, standard deviations, and minimum and maximum values. STATISTICA was used for analyzing the distribution of the data (Kolmogorov–Smirnov test) and for analysing the significance of differences between the time points (two-sided t-test for dependent samples for normally distributed data). The hypothesis of a normal distribution was accepted when there was a p-value > 0.05. Concerning the differences between the treatment situations and the points in time, in the case of a p value ≤ 0.05, a difference was accepted as statistically significant.

Statistical significance of differences was evaluated using the two-sided t-test for dependent samples as all analyzed data were normally distributed. The original data as well as data relative to baseline were analyzed.

Results

Subjects and Dropouts

One of the initially included 14 subjects dropped out of the study after 6 weeks of product usage due to noncompliance (intake of vitamin B12 and D). For this subject, only baseline data were obtained, and thus this subject was excluded from data analysis, as were four others designated as surrogates. A total of nine subjects, aged from 43.7 to 61.8 years (mean: 52.5 ± 5.9 years), completed the study according to the study protocol. No adverse events were recorded during the study.

Collagen Fiber Network

The analyzed data were normally distributed according to the Kolmogorov–Smirnov test.

At baseline, the collagen fiber network at the dermal side of the blister roof basement membrane of all samples exhibited varying degrees of damage depending on the age of the subjects. The size of the collagen fiber network, relative to the examined area, ranged from 22.34% to 27.22% with an average of 24.57% ± 1.59%.

Supplementation of the nutritional product for 12 weeks resulted in a statistically significant increase in collagen network size, which now ranged from 28.42 to 35.79%. The mean value increased by 8.49 ± 2.52% to 33.06 ± 2.16% (p < 0.0001). Compared with baseline, the supplementation led to a statistically significant average increase in collagen network size of 34.56%. Figure 1a illustrates the collagen fiber network of the basement membrane before product application. In Fig. 1b, a marked increase in collagen fiber cross-linking and fiber length following 12 weeks of supplementation can be seen. The comparison of both images also reveals improved cross-linking of collagen fibers and increased fiber length.

Fig. 1.

Collagen fiber network at the dermal side of the basement membrane before (a) and after 12-week intake of the nutritional supplement (b). Example shown for subject no. 6

Hyaluronic Acid Concentration in the Epidermis

Furthermore, supplement intake led to a statistically significant increase in epidermal hyaluronic acid concentration. At baseline, concentrations ranged from 36.39% to 41.20% in relation to the examined area. After 12-week supplementation, the values ranged from 41.59% to 48.40%. The mean values increased by 7.05% from 38.98% to 46.03% (p < 0.0001). Figure 2 illustrates the hyaluronic acid concentration (green) in the epidermis before (a) and after 12 weeks of collagen peptide administration (b). Cell nuclei are stained blue with DAPI staining. The increase in hyaluronic acid concentration is evident through enhanced green staining (right image), resulting in a more pronounced green coloration. On average, there was an 18.08% increase in hyaluronic acid concentration relative to baseline.

Fig. 2.

Hyaluronic acid concentration (green) in the epidermis before (a) and after 12-week intake of the nutritional supplement (b). Example shown for subject no. 6. Cell nuclei shown in blue (DAPI staining)

Quality of Collagen Fibers

The quality of collagen fibers, as determined by the quotient of mean length and mean diameter of fibers, also improved over the course of the study. The quotient ranged from 751 to 1516 at baseline and from 1367 to 2448 after 12-weeks product application (Table 1). The mean quotient increased by 80.02% from 1116 (± 245) to 2009 (± 351). Figure 3 presents detailed enlargements of colored collagen fibers before (t0) and after the 12-week product treatment (t1).

Table 1.

Individual values of the quotient of length and diameter of collagen fibers

Subject	t0 (before)	t1 (after)
1	1134	2118
2	1188	1660
4	1223	2203
6	948	1923
7	1379	2448
8	856	1367
9	1516	2285
10	1051	1792
11	751	2287
Mean	1116	2009
SD	245	351

Values before (t0) and after 12-week intake of the nutritional supplement (t1)

SD standard deviation

Fig. 3.

Mean quotient of length and diameter of collagen fibers before (t0) and after 12-week intake of the nutritional supplement (t1)

Discussion

This study visualized the quantitative and qualitative changes in the ultrastructure of the collagen fiber network in the human skin after supplementation with the collagen peptide-containing food supplement with the help of SEM on the underside of the suction blister roofs and analyzed the outcome parameters by image analysis [4]. The collagen fiber network at the dermal side of the basement membrane in the blister roofs was damaged in the aged skin of the probands as assessed at baseline. The size of the collagen fiber network relative to the examined area of 24.57% ± 1.59% increased after supplementation significantly by 8.49 ± 2.51% to 34.56 ± 2.16%. The marked increase in collagen fiber network size was associated with an enhanced cross-linking of the fibers and increased fiber length. The quality of collagen fiber network as determined by the quotient of mean length and mean diameter of fibers was also significantly improved after supplementation.

These findings confirm and extend the observations of Laing et al., 2020, on skin collagen structure using confocal laser scanning microscopy [11]. The collagen supplement improves not only skin structure, but also skin function [11, 16, 17]. The short chain collagen peptides increase collagen synthesis and extracellular matrix formation in human skin [13, 46]. The quality of the collagen fiber network also improved over the course of the study. The mean increase of 80.02% demonstrates the restoration of a large and dense collagen fiber network after supplementation with the special collagen peptides for 12 weeks as depicted in Fig. 1b. This contrasts with the diminished and damaged collagen fiber network at the beginning of the study.

A significant increase in epidermal hyaluronic acid concentration was also demonstrated. Hyaluronic acid is another very important component of healthy skin that can be increased by the collagen supplement [10, 46, 47]. Hyaluronic acid in the skin is known to maintain moisture, to support tissue repair, to preserve elasticity, and to provide antioxidant defence against environmental damage. The mean increase by 7.05% from 38.98% to 46.03% (p < 0.0001) confirms that the changes are not restricted to the collagen fibers. This relatively substantial increase in hyaluronic acid concentration by an average of 18.08% compared with the baseline, along with the demonstrated improvement in the collagen network, offers a plausible explanation for the visible skin improvements observed in previous studies. [11, 16, 17]

This pilot exploratory clinical study has several limitations including a limited number of participants resulting in low effect size and poor power, no randomized, placebo-controlled, double-blind design of a controlled clinical trial, and the use of descriptive rather that confirmative statistics using the two-sided t-test for dependent samples. The major drawback of the study is the lack of a control group that does not allow us to determine to what extend the supplement has contributed to the positive effects on the skin in the study participants. Another limitation of this study is the fact that we do not have any data and findings on the collagen fiber network of young probands. Furthermore, the supplement contains several ingredients that all could have contributed to the observed effects on the skin. However, this study can nevertheless contribute to our understanding of the ultrastructural morphological changes in the dermal collagen fiber network that can be elicited by a collagen supplement.

Future controlled studies will be conducted to confirm the data and findings on the improvement of the skin structure in large groups of volunteers. A longitudinal assessment of the age-dependent changes in skin ultrastructure can provide decisive insights into the time course of the development of degenerative changes to the collagen fiber network and how regeneration induced by nutrition can reverse the damage to its components. The suction blisters method combined with high-resolution SEM reveals the dynamic changes and characteristics of the collagen fiber network in the human skin, including the length and diameter of collagen fibers within the suction blister roof. Changes in hyaluronic acid can be imaged by immunohistochemistry and fluorescence light microscopy. Collagen fragmentation, clumping of the fragmented collagen, and a reduced quantity and quality of the collagen fiber network in aged and photodamaged skin can be reversed by specific supplementation [2–4, 6, 8]. Repair, regeneration, and rejuvenation is possible even when the collagen fiber network has been damaged and partially destroyed.

Baseline nutritional status and body mass index, which may affect the collagen fiber network in the skin, were not considered as inclusion and/or exclusion criteria. The dose and duration of the supplementation was chosen according to previous data and findings from clinical studies on the effects of these nutrients on the skin [11, 16, 17, 24, 29, 32]. These clinical studies and reviews demonstrated that collagen supplements have decisive effects on skin aging and physiology demonstrating the validity of this specific approach to improve the skin collagen fiber network [11, 16, 17, 24, 29, 32]. The positive effects of the collagen supplement were confirmed in the randomized, placebo-controlled, blind studies of Laing et al., 2021 and Bolke et al., 2021, by assessing skin hydration, elasticity, roughness, and density [11, 17]. Therefore, the oral application of the dermonutrients for 12 weeks allows for a significant long-lasting repair and regeneration of the skin that is clearly visible and cosmetically relevant.

Since this study was not designed as a controlled interventional trial but constitutes a first exploratory observational study without control group, future studies that include diet-controlled placebo groups are necessary to allow firm conclusions on the efficacy of such supplements in reversing skin aging on the ultrastructural level of the collagen fiber network [48–51]. Such studies can also rigorously assess the effects of collagen supplements on skin burns, pressure ulcers, and skin aging.

Conclusion

Following a 12-week supplementation with collagen peptides, the results of this pilot study overall provide compelling evidence of a substantial and statistically significant enhancement in the consolidation and fortification of the skin’s collagen network, along with an increase in the skin’s hyaluronic acid concentration. Utilizing an innovative approach, it could be demonstrated that a targeted dietary supplement is able to effectively restore the ultrastructural integrity of the dermal collagen network, which is disrupted by the natural aging process of the skin. These findings not only validate existing data on the positive effects of oral collagen peptides on skin structure and function but also extend our understanding of the ultrastructural morphological aspects of the skin’s collagen network and hyaluronic acid content influenced by the investigational supplement, building upon previous experimental research and randomized, placebo-controlled clinical trials.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dorothee Dähnhardt, Stephan Dähnhardt-Pfeiffer, and Dörte Segger. The first draft of the manuscript was written by Gunter Lemmnitz and Burkhard Poeggeler and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Corresponding author: Gunter Lemmnitz.

Funding

Sponsorship for this study and Rapid Service Fee were funded by QUIRIS Healthcare GmbH & Co. KG, Gütersloh, Germany.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of Interest

Dorothee Dähnhardt, Stephan Dähnhardt-Pfeiffer, and Dörte Segger are scientists of Contract Research Organizations. Both declare that they have no conflicts of interest. Burkhard Poeggeler and Gunter Lemmnitz are employees of the sponsor (QUIRIS Healthcare). The findings and conclusions presented in this manuscript are the result of independent and objective scientific research. The analyses, interpretations and conclusions set out herein have not been influenced by the commercial interests of the sponsor and are the sole responsibility of the authors.

Ethical Approval

Ethical approval was granted by the International Medical and Dental Ethics Commission GmbH (general approval issued on 17 January 2023; SGS ref. 307-01-0001, IRB ref. no.: 2023/101), and all research activities adhered to the Declaration of Helsinki. Prior to study commencement, written informed consent was obtained from all study participants. The subjects consented to the participate in the study and give their permission to the publication of the data and findings obtained.