CED
Clinical dermatology • Original article
Clinical and Experimental Dermatology
Effect of high advanced-collagen tripeptide on wound healing and skin recovery after fractional photothermolysis treatment S. Y. Choi,1 W. G. Kim,2 E. J. Ko,1 Y. H. Lee,1 B. G. Kim,3 H. J. Shin,3 Y. S. Choi,4 J. Y. Ahn,5 B. J. Kim1 and H. J. Lee2 1
Department of Dermatology, Chung-Ang University College of Medicine, Seoul, South Korea; 2Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea; 3Health Science Research Institute, Amorepacific Corporation Research and Development Center, Gyeonggi-do, South Korea; 4Department of Dermatology, Ulsan University Hospital, Ulsan, South Korea; and 5Department of Dermatology, National Medical Center, Seoul, South Korea doi:10.1111/ced.12405
Summary
Background. Collagens have long been used in pharmaceuticals and food supplements for the improvement of skin. Aim. We evaluated the efficacy of high advanced-collagen tripeptide (HACP) on wound healing and skin recovery. Methods. Using an in vitro model, we performed HaCaT cell migration assays and collagen gel contraction assays using HACP concentrations of 1, 10 and 100 lg/mL. In this pilot study, eight healthy volunteers were randomly divided into two groups. Both the control and experimental groups received fractional photothermolysis treatment, but in the experimental group, four subjects received 3 g/day of oral collagen peptide (CP) for 4 weeks. To assess transepidermal water loss in each patient before and after the treatment, we used a Corneometer and a Cutometer, and we also assessed the patient’s Erythema Index. Results. The cell migration assay showed that HACP enhanced wound closure, but not in a dose-dependent manner. The collagen gel contraction assay showed increased contractility when patients were treated with 100 lg/mL HACP, but the results were not significantly different from those of controls. We found that post-laser erythema resolved faster in the experimental group than in the control group (P < 0.05). In addition, the recovery of skin hydration after fractional laser treatment was greater in the experimental group than in the control group by day 3 (P < 0.05), and the experimental group showed significantly improved post-treatment skin elasticity compared with the controls by day 14 (P < 0.05). Conclusions. Collagen tripeptide treatment appears to be an effective and conservative therapy for cutaneous wound healing and skin recovery after fractional photothermolysis treatment.
Correspondence: Dr Hyong Joo Lee, WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. The first two authors contributed equally, and should be considered joint first authors. Accepted for publication 11 January 2014
874
Clinical and Experimental Dermatology (2014) 39, pp874–880
Introduction In recent years, there has been increasing interest in the use of nutritional supplements to improve human skin. Researchers have explored peptides derived from protein hydrolysates as potential nutraceuticals and also for their use in the development of functional foods.1 One supplement of interest, collagen, is one of the major constituents of the extracellular matrix. Collagen is found in skin, bone, cartilage and tendons,
ª 2014 British Association of Dermatologists
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
supporting their structure and function. It is well known that reduction of collagen synthesis, which is associated with ageing, causes functional decline of the skin and internal organs.2 It was reported that, when administered as an oral supplement, collagen hydrolysate was adsorbed from the intestine in its high molecular weight peptide form.3 Orally administered collagen or CP provides beneficial effects on skin properties and skin ageing. A recent preclinical trial suggested that daily ingestion of CP improves the skin properties of women in the winter months.4 In addition, one pilot study revealed that daily collagen supplementation affects the physiological processes associated with ageing, and reduces its facial signs.5 However, the currently marketed collagen products are widely variable in their composition and molecular weight, because of differences in collagen sources and preparation methods, which can make their effects less predictable. The purpose of this study was to evaluate the effectiveness of high advanced-collagen tripeptide (HACP) on wound healing in vitro, and to assess its effects on skin recovery after fractional photothermolysis treatment in humans.
Methods Cell migration assay
HaCaT cells were grown to confluence in 12-well plates. The medium was then replaced with serum-free Dulbecco modified Eagle medium (DMEM). After 24 h, a yellow pipette tip was used to create a single linear scar in the monolayer. The cells were washed three times with phosphate-buffered saline (PBS), and fresh medium containing 2% fetal bovine serum (FBS) with or without HACP at concentrations of 1, 10 and 100 lg/mL was added to the appropriate plates. Photographs of the wounds were taken at 0, 24 and 48 h by microscopy, and the length of the migrated area was measured by the Pax-it image analysis program (Midwest Information Systems, Inc. Villa Park, IL, USA). Collagen gel contraction assay
Collagen gel contraction assays have been used as in vitro models for investigating the effects of various compounds used to enhance wound healing. We evaluated the effect of HACP on collagen gel contraction activity. Type I collagen solution (3.75 mg/mL) from rat tail (BD Biosciences, Bedford, MA, USA) was mixed with an equal volume of 100 mmol/L HEPES and 2 9 PBS.
ª 2014 British Association of Dermatologists
Serum-free DMEM containing 6 9 105 cells/mL was then added until the final concentration of collagen was 1.25 mg/mL. A 500 lL collagen/cell mixture was poured into a 12-well plate, which was placed in a cell incubator (37 °C, 5% CO2) for 30 min to solidify. After incubation, 500 lL of serum-free DMEM containing HACP was added and left for 48 h. The final concentrations of HACP were 1, 10 and 100 lg/mL. The gels were detached from the side of the dish with a pipette tip, and imaged at 0, 7, 24 and 30 h. The areas of the gel were calculated using the CellSans image analysis programme (Olympus, Tokyo, Japan). Pilot study
The institutional review board approved the clinical study protocol, and written informed consent was obtained from all subjects before treatment. Eight healthy Korean women (mean SD age 37.25 3.15 years, range 34–44) were enrolled in the study. The exclusion criteria included any cutaneous diseases, other systemic diseases, pregnancy or lactation, and any history of any ablative or non-ablative laser resurfacing within the 6 months preceding the study. Subjects were randomly assigned to one of two groups. Both the control and experimental groups received nonablative fractional photothermolysis laser treatments (Sellas, Dinona Inc., Seoul, Korea). The fractional laser treatments were delivered using a wavelength of 1550 nm, a density of 100 microthermal zones (mtz)/cm2 and a fluence of 13 mJ/cm2 over the entire face. In the experimental group, four subjects received 3 g/day of oral CP for 2 weeks before and 2 weeks after treatment. Commercially available CPs were used (Amorepacific Co., Yongin, Korea). The CP supplied in this study was a 15% tripeptide form of HACP (Jellice Co. Ltd., Sendai, Japan). The CP also contained 3% glycine–proline–hydroxyproline (Gly-ProHyp), with an average molecular weight of 1500 Da. The subjects visited our clinic 2 weeks before treatment and at 1, 3, 7 and 14 days after treatment. The subjects were instructed to use standard skin toners and moisturizers during the treatment period. During the pilot study, they did not use any sunscreens or regenerative creams. All skin property measurements were assessed on the subjects’ cheeks by a single, blinded investigator, and all measurements were performed in triplicate. Skin hydration was measured by a Corneometer, transepidermal water loss by a Tewameter, skin elasticity by a Tutometer, and Erythema Index (IE) by a Mexameter (all devices Courage+Khazaka Electronic
Clinical and Experimental Dermatology (2014) 39, pp874–880
875
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
GmbH, Cologne, Germany). At the end of the study, the participants documented their degree of satisfaction on a four-point scale (4 = very satisfied, 3 = satisfied, 2 = slightly satisfied, 1 = dissatisfied). Statistical analysis
All data were statistically analyzed by paired t-tests and Wilcoxon signed rank tests, using SPSS software (version 18.0 for Windows; SPSS Inc. Chicago, IL, USA). In all cases, differences were considered statistically significant at P < 0.05.
Results Figure 2 Effect of high advanced-collagen tripeptide (HACP) on
Cell migration assay
The cell migration assay showed that HACP supplementation enhanced wound closure. The migrated distance was significantly increased at 48 h with both the 10 and 100 lg/mL concentrations of HACP (Fig. 1). However, the effect of wound closure was not dose-dependent, with the 10 and 100 lg/mL concentrations showing comparative effects. Collagen gel contraction assay
Samples treated with 100 lg/mL HACP tended to show improved contractility at 24 and 30 h compared with the control samples. However, these differences in contractility between the two groups were not significant (Fig. 2).
Figure 1 Effect of high advanced-collagen tripeptide (HACP) on
HaCaT cell migration. (a) The migrated distances were then calculated with an image analysis program. The values shown are the mean SEM of three independent experiments. *Compared with the control group, P < 0.05.
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Clinical and Experimental Dermatology (2014) 39, pp874–880
collagen gel contraction by normal human dermal fibroblasts. Collagen gels were prepared as described in Methods. (a) The surface area of collagen gels was measured at 7, 24 and 30 h after detachment. Values are expressed as the mean percentage contraction relative to the noncontracted state from three independent experiments. Error bars represent the standard deviation.
Pilot study
All eight subjects completed the full course of treatment and follow-up. The average age of the subjects in the experimental and control group was not significantly different. Photographs of representative cases are shown in Fig. 3. Mean EI values increased immediately after treatment but returned to baseline by 1 week after treatment in both groups. However, the post-laser erythema resolved faster in the experimental group than in the control group, with the experimental group showing significantly greater improvement in erythema than the control group by day 3 (P < 0.05) (Fig. 4a). The mean TEWL values started to decrease on day 3 and returned to baseline by 1 week after the treatment in both groups. However, there were no significant differences between the two groups at any point (Fig. 4b). Mean Corneometer values decreased immediately after treatment, and returned to baseline levels within 1 week in both groups. Recovery of skin hydration after the fractional laser treatment was faster in the experimental group than the control group, with the experimental group showing significantly improved hydration measures compared with the control group by day 3 (P < 0.05) (Fig. 5a). Mean Cutometer values started to increase on day 1, but returned to baseline by day 3 in both
ª 2014 British Association of Dermatologists
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
(c)
(e)
(f)
(g)
(d)
(h)
Figure 3 Photographs of representative patients from (a–d) the control group and (e–h) the experimental group: (a,e) immediately after
full-face fractional laser treatment and at (b,f), 1, (c,g) 3 and (d,h) 7 days post-treatment.
groups. The difference in the improvement of posttreatment skin elasticity between the two groups reached statistical significance on day 14 (P < 0.05) (Fig. 5b), with the experimental group showing higher levels of skin elasticity than the control group. At the final visit, patients were asked to assess their treatment results on a four-point scale (very satisfied, satisfied, slightly satisfied, or dissatisfied). In the experimental group, 75% of the patients either satisfied or very satisfied, compared with 50% of patients in the control group. None of the patients experienced any serious facial side effects during or after the fractional laser treatment, and no adverse gastrointestinal effects attributable to the daily intake of CPs were reported during the study.
Discussion Collagen has an amino acid sequence of Gly-X-Y, and glycines in the amino acid sequence are regularly repeated. The most frequent tripeptide unit is GlyPro-Hyp, which contributes to the maximal stability of
ª 2014 British Association of Dermatologists
the collagen triple helix and to its bioactivity.6 The Gly-Pro-Hyp tripeptide is partially hydrolyzed on intestinal apical membranes, and the Pro-Hyp dipeptide, which is highly resistant to hydrolysis by intestinal proteases, is then absorbed in the intestine.7 Because of the oral bioavailability of collage, we used HACP in this study, which is a CP and is a soluble powder with a tripeptide unit, Gly-X-Y, which is easily absorbed by the intestine. In an in vitro model, we found that high concentrations of HACP enhanced wound closure and might enhance the wound healing process. In addition, we found HACP administration to be efficacious in skin recovery after photothermolysis laser treatments in humans. Generally, wound-healing agents have the ability to enhance the deposition of collagen, which provides strength to the tissues and forms cross-linkages between collagen fibres. The healing process depends to a large extent on the regulated biosynthesis and deposition of new collagens and their subsequent maturation. Collagen is produced by fibroblasts and helps the wound gain tensile strength during repair.8 It is likely that the oral intake of CPs might enhance
Clinical and Experimental Dermatology (2014) 39, pp874–880
877
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
Figure 4 (a) The erythema index was
assessed using a Mexameter. The differences in erythema improvement were statistically significant (P < 0.05) by day 3, with the collagen supplement group showing a faster improvement time than the control group. (b) Transepidermal water loss (TEWL) was assessed using a Tewameter, and was not significantly different between the two groups.
the wound-healing process by improving the deposition of collagen in the skin, as has been suggested by the results of several studies. For example, oral marine CPs enhanced cutaneous wound healing and angiogenesis in an animal model,9 while in one human study, daily collagen supplementation led to a significant reduction in facial ageing signs and a significant increase in the collagen content in the dermis.5 In the present study, we did not establish the exact molecular mechanism by which the HACP supplement affects collagen deposition and wound healing or the skin-recovery process. However, many previous studies have reported on some of the potential mechanisms of beneficial effects of CP on the skin both in vitro and in vivo. It was shown that CP has chemotactic activity to human skin fibroblasts,10 and CP ingestion induced significant increases in the diameter and density of col-
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Clinical and Experimental Dermatology (2014) 39, pp874–880
lagen fibrils, as well as the density of pig skin fibroblasts.11 Pro-Hyp stimulated the growth and migration of mouse skin fibroblasts,12 and in another murine study, CP ingestion increased expression of collagentypes I and IV and suppressed matrix metalloproteinase 2, which degrades type IV collagen.13 In addition to its possible effects on promoting collagen deposition in the skin, we suggest that oral CP supplementation enhances the skin recovery process after fractional laser treatment by increasing skin hydration and elasticity. For example, CP enhanced hyaluronic acid production in human dermal fibroblasts in vitro and in murine skin in vivo.14 A preclinical trial showed that daily ingestion of CPs significantly improved the moisture content and viscoelastic properties of skin.4 Many patients who receive fractional photothermolysis laser report post-laser dryness, erythema and oedema. Therefore, CP
ª 2014 British Association of Dermatologists
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
Figure 5 (a) Skin hydration was assessed
using a Corneometer. Recovery of skin hydration after fractional laser treatment was faster in the experimental group than in the control group, with the experimental group showing significantly (P < 0.05) improved skin hydration compared with the control group by posttreatment day 3. (b) Skin elasticity was assessed using a Cutometer. The differences in skin elasticity were statistically significant on day 14 (P < 0.05), with the collagen supplement group showing improved measures of skin elasticity compared with the control group.
supplements appear to be an effective and conservative therapy for skin recovery after nonablative 1550 nm fractional photothermolysis laser treatments. This study had several limitations. It was an exploratory study with a small sample size, and the length of HACP supplementation before fractional photothermolysis treatment was short.
What’s already known about this topic? ● In recent years, there has been increasing
interest in the use of nutritional supplements to improve human skin.
Conclusion HACP supplementation may be a beneficial option to improve cutaneous wound healing and skin recovery after laser treatments. However, more in vitro studies and controlled long-term human studies with large numbers of participants will be necessary to verify the effects of CPs and to better understand their mechanism of action.
ª 2014 British Association of Dermatologists
What does this study add? ● HACP supplementation may be a beneficial
option that improves cutaneous wound healing and skin recovery after laser treatment.
Clinical and Experimental Dermatology (2014) 39, pp874–880
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Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
References 1 Zague V. A new view concerning the effects of collagen hydrolysate intake on skin properties. Arch Dermatol Res 2008; 300: 479–83. 2 Miyahara T, Murai A, Tanaka T et al. Age-related differences in human skin collagen: solubility in solvent, susceptibility to pepsin digestion, and the spectrum of the solubilized polymeric collagen molecules. J Gerontol 1982; 37: 651–5. 3 Oesser S, Adam M, Babel W et al. Oral administration of (14)C labeled gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/ BL). J Nutr 1999; 129: 1891–5. 4 Matsumoto H, Ohara H, Ito K et al. Clinical effects of fish type I collagen hydrolysate on skin properties. ITE Lett 2006; 7: 386–90. 5 Schwartz SR, Park J. Ingestion of BioCell Collagen, a novel hydrolyzed chicken sternal cartilage extract; enhanced blood microcirculation and reduced facial aging signs. Clin Interv Aging 2012; 7: 267–73. 6 Berg RA, Prockop DJ. The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem Biophys Res Commun 1973; 52: 115–20. 7 Aito-Inoue M, Lackeyram D, Fan MZ et al. Transport of a tripeptide, Gly-Pro-Hyp, across the porcine intestinal brush-border membrane. J Pept Sci 2007; 13: 468–74.
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8 de F atima A, Modolo LV, Sanches AC et al. Wound healing agents: the role of natural and non-natural products in drug development. Mini Rev Med Chem 2008; 8: 879–88. 9 Zhang Z, Wang J, Ding Y et al. Oral administration of marine collagen peptides from Chum Salmon skin enhances cutaneous wound healing and angiogenesis in rats. J Sci Food Agric 2011; 91: 2173–9. 10 Postlethwaite AE, Seyer JM et al. Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci USA 1978; 75: 871–5. 11 Matsuda N, Koyama Y, Hosaka Y et al. Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis. J Nutr Sci Vitaminol 2006; 52: 211–15. 12 Shigemura Y, Iwai K, Morimatsu F et al. Effect of prolyl-hydroxyproline (Pro-Hyp), a food-derived collagen peptide in human blood, on growth of fibroblasts from mouse skin. J Agric Food Chem 2009; 57: 444–9. 13 Zague V, de Freitas V, da Costa Rosa M et al. Collagen hydrolysate intake increases skin collagen expression and suppresses matrix metalloproteinase 2 activity. J Med Food 2011; 14: 618–24. 14 Okawa T, Yamaguchi Y, Takada S et al. Oral administration of collagen tripeptide improves dryness and pruritus in the acetone-induced dry skin model. J Dermatol Sci 2012; 66: 136–43.
ª 2014 British Association of Dermatologists
Clinical dermatology • Original article
Clinical and Experimental Dermatology
Effect of high advanced-collagen tripeptide on wound healing and skin recovery after fractional photothermolysis treatment S. Y. Choi,1 W. G. Kim,2 E. J. Ko,1 Y. H. Lee,1 B. G. Kim,3 H. J. Shin,3 Y. S. Choi,4 J. Y. Ahn,5 B. J. Kim1 and H. J. Lee2 1
Department of Dermatology, Chung-Ang University College of Medicine, Seoul, South Korea; 2Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea; 3Health Science Research Institute, Amorepacific Corporation Research and Development Center, Gyeonggi-do, South Korea; 4Department of Dermatology, Ulsan University Hospital, Ulsan, South Korea; and 5Department of Dermatology, National Medical Center, Seoul, South Korea doi:10.1111/ced.12405
Summary
Background. Collagens have long been used in pharmaceuticals and food supplements for the improvement of skin. Aim. We evaluated the efficacy of high advanced-collagen tripeptide (HACP) on wound healing and skin recovery. Methods. Using an in vitro model, we performed HaCaT cell migration assays and collagen gel contraction assays using HACP concentrations of 1, 10 and 100 lg/mL. In this pilot study, eight healthy volunteers were randomly divided into two groups. Both the control and experimental groups received fractional photothermolysis treatment, but in the experimental group, four subjects received 3 g/day of oral collagen peptide (CP) for 4 weeks. To assess transepidermal water loss in each patient before and after the treatment, we used a Corneometer and a Cutometer, and we also assessed the patient’s Erythema Index. Results. The cell migration assay showed that HACP enhanced wound closure, but not in a dose-dependent manner. The collagen gel contraction assay showed increased contractility when patients were treated with 100 lg/mL HACP, but the results were not significantly different from those of controls. We found that post-laser erythema resolved faster in the experimental group than in the control group (P < 0.05). In addition, the recovery of skin hydration after fractional laser treatment was greater in the experimental group than in the control group by day 3 (P < 0.05), and the experimental group showed significantly improved post-treatment skin elasticity compared with the controls by day 14 (P < 0.05). Conclusions. Collagen tripeptide treatment appears to be an effective and conservative therapy for cutaneous wound healing and skin recovery after fractional photothermolysis treatment.
Correspondence: Dr Hyong Joo Lee, WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. The first two authors contributed equally, and should be considered joint first authors. Accepted for publication 11 January 2014
874
Clinical and Experimental Dermatology (2014) 39, pp874–880
Introduction In recent years, there has been increasing interest in the use of nutritional supplements to improve human skin. Researchers have explored peptides derived from protein hydrolysates as potential nutraceuticals and also for their use in the development of functional foods.1 One supplement of interest, collagen, is one of the major constituents of the extracellular matrix. Collagen is found in skin, bone, cartilage and tendons,
ª 2014 British Association of Dermatologists
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
supporting their structure and function. It is well known that reduction of collagen synthesis, which is associated with ageing, causes functional decline of the skin and internal organs.2 It was reported that, when administered as an oral supplement, collagen hydrolysate was adsorbed from the intestine in its high molecular weight peptide form.3 Orally administered collagen or CP provides beneficial effects on skin properties and skin ageing. A recent preclinical trial suggested that daily ingestion of CP improves the skin properties of women in the winter months.4 In addition, one pilot study revealed that daily collagen supplementation affects the physiological processes associated with ageing, and reduces its facial signs.5 However, the currently marketed collagen products are widely variable in their composition and molecular weight, because of differences in collagen sources and preparation methods, which can make their effects less predictable. The purpose of this study was to evaluate the effectiveness of high advanced-collagen tripeptide (HACP) on wound healing in vitro, and to assess its effects on skin recovery after fractional photothermolysis treatment in humans.
Methods Cell migration assay
HaCaT cells were grown to confluence in 12-well plates. The medium was then replaced with serum-free Dulbecco modified Eagle medium (DMEM). After 24 h, a yellow pipette tip was used to create a single linear scar in the monolayer. The cells were washed three times with phosphate-buffered saline (PBS), and fresh medium containing 2% fetal bovine serum (FBS) with or without HACP at concentrations of 1, 10 and 100 lg/mL was added to the appropriate plates. Photographs of the wounds were taken at 0, 24 and 48 h by microscopy, and the length of the migrated area was measured by the Pax-it image analysis program (Midwest Information Systems, Inc. Villa Park, IL, USA). Collagen gel contraction assay
Collagen gel contraction assays have been used as in vitro models for investigating the effects of various compounds used to enhance wound healing. We evaluated the effect of HACP on collagen gel contraction activity. Type I collagen solution (3.75 mg/mL) from rat tail (BD Biosciences, Bedford, MA, USA) was mixed with an equal volume of 100 mmol/L HEPES and 2 9 PBS.
ª 2014 British Association of Dermatologists
Serum-free DMEM containing 6 9 105 cells/mL was then added until the final concentration of collagen was 1.25 mg/mL. A 500 lL collagen/cell mixture was poured into a 12-well plate, which was placed in a cell incubator (37 °C, 5% CO2) for 30 min to solidify. After incubation, 500 lL of serum-free DMEM containing HACP was added and left for 48 h. The final concentrations of HACP were 1, 10 and 100 lg/mL. The gels were detached from the side of the dish with a pipette tip, and imaged at 0, 7, 24 and 30 h. The areas of the gel were calculated using the CellSans image analysis programme (Olympus, Tokyo, Japan). Pilot study
The institutional review board approved the clinical study protocol, and written informed consent was obtained from all subjects before treatment. Eight healthy Korean women (mean SD age 37.25 3.15 years, range 34–44) were enrolled in the study. The exclusion criteria included any cutaneous diseases, other systemic diseases, pregnancy or lactation, and any history of any ablative or non-ablative laser resurfacing within the 6 months preceding the study. Subjects were randomly assigned to one of two groups. Both the control and experimental groups received nonablative fractional photothermolysis laser treatments (Sellas, Dinona Inc., Seoul, Korea). The fractional laser treatments were delivered using a wavelength of 1550 nm, a density of 100 microthermal zones (mtz)/cm2 and a fluence of 13 mJ/cm2 over the entire face. In the experimental group, four subjects received 3 g/day of oral CP for 2 weeks before and 2 weeks after treatment. Commercially available CPs were used (Amorepacific Co., Yongin, Korea). The CP supplied in this study was a 15% tripeptide form of HACP (Jellice Co. Ltd., Sendai, Japan). The CP also contained 3% glycine–proline–hydroxyproline (Gly-ProHyp), with an average molecular weight of 1500 Da. The subjects visited our clinic 2 weeks before treatment and at 1, 3, 7 and 14 days after treatment. The subjects were instructed to use standard skin toners and moisturizers during the treatment period. During the pilot study, they did not use any sunscreens or regenerative creams. All skin property measurements were assessed on the subjects’ cheeks by a single, blinded investigator, and all measurements were performed in triplicate. Skin hydration was measured by a Corneometer, transepidermal water loss by a Tewameter, skin elasticity by a Tutometer, and Erythema Index (IE) by a Mexameter (all devices Courage+Khazaka Electronic
Clinical and Experimental Dermatology (2014) 39, pp874–880
875
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
GmbH, Cologne, Germany). At the end of the study, the participants documented their degree of satisfaction on a four-point scale (4 = very satisfied, 3 = satisfied, 2 = slightly satisfied, 1 = dissatisfied). Statistical analysis
All data were statistically analyzed by paired t-tests and Wilcoxon signed rank tests, using SPSS software (version 18.0 for Windows; SPSS Inc. Chicago, IL, USA). In all cases, differences were considered statistically significant at P < 0.05.
Results Figure 2 Effect of high advanced-collagen tripeptide (HACP) on
Cell migration assay
The cell migration assay showed that HACP supplementation enhanced wound closure. The migrated distance was significantly increased at 48 h with both the 10 and 100 lg/mL concentrations of HACP (Fig. 1). However, the effect of wound closure was not dose-dependent, with the 10 and 100 lg/mL concentrations showing comparative effects. Collagen gel contraction assay
Samples treated with 100 lg/mL HACP tended to show improved contractility at 24 and 30 h compared with the control samples. However, these differences in contractility between the two groups were not significant (Fig. 2).
Figure 1 Effect of high advanced-collagen tripeptide (HACP) on
HaCaT cell migration. (a) The migrated distances were then calculated with an image analysis program. The values shown are the mean SEM of three independent experiments. *Compared with the control group, P < 0.05.
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Clinical and Experimental Dermatology (2014) 39, pp874–880
collagen gel contraction by normal human dermal fibroblasts. Collagen gels were prepared as described in Methods. (a) The surface area of collagen gels was measured at 7, 24 and 30 h after detachment. Values are expressed as the mean percentage contraction relative to the noncontracted state from three independent experiments. Error bars represent the standard deviation.
Pilot study
All eight subjects completed the full course of treatment and follow-up. The average age of the subjects in the experimental and control group was not significantly different. Photographs of representative cases are shown in Fig. 3. Mean EI values increased immediately after treatment but returned to baseline by 1 week after treatment in both groups. However, the post-laser erythema resolved faster in the experimental group than in the control group, with the experimental group showing significantly greater improvement in erythema than the control group by day 3 (P < 0.05) (Fig. 4a). The mean TEWL values started to decrease on day 3 and returned to baseline by 1 week after the treatment in both groups. However, there were no significant differences between the two groups at any point (Fig. 4b). Mean Corneometer values decreased immediately after treatment, and returned to baseline levels within 1 week in both groups. Recovery of skin hydration after the fractional laser treatment was faster in the experimental group than the control group, with the experimental group showing significantly improved hydration measures compared with the control group by day 3 (P < 0.05) (Fig. 5a). Mean Cutometer values started to increase on day 1, but returned to baseline by day 3 in both
ª 2014 British Association of Dermatologists
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
(c)
(e)
(f)
(g)
(d)
(h)
Figure 3 Photographs of representative patients from (a–d) the control group and (e–h) the experimental group: (a,e) immediately after
full-face fractional laser treatment and at (b,f), 1, (c,g) 3 and (d,h) 7 days post-treatment.
groups. The difference in the improvement of posttreatment skin elasticity between the two groups reached statistical significance on day 14 (P < 0.05) (Fig. 5b), with the experimental group showing higher levels of skin elasticity than the control group. At the final visit, patients were asked to assess their treatment results on a four-point scale (very satisfied, satisfied, slightly satisfied, or dissatisfied). In the experimental group, 75% of the patients either satisfied or very satisfied, compared with 50% of patients in the control group. None of the patients experienced any serious facial side effects during or after the fractional laser treatment, and no adverse gastrointestinal effects attributable to the daily intake of CPs were reported during the study.
Discussion Collagen has an amino acid sequence of Gly-X-Y, and glycines in the amino acid sequence are regularly repeated. The most frequent tripeptide unit is GlyPro-Hyp, which contributes to the maximal stability of
ª 2014 British Association of Dermatologists
the collagen triple helix and to its bioactivity.6 The Gly-Pro-Hyp tripeptide is partially hydrolyzed on intestinal apical membranes, and the Pro-Hyp dipeptide, which is highly resistant to hydrolysis by intestinal proteases, is then absorbed in the intestine.7 Because of the oral bioavailability of collage, we used HACP in this study, which is a CP and is a soluble powder with a tripeptide unit, Gly-X-Y, which is easily absorbed by the intestine. In an in vitro model, we found that high concentrations of HACP enhanced wound closure and might enhance the wound healing process. In addition, we found HACP administration to be efficacious in skin recovery after photothermolysis laser treatments in humans. Generally, wound-healing agents have the ability to enhance the deposition of collagen, which provides strength to the tissues and forms cross-linkages between collagen fibres. The healing process depends to a large extent on the regulated biosynthesis and deposition of new collagens and their subsequent maturation. Collagen is produced by fibroblasts and helps the wound gain tensile strength during repair.8 It is likely that the oral intake of CPs might enhance
Clinical and Experimental Dermatology (2014) 39, pp874–880
877
Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
Figure 4 (a) The erythema index was
assessed using a Mexameter. The differences in erythema improvement were statistically significant (P < 0.05) by day 3, with the collagen supplement group showing a faster improvement time than the control group. (b) Transepidermal water loss (TEWL) was assessed using a Tewameter, and was not significantly different between the two groups.
the wound-healing process by improving the deposition of collagen in the skin, as has been suggested by the results of several studies. For example, oral marine CPs enhanced cutaneous wound healing and angiogenesis in an animal model,9 while in one human study, daily collagen supplementation led to a significant reduction in facial ageing signs and a significant increase in the collagen content in the dermis.5 In the present study, we did not establish the exact molecular mechanism by which the HACP supplement affects collagen deposition and wound healing or the skin-recovery process. However, many previous studies have reported on some of the potential mechanisms of beneficial effects of CP on the skin both in vitro and in vivo. It was shown that CP has chemotactic activity to human skin fibroblasts,10 and CP ingestion induced significant increases in the diameter and density of col-
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lagen fibrils, as well as the density of pig skin fibroblasts.11 Pro-Hyp stimulated the growth and migration of mouse skin fibroblasts,12 and in another murine study, CP ingestion increased expression of collagentypes I and IV and suppressed matrix metalloproteinase 2, which degrades type IV collagen.13 In addition to its possible effects on promoting collagen deposition in the skin, we suggest that oral CP supplementation enhances the skin recovery process after fractional laser treatment by increasing skin hydration and elasticity. For example, CP enhanced hyaluronic acid production in human dermal fibroblasts in vitro and in murine skin in vivo.14 A preclinical trial showed that daily ingestion of CPs significantly improved the moisture content and viscoelastic properties of skin.4 Many patients who receive fractional photothermolysis laser report post-laser dryness, erythema and oedema. Therefore, CP
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Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
(a)
(b)
Figure 5 (a) Skin hydration was assessed
using a Corneometer. Recovery of skin hydration after fractional laser treatment was faster in the experimental group than in the control group, with the experimental group showing significantly (P < 0.05) improved skin hydration compared with the control group by posttreatment day 3. (b) Skin elasticity was assessed using a Cutometer. The differences in skin elasticity were statistically significant on day 14 (P < 0.05), with the collagen supplement group showing improved measures of skin elasticity compared with the control group.
supplements appear to be an effective and conservative therapy for skin recovery after nonablative 1550 nm fractional photothermolysis laser treatments. This study had several limitations. It was an exploratory study with a small sample size, and the length of HACP supplementation before fractional photothermolysis treatment was short.
What’s already known about this topic? ● In recent years, there has been increasing
interest in the use of nutritional supplements to improve human skin.
Conclusion HACP supplementation may be a beneficial option to improve cutaneous wound healing and skin recovery after laser treatments. However, more in vitro studies and controlled long-term human studies with large numbers of participants will be necessary to verify the effects of CPs and to better understand their mechanism of action.
ª 2014 British Association of Dermatologists
What does this study add? ● HACP supplementation may be a beneficial
option that improves cutaneous wound healing and skin recovery after laser treatment.
Clinical and Experimental Dermatology (2014) 39, pp874–880
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Effect of collagen peptide on wound healing and skin recovery S. Y. Choi et al.
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8 de F atima A, Modolo LV, Sanches AC et al. Wound healing agents: the role of natural and non-natural products in drug development. Mini Rev Med Chem 2008; 8: 879–88. 9 Zhang Z, Wang J, Ding Y et al. Oral administration of marine collagen peptides from Chum Salmon skin enhances cutaneous wound healing and angiogenesis in rats. J Sci Food Agric 2011; 91: 2173–9. 10 Postlethwaite AE, Seyer JM et al. Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci USA 1978; 75: 871–5. 11 Matsuda N, Koyama Y, Hosaka Y et al. Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis. J Nutr Sci Vitaminol 2006; 52: 211–15. 12 Shigemura Y, Iwai K, Morimatsu F et al. Effect of prolyl-hydroxyproline (Pro-Hyp), a food-derived collagen peptide in human blood, on growth of fibroblasts from mouse skin. J Agric Food Chem 2009; 57: 444–9. 13 Zague V, de Freitas V, da Costa Rosa M et al. Collagen hydrolysate intake increases skin collagen expression and suppresses matrix metalloproteinase 2 activity. J Med Food 2011; 14: 618–24. 14 Okawa T, Yamaguchi Y, Takada S et al. Oral administration of collagen tripeptide improves dryness and pruritus in the acetone-induced dry skin model. J Dermatol Sci 2012; 66: 136–43.
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