Authors: Mathias C. Aust, M.D. Kerstin Reimers, Ph.D. Claudia Repenning, Ph.D. Frank Stahl, Ph.D. Sabrina Jahn, B.A. Merlin Guggenheim, M.D. Nina Schwaiger, M.D. Andreas Gohritz, M.D. Peter M. Vogt, M.D.
Hannover, Germany; Zurich, Switzerland; and Graz, Austria
Background: Photoaging is generally treated by ablative procedures that injure the epidermis and basal membrane and lead to fibrosis of the papillary dermis. Damaging the epidermis significantly can cause potential adverse effects such as dyspigmentation. It was recently shown in clinical trials that percutaneous collagen induction therapy is an alternative for safely treating wrinkles and scars and for smoothening the skin without the risk of dyspigmentation.
Methods: The purpose of this study was to increase current knowledge regarding whether percutaneous collagen induction therapy presents an effective means for skin rejuvenation without risk of dyspigmentation, as the authors’ clinical data suggested. Fifty-six rats were assigned to three groups: group A (n 24), percutaneous collagen induction therapy plus skin care; Group B (n 24), skin care; and Group C (n 8) controls. The authors evaluated the effect of percutaneous collagen induction therapy on the epidermis, melanocytes, and the pigmentation markers interleukin-10 and melanocyte-stimulating hormone.
Results: Percutaneous collagen induction therapy left the epidermis intact without any damage to the stratum corneum, any other layers of the epidermis, or the basal membrane. No signs of dermabrasive reduction of epidermal thickness were evident 24 hours after the procedure. The number of melanocytes neither increased nor decreased in any of the groups. DNA microarray experiments demonstrated that interleukin-10 was increased in percutaneous collagen induction therapy–treated skin after 2 weeks. Concerning the MC1R (melanocyte-stimulating hormone) gene, gene expression microarray analysis indicated a faint down-regulation both 24 hours and 2 weeks after percutaneous collagen induction therapy.
Conclusion: Percutaneous collagen induction therapy offers a modality with which to rejuvenate and improve skin appearance and quality without risk of dyspigmentation. (Plast. Reconstr. Surg. 122: 1553, 2008.)
Percutaneous Collagen Induction: Minimally Invasive Skin Rejuvenation without Risk of Hyperpigmentation—Fact or Fiction?
Ablative laser resurfacing is generally considered-
From the Klinik fu ̈r Plastische, Hand- und Wiederherstel- lungschirurgie, Medizinische Hochschule Hannover, the De- partment of Technical Chemistry, University of Hannover, the Klinik fu ̈r Wiederherstellungschirurgie, Universita ̈tsspi- tal Zurich, and the Klinische Abteilung fu ̈r Plastische, Re- konstruktive und A ̈sthetische Chirurgie, Universita ̈tsklinik Graz.
Received for publication September 9, 2007; accepted May 9, 2008.
Copyright ©2008 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0b013e318188245e
This change can be associated with potentially
adverse effects, which include erythema, scarring,
2–4 and pigmentation problems.
Any type of trauma can lead to postinflammatory skin hyperpigmentation. Investigators have theorized that some people have an inherited tendency for a weak melanocyte response to inflammation, resulting in decreased melanin production, or for a strong response, characterized by increasing mela–
Disclosures: Matthias Aust, M.D., is a Medical Consultant for Care Concept, Distributors for Envi- ron Skin Care Products and Roll-CitR in Germany. None of the other authors has any sources of funds supporting the work or any financial interest.
www.PRSJournal.com 1553
in production.5 Evidence that a paracrine melanogenic cytokine network exists between melanocytes and other skin cells, including keratinocytes and fibroblasts, which regulate melanocyte function, has been discovered.6 –10 This network plays a key role in hyper-pigmentation and hyperpigmentary disorders.11–13
The ideal treatment for skin rejuvenation would be to preserve the epidermis and promote normal collagen formation in the dermis, thereby decreasing the risk of dyspigmentation. More recently, the authors introduced percutaneous collagen induction therapy, a new concept of cutaneous remodeling designed to approach this ideal objective: Dermal pricking allows reepithelialization within less than 24 hours, with only minimal loss of epidermal barrier function. Clinical long-term results suggest that complications, including postinflammatory hyperpigmentation, can be minimized in patients with all skin types.14 Recent studies have explored the changes in essential promoters of melanin formation in pigmentation disorders, such as interleukin-10, adrenocorticotropic hormone, and melanocyte-stimulating hormone. Nevertheless, no comparative studies have been performed on the effect of percutaneous collagen induction therapy on melanocytes and pigmentation markers.
MATERIALS AND METHODS
Percutaneous Collagen Induction
Percutaneous collagen induction therapy has been well described previously. In brief, it originated from the combined ideas of Orentreich and Orentreich,15 Fernandes,16 and Camirand and Doucet17 and involves pricking the skin and inducing new connective tissue formation. Percutaneous collagen induction therapy stimulates the natural posttraumatic inflammatory cascade by rolling a specially designed device vertically, horizontally, and diagonally16 (Fig. 1) over a skin area to create thousands of closely neighboring micro-wounds in the dermis that results in a confluent zone of very superficial inflammation, triggering the release of growth factors that ultimately results in increasing the patient’s own normal woven collagen.
Animals and Experimental Groups
Fifty-six male Sprague-Dawley rats (350 to 375 g), aged 4 months, were assigned randomly to three groups as follows (Table 1): group A (n 24), needling plus skin care (Fig. 2, above); group B (n 24), skin care only (Fig. 2, below); and group C (n 8), controls.
Fig.1. (Above)MedicalRoll-Cit(made by VividaC.C.Renaissance Body Science Institute, Cape Town, South Africa). (Below) Schematic image of the procedure. (Adapted from Aust, M. C., Fernandes, D., Kolokythas, P., Kaplan, H. M., and Vogt, P. M. Collagen induction therapy: An alternative treatment for scars, wrinkles and skin laxity. Plast. Reconstr. Surg. 121: 1421, 2008.)
Each rat in group A received a 30 percent total body surface area skin needling under general anesthesia and analgesia [Rompun (Bayer Health Care, Leverkusen, Germany), 0.3 ml/kg body weight; Ketanest (Park Davies GmbH, Karlsruhe, Germany), 1.1 ml/kg body weight]. Rats were anesthetized and shaved and received a 30 percent total body surface area scald needling (10 minutes) to induce percutaneous collagen, using a medical needling instrument (Environ Medical Roll-CIT; Vivida SA cc, Cape Town, South Africa).
Application of Vitamins A and C
After the needling, the rats in group A and the unoperated animals in group B were treated im- mediately with high levels of vitamin A cream [reti- nyl palmitate (Environ Original; Environ Skin Care (Pty) Ltd, Cape Town, South Africa)] and vitamin C cream [ascorbyl tetra-isopalmitate (En- viron C-Boost; Environ Skin Care)]. Both vitamin
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Volume 122, Number 5 • Percutaneous Collagen Induction Table1. ExperimentalGroups
Group (Biopsy)
A (needling and skin care) B (skin care)
C (controls)
Set I (24 hr)
6 6 2
Set II (2 wk)
Set III (4 wk)
6 6 2
Set IV (8 wk)
Total
6 6 2
6 24 6 24 2 8
Fig.2. Photographsofaratwithashavedback.(Above)Preoperative, untreated skin. (Below) Photograph obtained 1 hour postoperatively. Intradermal bleeding and bruising is visible.
creams were thereafter applied topically after cleaning once per day. This was performed to both maximize initial collagen production and maintain the homeostasis between collagenesis and collagenolysis at the new level induced by the procedure.
The control group (group C) rats received no injury, no skin care, no treatment, no anesthesia, and no analgesia and were killed at the same time points as the treated animals of groups A and B. These rats served as unneedled, untreated, time-matched sham rats to establish baseline levels for the present study. All animals in each group were killed in four sets (24 hours, 2 weeks, 4 weeks, and
8 weeks) after needling by an overdose of anesthesia. Biopsy specimens of their skin were collected and stored either at –73°C or in formalin for analysis as described below.
Histologic Examination
The skin biopsy specimens were fixed in 4% buffered formalin, dehydrated in ethanol, embedded in paraffin, and cut into 5-m sections on a Microm MH510 cryostat rotary microtome (Microm, Walldorf, Germany). The sections were deparaffinized and treated with descending ethanol. Afterward, the slices were stained with hematoxylin and eosin (Merck, Darmstadt, Germany) or Mas- son’s trichrome (Merck) or subjected to in situ hybridization and examined with an Olympus micro-scope (Hamburg, Germany).
Immunofluorescence
For immunofluorescence microscopy, sections were fixed in 4% paraformaldehyde/phos- phate-buffered saline at room temperature for 20 minutes. Each specimen was washed three times in phosphate-buffered saline (pH 7.3), and then 0.2% Triton-X-100/phosphate-buffered saline was applied for 10 minutes, after which the specimens were washed again for 5 minutes, three times, in phosphate-buffered saline. Then, 2% fetal calf serum in phosphate-buffered saline was applied for 40 minutes at room temperature for blocking. Primary antibody was applied for 60 minutes at 37°C in a humidified atmosphere. The primary antibodies against rat interleukin-10, melanocyte- stimulating hormone, and S100 was used at 1:100 dilution in phosphate-buffered saline plus 1% fetal calf serum (Sigma, St. Louis, Mo.). The secondary antibody was fluorescein isothiocyanate– conjugated or rhodamine-conjugated goat anti-rabbit Alexa antibody and was used at a 1:200 dilution in phosphate-buffered saline plus 1% fetal calf serum. The samples were washed as described above and mounted in Vectashield (Vector Laboratories, Inc., Burlingame, Calif.) supplemented with 4’,6- diamidino-2-phenylindole. Specimens were viewed on a Zeiss microscope (Go ̈ttingen, Germany) fitted with epifluorescence optics.
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In Situ Hybridization
Sections were fixed in 2.5% glutaraldehyde/ phosphate-buffered saline (Merck) for 30 minutes. The sections were washed with 4.5% sucrose/ phosphate-buffered saline for 1 hour with four changes of the incubation solution. Then, the sections were treated with 0.3% Triton-X-100/phos- phate-buffered saline for 15 minutes, followed by proteinase K (5 g/ml phosphate-buffered saline; Sigma-Aldrich, Taufkirchen, Germany) for 15 minutes, a postfixation step with 4% p-formalde- hyde/phosphate-buffered saline (Sigma) for 5 minutes, and 0.25% acetic anhydride in 0.1 M triethanolamine (Sigma) for 10 minutes. Between each step, the samples were washed with phos- phate-buffered saline for 5 minutes. After incubation in 50% formamide in 2 saline sodium citrate (Sigma) for 20 minutes, the samples were hybridized with a bio-tinylated gene fragment of 421 base pairs from interleukin-10 at 37°C over-night. The biotinylation reaction was carried out with Psoralen-Biotin from Ambion, Inc. (Austin, Texas). After hybridization, the sections were washed with 4 saline sodium citrate three times for 15 minutes and blocked with 1% bovine serum albumin (Sigma) and 10% normal horse serum (Biochrom, Berlin, Germany) in Tris-buffered saline. Biotin was detected by incubation with mouse monoclonal anti-biotin (1:30 in Tris-buffered saline; DAKO, Glostrup, Denmark) for 30 minutes, followed by bio-tinylated horse anti-mouse antibody (1:100 in Tris-buffered saline; Vector Laboratories) for 10 minutes, and a second layer with anti-biotin followed by anti-mouse antibody, each for 10 minutes at a dilution of 1:100 in Tris-buffered saline. The samples were treated with strepta- vidin-peroxidase (1:10; DAKO) for 5 minutes. Between incubation steps, the sections were washed with Tris-buffered saline. The peroxidase activity was detected with HistoGreen (Vector). All sections were examined under an inverse light microscope (Olympus).
Microarray Data Analysis
The scanning process of the hybridized chips included a six-fold scan of each chip at different settings, altering both percutaneous collagen induction therapy and laser power settings. For this last experimental step, the Axon 4000B scanner (Axon Instruments, Inc., Hawthorn East, Victoria, Australia) was used. The following primary analysis served as a quantification method and was performed with the Gene Pix Pro 6.0 (Axon) software tool.
The secondary analysis was conducted subsequently using the data from the primary analysis. Therefore, data from different scans was first normalized by using the median of all background intensities, which were therefore checked for outliers.
Nine replicates of each gene were tested for outliers. Outliers among the gene replicates were eliminated according to the outlier test by Nali-mov. A t test was applied to detect differences in gene expression between the sample groups. The independent t test was used to determine the statistical significance of the differences between the staining patterns and of the values for histologic measurements in treated and untreated skin. All p values were two-tailed, and differences were considered significant for values of p 0.05. Summary data are expressed as mean and standard error of the mean. This study was reviewed and approved by the Lower Saxony District Government (Hannover, Germany), ensuring that all animals received humane care.
RESULTS
Epidermal and Dermal Effects
The epidermal changes were observed by hematoxylin and eosin staining. Percutaneous collagen induction therapy left the epidermis intact without any indication of damage to the stratum corneum, any other layers of the epidermis, or its basal membrane. In particular, no signs of dermabrasive reduction of epidermal thickness were evident 24 hours after percutaneous collagen induction therapy (Fig. 3). The rete ridge pattern was as obvious in all the needled specimens as in the unneedled ones and the control group. No changes were observed in the elastin and collagen fibers by using Masson’s trichrome staining histologies (Fig. 4).
Gene Expression Analysis
DNA microarray experiments were performed to evaluate whether the genes coding for inter- leukin-10 and MC1R are regulated in treated (needling plus skin care) rat skin 2, 4, and 8 weeks after surgery. The results demonstrate that interleu- kin-10 is two times up-regulated in treated skin in comparison with the control samples after 2 weeks. After 4 and 8 weeks, there is no significant up-regulation detectable. Concerning the MC1R gene, t test calculation of the gene expression microarray analysis indicated a faint down-regulation 2 weeks after needling that increased at the later time points (e.g., 3.5 times after 8 weeks). The entire data set is represented on the Platform
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Fig. 3. Microphotographs taken of skin samples stained with hematoxylin and eosin. (Above) Control group: regular and intact epidermis. (Below) Microphotograph obtained 24 hours after percutaneous collagen induction therapy showing intact epidermis without any indication of damage to the stratum corneum, any other layers of the epidermis, or its basal membrane. There are no signs of dermabrasive reduction of epidermal thickness. Scale bar 100 m (representative example).
ID GPL5462 in the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database (Fig. 5 and Table 2).
In Situ Hybridization
Interleukin-10 gene expression in treated and unneedled skin 2 weeks after surgery was confirmed by in situ hybridization using a biotinylated probe directed against interleukin-10. In both skin types, a blue color was observed, indicating the presence of interleukin-10 mRNA, whereas the controls remained without blue (Fig. 6). Although the sections are somewhat difficult to compare directly concerning the amount of interleukin-10 because of the higher cellular density in needled skin, the number of interleukin-10 – expressing.
Fig. 4. Masson’s trichrome staining. (Above) Control group: regular and intact dermis. (Below) Microphotograph obtained 24 hours after percutaneous collagen induction therapy. No changes were observed in the elastin and collagen fibers after needling. Scale bar 50 m (representative example).
cells seems to be increased and more evenly distributed (Fig. 6, below, left).
Immunohistochemistry (Interleukin-10)
We stained samples with antibodies directed against interleukin-10. Green fluorescence indicated the presence of the interleukin-10 protein. The rate of synthesis of this anti-inflammatory cy- tokine was analyzed 2 weeks after the operation both in skin samples from treated animals and in control samples from unneedled animals (Fig. 7). Interleukin-10 was present throughout the entire skin samples of the treated and the untreated groups. However, interleukin-10 synthesis was increased in all needled groups compared with their controls (Fig. 7, left). The higher gene expression rates illustrated above was thus reflected by an equal increase in protein synthesis.
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Fig.5. Selectedgenesofneedledratsagainstuntreatedcontrolrats.Shownareratiosoftheexpression values of different time points after needling against untreated rats. Ratios between 0 and 1 have been transformed to negative values for a better comparison. Standard errors are marked by error bars. Please see significant regulation (calculated by t test) in Table 2.
Table2. RegulationofNeedledRatsComparedwithControlAnimalsatDifferentTimePoints*
2 Weeks 4 Weeks 8 Weeks
Gene Regulation Ratio SEM p Value Regulation Ratio SEM p Value Regulation Ratio SEM p Value
IL-10 1 2.13 0.3 0.00125 0 1.57 0.27 0.01895 0 1.13 0.62 0.32191 MC1R 1 1.27 0.07 0.00065 1 1.75 0.1 0.00001 1 3.41 0.1 0.00001
IL, interleukin.
*The regulation has been calculated with the t test (p 0.05): 0 means unregulated when comparing the corresponding time point to untreated control rats; 1 means that the gene is regulated. The ratio gives the amount of regulation. The last column shows the standard error. If genes could not be detected because they were not expressed, values are given with zeros.
Melanocyte Staining (Immunofluorescence)
To determine the distribution of melanocytes in the needled samples and the controls, we stained them with antibodies directed against S100, which is specific for melanocytes in the mammalian skin, followed by secondary antibody treatment conjugated to Alexa564. Skin samples from treated animals and control samples from unneedled animals were analyzed 8 weeks after the operation to evaluate the influence of percutaneous collagen induction therapy on melanocytes. Melanocytes in the skin of the treated and the untreated groups were found to be present throughout the entire epidermis. However, no increase of their number in any of the needled groups when compared with their controls (Fig. 8) was detected.
DISCUSSION
Ablative laser skin resurfacing can produce excellent cosmetic results and is considered to be the most effective option for skin rejuvenation.2,18,19
Damaging the epidermis significantly, however, can cause potentially adverse effects, such as dyspigmentation.5 Moreover, skin tightening frequently results from the significant damage to the epidermis and, even more importantly, its basal membrane and consequent inflammation accompanying the wound-healing process.18 This trauma to the epidermis can lead to postinflammatory skin hyperpigmentation in persons of any age or skin type.5 This side effect is the predominant reason why ablative laser resurfacing has become less pop- ular and the focus has shifted toward non-ablative skin rejuvenation because of its decreased risk of adverse effects and downtime. Although non-ablative laser or light procedures improve skin texture and pigmentary disorders, the degree of tightening is sometimes less than desired.
It was recently shown in a retrospective analysis of 480 patients that percutaneous collagen induction therapy is a safe and successful method for skin rejuvenation, and none of the patients devel-
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Fig.6. Insituhybridization.interleukin-10geneexpressionintreatedandunneedledskin2weeksaftersurgery.(Left) The blue color indicates the presence of interleukin-10 mRNA. Although the sections are somewhat difficult to compare directly con- cerning the amount of interleukin-10 because of the higher cellular density in needled skin, the number of interleukin-10 – expressing cells seems to be increased and more evenly distributed. (Right) Controls: remains without blue color. Scale bar 200 m (representative example).
oped postoperative dyspigmentation. Van Gieson staining showed a considerable increase in collagen deposition at 6 months postoperatively. The collagen also appears to have been laid down in a normal lattice pattern rather than in parallel bundles, as seen in scar tissue. Similarly, elastica stains showed an increase in elastin at 6 months post-operatively. Hematoxylin and eosin stains demonstrated a normal stratum corneum, thickened epidermis (40 percent thickening of the stratum granulosum), and normal rete ridges at 1 year postoperatively.14
The purpose of this study was to increase our current knowledge of whether percutaneous collagen induction therapy presents an effective means of skin rejuvenation without risk of dyspigmentation, as suggested by these clinical data. Therefore, we evaluated the effect of percutaneous collagen induction therapy on the epidermis, the melanocytes, and the pigmentation markers interleukin-10 and melanocyte-stimulating hormone in an animal model.
Epidermal Changes
Recently, we were able to show (data in sub-mission) on histologic samples taken 24 hours after percutaneous collagen induction therapy that the epidermis heals completely within this time, without any further indication of stratum corneum, epidermal, or basal membrane damage, and particularly with no signs of any dermabrasion (Fig. 3). The rete ridge pattern was as obvious in all the needled specimens as in the unneedled ones.20 The needles of the needling device puncture the epidermis and cause a bleeding in the
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papillary dermis, without ablation of the basal membrane and its melanocytes. The basal membrane remains intact, and the melanocytes are not removed. Ruiz-Maldonado and Orozco-Covarru- bias showed that some people have an inherited tendency for a weak melanocyte response to inflammation by decreasing melanin production or a strong melanocyte response by increasing melanin production.5 Our experiments show that melanocytes remained evenly distributed throughout the epidermis of the needled animals; however, their numbers neither increased nor decreased compared with the unneedled controls. In contrast to ablative laser treatment, percutaneous collagen induction therapy does not induce the process of reepithelialization and regeneration of the basal membrane and their melanocytes out of hair follicles in the postinflammatory response, which is where the risk for dyspigmentation lies.
Pigmentation Markers
In vivo studies directed toward identifying intrinsic paracrine cytokines involved in hyperpigmentary disorders have been difficult to interpret in the absence of information about whether the up- or down-regulation of some cytokines or chemokines in the lesional skin is responsible for the constitutive activation or in-activation of lesional melanocytes. This is attributable in part to a wide variety of cytokines or chemokines not directly related to melanocyte stimulation that is highly expressed in hyperpigmentary disorders because of the concomitant presence of other types of abnormal epidermal cells in addition to melanocytes.21
Melanocyte-stimulating hormone is a neuro-immunomodulating peptide that was recently de-tested in many non-pituitary tissues, including the skin. Accordingly, epidermal cells such as keratin-
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Fig.7. Immunohistochemistry(interleukin-10)green fluorescence indicated the presence of the interleukin-10protein.The rate of synthesis of this anti-inflammatory cytokine was analyzed 2 weeks after the operation both in skin samples from treated animals and in control samples from unneedled animals. (Left) Interleukin-10 synthesis was increased in all needled groups. (Right) Controls: no increase of interleukin-10 synthesis. Scale bar 200 m (representative example).
Volume 122, Number 5 • Percutaneous Collagen Induction
Fig.8. Microphotographsdemonstratingmelanocytestaining(immunofluorescence)withantibodiesdirectedagainstS100. Skin samples from treated animals and control samples from unneedled animals were analyzed 8 weeks after the operation. Melanocytes in the skin of the treated and the untreated groups were found to be present throughout the entire epidermis. No increase of their number in any of the needled groups was observed when compared with their controls. Scale bar 100 m (representative example).
oocytes and melanocytes (and dermal cells such as fibroblasts and endothelial cells), after stimulation with proinflammatory cytokines or ultraviolet light, synthesize and release melanocyte-stimulating- ing hormone. The effects of these peptides are mediated through specific melanocortin receptors that can be detected on immunocompetent and inflammatory cells and on keratinocytes,- melanocytes, fibroblasts, and endothelial cells. In addition to its well-known pigment-inducing capacity, the melanocyte-stimulating hormone is able to modulate keratinocyte proliferation and differentiation. Endothelial cell production, fibroblast cytokine production, and fibroblast collagenase production are also regulated by melanocyte-stimulating hormone. The immunosuppressive capacity of mela- nocyte-stimulating hormone is mediated mainly through its effects on monocyte and macrophage functions. Accordingly, melanocyte-stimulating
hormone down-regulates the production of proinflammatory cytokines and accessory molecules on antigen-presenting cells. The production of suppressor factors such as interleukin-10, however, is up-regulated by melanocyte-stimulating hormone. The in vivo relevance of these data is documented by the finding that the systemic application of melanocyte-stimulating hormone inhibits the induction and elicitation of murine contact hy- hypersensitivity and induces hapten-specific tolerance. These findings indicate that melanocyte-stimulating hormone is part of the mediator network that regulates cutaneous inflammation and hyperproliferative skin diseases.22 Using microarray analysis, our objectives were to determine relative mRNA levels of the genes coding for in-terleukin-10 and MC1R (melanocyte-stimulating hormone) after percutaneous collagen induction therapy treatment compared with normal un-
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treated skin of control rats. We were thus able to demonstrate that percutaneous collagen induction therapy modulates changes in the expression of these genes. The results support the hypothesis that the production of suppressor factors such as in- interleukin-10 is up-regulated by melanocyte-stimulating hormone because the expression levels of interleukin-10 increase during the first 2 weeks after percutaneous collagen induction therapy treatment. In addition, down-regulation of the MC1R gene suggests that needling reduces the risk of dyspigmentation through significant down-regulation of melanocyte-stimulating hormone in the postinflammatory response. Our data suggest that percutaneous collagen induction therapy does not induce postoperative dyspigmentation.
Concerning the microarray experiment, our objectives were to determine mRNA levels of the genes coding for interleukin-10 and MC1R after percutaneous collagen induction therapy treatment compared with normal untreated skin of control rats. The percutaneous collagen induction therapy method modulates changes in gene expression in both genes. The results support the hypothesis that the production of suppressor factors such as interleukin-10, however, is up-regulated by melanocyte-stimulating hormone because the expression level of interleukin-10 increases 2 weeks after percutaneous collagen induction therapy treatment. In addition, the down-regulation of the MC1R gene over all time points after surgery supports the hypothesis that percutaneous collagen induction therapy does not induce more postop-active depigmentation.
Microarray analysis promises dynamic snapshots of cell activity, but microarray results are unfortunately not straightforward to interpret. This means that, along with the generation of complicated data sets and the difficulty of interpreting them, the success with microarray approaches requires first a sound experimental design and particularly a coordinated and appropriate use of statistical methods. Tools for the efficient integration and interpretation of large data sets are needed. To obtain a standardized data set, several statistical methods have been applied to the raw data.
The fewer genes a low-density chip comprises, the more critical the normalization is because it cannot be assumed that the majority of genes are not regulated—such an assumption would allow for normalization methods such as the Loess regression. Therefore, a background-dependent normalization method has been applied. In contrast to the disadvantage of low-density chips contouring scan normalization, the advantage of this low-density chip consists of the possibility of offering many gene replicates, thereby allowing for a more statistical calculation of values for one gene. Thus, the variabilities generated by the microarray experiments could be kept within limits compared with the biological replicates and could, to a certain degree, be evened by the analysis.
The Rationale for Using Topical Vitamins A and C in the Study
The necessity for using vitamin A and C for percutaneous collagen induction therapy has been well described previously by Fernandes.16 Vitamin A, as retinoic acid, is an essential vitamin for the skin. It exercises its influence on more than 1000 genes that control proliferation, differentiation, and maturation of all the major cells in the epidermis, and dermis.23,24 Vitamin C is also essential for the production of normal collagen.16,25 Percutaneous collagen induction therapy and vitamin A induce fibroblasts to produce collagen and, therefore increase the need for vitamin C.
Human Skin in Comparison with Sprague-Daw- ley Rat Skin
To document age-related histologic morpho-metric changes in rat skin, Thomas published a study in 2005 of 344 rats in three age groups (young, 4 months; adult, 1 year; and old, 24 months). Sprague-Dawley rat melanocytes, however, produce no melanin. Rat skin, much like human skin, consists of keratinocytes, Langerhans cells, fibroblasts, and, essential for our trial, melanocytes.26 In the present study, the effect of percutaneous collagen induction therapy on melanocytes and pigmentation markers of 4-month-old rats was measured 1, 14, 28, and 56 days after percutaneous collagen induction therapy.
CONCLUSIONS
Postinflammatory dyspigmentation is a complex biological phenomenon. Ablative laser treatments are generally used to improve the skin aging processes, injures or destroys the epidermis, and leads to fibrosis of the percutaneous collagen induction therapy dermis, with the potentially adverse effect of depigmentation. We have shown that percutaneous collagen induction therapy may be preferable for skin rejuvenation by inducing the following improvements: percutaneous collagen induction therapy leaves the epidermis completely intact without any damage to the stratum corneum, any other layers of the epidermis, or its
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basal membrane. No signs of dermabrasive reduction of epidermal thickness were evident 24 hours after percutaneous collagen induction therapy. The number of melanocytes in the skin did not change postoperatively. Furthermore, DNA microarray experiments demonstrated that internet- kin-10 is faintly increased in treated skin after 2 weeks in comparison with control samples. Concerning the MC1R (melanocyte-stimulating hormone) gene, gene expression microarray analysis indicates a slight down-regulation 24 hours and 2 weeks after needling. Our data suggest that per-cutaneous collagen induction therapy does not induce postoperative dyspigmentation. With this study, we present an explanation in support of our previous clinical experience14 with regard to why percutaneous collagen induction therapy offers a modality for rejuvenating and improving skin ap- appearance and quality without the risk of dyspigmentation.
Matthias Aust, M.D.
Klinik fu ̈r Plastische, Hand und Wiederherstellungschirurgie Medizinische Hochschule Hannover Carl-Neuberg Strasse 1 30625 Hannover, Germany aust_matthias@gmx.de
ACKNOWLEDGMENT
The technical assistance provided by A. Lazaridis is greatly appreciated.
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9. Imokawa,G.,Yada,Y.,and Higuchi, K.Purification- and characterization of an allergy-induced melanogenic stimulating factor in brownish guinea pig skin. J. Biol. Chem. 273: 1605, 1998.
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11. Bernard, F. X., Pedretti, N., Rosdy, M., and Dequercy, A. Comparison of gene expression profiles in human keratin- oocyte mono-layer cultures, reconstituted epidermis and normal human skin: Transcriptional effects of retinoid treatments in reconstituted human epidermis. Exp. Dermatol. 11: 59, 2002.
12. Rosdahl, I., Andersson, E., Ka ̊ pedal, B., and To ̈ rma ̈ , H. Vitamin A metabolism and mRNA expression of retinoid-binding protein and receptor genes in human epidermal melanocytes and melanoma cells. Melanoma Res. 7: 267, 1997.
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