Percutaneous collagen inductioneRegeneration in place of cicatrisation?

M.C. Aust a,*, K. Reimers a, H.M. Kaplan b, F. Stahl c, C. Repenning c, T. Scheper c, S. Jahn a, N. Schwaiger d, R. Ipaktchi a, J. Redeker a, M.A. Altintas a, P.M. Vogt a

a Department for Plastic, Hand and Reconstructive Surgery, Medizinische Hochschule Hannover, Hannover, Germany b Department of Biomedical Engineering University of Southern California, Los Angeles, CA, USA
c Department of Technical Chemistry University of Hannover, Hannover, Germany
d Clinic for Hand and Plastic Surgery Friederikenstift, Hannover, Germany

Received 19 May 2009; accepted 14 March 2010

Summary Background: Ablative procedures that are used for the improvement of a degener- ative process that leads to a loss of skin elasticity and integrity, injure or destroy the epidermis and its basement membrane and lead to fibrosis of the papillary dermis. It was recently shown in clinical and laboratory trials that percutaneous collagen induction (PCI) by multiple needle application is a method for safely treating wrinkles and scars and smoothening the skin without the risk of dyspigmentation. In our study, we describe the effect of PCI on epidermal thickness and the induction of genes relevant for regenerative processes in the skin in a small animal model.

Methods: The purpose of this study in a rat model was to determine the effects of PCI on the skin both qualitatively and quantitatively. The epidermal and dermal changes were observed by histology and immunofluorescence. The changes in gene expression were measured by array analysis for cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-7, epidermal growth factor (EGF) and extracellular matrix molecules such as collagen type I and type III.

Results: The present study showed that PCI with topical vitamins resulted in a 140% increase in epidermal thickness; an increase in gene and protein expression of collagen I, glycosami- noglycans (GAGs) and growth factors such as VEGF, EGF and FGF7. The collagen fibre bundles were increased, thickened, and more loosely woven in both the papillary and reticular dermis.

* Corresponding author. Klinik fu ̈r Plastische, Hand und Wiederherstellungschirurgie Medizinische Hochschule Hannover, Carl-Neuberg Straße 1, 30625 Hannover, Germany. Tel.: þ49 511 5328864; fax: þ49 511 5328860.

E-mail address: aust_matthias@gmx.de (M.C. Aust).
1748-6815/$ – see front matter a 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights

reserved. doi:10.1016/j.bjps.2010.03.038

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During the last decade, substantial progress has been made in understanding cellular and molecular mechanisms that bring about chronological ageing and photoageing.1e3 This emerging information reveals that chronological ageing and photoageing share fundamental molecular pathways. These new insights regarding convergence of the molecular basis of chronological ageing and photoageing provide exciting new opportunities for the development of skin regeneration therapies.

The ideal treatment of skin regeneration should increase gene and protein expression responsible for skin regeneration without significant damage to the skin. Regeneration rather than cicatrisation could offer patients the better results.

The authors have recently shown that percutaneous collagen induction (PCI) by multiple needle application is a method for safely treating wrinkles and scars without the risk of dyspigmentation.4,5 These studies did not, however, provide any morphometric evaluation of epidermal changes, nor describe the quantitative and qualitative alterations in the dermis.

Materials and methods

PCI has been well described previously.5 It originated from the combined ideas of Orentriech and Orentriech, Fer- nandes and Camirand and Doucet and involves pricking the skin by rolling a specially designed device (Figure 1a and b)

over a skin area to create thousands of microwounds in the dermis that result in a confluent zone of very superficial inflammation, triggering the release of growth factors and inducing new connective tissue formation.6e8

Experimental groups

Sixty male SpragueeDawley rats (350e375g), age 4 months, were randomly assigned into four groups: group (A) (n Z 6: control); group (B) (n Z 18: skincare only); group (C) (n Z 18: needling only) and group (D) (n Z 18: needling plus skincare).

Group (A) was left untreated, and group (B) was anaes- thetised, shaved and treated additionally with skincare in the same way as group (D). Groups (C and D) received a 30% total body surface area skin needling (10min) on the shaved back skin under general anaesthesia and analgesia (Rompun 0.3 ml kg-1 of body weight, Ketanest 1.1 ml kg1 of body weight) to induce percutaneous collagen, using a medical needling instrument (Environ! Medical Roll- CITTM, Vivida SA cc, Cape Town, South Africa) (Figure 1c and d). Shaving was done only once in groups (B), (C) and (D). The length of the needles was 3 mm which penetrates right through the epidermis down to the dermis. The end point was achieved after rolling all animals for 10 min to create thousands of microwounds in the dermis that result in a confluent zone of haematoma.

Conclusion: We were able to show that PCI modulates gene expression in skin of those genes that are relevant for extracellular matrix remodelling.
a 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Figure 1 a) Medical Roll-Cit (made by Vivida C.C. Renaissance Body Science Institute, Cape Town South Africa). (b) Schematic image of the procedure. (c) Rat with shaved back: Preoperative: untreated skin. (d) Rat with shaved back: 1 h postoperative. An intradermal bleeding and bruising is visible.

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Application of vitamin A and C

Groups (B) and (D) were prepared with high levels of vitamin A cream (retinyl palmitate 1% as Environ! Original, Environ Skin Care (Pty) Ltd, Cape Town, South Africa) and vitamin C cream (ascorbyl tetra-isopalmitate 10% as

Environ! C-Boost, Environ Skin Care Ltd, Cape Town, South Africa), applied directly after needling and shaving, applied once daily. This was performed to both maximise initial collagen production and to maintain the homeostasis between collagenesis and collagenolysis. Group (C) received needling only. The control group (A) rats served as

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Figure 2 Microphotographs taken of skin samples stained with HaematoxylineEosin. The size of the scale bar is 200 mm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare. The epidermal thickening (up to 140% after 8 weeks in group (D)) coincided with increased thickening of the granular layer, an increased number of epidermal cell layers and a more compact stratum corneum. The control group (A) showed a uniform mean epidermal thickness of 13.0 mm through the entire set.

Please cite this article in press as: Aust MC, et al., Percutaneous collagen inductioneRegeneration in place of cicatrisation?, J Plast Reconstr Aesthet Surg (2010), doi:10.1016/j.bjps.2010.03.038

unneedled, untreated, time-matched rats to establish baseline levels.

Animals in each group were sacrificed in three sets (at 14, 28 and 56 days).

Histology

Skin biopsies were cut into 5 mm sections and stained with HaematoxylineEosin (Merck, Darmstadt, Germany) or Masson’s Trichrome (Merck, Darmstadt, Germany) as recommended by

the manufacturer. Measurements were carried out using CellD software on 15 representative slides of the same region for each individual. Periodic Acid Schiff’s (PAS) staining was performed using the Periodic Acid Schiff’s staining Kit (Roth, Germany) as recommended by the manufacturer.

Epidermal thickness

Measurement of the epidermal thickness was done using the CellD programme on an Olympus microscope (Hamburg,

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Figure 3 Masson’s Trichrome staining. The size of the scale bar is 2050 mm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare Collagen fibere bundles were increased, thickened, and more loosely woven in both the papillary and reticular dermis most prominently in the needled plus skincare group (D). Elastin fibres in the dermis highly linear and the epidermaledermal interface showed regular dermal papillae; cellular polarity and normal epidermal differentiation appeared to be maintained; and the elastin network within the reticular dermis was regularly thickened and organized in all groups.

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Selected genes of group (D) against untreated control rats (group (A)). Shown are ratios of the expression 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. A bold border shows the significant change in gene expression. Please see also significant modification (calculated by t-test) in Table 2. Collagen I levels were quantitatively increased in treated skin (group (D)) in comparison to normal untreated samples (group (A)) with very high significance (p value less than 10e-5) at all time points. In contrast to that collagen III is only significantly increased at time point 4 weeks (p-value Z 0.00183) and not significantly changed 2 and 8 weeks after treatment. FGF7 was found to be strongly increased in group (D) 2 weeks after the treatment and modestly increased 4 weeks and not significantly changed 8 weeks after needling, whereas EGF was not significantly increased. The results support our hypothesis that the expression levels of collagens qualitatively increase after PCI treatment.

Germany). Three random slides of the same region, representative for each individual were measured by drawing a vertical bar from the end of the epidermis to the basal lamina. To analyse the statistical significance of the epidermal thickness, three random zones per histological slide were examined by the program analysis Soft Imaging System! (Muenster, Germany).

Immunofluorescence

Skin samples were cut into 20 mm cryostat sections. Primary antibodies against rat collagen I, collagen III, fibronectin, glycosaminoglycans (GAGs) and vascular endothelial growth factor (VEGF) were used at 1:100 dilution (Sigma, St. Louis, USA). The secondary antibodies were Alexa488-conjugated or Alexa546-conjugated goat anti-mouse or goat anti-rabbit antibodies (Molecular Probes, Leiden, the Netherlands). The samples were washed and mounted in Vectashield (Vector Laboratories, Burlingame, USA) supplemented with 4’,6-dia- midino-2-phenylindole (DAPI). Specimens were viewed and photographed on a Zeiss microscope (Go ̈ttingen, Germany).

Gene expression analysis by array

Total RNA (mg) was isolated using the Aurum total fatty and fibrous tissue kit (Biorad, Munich, Germany). During reverse

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Percutaneous collagen inductioneRegeneration in place of cicatrisation? 5

Figure 4

transcription, RNA was converted into fluorescein (Fl) and biotin (B) labelled cDNA. Specifically bound Fl and B labelled cDNAs were sequentially detected with a series of conjugate reporter, ultimately with Tyramide-Cy3 and Tyramide-Cy5.

The hybridised chips were scanned six times with an Axon 400B” scanner at different settings. Primary image analysis was performed using the software tool Gene Pix Pro 6.0″.

Statistical analysis

The averaged values for each gene of gene expression anal- ysis comprise nine replicates. Outliers amongst the gene replicates were eliminated according to the outlier test by Nalimov. The independent Student’s t-test was used to determine the statistical significance of the differences between the staining patterns and the values for histological measurements in needled skin and in unneedled skin. All p values were two-tailed, and differences were considered significant for p values 0.05. Summary data are expressed as the mean and standard error of the mean (SEM).

Ethics

This study was reviewed and approved by the Lower Saxony District Government (Hannover, Germany), ensuring that all animals received humane care.

Results

Epidermal changes

(Table 1) In comparison to control group (A), group (B) showed an increase of 22% in thickness of epidermis over 8 weeks due to topical vitamins alone. In all sets, the mean epidermal thickness in the needled groups (C and D) was found to be significantly thicker than the control group (A) and skincare only group (B) and continually progressed postoperatively over the entire time of observation. This difference was statistically highly significant (p 0.00001). The epidermal thickening (up to 140% after 8 weeks in group (D)) coincided with increased thickening of the granular layer, an increased number of epidermal cell layers and a more compact stratum corneum. (Figure 2aed). The control group (A) showed a uniform mean epidermal thick- ness of 13.0 mm through the entire set.

Connective tissue changes

Connective tissue fibre bundles were found to be qualita- tively increased, thickened and more loosely woven in both the papillary and reticular dermis of the needled only group (C) in set I, II and III, than in the unneedled groups (A) and (B). The most prominent increase in connective tissue fibre density was observed in the needled plus skincare group (D) compared to every other group in all sets (Figure 3aed).

Gene expression analysis

(Table 2) The aim of the gene expression analysis was to analyse the observed differences between the

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Figure 5 Immunofluorescence visualization of collagen I: Staining with antibodies directed against Collagen I (Alexa488) and DAPI. The size of the scale bar is 100 mm (representative example). (a) Unneedled animal with 8 weeks of skincare areas of the dermis failed to react with the antibodies. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare abundant collagen present throughout the dermis. (d) Needled animal with 8 weeks of skincare stained without primary antibody. The amount of type I collagen was qualitatively increased in treated group (Figure 5c) compared to their controls judged by the brighter fluorescence.

experimental groups on a gene regulatory level. The microarray design comprised genes coding for extracellular matrix proteins and growth factors. The results for the extracellular matrix proteins showed that relative collagen I levels were quantitatively increased in treated skin (group (D)) in comparison to normal untreated samples (group (A)) at the given times. With very high significance (p value less than 10e-5), collagen I expression is increased at all time points. By contrast, collagen III is only significantly changed at a time point of 4 weeks (p value Z 0.00183) and not significantly changed 2 and 8 weeks after treatment. Collagen I and III increased in all needling groups.

Analysis of the expression of several growth factors of regenerative relevance showed that FGF7 was found to be strongly increased in group (D) 2 weeks after treatment and modestly increased 4 weeks after needling and not signifi- cantly changed 8 weeks after needling. (Figure 4, Table 2)

The whole dataset is represented by the Series id GS GPL5462 in the Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/geo/) database.

Type I collagen changes

The type I collagen fibres in the skin in all groups were found to be distributed throughout the whole dermis but tended to be more condensed just beneath the epidermis. Abundant collagen was present throughout the dermis of the needled skin group (D), while in the unneedled skin group (A), many areas of the dermis failed to react with the antibodies. The

amount of type I collagen was qualitatively increased in group (D) compared with their controls judged by the brighter fluo- rescence in the equally treated samples. The greatest amount of type I collagen was found in group (D) of set III (8 weeks), followed by sets II and I respectively (Figure. 5aed).

Type III collagen changes

Interestingly, collagen III gene expression showed a signifi- cant gene expression regulation only 4 weeks after the treatment; however we could observe that in immunoflu- orescence analysis, differing staining patterns in needled and unneedled skin samples could also be observed as long as 8 weeks after treatment. While the amount of type III collagen qualitatively increased particularly in the needled group of set I, followed by set II, compared to their controls (data not shown), only small amounts of type III collagen were found in set III (Figure 6aed). In the unneedled sample, collagen III was still detectable 8 weeks after the start of the experiment.

GAG changes

PAS staining was used to stain for the mucopolysaccharides of the glycosaminoglycans (GAGs). The staining intensity observed in samples derived from needled skin (groups C and D) was deepened and more regularly patterned than the unneedled samples (groups A and B) (Figure 7aed). The

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Figure 6 Immunofluorescence visualization of collagen III demonstrated in the dermal zone just beneath the epidermis (Alexa488-conjugated antibody) and DAPI. The size of the scale bar is 100 mm (representative example). (a) Unneedled animal with 8 weeks of skincare Collagen III is still detectable 8 weeks after treatment. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare only small amounts of type III collagen were found 8 weeks after needling. (d) Needled animal with 8 weeks of skincare stained without primary antibody. While the amount of type III collagen was qualitatively increased particularly in the needled group 2 weeks and 4 weeks after the operation compared to their controls (data not shown), only small amounts of type III collagen were found in set 8 weeks postoperatively (Figure 6c). In the unneedled sample collagen III was still detectable 8 weeks after treatment (Figure 6a).

staining pattern was most regular and intensified in the needling plus skincare group (Figure 7d).

A chondroitin sulphate antibody was used to further qualify the expression of GAGs. GAGs were readily observed in needled skin, appearing at the dermaleepidermal junction and inter- spersed between the abundant collagen bundles in the papil- lary and reticular dermis. Unneedled skin had also copious amounts of GAGs. Nevertheless, these GAGs showed dense deposits occupying much of the dermis, leaving only isolated collagen bundles visible (Figure 8aed). A marked increase in the amount of GAGs was observed throughout the different needled groups compared with the unneedled groups.

Fibronectin changes

Positive staining for fibronectin, a fibroblast differentiation protein, was observed in the papillary dermis of all groups with a more intense staining in the needled samples than the unneedled ones. Together with the quantitative data, this indicates that fibronectin expression was markedly increased in the dermis of all needled groups than in the unneedled groups (Figure 9aed).

VEGF changes

Positive staining for VEGF was observed in the dermis of all groups including the control group (A). VEGF showed

a membranous staining pattern along the intercellular junctions in the basal and suprabasal layers of the epidermis, excluding the stratum corneum. A brighter fluorescence indicates that the amount of VEGF in the dermis is augmented in the different needled groups compared with the unneedled groups (Figure 10aed).

Discussion

Human skin in comparison with SpragueeDawley rat skin

To document age-related histological morphometric changes of 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). Rat skin, much like human skin, consists of keratinocytes, Langerhans cells, fibroblasts and melanocytes.9 In the present study, the effect of PCI therapy on collagen production of 4-month-old rats was measured 14, 28 and 56 days after needling therapy.

The effect on the epidermis and the rationale for using topical vitamins A and C

Vitamin A and C are known as anti-ageing products and are essential for the production of normal collagen and

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Figure 7 Microphotographs taken of skin samples stained with PAS. The size of the scale bar is 2100 mm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare. The epidermal thickening coincided with increased thickening of the granular layer, an increased number of epidermal cell layers, and a more compact stratum corneum. The staining intensity observed in samples derived from needled skin (7c and 7d) was deepened compared to the unneedled samples (7a and 7b). The staining pattern was most regular and intensified in the needling plus skincare group (7d).

proliferation and differentiation of epidermal and dermal cells.6,10e13 Our results have demonstrated that the mean epidermal thickness increased progressively over time in the needled groups (sets I, II and III) compared with the unneedled groups. Our results are in agreement with Gilchrest’s findings of remarkable increases in epidermal thickness with the topical use of vitamin A.14

The effect on the dermis

PCI aims to stimulate collagen production by employing the normal inflammatory wound healing cascade that ensues after any trauma. Platelets and neutrophils release growth factors such as transforming growth factor (TGF)- a, TGF-b, platelet derived growth factor (PDGF), etc., which work in concert to increase the production of the intercellular matrix. A fibronectin matrix forms with an alignment of fibroblasts that determines the ultimate deposition of collagen. Eventually, collagen III is con- verted into collagen I. Due to this conversion, the collagen tightens naturally over a few months. We hypothesise that the controlled wound milieu created during PCI, by mini- mising the usual stresses such as exposure to air, infec- tion, mechanical tension, etc., may bring us closer to regenerative healing.

In the present study, the regeneration processes after PCI have been observed to qualify and quantify the changes

in type I collagen. We have shown that no needled skin sample demonstrated thickened, curled or amorphous fibres that would appear to replace the collagen in the papillary and upper reticular dermis.

The effect on important anti-ageing markers

GAGs are composed of specific repeating disaccharide units and are found widely distributed throughout the skin.15 They are especially important in skin because they are extremely hydrophilic, binding up to 1000 times their volume in water. Therefore, skin hydration is closely connected to the content and distribution of dermal GAGs. Unneedled skin had copious amounts of GAGs with dense deposits occupying much of the dermis at the dermaleepidermal junction. These results seem to show that the increase of GAGs after needling scatter diffusely on the elastinecollagen matrix as occurs in young skin.

Although VEGF was initially thought to be expressed exclusively by endothelial cells, recent studies have found that keratinocytes in the epidermis also express VEGF.16,17 These results suggest that VEGF plays an important bio- logical role in keratinocyte function and that may be differing from its traditional contribution in angiogenesis. Our results observed positive staining for VEGF in the epidermis and dermis of all groups but with a marked

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Figure 8 Immunofluorescence visualization of GAGs (Alexa488-conjugated) and DAPI. The size of the scale bar is 100 mm (representative example). (a) Unneedled animal with 8 weeks of skincare. GAGs showed dense deposits occupying much of the dermis, leaving only isolated collagen bundles visible (white arrows). (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare GAGs were observed, appearing at the dermaleepidermal junction and interspersed between the abundant collagen bundles in the papillary and reticular dermis (white arrows). (d) Needled animal with 8 weeks of skincare stained without primary antibody. As observed in the PAS staining a marked increase in the amount of GAGs was observed throughout the different needled groups in comparison to the unneedled groups.

increase throughout the different needled specimens. These data underline the importance of PCI on stimulating VEGF as yet another potential anti-ageing marker.

Fibronectin (a tension-related protein) is a differentia- tion marker protein for fibroblasts. Our results revealed significant increases in the expression of fibronectin in the needled specimens than in the unneedled ones. Taken together with the observation of high-intensity stained sections, this might indicate an overall increase in cellular skin components in accordance with the increase in extra- cellular matrix as well as an ongoing differentiation process in the needled skin.

Microarray results

The authors decided to compare the strongest group (group D) with the control group (group A) to show the most significant contrast.

The results for the extracellular matrix proteins show that relative collagen I levels were significantly increased in treated skin in comparison to untreated samples at all time points. Furthermore, collagen III is slightly increased 4 weeks after treatment. Analysis of the expression of several growth factors of regenerative relevance showed that FGF-7 was found to be highly increased in the needled group 2 weeks after the treatment.

PCI modulates changes in gene expression of several genes encoding for cytokines and extracellular matrix

proteins. Using microarray analysis, rat model genes, which may be implicated to the effects of PCI, were measured. The results support our hypothesis that the expression levels of collagens qualitatively increase after PCI treat- ment. These findings could be confirmed through immuno- cytochemistry antibody staining.

We used a small animal model to show that needling skin with topical vitamins A, C and antioxidants results in the following changes (as compared to unneedled skin): 140% increase in epidermal thickness; furthermore, an increase in collagen I, GAGs, VEGF and fibronectin as well as an increase of growth factors relevant for skin regeneration. The collagen fibre bundles were found to be qualitatively increased, thickened and more loosely woven in both the papillary and reticular dermis of the needled group. The epidermaledermal interface showed regular dermal papillae; cellular polarity and normal epidermal differen- tiation appeared to be maintained; and the connective tissue network within the reticular dermis was regularly thickened and organised. Our results suggest that PCI offers an anti-ageing modality to rejuvenate and improve the appearance of skin. We can now improve skin from the inside out, not just from the surface. In addition, PCI has proven to be very effective in skin rejuvenation and might bring us closer to the idaea of regenerating healthy skin in vivo rather than producing scars. As opposed to ablative laser treatments, the epidermis remains intact and is not removed.

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Figure 9 Immunofluorescence visualization of fibronectin in the epidermis and dermis (Alexa488-conjugated) and DAPI. The size of the scale bar is 100 mm (representative example). (a) unneedled animal with 8 weeks of skincare. (b) unneedled animal with 8 weeks of skincare stained without primary antibody. (c) needled animal with 8 weeks of skincare increase in the amount of fibronectin in the epidermis and dermis. (d) needled animal with 8 weeks of skincare stained without primary antibody. Positive staining for fibronectin was observed in the papillary dermis of all groups, but again there is an intense staining in the needled samples and a comparably weaker signal in the unneedled ones. Together with the quantitative data this indicates that fibronectin expression was markedly increased in the dermis of all needled groups compared to the unneedled groups.

Figure 10 Immunofluorescence visualization of VEGF observed in the epidermis and dermis (Alexa488-conjugated) and DAPI. The size of the scale bar is 100 mm (representative example). (a) Unneedled animal with 8 weeks of skincare. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare increase in the amount of VEGF in the epidermis and dermis. (d) Needled animal with 8 weeks of skincare stained without primary antibody. VEGF showed a membranous staining pattern along the intercellular junctions in the basal and suprabasal layers of the epidermis. A brighter fluorescence indicates that the amount of VEGF in the dermis is augmented in the needled groups compared to the unneedled groups.

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Acknowledgement

The technical assistance by A. Lazaridis is greatly appreciated.

Conflict of interest

None.

Funding

None.

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Percutaneous collagen inductioneRegeneration in place of cicatrisation? 11

Please cite this article in press as: Aust MC, et al., Percutaneous collagen inductioneRegeneration in place of cicatrisation?, J Plast Reconstr Aesthet Surg (2010), doi:10.1016/j.bjps.2010.03.038