Journal of Plastic, Reconstructive & Aesthetic Surgery (2011) 64, 97e107

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

KEYWORDS Summary Background: Ablative procedures that are used for the improvement of a degener-
Percutaneous collagen ative process that leads to a loss of skin elasticity and integrity, injure or destroy the epidermis
induction; and its basement membrane and lead to fibrosis of the papillary dermis. It was recently shown
Skin-rejuvenation; in clinical and laboratory trials that percutaneous collagen induction (PCI) by multiple needle
Collagen; application is a method for safely treating wrinkles and scars and smoothening the skin without
Regeneration 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 für 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: [email protected] (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
98 M.C. Aust et al.

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.

During the last decade, substantial progress has been made over a skin area to create thousands of microwounds in the
in understanding cellular and molecular mechanisms that dermis that result in a confluent zone of very superficial
bring about chronological ageing and photoageing.1e3 This inflammation, triggering the release of growth factors and
emerging information reveals that chronological ageing and inducing new connective tissue formation.6e8
photoageing share fundamental molecular pathways. These
new insights regarding convergence of the molecular basis Experimental groups
of chronological ageing and photoageing provide exciting
new opportunities for the development of skin regeneration
Sixty male SpragueeDawley rats (350e375 g), age 4
therapies.
months, were randomly assigned into four groups: group (A)
The ideal treatment of skin regeneration should increase
(n Z 6: control); group (B) (n Z 18: skincare only); group
gene and protein expression responsible for skin regeneration
(C) (n Z 18: needling only) and group (D) (n Z 18: needling
without significant damage to the skin. Regeneration rather
plus skincare).
than cicatrisation could offer patients the better results.
Group (A) was left untreated, and group (B) was anaes-
The authors have recently shown that percutaneous
thetised, shaved and treated additionally with skincare in
collagen induction (PCI) by multiple needle application is
the same way as group (D). Groups (C and D) received a 30%
a method for safely treating wrinkles and scars without the
total body surface area skin needling (10 min) on the
risk of dyspigmentation.4,5 These studies did not, however,
shaved back skin under general anaesthesia and analgesia
provide any morphometric evaluation of epidermal
(Rompun 0.3 ml kg-1 of body weight, Ketanest 1.1 ml kg1 of
changes, nor describe the quantitative and qualitative
body weight) to induce percutaneous collagen, using
alterations in the dermis.
a medical needling instrument (Environ Medical Roll-
CITTM, Vivida SA cc, Cape Town, South Africa) (Figure 1c
Materials and methods and d). Shaving was done only once in groups (B), (C) and
(D). The length of the needles was 3 mm which penetrates
PCI has been well described previously.5 It originated from right through the epidermis down to the dermis. The end
the combined ideas of Orentriech and Orentriech, Fer- point was achieved after rolling all animals for 10 min to
nandes and Camirand and Doucet and involves pricking the create thousands of microwounds in the dermis that result
skin by rolling a specially designed device (Figure 1a and b) in a confluent zone of haematoma.

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.
Percutaneous collagen inductioneregeneration in place of cicatrisation? 99

Table 1 Epidermal thickness in skincare only vs. needling only vs. needling plus skincare skin in different sets (mm). 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). Group C showed an increase of 112% in epidermal thickness 8 weeks after
treatment, and there was an increase up to 140% in group D. This difference was statistically highly significant (p  0.00001).

p-value  0.00001; skincare only vs. needling only vs. needling plus skincare skin.

Application of vitamin A and C Environ C-Boost, Environ Skin Care Ltd, Cape Town, South
Africa), applied directly after needling and shaving, applied
Groups (B) and (D) were prepared with high levels of once daily. This was performed to both maximise initial
vitamin A cream (retinyl palmitate 1% as Environ Original, collagen production and to maintain the homeostasis
Environ Skin Care (Pty) Ltd, Cape Town, South Africa) and between collagenesis and collagenolysis. Group (C)
vitamin C cream (ascorbyl tetra-isopalmitate 10% as received needling only. The control group (A) rats served as

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.
100 M.C. Aust et al.

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.

unneedled, untreated, time-matched rats to establish the manufacturer. Measurements were carried out using CellD
baseline levels. software on 15 representative slides of the same region for each
Animals in each group were sacrificed in three sets (at individual. Periodic Acid Schiff’s (PAS) staining was performed
14, 28 and 56 days). using the Periodic Acid Schiff’s staining Kit (Roth, Germany) as
recommended by the manufacturer.
Histology
Epidermal thickness
Skin biopsies were cut into 5 mm sections and stained with
HaematoxylineEosin (Merck, Darmstadt, Germany) or Masson’s Measurement of the epidermal thickness was done using
Trichrome (Merck, Darmstadt, Germany) as recommended by the CellD programme on an Olympus microscope (Hamburg,

Table 2 Table 2 shows the changes in gene expression of group (D) against control animals at different time points. The
changes have been calculated with the t-test (p-value Z 0.05): 0 means unchanged when comparing the according time point to
untreated control rats. 1 means that the gene expression is changed. The ratio gives the amount of modification in gene
expression. The last column shows the standard error. If genes could not be detected because they were not expressed, all
values are given with zeros. With very high significance (p value less than 10e-5) Collagen I expression in treated skin (group (D))
is increased at all time points in comparison to untreated samples. In contrast to that Collagen III is only significantly increased
at time point 4 weeks (p-value Z 0.00183). FGF7 was found to be strongly increased in group (D) 2 weeks after the treatment,
but not significantly changed 8 weeks after needling.
Gene ID 2 weeks 4 weeks 8 weeks
Modification Ratio SEM p-Value Modification Ratio SEM p-Value Modification Ratio SEM p-Value
Collagen 1a1 1 5.82 1.01 <0.00001 1 5.91 0.26 <0.00001 1 6.91 0.88 <0.00001
Collagen 3a1 0 1.34 0.23 0.03457 1 1.54 0.16 0.00183 0 1.77 0.24 0.02796
EGF 0 -1.02 0.94 0.49055 0 1.42 1.77 0.00001 0 1.42 1.95 <0.00001
FGF7 (KGF) 1 13.47 1.72 0.00146 1 3.92 0 0 0 1.77 0 0
Percutaneous collagen inductioneregeneration in place of cicatrisation? 101

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
Figure 4 Selected genes of group (D) against untreated control between the staining patterns and the values for histological
rats (group (A)). Shown are ratios of the expression values of measurements in needled skin and in unneedled skin. All p
different time points after needling against untreated rats. Ratios values were two-tailed, and differences were considered
between 0 and 1 have been transformed to negative values for significant for p values  0.05. Summary data are expressed
a better comparison. Standard errors are marked by error bars. A as the mean and standard error of the mean (SEM).
bold border shows the significant change in gene expression.
Please see also significant modification (calculated by t-test) in Ethics
Table 2. Collagen I levels were quantitatively increased in treated
skin (group (D)) in comparison to normal untreated samples (group This study was reviewed and approved by the Lower Saxony
(A)) with very high significance (p value less than 10e-5) at all time District Government (Hannover, Germany), ensuring that all
points. In contrast to that collagen III is only significantly increased animals received humane care.
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
Results
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 Epidermal changes
results support our hypothesis that the expression levels of
collagens qualitatively increase after PCI treatment. (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
Germany). Three random slides of the same region, epidermal thickness in the needled groups (C and D) was
representative for each individual were measured by found to be significantly thicker than the control group (A)
drawing a vertical bar from the end of the epidermis to the and skincare only group (B) and continually progressed
basal lamina. To analyse the statistical significance of the postoperatively over the entire time of observation. This
epidermal thickness, three random zones per histological difference was statistically highly significant (p  0.00001).
slide were examined by the program analysis Soft Imaging The epidermal thickening (up to 140% after 8 weeks in group
System (Muenster, Germany). (D)) coincided with increased thickening of the granular
layer, an increased number of epidermal cell layers and
Immunofluorescence 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.
Skin samples were cut into 20 mm cryostat sections. Primary
antibodies against rat collagen I, collagen III, fibronectin,
glycosaminoglycans (GAGs) and vascular endothelial growth Connective tissue changes
factor (VEGF) were used at 1:100 dilution (Sigma, St. Louis,
USA). The secondary antibodies were Alexa488-conjugated or Connective tissue fibre bundles were found to be qualita-
Alexa546-conjugated goat anti-mouse or goat anti-rabbit tively increased, thickened and more loosely woven in both
antibodies (Molecular Probes, Leiden, the Netherlands). The the papillary and reticular dermis of the needled only group
samples were washed and mounted in Vectashield (Vector (C) in set I, II and III, than in the unneedled groups (A) and
Laboratories, Burlingame, USA) supplemented with 4’,6-dia- (B). The most prominent increase in connective tissue fibre
midino-2-phenylindole (DAPI). Specimens were viewed and density was observed in the needled plus skincare group (D)
photographed on a Zeiss microscope (Göttingen, Germany). compared to every other group in all sets (Figure 3aed).

Gene expression analysis by array Gene expression analysis

Total RNA (mg) was isolated using the Aurum total fatty and (Table 2) The aim of the gene expression analysis was to
fibrous tissue kit (Biorad, Munich, Germany). During reverse analyse the observed differences between the
102 M.C. Aust et al.

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 amount of type I collagen was qualitatively increased in group
microarray design comprised genes coding for extracellular (D) compared with their controls judged by the brighter fluo-
matrix proteins and growth factors. The results for the rescence in the equally treated samples. The greatest amount
extracellular matrix proteins showed that relative collagen of type I collagen was found in group (D) of set III (8 weeks),
I levels were quantitatively increased in treated skin (group followed by sets II and I respectively (Figure. 5aed).
(D)) in comparison to normal untreated samples (group (A))
at the given times. With very high significance (p value less
Type III collagen changes
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 Interestingly, collagen III gene expression showed a signifi-
significantly changed 2 and 8 weeks after treatment. cant gene expression regulation only 4 weeks after the
Collagen I and III increased in all needling groups. treatment; however we could observe that in immunoflu-
Analysis of the expression of several growth factors of orescence analysis, differing staining patterns in needled
regenerative relevance showed that FGF7 was found to be and unneedled skin samples could also be observed as long
strongly increased in group (D) 2 weeks after treatment and as 8 weeks after treatment. While the amount of type III
modestly increased 4 weeks after needling and not signifi- collagen qualitatively increased particularly in the needled
cantly changed 8 weeks after needling. (Figure 4, Table 2) group of set I, followed by set II, compared to their controls
The whole dataset is represented by the Series id GS (data not shown), only small amounts of type III collagen
GPL5462 in the Gene Expression Omnibus (GEO, http:// were found in set III (Figure 6aed). In the unneedled
www.ncbi.nlm.nih.gov/geo/) database. sample, collagen III was still detectable 8 weeks after the
start of the experiment.
Type I collagen changes
GAG 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 PAS staining was used to stain for the mucopolysaccharides
more condensed just beneath the epidermis. Abundant of the glycosaminoglycans (GAGs). The staining intensity
collagen was present throughout the dermis of the needled observed in samples derived from needled skin (groups C
skin group (D), while in the unneedled skin group (A), many and D) was deepened and more regularly patterned than
areas of the dermis failed to react with the antibodies. The the unneedled samples (groups A and B) (Figure 7aed). The
Percutaneous collagen inductioneregeneration in place of cicatrisation? 103

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 a membranous staining pattern along the intercellular
needling plus skincare group (Figure 7d). junctions in the basal and suprabasal layers of the
A chondroitin sulphate antibody was used to further qualify epidermis, excluding the stratum corneum. A brighter
the expression of GAGs. GAGs were readily observed in needled fluorescence indicates that the amount of VEGF in the
skin, appearing at the dermaleepidermal junction and inter- dermis is augmented in the different needled groups
spersed between the abundant collagen bundles in the papil- compared with the unneedled groups (Figure 10aed).
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 Discussion
collagen bundles visible (Figure 8aed). A marked increase in
the amount of GAGs was observed throughout the different Human skin in comparison with SpragueeDawley
needled groups compared with the unneedled groups. rat skin

Fibronectin changes To document age-related histological morphometric

changes of rat skin, Thomas published a study in 2005 of 344
Positive staining for fibronectin, a fibroblast differentiation rats in three age groups (young, 4 months; adult, 1 year;
protein, was observed in the papillary dermis of all groups and old, 24 months). Rat skin, much like human skin,
with a more intense staining in the needled samples than consists of keratinocytes, Langerhans cells, fibroblasts and
the unneedled ones. Together with the quantitative data, melanocytes.9 In the present study, the effect of PCI
this indicates that fibronectin expression was markedly therapy on collagen production of 4-month-old rats was
increased in the dermis of all needled groups than in the measured 14, 28 and 56 days after needling therapy.
unneedled groups (Figure 9aed).
The effect on the epidermis and the rationale for
VEGF changes using topical vitamins A and C

Positive staining for VEGF was observed in the dermis of all Vitamin A and C are known as anti-ageing products and are
groups including the control group (A). VEGF showed essential for the production of normal collagen and
104 M.C. Aust et al.

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 in type I collagen. We have shown that no needled skin
cells.6,10e13 Our results have demonstrated that the mean sample demonstrated thickened, curled or amorphous
epidermal thickness increased progressively over time in fibres that would appear to replace the collagen in the
the needled groups (sets I, II and III) compared with the papillary and upper reticular dermis.
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 important anti-ageing markers

GAGs are composed of specific repeating disaccharide

The effect on the dermis units and are found widely distributed throughout the
skin.15 They are especially important in skin because
PCI aims to stimulate collagen production by employing they are extremely hydrophilic, binding up to 1000 times
the normal inflammatory wound healing cascade that their volume in water. Therefore, skin hydration is
ensues after any trauma. Platelets and neutrophils release closely connected to the content and distribution of
growth factors such as transforming growth factor (TGF)- dermal GAGs. Unneedled skin had copious amounts of
a, TGF-b, platelet derived growth factor (PDGF), etc., GAGs with dense deposits occupying much of the dermis
which work in concert to increase the production of the at the dermaleepidermal junction. These results seem to
intercellular matrix. A fibronectin matrix forms with an show that the increase of GAGs after needling scatter
alignment of fibroblasts that determines the ultimate diffusely on the elastinecollagen matrix as occurs in
deposition of collagen. Eventually, collagen III is con- young skin.
verted into collagen I. Due to this conversion, the collagen Although VEGF was initially thought to be expressed
tightens naturally over a few months. We hypothesise that exclusively by endothelial cells, recent studies have found
the controlled wound milieu created during PCI, by mini- that keratinocytes in the epidermis also express VEGF.16,17
mising the usual stresses such as exposure to air, infec- These results suggest that VEGF plays an important bio-
tion, mechanical tension, etc., may bring us closer to logical role in keratinocyte function and that may be
regenerative healing. differing from its traditional contribution in angiogenesis.
In the present study, the regeneration processes after Our results observed positive staining for VEGF in the
PCI have been observed to qualify and quantify the changes epidermis and dermis of all groups but with a marked
Percutaneous collagen inductioneregeneration in place of cicatrisation? 105

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. proteins. Using microarray analysis, rat model genes, which
These data underline the importance of PCI on stimulating may be implicated to the effects of PCI, were measured.
VEGF as yet another potential anti-ageing marker. The results support our hypothesis that the expression
Fibronectin (a tension-related protein) is a differentia- levels of collagens qualitatively increase after PCI treat-
tion marker protein for fibroblasts. Our results revealed ment. These findings could be confirmed through immuno-
significant increases in the expression of fibronectin in the cytochemistry antibody staining.
needled specimens than in the unneedled ones. Taken We used a small animal model to show that needling skin
together with the observation of high-intensity stained with topical vitamins A, C and antioxidants results in the
sections, this might indicate an overall increase in cellular following changes (as compared to unneedled skin): 140%
skin components in accordance with the increase in extra- increase in epidermal thickness; furthermore, an increase
cellular matrix as well as an ongoing differentiation process in collagen I, GAGs, VEGF and fibronectin as well as an
in the needled skin. increase of growth factors relevant for skin regeneration.
The collagen fibre bundles were found to be qualitatively
Microarray results increased, thickened and more loosely woven in both the
papillary and reticular dermis of the needled group. The
The authors decided to compare the strongest group epidermaledermal interface showed regular dermal
(group D) with the control group (group A) to show the papillae; cellular polarity and normal epidermal differen-
most significant contrast. tiation appeared to be maintained; and the connective
The results for the extracellular matrix proteins show tissue network within the reticular dermis was regularly
that relative collagen I levels were significantly increased in thickened and organised. Our results suggest that PCI offers
treated skin in comparison to untreated samples at all time an anti-ageing modality to rejuvenate and improve the
points. Furthermore, collagen III is slightly increased 4 appearance of skin. We can now improve skin from the
weeks after treatment. Analysis of the expression of inside out, not just from the surface. In addition, PCI has
several growth factors of regenerative relevance showed proven to be very effective in skin rejuvenation and might
that FGF-7 was found to be highly increased in the needled bring us closer to the idaea of regenerating healthy skin in
group 2 weeks after the treatment. vivo rather than producing scars. As opposed to ablative
PCI modulates changes in gene expression of several laser treatments, the epidermis remains intact and is
genes encoding for cytokines and extracellular matrix not removed.
106 M.C. Aust et al.

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.
Percutaneous collagen inductioneregeneration in place of cicatrisation? 107

Acknowledgement 6. Fernandes D. Percutanous collagen induction: an alternative to

laser resurfacing. Aestetic Surg 2002;22:315e7.
7. Orentreich DS, Orentreich N. Subcutaneous incisionless (sub-
The technical assistance by A. Lazaridis is greatly appreciated. cision) surgery for the correction of depressed scars and
wrinkles. Dermatol Surg 1995;21:543e9.
Conflict of interest 8. Camirand A, Doucet J. Needle dermabrasion. Aesthetic Plast.
Surg 1997;21:48e51.
9. Thomas JR. Effects of age and diet on rat skin histology.
None. Laryngoscope 2005;115:405.
10. Chapellier B, Mark M, Messaddeq N, et al. Physiological and
Funding retinoid-induced proliferations of epidermis basal keratino-
cytes are differently controlled. EMBO J 2002;21:3402e13.
11. Ghyselinck NB, Chapellier B, Calleja C, et al. Genetic dissec-
None.
tion of retinoic acid function in epidermis physiology. Ann
Dermatol Venereol 2002;129:793e9.
References 12. Gilchrest BA. Treatment of photodamage with topical
tretinoin: an overview. J Am Acad Dermatol 1997;36:27e36.
1. El Domyati M, Attia S, Saleh F, et al. Intrinsic aging vs. pho- 13. Nusgens BV, Humbert P, Rougier A, et al. Topically applied
toaging: a comparative histopathological, immunohistochem- vitamin C enhances the mRNA level of collagens I and III, their
ical, and ultrastructural study of skin. Exp Dermatol 2002;11: processing enzymes and tissue inhibitor of matrix metal-
398e405. loproteinase 1 in the human dermis. J Invest Dermatol 2001;
2. Dzubow. The aging face. In: Coleman WP, Hanke WC, Alt TH, 116:853e9.
Asken S, editors. Cosmetic surgery of the skin. Philadelphia PA: 14. Gilchrest BA. A review of skin ageing and its medical therapy.
B.C.Decker Inc.; 1991. p. 1e12. Br J Dermatol 1996 Dec;135:867e75.
3. Uitto J, Bernstein EF. Molecular mechanisms of cutaneous 15. Bernstein EF, Underhill CB, Hahn PJ, et al. Chronic sun expo-
aging: connective tissue alterations in the dermis. J Investig sure alters both the content and distribution of dermal
Dermatol Symp Proc 1998;3:41e4. glycosaminoglycans. Br J Dermatol 1996;135:255e62.
4. Aust MC, Fernandes D, Kaplan HM, et al. Collagen induction 16. Man XY, Yang XH, Cai SQ, et al. Immunolocalisation and
therapy an alternative treatment for scars, wrinkles and skin expression of vascular endothelial growth factor receptors
laxity. Plast Reconstr Surg 2008;121:1421e9. (VEGFRs) and neutrophilins (NPRs) on keratinocytes in human
5. Aust MC, Reimers K, Repenning C, et al. Percutaneous collagen epidermis. Mol Med 2006;12:127e36.
induction: minimally invasive skin rejuvenation without risk of 17. Wilgus TA, Matthies AM, Radek KA, et al. Novel function for
hyperpigmentation-fact or fiction? Plast Reconstr Surg 2008; vascular endothelial growth factor receptor-1 on epidermal
122:1553e63. keratinocytes. Am J Pathol 2005;167:1257e66.