Research

Aesthetic Surgery Journal

Effect of Use of Platelet-Rich Plasma (PRP) in 2017, 1–8

© 2017 The American Society for
Aesthetic Plastic Surgery, Inc.
Skin with Intrinsic Aging Process Reprints and permission:
journals.permissions@oup.com
DOI: 10.1093/asj/sjx137
www.aestheticsurgeryjournal.com

Luiz Charles-de-Sá, MD; Natale Ferreira Gontijo-de-Amorim, PhD;

Christina Maeda Takiya, PhD; Radovan Borojevic, PhD;
Donatella Benati, MD; Paolo Bernardi; Andrea Sbarbati, PhD; and
Gino Rigotti, MD

Abstract
Background:  In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or
expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in
the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous
component.
Objectives:  The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the
retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results.
Methods:  This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injec-
tion was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron
microscopy.
Results:  After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with
a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found
at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed.
Conclusions:  Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and
microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process.

Level of Evidence: 4

Editorial Decision date: June 28, 2017.

The aging process of the skin is a complex biological phe-

nomenon that can be divided into intrinsic and extrin-
sic aging. The facial skin aging process is a degenerative
process that affects the skin and deep structures of the
face, resulting from the action of the intrinsic factor (age)
associated with the extrinsic factor, particularly exposure
to ultra-violet radiation. Intrinsic aging, which is largely
genetically determined, affects the skin in a manner similar
to that of most internal organs1 through a slow and partly
reversible degeneration of connective tissue. Otherwise,
extrinsic aging, more commonly termed photoaging,
caused by environmental exposure, primarily ultraviolet
Dr Charles-de-Sá is a PhD student, Dr Takiya is a Researcher and
Professor, and Dr Borojevic is a Researcher, Postgraduate Program
in Surgical Science, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil. Dr Gontijo-de-Amorim is an Invited Researcher, Drs
Benati and Bernardi are Researchers, and Dr Sbarbati is a Professor
and Chief, Department of Neurological and Motor Science, Section
of Anatomy and Histology, University of Verona, Verona, Italy.
Dr Rigotti is the Chief, Regenerative Surgery Unit, San Francesco
Clinic, Verona, Italy.
Corresponding Author:
Dr Gino Rigotti, Clinica San Francesco, Via Monte Urtigara 21/B
37127, Verona, Italy.
Email: ginorigotti@libero.it
2
radiation, leads to a premature aging phenotype even in
young individuals.2 In sun-exposed areas, these two pro-
cesses, intrinsic and extrinsic, are superimposed; there is
evidence3 that they have at least in part overlapping, bio-
logical, biochemical, and molecular mechanisms.4
The connective tissue of the skin is composed mostly
of collagen and elastin. Collagen makes up 70% to 80% of
the dry weight of the skin and gives the dermis its mechan-
ical and structural integrity. Elastin is a minor component
of the dermis, but it has an important function in providing
the elasticity of the skin, and elastin accounts for 2% to 4%
of the extracellular matrix.5 In the photoaged skin, there is
a progressive destruction of the entire network of elastin in
the dermis. Elastic fibers become thickened, tangled, tortu-
ous, degraded, and dysfunctional, with increased density
of the elastic material, resulting in a cluster of amorphous
and dystrophic elastotic materials throughout the dermis,
setting up solar elastosis, the most important feature of
photoaging.5 This is caused by the marked loss of collagen
and the thickening of elastic fibers, causing a higher level
of the elastic component in the elderly than the young.6
Regenerative medicine is an emerging field whose focus
is on cell- and tissue-based therapies applicable to plastic
surgery procedures, including the aging process of skin.6
The mechanism transforms undifferentiated stem cells
into the differentiated cells that ultimately form the spe-
cialized tissues that are required for reconstruction, repair,
and restoration, including the role of the surrounding envi-
ronment.7 Moreover, a stem cell has paracrine effects and
secretes a wide range of growth factors, cytokines, and bio-
active molecules that can stimulate tissue regeneration.7
In this sense, platelet-rich plasma (PRP) has also been
described as having a possible regenerative effect due to its
growth factors and cytokines, however empirical, without
scientific evidence until now.8-11
In a previous paper,6 we demonstrated that the treat-
ment of human photoaged skin (preauricular area) with
the autologous lipidic component plus stromal vascular
fraction rich in adipose-derived stem cells (SVF-enriched
fat) or expanded adipose-derived stem cells (ADSCs)
showed a decrease in the elastic fiber network (elasto-
sis) and the appearance of new oxytalan elastic fibers in
papillary dermis. The ultrastructural examination showed
a modified 3-dimensional architecture of the reticular
dermis and the presence of a richer microvascular bed,
representing a skin rejuvenation effect. Considering that
expanded stem cells require a cell factor, we developed
another study12 in which we wished to assess similar
results replacing the ADSC by PRP. PRP is easier to obtain
and could be utilized in an ambulatory environment. We
aimed to determine if PRP injection could replace the cuta-
neous regenerative effect of ADSCs when injected together
with fat in the preauricular area, with photoaging charac-
teristics. We observed that the use of fat plus PRP led to
Aesthetic Surgery Journal
the presence of more pronounced inflammatory infiltrates
and a greater vascular reactivity, increasing in the vascular
permeability and a certain reactivity of the nervous com-
ponent. The addiction of PRP to fat did not improve the
regenerative effect and did not present significant advan-
tages over the use of expanded ADSC or SVF-enriched fat
in skin rejuvenation.12
The purpose of this new article was to analyze the his-
tologic and ultrastructural changes of skin after the injec-
tion of only PRP in the retroauricular area (mastoidea area)
that normally suffered only the intrinsic aging process,
because it was not exposed to sun and did not present the
photoaging process. We observed the histologic alterations
due to the intrinsic aging process and observed the action
of PRP in this area.
METHODS
The study was executed in 13 patients, 11 females and 2
males, who were candidates for facial rejuvenation surgery
(facelift), whose ages ranged between 45 and 65 years
with no history of chronic viral, metabolic, ischemic, or
autoimmune diseases, or other systemic pathologies. This
study also excluded patients who reported a smoking
habit. The study period ranged from May 2012 through
May 2016.
The patients were subject to the sampling of blood to
prepare the PRP, as described below. The injection of PRP
was performed in the retroauricular area. Prior to injec-
tion, with the patient under local anesthesia, a fragment of
skin and subcutaneous tissue was removed from the mas-
toidea area and sent to the Section of Human Anatomy and
Histology of Verona (Italy) and the Federal University of
Rio de Janeiro (Brazil) for morphological analysis through
optical and electron microscopy. At histology, the anal-
ysis of skin biopsies was performed through hematoxy-
lin and eosin (HE) staining, picrosirius red staining (for
visualization of collagen), and orcein staining (for visual-
ization of elastic fibers). The morphological examination
was repeated after 3 months, taking a piece of skin and
subcutaneous tissue in the same area where the PRP was
injected. After this second biopsy, the facelift was per-
formed. The regenerative anti-aging activity was evaluated
by comparing the morphology of the skin and subcutane-
ous area before and after treatment.
The biopsy specimens, consisting of fragments of skin
measuring 0.5 cm × 1 cm, were limited inside of the 1 cm2
retroauricular area (mastoid) and split into two equal parts
of 0.5 cm × 0.5 cm. One half was sent in a plastic bottle
immersed in 4% formaldehyde to the Institute of Biophysics
Carlos Chagas Filho, Laboratory of Immunology, Federal
University of Rio de Janeiro (Rio de Janeiro, Brazil) for
histologic study. The other half (0.5 cm × 0.5 cm) was
sent in a plastic bottle with a solution of buffered 2%
Charles-de-Sá et al 3

glutaraldehyde to the Section of Human Anatomy and process, samples were positioned in a multi-well grid for
Histology of Verona (Verona, Italy) to be subjected to electron microscopy and observed through the use of a
histomorphometric analysis with electron microscopy. The TEM Morgagni 268D (FEI Philips).
skin biopsies (untreated and treated skin biopsies) were
performed under local anesthesia with lidocaine 0.5% and
1:200.000 epinephrine, on an outpatient basis.
Scanning Electron Microscopy (SEM)
All patients consented under approved guidelines set The specimens were fixed in 2% glutaraldehyde in a phos-
forth by the human clinical trial to the Brazilian investiga- phate buffer for 2 to 4 hours, post-fixed in 1% osmium
tion ethical committee board (protocol no. 28063) and the tetroxide in the same buffer for 1 hour, and dehydrated in
Brazilian Clinical Trials Registry (Universal Trial Number, graded concentrations of acetone. The samples were then
U1111-1145-3081). treated by critical point dryer (CPD 030, Balzers Vaduz,
Liechtenstein), mounted on metallic specimen stubs, and
sputter-coated with gold (MED 010 Balzers). SEM Imaging
Platelet-Rich Plasma Preparation
was performed with XL30 ESEM (FEI-Philips Eindhoven,
Peripheral blood was collected in blood collection tubes Netherlands).
containing 0.5 ml 3.2% sodium citrate solution. The PRP
preparation procedure consisted of two centrifugation
steps. All steps were performed in a refrigerated centrifuge RESULTS
(certified Jouan Br4i, Saint-Herblain, Loire-Atlantique,
The present study was executed in 13 patients, 11 females
France). Whole blood was centrifuged at 300 g for 5 min-
and 2 males, age ranging between 45 and 65 years old
utes at 18°C. After the first centrifugation, the whole
(mean, 56.2 years), through the analysis of the retroauric-
plasma above the buffy coat was collected, separating
ular skin (mastoidea area) that normally is protected from
platelets from red blood cells and leukocytes (PRP1). PRP1
the sun. No elastosis was observed before the use of PRP.
was centrifuged at 700 g for 17 minutes at 18°C. After
Therefore, we can conclude that there is only an intrinsic
the second centrifugation step, the platelet pellet was
aging process in this area (Figure 1).
suspended in 300 μl of platelet-poor plasma (PPP; new
As described in the introduction, we wrote two previ-
fraction named PRP2). Platelet activation was performed
ous papers pertaining to preauricular skin. This figure of
adding 20 mM CaCl2 (PRP2-Ca) and 25 IU/ml human
preauricular skin was utilized in the present paper with
plasma thrombin (PRP2-Thr, Ref: T6884; Sigma-Aldrich,
the aim of comparing the photoaged skin (preauricular)
St. Louis, Missouri, USA), incubating at 37°C for 1 hour
with the protected skin, from the retroauricular area,
and at 4ºC for 16 hours. To recover the activated PRP2, all
of the same patient. Thus, this information is import-
of the treated samples were centrifuged at 3,000 × g for 20
ant to readers understand the cutaneous quality of the
minutes at 18°C. All samples were stored at –80°C.8
retroauricular area.
Different PRP preparations have been published, elic-
The treatment with PRP induced modifications mainly
iting variable responses that cannot be compared, due to
in the reticular dermis. After the use of PRP, a great
the lack of the final platelet quantification. This prepara-
increase of the thickness of the reticular dermis, which
tion, published by Amable et al,8 optimized PRP prepa-
resulted from the horizontal deposition of layers of mature
ration and maximized platelet recovery, 46.9% to 69.5%
elastic fibers and collagen, was observed (Figure 2). In
of initial platelets, and the platelets were highly purified.
this layer, activated fibroblasts were also visible. The great
The blood sample was collected in a 5-ml tube containing
increase of elastic and collagen fibers and the morphology
3.2% sodium citrate. A blood sample for all 13 patients
of the fibroblasts suggest that in some areas a fibrotic reac-
was stored and then analyzed in the laboratory before
tion occurs.
injection in the patient. It was injected in intradermal level
At the dermohypodermal junction, before the treatment
of the retroauricular skin.
with PRP, the adipocytes located at the boundary of the
dermis and the subcutaneous tissue were organized in lob-
ules, and the lobules were often vertically directed toward
Transmission Electron Microscopy (TEM)
the surface of the skin. These lobules formed adiposae
Different samples were fixed with glutaraldehyde 2% in papillae, in which a vascular peduncle was often seen. The
a Sorensen buffer pH 7.4 for 2 hours, post-fixed in 1% papillae, in their apical portion, often showed a relation-
osmium tetroxide in aqueous solution for 2 hours, dehy- ship with sebaceous or sweat glands. At this level, the adi-
drated in graded concentrations of acetone, embedded in pocytes were covered by a dense basket of collagen strictly
Epon-Araldite and cut with an Ultracut E ultramicrotome adherent to their plasma membrane. After the use of PRP,
(Reichert, Wien, Austria). At the end of the dehydrating the relationship between subcutaneous fat and sebaceous
4 Aesthetic Surgery Journal

A B

Figure 1.  Light microscopy, orcein stain. The figure shows the structural differences between the preauricular skin
(photoaging skin) and the retroauricular skin (protected skin). (A) Preauricular skin with the presence of elastic fibers stained
in black (asterisk). (B) The retroauricular skin of the same patient appears poor in elastic fibers (lesser material stained in
black). Scale bars: 150 µm.

or sweat glands appears less close, after the reticular der-
mis increases and forms a kind of barrier between these
elements (Figures 2-4).
At ultrastructural examination, after treatment with
PRP, perivascular areas with mononuclear cell infiltration
were visible in the reticular dermis and the dermohypo-
dermal junction (Figure 3). Often the blood vessels (blood
capillaries and arterioles) were surrounded by reduplicated
and fragmented layers of basal lamina (Figure 5). The
ultrastructural examination also confirmed the histologic
data, revealing a large amount of elastic fibers and colla-
gen mainly disposed parallel to the skin surface. Collagen
fibers were disposed in compact bundles. Ultrastructural
examination also revealed the presence of activated fibro-
blasts with a large amount of rough endoplasmic reticu-
lum and large lysosomes. These findings suggest that PRP
causes fibrosis in the area where it is injected (Figure 4
and 5).
DISCUSSION
The retroauricular skin has its own characteristics. It is a
thin and non-hair-bearing skin and has skin-color match-
ing. The retroauricular skin shows a thin stratum cor-
neum, the dermo-epidermal junction is rather flat, and the
connective papillae were poorly developed. The dermal
papillae are also thin and showed a scarce cellularity. The
reticular dermis is homogeneous and appeared as a regu-
lar network of collagen and elastin fibers disposed in thin
bundles. Scanning microscopy showed the elastin fibers as
thin ribbons with smooth surface. The subcutaneous adi-
pose tissue is present in a variable amount and generally
formed elongated lobules mainly disposed parallel to the
external surface. The pilo-sebaceous structures, as well as
the sweat glands, are scarce. In the male patients, a larger
amount of sebaceous glands is evident and appeared larger
in diameter than those visible in female patients.13,14
During aging, degenerative processes affecting the skin
and deep structures of the face commonly occur. This aging
process is the result of the action of intrinsic skin events
associated with extrinsic agents, especially exposure to
ultraviolet radiation. Normally, the retroauricular skin is
protected from the sun and does not suffer the extrinsic
aging process that leads to elastosis and actinic pathol-
ogy. The age-related modifications extensively described
in the elastic fibers of all organs and tissues may be largely
interpreted as resulting from progressive degradation of a
protein polymer that has been produced early in life.15-21
In previous papers,6,12 we demonstrated the regenera-
tive effect of SVF-enriched fat and expanded ADSC when
injected in preauricular skin with the photoaged process.
When the fat plus PRP was injected in the same place,
the presence of more pronounced inflammatory infiltrates
and a greater vascular reactivity was observed, increasing
Charles-de-Sá et al 5

A B

C D

Figure 2.  Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin
before and after PRP injection. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis with the
subcutaneous tissue organized in lobules, which are directed vertically toward the surface of the skin (stars). These lobules
form adiposae papillae, which in their apical portion show a relationship with sebaceous or sweat glands. (C, D) After the use
of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers
(asterisks) are increased. A small flogistic infiltrate is present (arrows). Scale bars: 300 µm.

in vascular permeability and a certain reactivity of the
nervous component. The addiction of PRP to fat did not
improve the regenerative effect and did not present sig-
nificant advantages over the use of expanded ADSCs or
SVF-enriched fat in skin rejuvenation.12 However, PRP has
been described as having a possible regenerative effect due
to its growth factors and cytokines, although the previous
evidence is empirical.8-11,22-24
Considering the results, we observed that treatment
with PRP induced modifications in the reticular dermis,
due to the primarily horizontal deposition of layers of
collagen and elastic fibers. The great increase of colla-
gen suggests a fibrotic reaction, because there are acti-
vated fibroblasts in this layer. This fibrosis creates a kind
of barrier between the sebaceous sweat glands and sub-
cutaneous fat tissue. At the dermohypodermal junction,
before treatment with PRP, the adipocytes located at the
boundary of the dermis and the subcutaneous tissue were
organized in lobules, which often were vertically directed
toward the surface of the skin. These lobules formed adi-
posae papillae, in which a vascular peduncle was often
seen. The papillae, in their apical portion, often showed a
relationship with sebaceous or sweat glands. At this level,
the adipocytes were covered by a dense basket of collagen
strictly adherent to their plasma membrane. At ultrastruc-
tural examination, after treatment with PRP, perivascu-
lar areas with mononuclear cell infiltration were visible
in the reticular dermis and the dermohypodermal junc-
tion. These areas showed blood capillaries and arterioles
surrounded by mononuclear infiltrates. Often the blood
vessels were surrounded by reduplicated and fragmented
layers of basal lamina. Elastic fibers and collagen are hor-
izontal and strongly attached, and the presence of lyso-
somes and activated fibroblasts suggests that PRP causes
fibrosis in the area where it is injected.
The limitation of this study is that PRP injections cur-
rently occur into photoaged skin, so this aspect, not inves-
tigated in this paper, could warrant new research.
Otherwise, we published a previous paper12 in photo-
aged skin that was treated with fat plus PRP, in which
we observed an inflammatory reaction and no regenerative
effect as the ADSC demonstrated.
6 Aesthetic Surgery Journal

A B

C D

Figure 3.  Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before
and after PRP injection. In a different patient, the results are similar to those visible in the patient illustrated in Figure 2,
indicating the reproducibility of the findings. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis,
with the subcutaneous tissue organized in lobules (stars), which are vertically directed toward the surface of the skin. A small
quantity of elastic fibers is present. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular
dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. Scale bars: 300 µm.

It is not possible to do quantitative analysis in small
fragments of skin tissue. Reproduction of the same mor-
phology and the same phenomena in all patients treated
with PRP allows us to justify our statements by comment-
ing on the results obtained.
CONCLUSIONS
After the treatment with PRP, we observed an increase of
reticular dermis thickness due to the deposition of elas-
tic fibers and collagen, with a fibrotic aspect. A modified
pattern of adipose tissue was also found at the dermohy-
podermal junction. Significative regenerative aspects were
not found at histologic and ultrastructural analysis in our
experimental conditions. The presence of long-term foci of
moderate inflammation and microangiopathy could lead
to trophic alteration of the skin and a precocious aging
process, because an inflammatory process induces fibro-
sis which is an antithetic condition to the morphological
integrity of each tissue and organ.
This paper is a part of a series of human face skin stud-
ies that we are conducting during recent years. The interest
in this approach arises from the fact that previous investi-
gations have been done on animals or human skin of other
districts, but not on the face. These extra face regions have
been found to be sensitive to stem cells or adipose tissue.
It is therefore of the utmost interest to know what changes
are induced by PRP injected into the face. The present
study provides important information on the biological
effect of PRP on this district. The most obvious evidence
is that we have not recorded very remarkable phenomena
both in its regenerative or degenerative sense. However, the
data derived from the ultrastructural examination reveal
phenomena that seem to indicate a stimulation of connec-
tive fiber deposition together with modest inflammatory
aspects. Our approach has obvious limits that arise from
the analysis of small samples. In addition, on the one hand,
the ultrastructural approach chosen gives an exact defini-
tion of the phenomena occurring at the dermal level, yet
on the other hand, this results in a substantial impossibility
Charles-de-Sá et al 7

A B

Figure 4.  Scanning electron microscopy. (A) Retroauricular skin after treatment with PRP at low (A) enlargement. The dense
dermis (square) is visible below the epidermis (e). (B) At medium enlargement, the deposition of elastic and collagen fibers
parallel to the skin surface is visible. (C) The fibers are organized in compact bundles (asterisk). Scale bars: A, 500µ; B, 200µ;
C, 5 µm.

A B

Figure 5.  Transmission electron microscopy. Retroauricular skin after the use of PRP. (A) Duplication of the vascular basal
membrane (d). (B) Activated fibroblasts (f) with a rich organular pattern surrounded by thick bundles of collagen fibers (c).
Scale bars: A, 2 µm; B, 4 µm.

to determine the exact amount of newly formed fibers.
However, the comparison of samples from the same patient
in the same area at different times on a significant pop-
ulation clearly leads to the ultimate conclusion that PRP
injected in the facial skin for regenerative purposes induces
modest inflammatory processes. In our view, this aspect
has to be further investigated to determine, for example,
whether these phenomena are lasting or temporary. And
just considering these two possibilities, we must therefore
investigate what could happen in both cases.
In conclusion, we have verified these phenomena and
we report them through a rigorous scientific approach.
8
Acknowledgments
The authors would like to thank the Institute of Biophysics
Carlos Chagas Filho, Laboratory of Immunology, Federal
University of Rio de Janeiro (Rio de Janeiro, Brazil) for insti-
tutional support.
Disclosures
The authors declared no potential conflicts of interest with
respect to the research, authorship, and publication of this
article.
Funding
The authors received no financial support for the research,
authorship, and publication of this article.
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