548212

research-article2014
AESXXX10.1177/1090820X14548212Aesthetic Surgery JournalNowacki et al

AL CON
ON

TR
INT ATI

IBUTION
ERN
Research

Aesthetic Surgery Journal

Filling Effects, Persistence, and Safety 2014, Vol. 34(8) 1261­–1269

© 2014 The American Society for
Aesthetic Plastic Surgery, Inc.
of Dermal Fillers Formulated With Reprints and permission:
http://www​.sagepub.com/

Stem Cells in an Animal Model journalsPermissions.nav

DOI: 10.1177/1090820X14548212
www.aestheticsurgeryjournal.com

Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020

Maciej Nowacki, MSc; Katarzyna Pietkun, MSc; Marta Pokrywczyńska, PhD;
Marta Rasmus, BSc; Karolina Warda, BSc; Tomasz Kloskowski, PhD;
Arkadiusz Jundziłł, MD, PhD; Maciej Gagat, MSc; Alina Grzanka, PhD;
Magdalena Bodnar, PhD; Andrzej Marszałek, MD, PhD; Tomasz Drewa, MD, PhD;
and Rafał Czajkowski, MD, PhD

Abstract
Background: Research is scarce regarding the effectiveness of dermal fillers containing autologous stem cells.
Objectives: The authors sought to determine the local and systemic effects of adipose-derived stem cells (ADSCs) as a component of dermal fillers
in an animal model.
Methods: Wistar rats were injected with 1 of the following dermal fillers: ADSCs combined with hyaluronic acid (ADSC-HA), ADSCs combined with
fish collagen (ADSC-COL), HA alone (CONTROL-HA), or COL alone (CONTROL-COL). Fillers were injected into the glabella, dorsum, and chest of each
animal. The ADSCs were labeled with PKH26 to assess cell migration. Filling effects (FEs) were measured immediately after injection and at 1.5 months
and 3 months after injection. Skin specimens were stained with hematoxylin and eosin to assess localization and persistence of ADSCs.
Results: Mean FEs in animals implanted with ADSCs were greater and persisted longer than those of controls. No inflammatory responses were
observed in any group. Three months after injection, PKH26-positive cells comprised nearly 70% of cells at the injection site in animals treated with ADSC-
HA. PKH26 fluorescence also was detected in the spleen but not in the brain, kidney, or lung.
Conclusions: Stem cells have the potential to improve the aesthetic effects and longevity of dermal fillers.

Keywords
dermal fillers, stem cells, soft-tissue augmentation, hyaluronic acid, animal model

Accepted for publication April 14, 2014.

Dermal fillers include a variety of specialized chemical and
biologic substances that are administered to improve the
appearance of the skin surface. In dermatology and aes-
thetic medicine, dermal fillers may be utilized when
patients wish to improve their appearance or for numerous
other clinically appropriate instances, such as nonsurgical
rhinoplasty or treatment for acne scaring. Treatment with
dermal fillers is a type of soft-tissue augmentation1-3 that
comprises 2 clinical categories, depending on the type of
application: (1) operative (invasive) applications of filling
substances or small implants, commonly performed by
plastic or craniofacial surgeons, and (2) injectable
preparations administered by dermatologists.4-9 The latter
category is the subject of the present study.
The first administration of a dermal filler was described
by a German physician, Gustav Adolf Neuber, at the 22nd
meeting of the Deutschen Gesellschaft für Chirurgie in
April 1893.4 Neuber presented an innovative method of
filling deep facial scars with fat grafted from the upper
arm. Numerous dermal fillers have been introduced since
then, but their clinical effects have varied. Some fillers
have been controversial and associated with complica-
tions, whereas others have produced insufficient or unsat-
isfactory clinical results.10 Paraffin and beeswax were
1262 Aesthetic Surgery Journal 34(8)

administered as fillers in the beginning of the 20th century,
and silicone formulations emerged in the mid-1950s.
Collagen fillers were employed in the late 1970s, and the
revolutionary hyaluronic acid (HA) products were intro-
duced in the early 1980s. Hyaluronic acid currently is
regarded as a first-line filling product.10-15 However, several
investigators have noted significant limitations of dermal
fillers, including foreign body reaction and longevity, and
have suggested that the ideal dermal filler does not yet
exist.16-19
Innovations in regenerative medicine and tissue engi-
neering have advanced numerous branches of science
Isolation, Validation, and Labeling of
ADSCs
Adipose-derived stem cells were isolated from adipose tis-
sue harvested from the retroperitoneal space of 5 donor
rats, as described previously.26 Briefly, adipose tissue (1 g)
was digested in collagenase type I solution (1 mg/mL;
Sigma-Aldrich, St Louis, Missouri) for 30 minutes at 37°C
with shaking. The reaction was stopped by the addition of
Dulbecco’s modified Eagle’s medium (DMEM; PAA,
Austria) supplemented with 10% fetal bovine serum (FBS;
PAA) and antibiotics (PAA). The cell suspension was fil-

Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020

and medicine and are applicable to many popular medi-
cal procedures. The implementation of stem cells has
evoked tremendous enthusiasm among clinicians.20-24 In
the present study, we aimed to determine the local and
systemic effects and utility of adipose-derived stem cells
(ADSCs) as a component of dermal fillers in an in vivo rat
model.
Methods
Animals
This study included twenty 10-week-old inbred Wistar rats
and 5 donor Wistar rats, from which ADSCs were isolated.
All experiments were approved by and conducted in accor-
dance with the local ethical committee associated with the
University of Technology and Life Sciences in Bydgoszcz,
Poland, which is a legally established official committee
for the Bydgoszcz district. Our work involving experimen-
tal animal models was conducted in compliance with the
guidelines of the European Union (Directive 2010/63/EU)
and other representative recommendations such as the
Guide for the Care and Use of Laboratory Animals of the
National Research Council.25
Animals were divided into 4 groups according to the
dermal filler received, as follows: ADSCs combined with
HA (ADSC-HA; n = 7), ADSCs combined with an experi-
mental filler prepared from fish collagen (ADSC-COL; n =
7), HA alone (CONTROL-HA; n = 3), or COL alone
(CONTROL-COL; n = 3).
tered through a 100-µm cell strainer (BD Biosciences,
Franklin Lakes, New Jersey) and was centrifuged at 350g
for 5 minutes. Viable cells were counted by trypan blue
exclusion and seeded on a 25-cm2 flask at a density of
15 000 cells/cm2. The ADSCs were cultured according to
standard protocols in DMEM supplemented with 10% FBS,
fibroblast growth factor (10 ng/mL; Sigma-Aldrich), peni-
cillin (100 U/mL), streptomycin (100 µg/mL), and ampho-
tericin B (5 µg/mL) (PAA) at 37°C, 5% CO2, and 95%
humidity until the third passage.
To confirm the phenotype of ADSCs, flow cytometric
analysis of cellular antigens was performed. Detached cells
from the third passage were washed and resuspended with
phosphate-buffered saline (PBS). Approximately 1 × 106
cells were incubated for 30 minutes with monoclonal anti-
bodies against CD34 (Santa Cruz Biotechnology, Santa
Cruz, California; catalog no. sc-7324 PE; 20 μl/sample),
CD44 (Millipore, Billerica, Massachusetts; catalog no.
CBL1508F; 10 μl/sample), CD45 (BD Biosciences; catalog
number 554877; 0.06 µg/sample), or CD90 (Millipore; cat-
alog number CBL1500F; 10 µL/sample) and conjugated
with phycoerythrin (PE) or fluorescein (FITC).
The expression of cell-surface markers was quantified
using an Epics XL flow cytometer (Beckman Coulter,
Fullerton, California). The ADSCs were induced to differ-
entiate into adipogenic, osteogenic, or chondrogenic lin-
eages by culturing in the appropriate media according to
the manufacturer’s instructions (Invitrogen, Carlsbad,
California). Negative-control cells were maintained in
DMEM/Ham’s F-12 medium supplemented with 10% FBS

Mr Nowacki is an MD and PhD student, Dr Jundziłł is a volunteer, Dr Pokrywczyńska and Dr Kloskowski are research fellows, and Ms Rasmus
and Ms Warda are MSc students in the Chair of Regenerative Medicine, Department of Tissue Engineering, Nicolaus Copernicus University
Collegium Medicum in Bydgoszcz, Poland. Mr Gagat is a research fellow and Dr Grzanka is Professor and Head of the Department of Histology
and Embryology, Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland. Dr Bodnar is a research fellow in the Department of
Clinical Pathomorphology, Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland, and Dr Marszałek is Professor and head of the
Department of Clinical Pathomorphology, Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland, and Department of Oncologic
Pathology, Poznan University of Medical Sciences and Greater Poland Oncology Center, Poznan, Poland. Ms Pietkun is an MD and PhD student in
the Chair of Regenerative Medicine, Department of Tissue Engineering and Dermatology, Sexually Transmitted Diseases and Immunodermatology,
Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland. Dr Czajkowski is a Professor and Head of the Department of Dermatology,
Sexually Transmitted Diseases and Immunodermatology, Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland. Dr Drewa is
Professor and Head of the Chair of Regenerative Medicine, Department of Tissue Engineering, Nicolaus Copernicus University Collegium Medicum
in Bydgoszcz, Poland, and Head of the Urology and Oncological Urology Department, Nicolaus Copernicus Hospital, Toruń, Poland.

Corresponding Author:
Mr Tomasz Kloskowski, Karlowicza 24, 85-092 Bydgoszcz, Poland.
E-mail: tomaszkloskowski@op.pl
Nowacki et al 1263
and antibiotics. Adipogenesis was assessed as the accumu-
lation of neutral lipids in fat vacuoles stained with Oil Red
O (Sigma-Aldrich). Osteogenesis was confirmed by von
Kossa staining (Bio-Optica, Milan, Italy). Chondrogenic
differentiation was assessed by anti–collagen type II immu-
nocytochemical staining (clone 6B3, 1:100; Millipore) for
16 hours at 4°C.
Prior to injection, ADSCs were labeled with the fluoro-
chrome PKH26 (labeling kit from Sigma-Aldrich) to assess
migration in vivo.
Preparation and Injection of Dermal
Fillers
To prepare the dermal fillers, 106 ADSCs were suspended
by reciprocal mixing in 1 mL of cross-linked HA with high
viscosity (Revanesse Ultra, Prollenium Medical Technologies,
Inc, Ontario, Canada) or in an experimental filler con-
taining fish collagen (Collife, Poznań, Poland). The ADSCs
were omitted from control fillers (CONTROL-HA,
CONTROL-COL).
Fillers and control substances were injected according
to current dermatologic and aesthetic medical standards of
practice and representative guidelines. Animals were pre-
pared for injection by shaving and disinfecting the skin
site, delineating the injection area with a red marker, and
applying EMLA cream per the manufacturer’s instructions
(lidocaine 2.5%, prilocaine 2.5%; AstraZeneca, Waltham,
Massachusetts). One researcher (M.N.) performed all
injections at the same time of day. Animals were injected
through a syringe connected to a sterilized 13-mm,
30-gauge needle into the glabella, dorsum, and chest
regions. All animals were administered the same dose of
filler (1 mL) and received injections at all 3 anatomic sites.
Fillers were injected slowly into the dermis, via the linear
tracking technique, with overcorrection for improved mac-
roscopic assessment (Figure 1). Overcorrection enabled us
to obtain accurate, noninvasive measurements with cali-
pers. To our knowledge, this is the most appropriate
approach for evaluating this type of rat model.
Assessment of Filling Effects
All animals were sacrificed by anesthesia overdose with
ketamine and xylazine (Sedazin and Bioketan, Biowet,
Puławy, Poland) after 3 months of follow-up. The monitor-
ing period was selected to maximize detection of PKH26
fluorescence, which has a half-life in vivo of approximately
100 days. The filling effects (FEs) of the injected substances
were observed macroscopically upon injection and at 1.5
months and 3 months after injection. FEs were represented
as the largest width of the overcorrected area measured
with standard calipers.
Histologic Analysis
Tissue specimens were fixed in 10% neutral-buffered for-
malin and embedded in paraffin. Cross sections of the ana-
lyzed areas were prepared and stained for histologic
analysis with hematoxylin and eosin.
Cell Migration Assay
Migration of ADSCs in the skin, brain, lungs, kidneys, and
spleen was determined immediately after animals were
sacrificed. Tissues were frozen in an optimal cutting
Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020
temperature compound (Tissue-TeK; Sakura, Torrance,
California), and 5-µm-thick sections were obtained.
Sections were washed with PBS, stained with DAPI (Sigma-
Aldrich) for visualization of cell nuclei, and mounted to
slides with Aqua-Poly/Mount coverslipping medium
(Polysciences, Warrington, Pennsylvania). Slides were
examined immediately after preparation by means of a C1
laser-scanning confocal microscope (Nikon Corp, Tokyo,
Japan) with a ×20 objective, a 405-nm diode laser, and a
543-nm HeNe laser. Double-labeled images were collected
with EZ-C1 software (v3.80; Nikon) at the brightest PKH26
signals. PKH26 is a red fluorochrome with excitation (551
nm) and emission (567 nm) characteristics similar to rho-
damine or phycoerythrin detection systems.
Statistical Analysis
Filling effects were represented as mean ± standard devia-
tion (SD) for each group. The t test was applied to compare
differences in FEs between groups and over time. Differences
among more than 2 groups were ascertained by 1-way
analysis of variance followed by Tukey’s post hoc test for
multiple comparisons. Statistical significance was defined
as P < .05.
Results
Following injection, no topical or systemic complication
was noted in any animal, and no atypical situations
occurred to necessitate medication or exclusion of any ani-
mal from the study. We did not observe any changes in
animal behavior or circadian rhythm. No pathologic
changes or lesions of the skin were noted.
Filling effects were calculated by averaging the caliper
measurements at all 3 anatomic sites in all animals of each
group. For both the ADSC-HA and CONTROL-HA groups,
the mean FE immediately after injection was 0.50 cm.
After 1.5 months and 3 months, the mean FEs in the
ADSC-HA group were 0.40 cm and 0.31 cm, respectively
(Table 1). In the CONTROL-HA group, the mean FEs at 1.5
and 3 months were 0.37 cm and 0.30 cm, respectively
(Table 1). For both the ADSC-COL and CONTROL-COL
1264 Aesthetic Surgery Journal 34(8)

Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020

Figure 1.  Injection of dermal fillers. (A) Delineation of glabella, dorsum, and (B) chest sites for injection. (C) Application
of EMLA cream to glabella before filler injection. (D) Injection of dermal filler into dorsum. (E) Overcorrected filling effects
immediately after injection into the dorsum, (F) chest, and (G) glabella. (H) Persistence of filling effects 1.5 months after
injection with a formulation of adipose-derived stem cells and hyaluronic acid.
Nowacki et al 1265

Table 1.  Persistence of Dermal Fillers

FE at 1.5 mo, mm FE at 3 mo, mm P Value

I. ADSC-HA 0.40 ± 0.07 0.31 ± 0.08 <.001

II. CONTROL-HA 0.37 ± 0.08 0.30 ± 0.07 <.01

III. ADSC-COL 0.27 ± 0.06 0.14 ± 0.04 <.001

IV. CONTROL-COL 0.12 ± 0.05 0.08 ± 0.04 .03

Groups Compared, 1.5 mo Groups Compared, 3 mo

I vs III, P < .001 I vs III, P < .001

Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020

I vs IV, P < .001 I vs IV, P < .001

II vs III, P < .001 II vs III, P < .001

II vs IV, P < .001 II vs IV, P < .001

III vs IV, P < .001 III vs IV, P = .016

Results are presented as means ± standard deviations. ADSCs, adipose-derived stem cells; COL, fish collagen; CONTROL, fillers in which ADSCs were omitted; FE, filling effect (ie, the largest
width measured with calipers at the injection site); HA, hyaluronic acid.

Figure 2.  Hematoxylin and eosin staining of dorsal skin from an animal treated with dermal filler composed of adipose-
derived stem cells (ADSCs) and hyaluronic acid (HA). (A) ADSCs (arrow) seeded in HA are evident in stacks beneath the thin
layer of muscle cells (original magnification ×4). (B) No inflammatory response was observed. Arrows indicate ADSCs seeded
in HA (original magnification ×10). (C) ADSCs (between arrows) are visible in the region treated with dermal filler (original
magnification ×20).

groups, the mean FE immediately after injection was 0.38
cm. The FEs for the ADSC-COL group were 0.27 cm at 1.5
months and 0.14 cm at 3 months. In the CONTROL-COL
group at 1.5 and 3 months, the FEs were 0.12 cm and 0.08
cm, respectively (Table 1). Animals injected with a mixture
of ADSCs and HA demonstrated significantly larger FEs at
1.5 and 3 months (P < .001 vs all other groups).
Histologic analysis confirmed that cell morphology was
well preserved. The ADSCs incorporated into HA were
stacked immediately below the thin layer of muscle cells,
and no inflammatory response was observed (Figure 2).
Results of the cell migration assay indicated that the
proportions of cells exhibiting PKH26 fluorescence differed
in skin and organ specimens across groups. Among ani-
mals receiving ADSC-HA, 68.61% of cells were positive for
PKH26 at the injection sites 3 months after treatment
(Figure 3A). In contrast, PKH26 fluorescence was observed
in 60.62% of cells sampled from ADSC-COL animals
(Figure 3B). PKH26 fluorescence was noted in the spleens
of both groups (Figure 3C) but could not be detected in the
lungs, kidney, or brain (Figure 3D-F), suggesting that
ADSCs do not migrate chaotically after injection into a der-
mal filler preparation.
Discussion
Currently, 80% to 85% of all aesthetic procedures are min-
imally invasive, many of which are dermal filler treat-
ments. Despite the growing popularity of these fillers, they
have been associated with limited effectiveness and other
problems in aesthetic medicine and dermatology.27-29
The development of novel dermal fillers has involved
the incorporation of specific cell types as primary or sup-
plementary constituents. However, experimental research
1266 Aesthetic Surgery Journal 34(8)

Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020

Figure 3.  Migration of adipose-derived stem cells (ADSCs) labeled with PKH26 and injected as a dermal filler formulation.
Images were obtained 3 months after injection. (A) Compared with other dermal fillers, PKH26-positive cells were most
abundant in animals treated with ADSCs and hyaluronic acid (HA), comprising 68.61% of detectable fluorescence at
the injection sites. (B) In specimens collected from the injection sites of animals treated with ADSCs and fish collagen,
PKH26-positive cells comprised 60.62% of detectable fluorescence. (C) PKH26 fluorescence comprised 5.86% of detectable
fluorescence in spleen specimens from animals treated with ADSCs and HA. PKH26 expression was not detected in (D) lung,
(E) kidney, or (F) brain specimens of animals treated with ADSCs and HA. Images were obtained by light microscopy (original
magnification ×20).
Nowacki et al 1267
is scarce regarding fibroblasts or other types of stem cells
as dermal fillers, and few investigators have assessed cell
migration or described the practical and clinical applica-
tions of this type of cell therapy.30-33 We maintain that the
in vivo effects of cell-based dermal fillers cannot be estab-
lished until the persistence and cell migration of these fill-
ers are understood.34-36
We evaluated ADSCs combined with 2 filler substances
because cells suspended in standard saline or PBS failed to
produce any FEs (data not shown). Our approach enabled
assessment of the utility of HA and COL as ADSC carriers.
We evaluated ADSCs rather than stromal vascular fraction
(SVF) cells because ADSCs are established homogeneous
cells with well-known properties.37 Adipose-derived stem
cells proliferate rapidly with a few passages and exhibit a
stable phenotype after the third passage. These properties
allowed us to obtain a large number of ADSCs with a low
risk of culture-induced chromosomal abnormalities or tera-
toma formation because the latter typically is not associ-
ated with mesenchymal stem cells.38,39 The ADSCs also
have the potential to differentiate into multiple lineages.40,41
Although SVF cells are easier to obtain, these cells have
been associated with adverse events in humans, such as
cyst formation and microcalcifications.38 Our ADSC filler
preparations allowed for precise control of types and
amounts of filler components. Although ADSCs have been
evaluated in several clinical trials, peer-reviewed data on
ADSCs have been limited in the field of aesthetic medi-
cine.37 Adipose-derived stem cells have been applied suc-
cessfully to maxillary reconstruction and to treat fistulas of
cryptoglandular origin with or without Crohn disease.42-45
In the latter application, ADSCs were more effective than
stromal vascular cells.43
Our findings regarding the persistence of dermal fillers
are consistent with those of other in vivo studies. We
observed decreases in overcorrected FEs with time in all
study groups. Similarly, other investigators have reported
that the effects of experimental dermal fillers diminish
with time in humans and animal models.46,47 Our results
indicate that ADSC-based formulations of dermal fillers
produce greater FEs that persist significantly longer than
dermal fillers prepared without ADSCs. Other researchers
have observed trophic changes and modulation of the tis-
sue environment when mesenchymal stem cells were
applied to a reconstructed bladder wall in a rat model or
injected into human skin as a filler component.48-50 ADSCs
and their soluble factors function in protective and regen-
erative roles in the skin, inducing collagen synthesis,
inhibiting melanogenesis, and recruiting and protecting
dermal fibroblasts.51
Our cell migration assays and histologic analyses indi-
cate that ADSCs in HA were maintained in the vicinity of
the injection sites during the 3-month observation period,
supporting their localized activity. This phenomenon may
be explained by the incorporation of stem cells into the
cross-linked HA. The absence of uncontrolled pathologic
cell migration to other organs (brain, kidneys, or lungs)
supports the potential safety of ADSC-based dermal fillers
for clinical applications. When mesenchymal stem cells
were injected into the circulatory system, cell migration to
sites of inflammation and injury was observed.52 However,
cell migration is minimized when cells are implanted
directly into the injury site,48 as demonstrated in the pres-
ent study.
Mesenchymal stem cells secrete various bioactive trophic
and proangiogenic factors that enhance tissue regeneration
Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020
and neoangiogenesis, downregulate the inflammatory
response,53-55 and inhibit apoptosis.56 Minimal ADSC migra-
tion combined with extended viability after implantation
could prolong release of these factors, thereby increasing
the therapeutic effects on surrounding tissues. In clinical
practice, ADSC-based fillers could potentially stimulate tis-
sue regeneration, obviate frequent treatments, and extend
cosmetic effects.
Although the Wistar rat model utilized in our in vivo
study was appropriate for our present objectives, a nude
animal model might be preferable for subsequent studies
due to better continuous observation and measurement
possibilities of FEs. We demonstrated that ADSC-based
dermal fillers were safe in our rat model; however, we did
not examine biologic effects on the skin layers. This topic
should be evaluated in a larger series of animals to obtain
greater statistical power and validate the effectiveness of
our methods. If safety and effectiveness are confirmed in a
large study of animals, a prospective clinical study of
ADSC-based dermal fillers should be conducted.
Our research complements existing data regarding the
biologic effects of ADSC implantation, and our novel for-
mulation for a dermal filler may be applicable to dermatol-
ogy and aesthetic medicine. We expect that the development
and evaluation of stem cell–based procedures will be vital
to improvements in aesthetic surgery and dermatology,
especially when noninvasive methods are preferred.
Conclusions
Stem cells have the potential to become an essential com-
ponent of dermal fillers, improving and prolonging cos-
metic results and decreasing the complications and
ineffectiveness associated with minimally invasive aes-
thetic procedures. Our findings in a rat model support the
safety and effectiveness of fillers formulated with ADSCs
and HA. Subsequent investigations are needed to assess
the immunohistologic properties of these fillers.
Disclosures
The authors declared no potential conflicts of interest with respect
to the research, authorship, and publication of this article.
1268 Aesthetic Surgery Journal 34(8)
Funding
The authors received no financial support for the research,
authorship, and publication of this article.
References
1. Lemperle G, Romano JJ, Busso M. Soft tissue augmenta-
tion with Artecoll: 10-year history, indications, techniques,
and complications. Dermatol Surg. 2003;29:573-587.
2. Vedamurthy M. Standard guidelines for the use of der-
mal fillers. Indian J Dermatol Venereol Leprol. 2008;
74(suppl):S23-S27.
3. Alijotas-Reig J, Fernández-Figueras MT, Puig L. Late-onset
inflammatory adverse reactions related to soft tissue filler
injections. Clin Rev Allergy Immunol. 2013;45:97-108.
4. Van De Graaf RC, Kortweg SFS. Gustav Adolf Neuber
(1850-1932) and the first report on fat auto-grafting in
humans in 1893. Hist Plast Surg. 2010;1:7-11.
5. Marwah M, Kulkarni A, Godse K, Abhyankar S, Patil S,
Nadkarni N. Fat fulfillment: a review of autologous fat
grafting. J Cutan Aesthetic Surg. 2013;6:132-138.
6. Sharma P, Sharma S. Comparative study of a new dermal
filler Uma Jeunesse® and Juvéderm®. J Cosmet Dermatol.
2011;10:118-125.
7. Klein AW, Elson ML. The history of substances for soft
tissue augmentation. Dermatol Surg. 2000;26:1096-1105.
8. Breitenfeldt N, Vaingankar NV. Hyaluronic acid der-
mal filler for the treatment of oral incompetence. Plast
Reconstr Surg. 2010;126:76e-78e.
9. Vedamurthy M, Vedamurthy A, Nischal K. Dermal fillers:
do’s and don’t’s. J Cutan Aesthetic Surg. 2010;3:11-15.
10. Gilbert E, Hui A, Meehan S, Waldorf HA. The basic sci-
ence of dermal fillers: past and present. Part II: adverse
effects. J Drugs Dermatol. 2012;11:1069-1077.
11. Carruthers J, Cohen SR, Joseph JH, Narins RS, Rubin M.
The science and art of dermal fillers for soft-tissue aug-
mentation. J Drugs Dermatol. 2009;8:335-350.
12. Figueiredo VM. A five-patient prospective pilot study of
a polycaprolactone based dermal filler for hand rejuvena-
tion. J Cosmet Dermatol. 2013;12:73-77.
13. Price RD, Berry MG, Navsaria HA. Hyaluronic acid: the
scientific and clinical evidence. J Plast Reconstr Aesthetic
Surg. 2007;60:1110-1119.
14. Baumann LS, Shamban AT, Lupo MP, et al. Comparison
of smooth-gel hyaluronic acid dermal fillers with cross-
linked bovine collagen: a multicenter, double-masked,
randomized, within-subject study. Dermatol Surg.
2007;33(suppl 2):S128-S135.
15. Gold MH. Use of hyaluronic acid fillers for the treatment
of the aging face. Clin Interv Aging. 2007;2:369-376.
16. Burgess CM. Principles of soft tissue augmentation for the
aging face. Clin Interv Aging. 2006;1:349-355.
17. Santoro S, Russo L, Argenzio V, Borzacchiello A.
Rheological properties of cross-linked hyaluronic acid
dermal fillers. J Appl Biomater Biomech. 2011;9:127-136.
18. Bank DE. A novel approach to treatment of the aging
hand with Radiesse. J Drugs Dermatol. 2009;8:1122-1126.
19. Gold M. The science and art of hyaluronic acid dermal filler use
in esthetic applications. J Cosmet Dermatol. 2009;8:301-307.
20. Kloskowski T, Kowalczyk T, Nowacki M, Drewa T. Tissue
engineering and ureter regeneration: is it possible? Int J
Artif Organs. 2013;36:392-405.
21. Diekman BO, Christoforou N, Willard VP, et al. Cartilage
tissue engineering using differentiated and purified
induced pluripotent stem cells. Proc Natl Acad Sci U S A.
2012;109:19172-19177.
22. Adamowicz J, Juszczak K, Bajek A, et al. Morphological
and urodynamic evaluation of urinary bladder wall regen-
eration: muscles guarantee contraction but not proper
function—a rat model research study. Transplant Proc.
2012;44:1429-1434.
23. Adamowicz J, Kowalczyk T, Drewa T. Tissue engineering
Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020
of urinary bladder—current state of art and future per-
spectives. Cent Eur J Urol. 2013;66:202-206.
24. Czajkowski R. BRAF, HRAS, KRAS, NRAS and CDKN2A
genes analysis in cultured melanocytes used for vitiligo
treatment. Int J Dermatol. 2011;50:180-183.
25. National Research Council. Guide for the Care and Use of
Laboratory Animals. 8th ed. Washington, DC: National
Academies Press; 2011.
26. Safford KM, Hicok KC, Safford SD. Neurogenic differenti-
ation of murine and human adipose derived stromal cells.
Biochem Biophys Res Commun. 2002;294:371-379.
27. Small R. Aesthetic procedures in office practice. Am Fam
Physician. 2009;80:1231-1237.
28. Vedamurthy M, Vedamurthy A. Dermal fillers: tips to
achieve successful outcomes. J Cutan Aesthetic Surg.
2008;1:64-67.
29. Funt D, Pavicic T. Dermal fillers in aesthetics: an over-
view of adverse events and treatment approaches. Clin
Cosmet Investig Dermatol. 2013;6:295-316.
30. Hoben G, Schmidt VJ, Bannasch H, Horch RE. Tissue aug-
mentation with fibrin sealant and cultured fibroblasts: a
preliminary study. Aesthetic Plast Surg. 2011;35:1009-1105.
31. Solakoglu S, Tiryaki T, Ciloglu SE. The effect of cultured
autologous fibroblasts on longevity of cross-linked hyal-
uronic acid used as a filler. Aesthetic Surg J. 2008;28:412-
416.
32. Bassetto F, Turra G, Salmaso R, Lancerotto L, Del Vecchio
DA. Autologous injectable dermis: a clinical and histologi-
cal study. Plast Reconstr Surg. 2013;131:589e-596e.
33. Yoon ES, Han SK, Kim WK. Advantages of the presence of
living dermal fibroblasts within Restylane for soft tissue
augmentation. Ann Plast Surg. 2003;51:587-592.
34. Johnsson C, Festin R, Tufveson G, Tötterman TH. Ex vivo
PKH26-labelling of lymphocytes for studies of cell migra-
tion in vivo. Scand J Immunol. 1997;45:511-514.
35. Mercer SE, Kleinerman R, Goldenberg G, Emanuel PO.
Histopathologic identification of dermal filler agents. J
Drugs Dermatol. 2010;9:1072-1078.
36. Fernández-Cossío S, León-Mateos A, Sampedro FG, Oreja
MT. Biocompatibility of agarose gel as a dermal filler:
histologic evaluation of subcutaneous implants. Plast
Reconstr Surg. 2007;120:1161-1169.
37. Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype
of human adipose-derived cells: temporal changes in stromal-
associated and stem cell–associated markers. Stem Cells.
2006;24:376-385.
Nowacki et al 1269
38. Casteilla L, Planat-Benard V, Laharrague P, Cousin B.
Adipose-derived stromal cells: their identity and uses in
clinical trials, an update. World J Stem Cells. 2011;3:25-33.
39. Bharadwaj S, Liu G, Shi Y, et al. Multipotential differ-
entiation of human urine-derived stem cells: poten-
tial for therapeutic applications in urology. Stem Cells.
2013;31:1840-1856.
40. Nae S, Bordeianu I, Stăncioiu AT, Antohi N. Human
adipose-derived stem cells: definition, isolation, tissue-
engineering applications. Rom J Morphol Embryol. 2013;
54:919-924.
41. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem
cells for regenerative medicine. Circ Res. 2007;100:1249-
1260.
42. Mesimäki K, Lindroos B, Törnwall J, et al. Novel maxillary
reconstruction with ectopic bone formation by GMP adi-
pose stem cells. Int J Oral Maxillofac Surg. 2009;38:201-209.
43. Garcia-Olmo D, Herreros D, Pascual M, et al. Treatment of
enterocutaneous fistula in Crohn’s disease with adipose-
derived stem cells: a comparison of protocols with and
without cell expansion. Int J Colorectal Dis. 2009;24:27-
30.
44. Garcia-Olmo D, Garcia-Arranz M, Herreros D. Expanded
adipose-derived stem cells for the treatment of complex
perianal fistula including Crohn’s disease. Expert Opin
Biol Ther. 2008;8:1417-1423.
45. Garcia-Olmo D, Herreros D, Pascual I, et al. Expanded
adipose-derived stem cells for the treatment of complex
perianal fistula: a phase II clinical trial. Dis Colon Rectum.
2009;52:79-86.
46. Hillel AT, Nahas Z, Petsche J, Unterman S, Elisseeff JH.
A novel animal model for hyaluronic acid filler longevity.
Laryngoscope. 2009;119(suppl 3):S307.
47. Pitaru S, Noff M, Blok L, et al. Long-term efficacy of a
novel ribose-cross-linked collagen dermal filler: a histo-
logic and histomorphometric study in an animal model.
Dermatol Surg. 2007;33:1045-1054.
48. Pokrywczynska M, Jundzill A, Bodnar M, et al. Do
mesenchymal stem cells modulate the milieu of recon-
structed bladder wall? Arch Immunol Ther Exp (Warsz).
2013;61:483-493.
49. Kim JH, Jung M, Kim HS, Kim YM, Choi EH. Adipose-
derived stem cells as a new therapeutic modality for age-
ing skin. Exp Dermatol. 2011;20:383-387.
50. Yoo G, Lim JS. Tissue engineering of injectable soft tissue
filler: using adipose stem cells and micronized acellular
Downloaded from https://academic.oup.com/asj/article/34/8/1261/2801401 by guest on 23 September 2020
dermal matrix. J Korean Med Sci. 2009;24:104-109.
51. Kim WS, Park BS, Sung JH. Protective role of adipose-
derived stem cells and their soluble factors in photoaging.
Arch Dermatol Res. 2009;301:329-336.
52. Rustad KC, Gurtner GC. Mesenchymal stem cells home to
sites of injury and inflammation. Adv Wound Care (New
Rochelle). 2012;1:147-152.
53. Ding DC, Shy WC, Lin SZ. Mesenchymal stem cells. Cell
Transpl. 2011;20:5-14.
54.
Yagi H, Soto-Gutierrez A, Parekkadan B, et al.
Mesenchymal stem cells: mechanisms of immunomodu-
lation and homing. Cell Transpl. 2010;19:667-679.
55. He J, Decaris ML, Leach JK. Bioceramic-mediated tro-
phic factor secretion by mesenchymal stem cells enhances
in vitro endothelial cell persistence and in vivo angiogen-
esis. Tissue Eng Part A. 2012;18:1520-1528.
56. Qi S, Wu D. Bone marrow–derived mesenchymal stem
cells protect against cisplatin-induced acute kidney
injury in rats by inhibiting cell apoptosis. Int J Mol Med.
2013;32:1262-1272.