Cell Tissue Bank (2016) 17:481–489

DOI 10.1007/s10561-016-9574-5

Characterization of pediatric microtia cartilage: a reservoir

of chondrocytes for auricular reconstruction using tissue
engineering strategies
Y. Melgarejo-Ramı́rez . R. Sánchez-Sánchez . J. Garcı́a-López .
A. M. Brena-Molina . C. Gutiérrez-Gómez . C. Ibarra .
C. Velasquillo

Received: 18 May 2016 / Accepted: 1 August 2016 / Published online: 26 August 2016
Ó Springer Science+Business Media Dordrecht 2016

Abstract The external ear is composed of elastic
cartilage. Microtia is a congenital malformation of the
external ear that involves a small reduction in size or a
complete absence. The aim of tissue engineering is to
regenerate tissues and organs clinically
implantable based on the utilization of cells and
biomaterials. Remnants from microtia represent a
source of cells for auricular reconstruction using tissue
engineering. To examine the macromolecular archi-
tecture of microtia cartilage and behavior of chondro-
cytes, in order to enrich the knowledge of this type of
cartilage as a cell reservoir. Auricular cartilage
remnants were obtained from pediatric patients with
Y. Melgarejo-Ramı́rez
R. Sánchez-Sánchez

A. M. Brena-Molina
C. Velasquillo (&)
Laboratorio de Biotecnologı́a, Centro Nacional de
Investigación y Atención de Quemados (CENIAQ),
Instituto Nacional de Rehabilitación, Calzada México-
Xochimilco No. 289, Col. Arenal de Guadalupe,
C.P. 14389 Mexico City, Mexico
e-mail: mvelasquillo@gmail.com;
mvelasquillo@inr.gob.mx
microtia undergoing reconstructive procedures. Extra-
cellular matrix composition was characterized using
immunofluorescence and histological staining meth-
ods. Chondrocytes were isolated and expanded in vitro
using a mechanical-enzymatic protocol. Chondrocyte
phenotype was analyzed using qualitative PCR.
Microtia cartilage preserves structural organization
similar to healthy elastic cartilage. Extracellular
matrix is composed of typical cartilage proteins such
as type II collagen, elastin and proteoglycans. Chon-
drocytes displayed morphological features similar to
chondrocytes derived from healthy cartilage, express-
ing SOX9, COL2 and ELN, thus preserving chondral
phenotype. Cell viability was 94.6 % during in vitro
expansion. Elastic cartilage from microtia has similar
characteristics, both architectural and biochemical to
healthy cartilage. We confirmed the suitability of
microtia remnant as a reservoir of chondrocytes with
potential to be expanded in vitro, maintaining pheno-
typical features and viability. Microtia remnants are an
accessible source of autologous cells for auricular
reconstruction using tissue engineering strategies.

J. Garcı́a-López
C. Ibarra Keywords Elastic cartilage
Microtia
Auricular

Unidad de Ingenierı́a de tejidos, terapia celular y medicina
remnant
Tissue engineering
Auricular
regenerativa, Instituto Nacional de Rehabilitación,
Calzada México-Xochimilco No. 289, Col. Arenal de reconstruction
Guadalupe, C.P. 14389 Mexico City, D.F., Mexico
Introduction
C. Gutiérrez-Gómez
División de cirugı́a plástica y reconstructiva, Hospital
General Dr. Manuel Gea González, Calz. De Tlalpan No.
Cartilage is an avascular tissue with a low metabolic
4800 Col. Sección XVI, C.P. 14080 Mexico City, Mexico rate, integrated by different cellular and

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extracellular components that provide unique char-
acteristics (Zhang et al. 2009). Histologically, there
are three kinds of cartilage: hyaline is the most
abundant and is located recovering joints; fibrocar-
tilage is found in intervertebral discs, and elastic
cartilage which is rich in elastic fibers and consti-
tutes the external ear (Umlauf et al. 2010). The
external ear is a unique piece of concave irregular
elastic cartilage, oriented downward and forward
with numerous protrusions and depressions, covered
with thin skin and joined to the skull by ligaments
and muscles (Zhang et al. 2011). Elastic cartilage is
flexible, able to maintain its shape and rich in elastic
fibers, type II collagen, interstitial fluid, proteogly-
cans, proteins and carbohydrates. Chondrocytes are
the cells responsible for cartilage formation; they
are suspended in spaces within the extracellular
matrix (ECM) forming lacunae; adapted to survive
in a low-tension oxygen environment in normal and
pathological conditions (Nabzdyk et al. 2009); they
also are numerous and more compact than those
found in other types of cartilage (Naumann et al.
2002). Chondrocytes express transcriptional factor
SOX9, which is an early chondrogenic molecular
marker. These cells also express type II collagen
(COL2) and aggrecan (ACAN), main components of
ECM (Cole 2011). Proteoglycans are the second
most abundant proteins in cartilage and; as a result
of the presence of glycosaminoglycans, it has the
ability to retain water and to resist mechanical
compression. Elastic cartilage also expresses elastin
(ELN), a characteristic protein that confers flexibil-
ity and elasticity to this kind of tissue (Frantz et al.
2010). Microtia is an external ear congenital
anomaly comprising from a minimal reduction of
size to a complete absence of the external ear
(Luquetti et al. 2012). Costal cartilage implant is the
traditional reconstruction technique for pediatric
patients suffering microtia; however, post operatory
complications such as pneumothorax, malformation
of thoracic cage may occur (Sabbagh 2011). In this
scenario, the development of alternative treatments
for microtia and other congenital and degenerative
diseases will potentially improve clinical results in
the field of auricular reconstruction surgery. Tissue
engineering brings together engineering and biolog-
ical sciences to develop biological substitutes able
to restore, maintain or improve biological and body
functions. This approach is based on the
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Cell Tissue Bank (2016) 17:481–489
combination of cells, molecular signals and bioma-
terials to induce tissue formation (Langer 2000). In
order to accomplish this task; identification of a
suitable source of cells is necessary. Cells must be
easy to isolate and expandable without altering cell
phenotype; preserving specialized functions and not
immunoreactive (Shieh and Vacanti 2005). Consid-
ering that the auricular remnant of microtia has been
described as a source of chondrocytes (Kamil et al.
2004); the aim of this work was to examine the
macromolecular architecture of microtia cartilage
and behavior of isolated chondrocytes cultured
in vitro, in order to enrich the knowledge of this
type of cartilage as a reservoir of chondrocytes with
potential for auricular reconstruction using tissue
engineering.
Materials and methods
Tissue samples
Microtia auricle remnants from pediatric patients
subjected to autologous costal cartilage reconstruction
were obtained, previous informed consent signature.
Patients were recruited from the microtia clinic at the
National Institute of Rehabilitation and from the
General Hospital Dr. Manuel Gea González in Mexico
City. Age range varied from 4 to 10 years old,
indistinct gender with unilateral microtia. Samples
weight was between 1.0 and 2.0 g. Healthy elastic
cartilage was obtained from otoplasties performed in
the same hospitals. Sample processing and culture
were carried out at the Laboratory of Biotechnology of
the National Institute of Rehabilitation in Mexico
City.
Histological analysis
Auricle remnant tissue was fixed for 24 h in 4 %
paraformaldehyde and embedded in paraffin. Histo-
logical slices of 5 lm were deparaffinized, rehydrated
and subjected to the following staining protocols:
hematoxylin and eosin (tissue morphology), Masson’s
trichromic (collagen content), Verhoeff’s stain (elastic
fibers content) and safranin O (proteoglycans content).
Micrographs were captured using a Carl Zeiss Axio-
vision LE microscope.
Cell Tissue Bank (2016) 17:481–489
Fig. 1 Gross morphology of microtia auricular remnants. Elastic cartilage after connective tissue was removed (a). Mechanically
disaggregated cartilage previous enzymatic digestion (b)
Immunofluorescence of elastin and type II
collagen
Histological slices were blocked for 20 min in 1.5 %
fetal bovine serum solution and incubated with
monoclonal antibodies against human ELN (1:50,
Abcam 9519) and COL2 (1:50, Abcam 3092). Shortly
after, slices were incubated with Alexa fluor 488
secondary antibody (1:500, Invitrogen) for 2 h.
Micrographs were captured using fluorescence
microscopy.
Auricular chondrocyte isolation and culture
Auricular remnants were kept in transport media
(DMEM-F12 and 10 % antibiotic–antimycotic) until
use. Connective tissue was removed and tissue was
mechanically and enzymatically disaggregated using
trypsin–EDTA 0.25 %, following type II collagenase
digestion (3 mg/mL) for five hours (Fig. 1). Isolated
chondrocytes were quantified and seeded at a cell
density of 1 9 104 cells/cm2 and kept under standard
culture conditions in DMEM-F12 supplemented with
10 % FBS and 1 % antibiotic during 3 weeks. Cells
were expanded during primary culture (PC) and first
subculture (P1).
Viability assay
Auricular chondrocytes from PC were washed and
incubated with 1 lM calcein and 2 lM ethidium
homodimer (Live/dead viability/cytotoxicity kit,
Invitrogen) for 45 min at 37 °C.
483
Identification of molecular markers for elastic
cartilage
RNA (1 lg) was extracted from remnant tissue, PC and
P1 cells (1 9 106 cells) using TrizolÒ according to
manufacturer’s protocol. CDNA was synthesized using
1st strand cDNA synthesis for RT-PCR (AMV) kit
(Roche). Expression of cartilage molecular markers was
determined using the following primers: human SRY (sex
determining region 1)-box 9 (SOX9, NM_000346.3),
elastin (ELN, NM_000501.2), type II collagen (COL2A1,
NM_1844.4/NM_033150.2), aggrecan (ACAN, NM_
001135.3/NM_013227.3), type I collagen (COL1A2,
NM_000089.3) and glyceraldehyde-3-phosphate-dehy-
drogenase (GAPDH) as loading control. Reactions were
carried out using a 2728 Thermal cycler (Applied
Biosystems). Products were visualized via electrophoresis
in a 1.2 % agarose gel and stained with ethidium bromide.
Statistical analysis
All experiments were independently repeated at least
three times. Viability and mortality percentages were
calculated counting individual cells in at least five fields
per experiment. Data were expressed as Mean ± SE of
the mean (MES) using Graph-Pad Prism 5.
Results
Tissue from microtia resembles normal elastic
cartilage
To analyze organization and composition of microtia
cartilage ECM, histological slices were stained under
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different protocols. Hematoxylin/eosin shows auric-
ular chondrocytes distributed individually or in pairs,
forming isogenic groups. Nuclei are shown in purple,
white spaces correspond to cell cytoplasm and purple
coloration corresponds to ECM and other compo-
nents. Intercellular spaces are reduced and consti-
tuted by compact abundant ECM. Chondrocytes turn
closer together and flatter as they approach to the
perichondrium (p) or superficial external layer that
covers cartilage (Fig. 2a, b). Safranin O staining
reveals a high content of proteoglycans in color red
(Fig. 2c, d). The presence of elastic fibers confers
biomechanical and elastic properties to elastic carti-
lage. Verhoeff’s staining shows dense and abundant
elastic fibers in steel gray color, surrounding chon-
drocytes and projecting themselves across ECM in a
uniform pattern. Noteworthy, elastic fibers were not
stained by safranin O (Fig. 2e, f). Masson’s tri-
chrome allows to distinguish between collagen fibers
and smooth muscle fibers. Blue stained areas indicate
that ECM has a high content of fine collagen fibers,
although we are not able to specify which type of
collagen. Chondrocyte cytoplasm was stained in red
(Fig. 2g, h).
ECM of microtia cartilage is constituted by elastin
and type II collagen
Once organization and composition of auricular
remnant tissue were determined, we analyzed pres-
ence of specific ECM proteins. Microtia cartilage has
an abundant presence of elastic fibers surrounding
chondrocytes. These fibers expand and intercalate
across intercellular spaces, forming a network that
covers the tissue and its location corresponds to those
fibers that were detected using Verhoeff’s staining
(Fig. 3a, b). Type II collagen is another typical protein
found in cartilage extracellular matrix. It constitutes
about 60 % of tissue dry weight and works as a
scaffold for the generation of an adequate ECM. We
determined that ECM of auricular remnant from
microtia is integrated by type II collagen surrounding
and secreted by chondrocytes. This matrix extends
along tissue until it reaches the perichondrium and
becomes more compact. We detected areas with a
richer content of collagen occupying intercellular
spaces; leaving black spaces corresponding to chon-
drocyte cytoplasm. DAPI stained nuclei were
observed in blue (Fig. 3c, d).
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Chondrocytes from microtia preserve typical
morphology
Chondrocytes from auricular remnant were isolated
and cultured as previously described. Along primary
culture (PC) auricular chondrocytes readily adhere to
the surface of culture flask during the first 24 h
forming a uniform monolayer and reaching confluence
after 2 weeks. During in vitro expansion, chondrocytes
presented typical polygonal shape with spiculated
cytoplasmic projections (Fig. 4d–f). Once chondro-
cytes reached 80 % confluence, they were trypsinized
to establish first subculture (P1). Chondrocytes
adhered within the first 12 h of culture and displayed
the same morphology to that observed in primary
culture (Fig. 4g–i). Morphological characteristics
were similar to those presented by auricular chondro-
cytes isolated from healthy auricular cartilage
(Fig. 4a–c).
Isolated chondrocytes are viable during in vitro
expansion
To determine in vitro viability of auricular chondro-
cytes isolated from microtia cartilage, we performed a
calcein assay by day 15 of culture. An intracellular
fluorescent signal indicates active cell metabolism and
provides a qualitative indicator of cell viabil-
ity (Fig. 5a, b). We determined that 94.6 % of
auricular chondrocytes remained viable after 15 days
of culture. Percentage of mortality was 5.37 % during
the same period of time. Statistical analysis demon-
strated data normality and reproducibility of assays.
Chondrocytes from microtia preserve phenotype
To determine chondral phenotype of expanded
chondrocytes, we analyzed expression of cartilage
molecular markers in tissue (T), primary culture (PC)
and first subculture (P1) using qualitative PCR. The
transcriptional factor SOX9 promotes type II colla-
gen expression in cartilage. We detected an increase
in SOX9 expression in PC chondrocytes than
observed in those from T and P1. COL2 expression
was significant and uniform from T and throughout
in vitro expansion (PC and P1), confirming the
relation between COL2 and SOX9 expression. ACAN
messenger codes for one of the most abundant
proteoglycans in cartilage and its expression is also
Cell Tissue Bank (2016) 17:481–489 485

Fig. 2 Histological analysis of microtia cartilage. Elastic (c, d), elastic fibers (e, f) and collagen (g, h). Scale bars 500 lm
cartilage from auricular remnant (a, b) preserves a similar (a, c, e, g) and 200 lm (b, d, f, h). Perichondrium (p) is visible as
organization to healthy cartilage. ECM is rich in proteoglycans an outer fibrous layer

related to SOX9. ACAN expression during PC and P1 elastic cartilage ECM. Finally, we detected expres-
was similar to that found in tissue. Noteworthy, sion of COL1 in T and subsequent stages during
isolated chondrocytes from microtic cartilage also in vitro culture and its presence will be further
maintain ELN expression, the main component in discussed (Fig. 6).

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Fig. 3 Microtia cartilage is rich in elastin and type II collagen.
Extracellular matrix is abundantly constituted by elastin fibers
(a, b) and type II collagen (c, d) forming a network surrounding
chondrocytes. Green signal corresponds to specific proteins and
Discussion
Tissue engineering is the best alternative to develop
biological substitutes to reestablish specific func-
tions. At present, research groups are using auricular
remnants from microtia as a source of chondrocytes
in order to generate tissue-engineered elastic carti-
lage. However, in order to achieve clinical applica-
tions; such as auricular reconstruction, information
about histological organization of microtia cartilage
and behavior of auricular chondrocytes during
in vitro expansion will support the feasibility of this
cellular source. Histological analysis of auricular
remnant confirmed that microtia cartilage possess a
similar tissue organization to healthy elastic cartilage
found in human, rat (Bradamante et al. 1991) and
rabbit (Naumann et al. 2002). Microtia cartilage
possesses a thin layer of young chondrocytes that
ensheath the tissue and forms the perichondrium.
Perichondrium cells are orientated in a parallel way
considering those found inside the tissue. Cartilage
from the remnant contains a dense population of
chondrocytes surrounded by an abundant ECM rich
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blue signal corresponds to nuclei stained with DAPI. Scale bars
correspond to 50 lm (a, d), 20 lm (b) and 100 lm (c). (Color
figure online)
in elastin and type II collagen (conferring mechanical
properties), as well as a high number of proteogly-
cans. These characteristics indicate that microtia
cartilage preserves the similar physical and biochem-
ical properties to healthy elastic cartilage. Previous
studies have isolated and expanded chondrocytes
from healthy tissue. During in vitro expansion, these
cells exhibit a polygonal morphology during early
stages of cell culture, which turns into a more
elongated and spiculated shape in later stages
(Ruszymah et al. 2007). Our protocol allowed
isolation of auricular chondrocytes from the microtia
remnant using mechanical and enzymatic methods.
During in vitro expansion, chondrocytes displayed
abundant cytoplasm with polygonal shape and main-
tained viable through monolayer culture; thus con-
firming the suitability of microtia remnant as a
reservoir of chondrocytes with potential to be
expanded in vitro, retaining their phenotypical fea-
tures and viability. Chondrocyte morphology turns
fibroblastoid in later stages of primary culture.
Fibroblastoid morphology has been considered as
an indicator of cellular dedifferentiation; however,
Cell Tissue Bank (2016) 17:481–489 487

Fig. 4 In vitro expansion of auricular chondrocytes from passage (P1, g–i). Cells displayed similar morphological
microtia. Chondrocytes isolated from auricular remnant, cul- features than auricular chondrocytes isolated from healthy
tured for 6, 9 and 15 days in primary culture (PC, d–f) and first cartilage (a–c). Scale bars correspond to 100 lm

Fig. 5 Viability of chondrocytes from microtia cartilage. viability by day 15 was 94.63 % ± 2.13 and cell mortality was
Green signal in cytoplasm corresponds to calcein, red signal 5.37 % ± 2.13. Scale bars correspond to 50 lm. (Color
corresponds to nuclei of dead cells (a, b). Graph shows that cell figure online)

this may not apply for elastic cartilage cells. It has elastic chondrocyte (Quatela et al. 1993). For this
been reported that fibroblasts are an intermediary cell reasons, we suggest that behavior and morphology of
between an undifferentiated mesenchymal cell and chondrocytes isolated from the microtia remnant is
the chondroblast, which then differentiates into similar to those found in healthy cartilage.

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Fig. 6 Expression of cartilage molecular markers in chondro-
cytes and microtia tissue. Auricular chondrocytes express
typical cartilage markers found in tissue (T), primary culture
(PC) and first passage (P1). Expression of transcriptional factor
Expression of molecular markers such as SOX9,
COL2 and ELN has been used to verify chondral
phenotype in these cells. Our results qualitatively
confirmed that chondrocytes from microtia cartilage
synthesize messengers required for production of
proteins found in ECM. Ruszymah et al. (2007) also
reported that auricular chondrocytes isolated from
healthy cartilage and cultured in DMEM-F12 express
COL2 during PC with a slight tendency to lose this
expression after several passages. The same effect is
observed with ELN and ACAN. According to our
results, chondrocytes from the auricular remnant
show the same tendency as articular chondrocytes to
lose expression of molecular markers; however,
expression levels after P1 might be sufficient for
their use. Presence of these markers indicates that
auricular chondrocytes from microtia cartilage main-
tain chondral phenotype independently of the pathol-
ogy they were derived. Therefore; we might say that
even when external ear from microtia patients suffer
a malformation, elastic cartilage still possess a
similar architecture and biochemical composition
than healthy cartilage. Noteworthy, isolated chon-
drocytes preserve their phenotype during the first
stages of expansion. For these reasons, chondrocytes
isolated from microtia cartilage are suitable cells for
autologous transplants. However, it is necessary to
develop efficient strategies to maintain chondral
phenotype during cell expansion. These strategies
will allow obtaining higher number of cells enough
for future clinical applications.
Conclusion
We determined that elastic cartilage from microtia has
similar characteristics, both architectural and bio-
chemical to healthy cartilage. We confirmed that this
tissue is a reservoir of chondrocytes for in vitro
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SOX9, type II collagen (COL2), elastin (ELN), aggrecan (ACAN)
and type I collagen (COLI) was analyzed. GAPDH was used as a
loading control
expansion and with high potential for auricular
reconstruction.
Acknowledgments The authors thank to IB Karina Martinez
for her technical assistance. This work was supported by Grants
CONACYT Sectoriales 114359 and Sectoriales 78798, from
Consejo Nacional de Ciencia y Tecnologia, Mexico.
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