PELLET CULTURE SYSTEM OF HUMAN STEM CELLS AS AN IN
VITRO MODEL FOR CARTILAGE ENGINEERING APPROACHES
Karina Ribeiro da Silva1, João Vitor Belizario dos Santos1,4, Carolina da Silva Gouveia
Pedrosa1,3, Ronaldo José Farias Correa do Amaral3, José Mauro Granjeiro1, Leandra Santos
Baptista1,4
1
Diretoria de Programas, Instituto Nacional de Metrologia Normalização e Qualidade Industrial, Duque de
Caxias (RJ), Brazil.
2
Programa de Pós-Graduação em Clínica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro (RJ),
Brazil.
3
Programa de Pós-Graduação em Ciências Morfológicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro
(RJ), Brazil.
4
Universidade Federal do Rio de Janeiro, Polo Xerém, Duque de Caxias (RJ), Brazil.
E-mail: ribeiro.ks@gmail.com
Abstract. Introduction: Much of our current knowledge concerning the regulation of chondrogenesis has been
acquired with in vitro studies using high-density cultures of undifferentiated stem cells stimulated to differentiate
into chondrocytes. Objective: The aim of this study is to set a model for studies of chondrogenesis, based on a
pellet three-dimensional (3D) system of cell culture, which could mimic molecular, cellular and spatial
organization of cartilage extracellular matrix (ECM). Methodology: Samples of human nasal septum cartilage
were collected during aesthetic surgeries, approved by the Research Ethics Committee of HUCFF. Cartilage
stem cells were isolated after collagenase digestion, expanded in vitro and cultured to form micro-sediments.
Cartilage fragments and micro-sediments were fixed and prepared for histological analysis. Sections were
stained with Safranin O for sulfated glicosaminoglycans detection or with phalloidin, collagen type I and II
antibodies for confocal fluorescence analysis. Transmission electron microscopy (TEM) was also performed to
observe the ultrastructure of ECM in 3D cultures. Results: Cells maintained on a 3D culture system for 3 weeks
showed reduced actin stress fibers when compared to 2D culture. Notably, cells in 3D cultures switched from
fibroblast-like morphology - observed in monolayer - to a rounded morphology, typical of chondrogenic lineage
cells in vivo, even without supplementation with specific growth factors. Besides that, cells synthesized ECM
components: Sediments showed an intense production of sulfated glycosaminoglycans and collagen
II. Conclusions: The 3D model described composed by stem cells from human nasal septum seems to reproduce
the tissue microenvironment of hyaline cartilage. It is effective for monitoring chondrogenesis faster than
typically described in the literature and could serve as an excellent model for other researches in the fields of
chondrogenesis, cartilage engineering and drugs testing. The impressive spontaneous chondrogenic potential of
these cells would be able to promote, in the future, successful cartilage engineering in vivo.
Palavras-chave: 3D cell culture, Cartilage stem cells, Chondrogenesis.
1.
INTRODUCTION
Regenerative medicine techniques are required to treat most hyaline cartilage defects,
as it is a tissue with low regenerative capacity. Tissue engineering and cell-based approaches
are the main strategies (Ahmed & Hincke, 2010). Cartilage tissue engineering aims to
produce in vitro a tissue with molecular, cellular and spatial organization of cells and cartilage
extracellular matrix, that could best mimic native cartilage.
Chondrocytes, the specialized cartilage cells responsible for the production of cartilage
extracellular matrix, were the first cell type used for cartilage tissue engineering. While some
favorable results were achieved in vivo, chondrocytes sometimes failed to long term tissue
maintenance in vivo, besides being difficult to expand and culture these cells in vitro, because
dedifferentiation occurs (Salgado et al., 2006).
Unlike terminally differentiated cells, adult stem cells are defined by self-renew
capacity and potential to differentiate into mature cells (Reya et al., 2001). For cartilage tissue
engineering, mesenchymal stem cells from bone marrow and adipose tissue seems to be good
candidates, as they exhibit chondrogenic potential under inducing stimuli in vitro (Pittenger et
al., 1999; Zuk et al, 2001). Although chondrogenic differentiation has been shown in
monolayer culture, 3D structures, where cells acquire a spherical morphology, better mimic
mesenchymal condensation, one of the primary events of chondrogenic differentiation
(Johnstone et al, 1998). On the other hand, it has been reported that chondrogenic
differentiation of mesesenchymal stem cells usually results in a significant proportion of
fibrocartilage (Steck et al., 2005). Stem cells from cartilage is advantageous because of their
high chondrogenic potential. However many issues concerning autologous hyaline cartilage
cell source still exists, as this tissue is not in abundance to be collected without jeopardizing
tissue function.
Our group have recently isolated stem cells from negligible nasal septum cartilage
fragments obtained by aesthetic surgery procedures, with spontaneous cartilage differentiation
potential (Amaral et al., 2012). The use of chondrogenic cells is advantageous due to this
spontaneous chondrogenic potential, but adipose mesenchymal stem cells are very attractive
for medicine regenerative as this tissue is abundant and easily accessed by lipoaspiration
procedures (Casteilla et al, 2005). However, there is no consensus in the scientific literature
about the chondrogenic potential of these and other stem cells to form a completely cartilage
construct. Therefore, it is necessary to set a standard model of chondrogenesis, which could
support differentiation potential comparisons.
Many strategies are available to induce chondrogenesis in vitro, like micromass
culture, pellet culture and association with some scaffolds as alginate (Seda Tigli et al., 2009).
Pellet cultures provide a microenvironment similar to that found in micromass cultures
(Bobick et al, 2009). These pellet cultures are simple to produce and have experimental
conditions easy to control, making them a good tool to investigate molecular issues involved
in chondrogenesis (Muraglia et al, 2003). Using high-density cultures of undifferentiated cells
stimulated to differentiate into chondrocytes, researchers have described many issues
concerning the regulation of cartilage formation (Bobick et al, 2009). Although molecular
analysis shows a initial differentiation to chondrogenesis, it does not certify the quality of the
tissue constructed. On the other hand, histological and quantitative biochemaical assays could
better evaluate the extracellular matrix production and organization (Kunisaki et al., 2007).
The aim of this research is to set a model for studies of chondrogenesis, based on a
pellet three-dimensional (3D) system of cell culture, which could best mimic hyaline cartilage
extracellular matrix and be used as a standard for chodrogenic differentiation studies and
drug testing.
2.
MATERIALS AND METHODS
Human cartilage
Cartilage fragments from nasoseptal were obtained from healthy donors from 25 to 40
years old (n=3), that underwent aesthetic surgery procedures, under approval of the Research
Ethics Committee of the Clementino Fraga Filho University Hospital, Federal University of
Rio de Janeiro, Brazil.
Isolation and culture of chondrogenic cells
Chondrogenic cells were isolated as described previously (Amaral et al, 2012). Briefly,
small fragments of cartilage were digesteded with collagenase 1A (Sigma Chemical Co)
under shaking at 37°C for two hours. Cells were harvested by centrifugation and plated in
tissue culture flasks with alpha-minimum essential medium (alpha-MEM, Sigma) containing
10% fetal bovine serum (FBS, LGC), 100 U/mL penicillin, and 100 μg/mL streptomycin.
Cultures were maintained in a humid atmosphere with 5% CO2 at 37°C, and the medium was
changed every 3–5 days until cell monolayer reached confluence. At nearly 90% confluence,
cells were harvested with 0.125% trypsin (Gibco) 0.78mM EDTA (Gibco) and re-seeded at a
density of 104 cells/cm2. Dissociation with trypsin followed by re-seeding for cell expansion
was denominated “passage”. Experiments were performed with cells at second passage.
Three- dimensional pellet culture
Aliquots of 2x105 cells were centrifuged at 300g for 10 min in 15mL conical
polypropylene tubes to form a pellet. Pellets were cultivated with a serum-free medium
consisting of alfa-MEM supplemented with 6.25μg/mL insulin, 6.25μg/mL transferrin,
1.25μg/mL of bovine serum albumin (BSA, - Sigma), 50μg/mL ascorbic acid (all reagents
from Sigma). Culture
medium was renewed twice per week, keeping the micromass intact up to 21 days of
cultivation. Immunofluorescence for phalloidin was used to evaluate the actin cytoskeleton.
Chondrogenesis was assessed histologically, by Safranin O staining and collagen type II
detection by immunofluorescence.
Immunofluorescence analysis
Pellet samples (n=3) were fixed in 4% buffered paraformaldehyde and incubated with
solutions of sacarose 15% and 30% for at least 6 hours each, for subsequent embedding in
OCT compound (Sakura Finetek) and freezing. Cryosections of 10μm were obtained on a
Leica C1850 cryostat and mounted on microscope slides. Antigen unmasking for type II
collagen antibody was done by treatment with hyaluronidase (4800U/ml Sigma diluted with
0.025mM NaCl and 0.05M of acetic acid, pH 5) for 2 hours at room temperature followed by
treatment with 0.4% pepsin (Sigma) diluted in10mM HCl for 30 min at 37°C. Blockade of
unspecific binding of immunoglobulins were performed by incubating sections with 5% BSA,
5% of goat serum and 0.5% Triton X-100 (Sigma) in phosphate buffer saline for 1.5 h.
Overnight incubation at 4°C with primary antibodies was done: type II collagen (1:50; Santa
Cruz Biotech) or TRITC conjugated phalloidin (1:100; Invitrogen). Secondary antibody
staining was performed for type II collagen antibody, using FITC conjugated anti-mouse IgG
(1:200; Invitrogen) for 1 hour at room temperature. Nuclei were stained with Sytox (1:500) or
TO-PRO3 (1:500; Invitrogen) for 10 minutes and slides were mounted in Vectashield (Vector
Lab). Stained sections were examined under a confocal fluorescence microscope (Leica TCS
SP5). Slides incubated only with the secondary antibody as used as negative control.
Safaranin O- Fast green staining
Detection of sulfated glicosaminoglicans was performed by safranin O staining. Pellet
samples (n=3, each cell type) and cartilage tissue fragments were fixed in 10% buffered
formaldehyde. Samples were dehydrated in graded ethanol (70%, 100%, 100%), cleared in
xylol and embedded in paraffin (all from Vetec). Two sections of 5μm cut at 50-μm intervals
on an American Optical microtome were stained with Safranin O (Sigma) to evaluate the
glycosaminoglycan content. Briefly, sections were dewaxed in xylene, hydrated in ethanol
(100%, 95%, 70%; 2 min each) and finally in distilled water for 3 min. Slides were immersed
in a solution of 0.2% safranin O (Sigma) in 1% acetic acid for 10 min followed by rinsing in
distilled water to remove excess dye. Sections were then counterstained with a solution of
0.04% Fast green (Sigma) in 0.2% acetic acid for15s and rinsed in distilled water. After
drying on a filter paper, slides were rinsed in absolute ethanol until excess of fast green was
removed. After 3 washes in xylene (3min each), slides were mounted with EntellanR and
examined under an optical microscope (Leica cDMI 6000 B) equipped with Leica DFC 500
digital camera.
Transmission Electron Microscopy
For transmission electron microscopy analysis, pellet cultures (n=3) were fixed in 2.5%
glutaraldehyde buffered with 0.1 M sodium cacodylate for 2 hours at room temperature and
post fixed with 1% OsO4 in the same buffer for 30 minutes (all from Electron Microscopy
Sciences). Dehydration was performed in graded acetone (Merck) and then pellets were
embedded in the Epon resin (Electron Microscopy Sciences). Sections of 70nm were obtained
on a ultramicrotome (EM UC6), being examined under a transmission electron microscope
(FEI- Tecnai Spirit 12).
3.
RESULTS AND DISCUSSION
Chondrogenic cells cultivated in monolayer showed a fibroblastoid morphology (Fig.
1A), with a spindle-shaped actin cytoskeleton (Fig.1C). Cells maintained in the high cellular
density pellet culture system for 3 weeks (Fig.1B) showed reduced actin stress fibers (Fig.
1D) comparing to 2D culture system, which is closer to a physiological model for efficient
chondrogenesis evaluation.
Switch from fibroblast-like morphology – observed in monolayer – to a rounded one was
more evident by analyzing sections of pellet cultures stained for Safranin O/Fast Green (Fig. 2).
After three weeks of 3D cultivation, cells acquired a morphology that resembles chondrocytes
(Fig. 2A), as observed in native hyaline cartilage (Fig. 2 – insert in A).
Besides that, Safranin O staining revealed the production of sulfated glicosaminoglicans by
cells culture in the 3D system (Fig. 2). Pink to orange color shows the glicosaminoglican content
of pellets after three weeks of cultivation even without addition of chondrogenic growth factors to
the culture medium. Some areas showed a higher degree of differentiation than others (Fig. 2 A
and B).
Figure 1. Cell culture behavior of human chondrogenic cells from nasal septum cartilage. (A) Monolayer
of chondrogenic cells cultured in the presence of 10% FBS. (B) Pellet culture of chondrogenic cells culture in
serum-free medium. Phase contrast microscopy — objective magnification: 10X (A,B). Confocal laser scanning
microscopy shows actin cytoskeleton on monolayer cultures - bar size, 25 μm (C) and on pellet cultures – bar
size, 10μm (D). Red: TRITC-phalloidin labeled actin filaments; Green: Sytox labeled cell nuclei).
Figure 2. Production of glicosaminoglicans evaluated by Safranin O staining. After 21 days of 3D culture,
chondrogenic cells produced an extracellular matrix rich in sulfated glicosaminoglicans, revealed by Safranin O
staining . (A) and (B) are representative areas of sections analyzed. (C) Image representative of cartilage tissue
stained for safranin O as a positive control. Objective lens magnification: 10X.
Transmission electron microscopy revealed that the extracellular matrix produced by
cells of pellet cultures was also enriched in collagen (Fig. 3A,B), which formed fibers through
the sediment. By immunolabeling, it was observed that this collagen content is enriched in
collagen type II, typical of hyaline cartilage (Fig 3C). Collagen type I, typical of
fibrocartilage, was less detected throughout the pellet (data not shown). Considering that
mesenchymal stem cells (Johnstone et al., 1998; Estes et al., 2010) and dedifferentiated
articular chondrocytes (Barbero at al., 2003) need to be cultured in a 3D system with
chondrogenic growth factors (as TGF- s and BMPs) for a successful chondrogenic
differentiation, it is impressive how nasoseptal chondrogenic cells adopts the full
chondrogenic phenotype, without the use of such factors. This chondrogenic commitment
makes these cells an advantageous product for tissue engineering approaches
Figure 3. Synthesis of collagen by chondrogenic cells of human nasal septum cartilage. Collagen content
observed by transmission electron microscopy analysis (A, B) showed to be enriched in collagen type II,
detected by imunofluorescence (C). Arrows in (A) and (B) indicates collagen fibers.
4.
CONCLUSIONS
The high-density three-dimensional cell culture system described in this study, using
chondrogenic cells isolated from human nasal septum cartilage, seems to reproduce the tissue
microenvironment of hyaline cartilage. It showed a complete chondrogenic differentiation
process, as cells switched from a fibroblastoid to an oval morphology, and an effective
production of hyaline cartilage extracellular matrix components. Besides, this system provides
a chondrogenic differentiation model with reduced both time and procedure costs, because it
abrogates the need to add growth factors in the cell culture medium, like TGF-3.
Therefore, it is suggested that the culture system proposed in the current study be
considered as a model of cartilage formation in vitro that can lead to the development of novel
cartilage tissue engineering strategies, by contributing to the knowledge of this complex
tissue microenvironment. In addition, this system also represents an efficient model for drug
testing, as it can be easily handled and could promptly respond to exogenous factors.
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