Engineering, 2012, 5, 110-113
doi:10.4236/eng.2012.410B028 Published Online October 2012 (http://www.SciRP.org/journal/eng)
Does Making Method of Alginate Hydrogel Influence the
Chondrogenic Differ entiation of Human Mesenchymal Stem
Cells?
Jessica Schiavi1, Naceur Charif2, Natalia de Isla2, D anièle Bensoussan2, Jean-François Stoltz2,
Nadia Benkirane-Jessel1, Céline Husel st e in2
1INSERM-UMR, Faculty of medicine, Strasbourg University, Strasbourg, France
2Université de Lorraine, CNRS-UMR– Campus Santé – Biopôle, Faculty of medicine, Lorraine University, Vandœuvre-lès-Nancy, France
Received 2012
ABSTRACT
To overco me cartila ge inju ry, strat egies have b een d eveloped in the last fe w years b ased on tissu e engineerin g to rebu ild the defects.
Cartilage en gin eerin g is pri ncip ally based o n three main b iolo gical facto rs: cells (n ative cell s (cho ndrocytes) or a more primitive ones
as mesenchymal stem cells), scaffolds and functionalization factors (growth factors, mechanical stimulation and/or hypoxia). Carti-
lage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to native zonal o rganiza-
tion of articular cartilage. The main objective of our work concerns the buildup of complex biomaterials aimed at reconstructing bio-
logical tissue with three dimensional cells construction for mimicking cartilage architecture. Ou r strategy is based on structures for-
mation by simple and progressive spraying of mixed alginate hydrogel and human mesenchymal stem cells (hMSC). In this work, the
comportment of cells and more precisely their chondrogenic differentiation potential is compared to a traditional making process: the
mold. We report here that spraying method allowed to product a scaffold with hMSC that confer a favorable environment for neocar-
tilage construction.
Keywords: Component: Cartilage Tissu e Engin eering; So dium Alginate; Sprayed method; Molded Method
1. Introduction
Healthy cartilage is a complex tissue with well-defined func-
tions allowing principally harmonious movements of the arti-
culations over the whole life. Acute or repetitive blunt trauma
and excessive impact loading can cause irreversible alterations
in articular cartilage matrix. The lack of blood vessels and the
inability of chondrocytes (cartilage cells) to repair significantly
tissue defects limit the response of cartilage injury. Tissue en-
ginee ring aims at repairing or replacing damaged or diseased
tissue which present a limited self-repair cap acit y like carti lag e.
Cells, scaffold materials or both form the basis of this approach,
[tritz1, 2]. For cartilage tissue engineering, the use of mature
cell types such as chondrocytes is associated with several
drawbacks. The limited availability, donor site morbidity, de-
differentiation and limited proliferative capacity urged re-
searchers to study other cell types, particularly mesenchymal
stem cells, [3]. Hydrogels provide advantages as their capacity
of shaping, supporting chondrocytic phenotype, transducing
mechanical loads to cells and their biodegradability, [4, 5].
Alginate hydrogel can be composed with different proteins,
GAG (hyaluronic acid), natural or synthetic polymer to mimic
tissue composition, have specific mechanical properties or in-
ducing differentiation, [6]. Whereas, most studies using these
scaffolds obtain homogenous structure without zonal variation
as cartilage in vivo, [7, 8]. Recently, investigations have consi-
dered cartilage organization in order to create stratified tissue
engineering which attempts a layered design, [5, 9-13]. How-
ever, up to now, few methodologies efficiently synthesizing
stratified structures including viable specific cells interacting
with a biofunctionalized environment has been proposed. An
easily applicable technique that is able to deposit multiple cell
types in 3-D without overt cellular damage is probably cell
spraying [14].
The goal of the present work is based on structures formation
by simple and progressive spraying of mixed alginate hydrogel
and human mesenchymal stem cells (hMSC) and to compare
their chondrogenic differentiation potential to a traditional
making process: the mold. The differentiation potential of hu-
man mesench ymal stem cells was characterized parti cularly for
their interest for cartilage engineering.
2. Materials and Methods
2.1. Extraction and Culture of Mesenc hymal Ste m
Cells
Mesenchymal Stem Cells from human donors were isolated
from bone marrow obtained from total hip or knee replacement
surgery. Bone marrow was aspirated and diluted in HBSS.
Then, mononuclear cells were counted and the suspension was
seeded at 50 000 mononuclear cells/cm2 with co mplete medium
(Dulbecco's Modified Eagles Medium low glucose supple-
mented with 1 0% F etal Bovin e Serum ( Gib co, Fran ce), P enicil-
lin at 100 U/mL /Streptomycin at 100 μg/mL (P/S), Amphoteri-
cin B (Amp) at 2.5 μg/mL, 2 mM of Glutamine, and 1 ng/mL of
FGF). hMSC were cultivated up to confluence, passage 0 (P0),

J. SC H IAVI ET AL.
111
and detached with trypsin and seeded with complete mediu m at
1 000 cells/cm2, P1. Cells expansion was done up to P3, then
cells were detached from the support and embedded in Algi-
nate/Hyaluronic Acid hydrogel (Alg/HA).
2.2. Construction of Scaf folds Seeded with Cells
Scaffolds were build-up with 1.5% Alginate/Hyaluronic Acid
(ratio 4:1, Alg/HA). Whatever the making method (spray or
mold), hMSC were seeded at 3x106cells/mL of Alg/HA hydro-
gel.
Spraying construction: The sraying method was previously
used and described with other cells type, [15,16]. In brief, cel ls
suspension was sprayed at 0.9 bar on a sterile glass plate with
an airbrush connected to a compressor. After the glass plate
deepi ng in a CaCl2 bath at 102 mM, the gelation was done dur-
ing 15 min, then the hydrogel was washed twice with a 0.9%
NaCl solution supplemented with 10 mM Tris at pH 7.4.
Molding construction: The first type of construction was
composed of alginate gel injected into a mold. This approach is
often used in cartilage bioengineering. [17,18] 1.5-2 mL of
sterile 1.5% sodium alginate solution are injected into molds
(thickness = 1.5 mm, diameter = 25 mm; Bern, Switzerland).
[17] Then, alginate suspension gelled immediately on contact
with the CaCl2 solution (102 mM), during 15 min.
Whatever scaffolds, cylinders were cut at 5 mm diameter
with a biopsy punch (Stiefel), and were cultivated up to use
with differentiation medium (DMEM high glucose, antibiotic, 2
mM o f L -glutamine, 100 µg/mL of sodium pyruvate, 40 µg/mL
of L-Proline, 50 µg/mL of L-acid ascorbic, 100 nM of dexame-
thasone and 1 mM of CaCl2), changed twice in a week.
2.3. Analyze of Cells Viabilit y and
Immunophénotyping by Flow Cytometry
Apopt osis and necr osis of cells were anal yzed by flow c yto me-
try using the Vybrant/Apoptosis™ kit based on Annexin
V/Propidium Iodide staining procedure (Molecular Probes,
France).
To determine the typical cell surface epitope profile after
monolayer expansion and after 3 days of culture in sprayed
Alg/HA hydrogels, standard flow cytometer techniques
(FACSCalibur; BD biosciences , France) were used.
2.4. Metabo lic A c t ivity
Mitochondrial activities were measured with Alamar Blue as-
say (AbD Serotec Ltd, UK) on sample (one cylinder of 5 mm
diameter) during the 28 days of culture. After 4h of scaffolds
incubation with Alamar Blue solution (10% v/v) at 37°C, the
solution was subsequently removed and Alamar Blue absor-
bance is read at wavelength 570 nm (reduced compound absor-
bance) and 600 nm (oxidized compound absorbance) with a
spectrophotometer (Varioskan Flash, Thermo Scientific). Re-
sults were standardized with the quantity of DNA present by
samp le .
2.5. Extracellular Mat rix Synthesis
Cells were extracted from hydrogel by chelating Ca2+ in algi-
nate eggbox bindings and were washed with PBS 3 fold. First,
RNA extractio n was done with the RNeasy extr action kit (Qia-
gen), and purified with a DNase kit (Qiagen). Then, cDNA
were obtained thanks to the reverse-transcription of mRNA
with the iscript kit (Biorad). Finally, a qRT PCR was done with
the Quantitec SYBR Green PCR kit (Qiagen) and cDNA ob-
tained from samples, on a Light Cycler system (Roche Diag-
nost ics) during 45 cycles to analyze quantitat ively gene expres-
sion. All gene expression was reported to the housekeeping
gene RP-29.
2.6. Statistical Analysis
All data are presented as means ± standard error means (SEM)
of at least three independent experiments. A second way
ANOVA was used to determine if significant differences ex-
isted for the mixed-population experiment and a Bonferroni
posttest was performed to evaluate significance for all experi-
ments.
3. Results
To examine surface molecule expression on hMSC, flow cyto-
metric analysis was used to show the proportion of cells posi-
tive for hMSC markers (CD166, CD105, CD44 and CD73) at
the end of monolayer culture (P3) and after 3 days of culture in
sprayed Alg/HA hydrogels (Figure 1). It appeared that the
proportion of positive cells for CD105 significantly decreased
when cells were in a 3D matrix compared to the monolayer
culture. On the other hand, the proportion of cells expressing
CD73, CD44 and CD166 markers increased significantly in
sprayed Alg/HA hydrogel compared to the monolayer culture.
Whatever the kinds of culture, hMSC were negative for leuko-
cyte marker CD45 , hematop oieti c stem cell markers CD34 thus
verifying the lack of contaminating hematopoietic cells. Cells
were also negative for HLA-DR.
hMSC phenoty pe
CD44 CD105 CD166 CD73
0
50
100
150
Monolayer
Spr ay A lg/HA hy drogel
***
***
**
Positive Cells for a marker (%)
A
B
Figure 1. Flow cytometry analysis for the expression of cell surface
markers related to mesenchymal stem cells (CD44, CD73, CD105
and CD166; Fig. A), hematopoietic stem cells (CD34, Fig. B), leu-
kocytes (CD45, Fig. B), and HLA-DR (Fig. B) on hMSC at the end
of monolayer culture (P3) and in sprayed Alg/HA hydrogels (on
day 3). Data are presented as mean ±SEM.

J. SC H IAVI ET AL.
112
hMSC viability in sprayed Alg/HA hydrogels was followed
during 28 days of culture, and was compared to cell viability in
molded Alg/HA hydrogels (Figure 2). After 3 days of culture,
whatever the scaffold, cell viability was always higher than
70%. Alamar Blue test was done to measure mitochondrial
activity of samples and metabolic activity was obtained by
taking account the DNA content. During the first week of cul-
ture, metabol ic acti vit y was h omogen ou s b etween each sca ffold
and still weak. After 14 days of culture, hMSC embedded in the
sprayed hydrogels were more active than hMSC embedded in
the molded ones. Then, metabolic activity increased up to the
end of the experiment in sprayed hydrogels, with an increase
2.8 times higher than the molded hydrogels.
Quantitative RT-PCR was performed for gene expression
analysis on sprayed and molded Alg/HA hydrogels after 3 and
14 days of culture. The mean relative gene expression of ag-
grecan, collagen I, collagen II, Cartilage Oligomeric Matrix
Protein (CO MP), Sox9, i n the s c af folds is pr es e nte d in Figu re 3.
After 3 days of culture, any difference was observed for the
mean mRNA expression of aggrecan, collagen I, COMP, Sox9
in Alg/HA hydrogels, whatever the making method. In contrast,
after 14 days of culture, the mRNA expression levels of ag gre-
can, COMP, collagen I and Sox9 were higher in hMSC em-
bedded in sprayed hydrogels than in molded ones.
4. Discussion
Alginate hydrogel is a polymer largely employed for it is very
easy to use in cartilage engineering. [19,20] However, few in-
vestigations considered the organization of cartilage as a basis
for the development of the stratified tissue engineering. From
this viewpoint, complex 3-D structu res of cartilage can a priori
be built layer by layer using a large number of different com-
ponents, including hydrogel with various cell types, drugs,
proteins, peptides or DNA. However, few methodologies effi-
ciently synthesizing stratified structures including viable spe-
cific cells interacting with a biofunctionalized environment has
been proposed. An easily applicable technique that is able to
deposit multiple cell types in 3-D without over cellular damage
is prob ably cell spraying. Recen tly, we have repo rted the spray
formation of complex 3D multilayers, composed of alternating
calcium-alginate gel d omains contain ing cells [14,16].
In this study, we compared the effect of sprayed method on
hMSC behavior (phenotype, viability, metabolic activity) and
differentiation potential in chondrogenic lineage (Sox9 and
extracellular matrix markers expressions) to a more traditional
making process: the mold [17]. hMS C are l argel y used in ti ssu e
engineering in particular in cartilage engineering. Indeed, na-
tive cartilage cells, chondrocytes, are a limited source and can
induce fibrocartilage in the taking area, thus we use hMSC for
the easier ability to obtain a large number of cells, as other
research wa ys, [7,8]. Ou r data showed that, after 3 days of cul-
ture in sprayed Alg/HA hydrogels, hMSC obtained expressed a
panel of conventionally used mesenchymal stem cell surface
markers. Moreover, after 3 days of culture, it seems that cells
were adapted to their environment without any detectable
damage. This hypothesis could be confirmed by cell metabol-
ism results. Indeed, we observed, from D14 to D28, significant
increase of cell metabolism when cells were embedded in
sprayed hydrogel. This metabolism is higher than in molded
hydrogels. In addition, the differentiation of the hMSC in
chond rocyte seems to begin because relative e xpression o f car-
tilage genes (Sox9, COMP, Aggrecan, Collagen II) increases
significantly up to 14 days in sprayed Alg/HA hydrogels. In a
previous study, we showed that making method influence the
scaffolds mechanical behavior. In fact, the mechanical perfor-
mances of sprayed hydrogels was enhanced compared to
molded ones [14]. It is well-known that cell mechanical envi-
ronment is important for various cell activities, such as survival,
migration, signal transduction but also cell differentiation. This
new making method allowed having a tridimensional structure
and a mechanical environment in favor of chondrogenic diffe-
rentiation.
Viab ility
314 28
0
20
40
60
80
100
120
Time (D ays)
Cells (%)
Metaboli c Activit y
314 28
0
50
100
150
Molded MethodSprayed Method
*
Time (D ays)
Alamar Blue Re duction / DNA
(%/µ g)
Figure 2. hMSCs viability and metabolism during 14 days of cul-
ture in alginate scaffolds obtained either by sprayed or molded
method. Data are presented as mean ±SEM.
Aggrecan
314
0.0
0.2
0.4
0.6
**
Time (D a ys)
Agg/RP29
Collagen II
314
0.0
0.1
0.2
0.3
0.4
*
Time ( D ays )
Col2/RP29
COMP
314
0.0
0.2
0.4
0.6
Time ( D ays )
COMP/RP29
Collagen I
314
0
10
20
30
Sprayed Method
Molded Method
Time (D ays)
Col 1 / RP29
Sox 9
314
0.0
0.5
1.0
1.5
2.0
Time (Days)
Sox9/RP29
Figure 3. Real-time PCR analysis of cDNA derived from hMSC embedded in Alg/HA hydrogels. 3D hydrogels were performed with sprayed
or molded method and maintened 14 days in culture. Data are presented as mean ±SEM.

J. SC H IAVI ET AL.
113
The proposed way will allow to control from the surface to
th e in-depth of the distribution of the different needed elements
(matrix and cells). This strategy is important for chondrogene-
sis induction of hMSC without growth factor, [8], it will permit
also building up a scaffold and gives configuration to mimic
cartilage structu re.
5. Acknowledgements
This work was supported by the "Lorraine region" grant. N.
Jessel is indebted to CHU de Nancy, Hôpital Central, ortho-
pedic surgery (Contrat d’interface INSERM-CHU).
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