In vitro evaluation of an agarose-alginate-based hydrogel for chondrocytes implantation

1
Barnouin, L; +1 Tan, N; 1Duverneuil, L; 1Gauduin, A; 1Laganier, L;
+1Tissue Bank of France, Mions, France
laurence.barnouin@tbf-lab.com
INTRODUCTION
Because of its avascularity and low cellularity, articular cartilage
tissue has very poor self-repair capacity. Marrow stimulation, such as
microfracture, and mosaicplasty are the most readily available
techniques, although not completely satisfactory. That is why tissue
engineering strategies are now employed with the goal of improving the
quality and longevity of the repair tissue. Brittbergs technique,
implantation of a chondrocytes suspension under a periosteal flap,
proved long term cartilage repair but gave non-homogenous results due
to cells leaking and chondrocytes differentiation. To avoid these
complications and repair deeper osteochondral defects, current strategies
aim at introducing chondrocytes into a biological scaffold. The
biomaterial must fulfill both the mechanical capability to withstand the
high contact stresses and strains of articular joint environment as well as
the functional property allowing tissue growth. To this end, an agarosealginate hydrogel has been developed for chondrocytes implantation.
METHODS
Hydrogel is composed of 1.5% agarose and 1% alginate in Earles
saline solution.
Biocompatibility tests were carried out according to ISO 10993
standard : genotoxicity (Ames test), acute systemic toxicity on mice and
maximized sensitization test on guinea pig. Pyrogen test was performed
on rabbits according to European pharmacopoeia. In order to increase
the sensitivity of all tests, an hydrogel containing 2% agarose and 2.5%
alginate was used.
Cartilage was harvested from the non-weight-bearing area of the
femoral condyle of living donors. It was washed several times in EDTA
and trypsin and finely minced and then digested with 0.2% collagenase.
The chondrocytes were cultured in monolayer and then suspended in the
hydrogel at a final concentration of 10 x 106 cells/mL and moulded. The
cell-scaffold combination was gelled by cooling down and incubation in
calcium chloride solution. Then, the hydrogel containing chondrocytes
was incubated at 37C under orbital agitation in expansion medium.
Cell distribution assay was performed on 90 areas distributed in 6
sections per sample. Statistical difference (Student t test) between top
and bottom and between middle and edge of the hydrogel was evaluated.
The level of significance was set at 0.01.
Viability assay was performed by incubation of cell-scaffold
combinations in a propidium iodide solution. Living versus dead cells
were quantified on 100 areas distributed in 2 sections per sample.
Redifferentiation of chondrocytes following incubation in the
hydrogel was analyzed through cell phenotype using immunohistochemistry (type II collagen and aggrecan) and quantitative RT-PCR
(types I and II collagens, and aggrecan) techniques.
RESULTS
Agarose-alginate hydrogel gelation occurs through both thermal and
chemical process, leading to three-dimensional network formation. At
low temperature, agarose molecules association results in a reversible
hydrogel, while in calcium chloride solution, alginate forms stable crosslinked junctions. Association of agarose and alginate allows gel molding
to manufacture grafts adapted to the defect thanks to their defined shape
and size.
This agarose-alginate hydrogel is not pyrogenic and not genotoxic. It
did not induce any acute systemic toxicity or delayed hypersensitization.
Cell distribution assay showed no statistical difference between top
and bottom and between middle and edge of the scaffold (t test, p0.01)
(n=7). Cells were homogeneously distributed within the scaffold.
Chondrocytes viability following incubation in agarose-alginate
hydrogel was between 72 and 96% after 1 month (n=8), and over 85%
after 3 months (n=5), 6 months (n=2) and 17 months (n=1).
Histological sections prepared for immunohistochemistry analysis of
type II collagen and aggrecan expression showed chondrocytes
characterized with a spherical morphology close to their native aspect in
cartilage, whereas a fibrobastic-like morphology was observed in
monolayer culture. The chondrocytes differentiated phenotype was
defined by medium to strong staining intensity in more than 25% of the
cells reflecting type II collagen and aggrecan expression. After a two-

week incubation period in agarose-alginate hydrogel, chondrocytes of all

samples (n=9) showed a differentiated phenotype. In 5 out of these 9
samples, more than 75% of the chondrocytes were redifferentiated. After
long-term incubation, either 1 month (n=9), 3 months (n=6), 6 months
(n=2) or 17 months (n=1), most of the samples showed chondrocytes
with redifferentiated phenotype. Extracellular staining was found in few
samples. A kinetics study allowed to analyze data after 1 month, 3
months and either 6 months (n=2) or 17 months (n=1). In 2 samples, the
number of differentiated chondrocytes increased with time whereas it
decreased in 1 out of the 3 samples.
In order to study the effect of incubation in agarose-alginate hydrogel
on the phenotype of monolayer-expanded chondrocytes, aggrecan, type
II collagen and type I collagen expressions were quantified by RT-PCR.
During monolayer expansion, chondrocytes phenotype was modified.
Type II collagen rate decreased and was no longer detectable after 3 to 4
passages (n=21), while type I collagen rate increased by 3 fold (n=27).
Aggrecan expression was quite stable during monolayer expansion
(n=26). Incubation in agarose-alginate hydrogel led to chondrocytes
redifferentiation. Type II collagen expression was detectable in 7
samples out of 21 after 2 weeks and in 20 out of 21 after 3 months with a
10,000 fold increase. Type I collagen median expression decreased
gradually down of about 10 fold in 3 months. Aggrecan median
expression was not modified by incubation in agarose-alginate hydrogel.
Catabolic mediators such as MMP-1, MMP-13 and ADAMTS-5 were
then studied. MMP-1 expression decreased of about 3 fold during
monolayer expansion (n=11) and increased of about 7 fold after a twoweek incubation period (n=21). Then it increased very slightly at 3
months (n=8). MMP-13 expression followed the same pattern but with a
higher increase of about 16 fold after 2 weeks of incubation (n=21) and a
slight decrease during the period between 2 weeks and 3 months (n=9).
On the contrary, ADAMTS-5 expression made a 3-fold increase during
monolayer expansion (n=16) and decreased of more than 100 fold
(n=16) after 2 weeks of incubation. It was increased of 15 fold after a 3month incubation period (n=7). Incubation in agarose-alginate hydrogel
reversed chondrocytes monolayer expression pattern of catabolic
mediators.
DISCUSSION
This agarose-alginate-based hydrogel shows interesting properties for
cartilage repair. It is very easy to handle for the surgeon with a press-fit
fixation, requiring no periosteal flap or suturing. It can support
chondrocytes transplantation and do not hinder tissue repair. Cells are
homogeneously distributed inside the scaffold with a good viability until
a 17-month period of incubation. This agarose-alginate hydrogel allows
redifferentiation of chondrocytes, which have lost their phenotype due to
monolayer expansion. Matrix metalloproteinases increased expression in
the scaffold may be due to hydrogel degradation by chondrocytes that
would replace it gradually by their own matrix proteins. This hypothesis
is supported by the results of the phase II clinical study, showing that the
scaffold was completely substituted by a neomatrix [1]. Chondrocytes
embedded in extracellular matrix are protected against immunogenic
reactions [2-4]. As incubation in agarose-alginate allows chondrocytes to
produce their own matrix proteins, the agarose-alginate hydrogel could
be used for allogenic chondrocytes implantation without rejection of the
graft, in the same way as osteochondral allografts.
REFERENCES
1. Selmi TA, et al. Autologous chondrocyte implantation in a novel
alginate-agarose hydrogel: outcome at two years. J Bone Joint Surg Br
2008 May;90(5):597-604.
2. Yao X, et al. Chondrocyte allografts for repair of full-thickness
defects in the condylar articular cartilage of rabbits. Chin J Dent Res
2000 Nov;3(3):24-30.
3. Fragonas E, et al. Articular cartilage repair in rabbits by using
suspensions of allogenic chondrocytes in alginate. Biomaterials 2000
2000 Apr;21(8):795-801.
4. Rahfoth B, et al. Transplantation of allograft chondrocytes embedded
in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage
1998;6(1):50-65.

Poster No. 1195 56th Annual Meeting of the Orthopaedic Research Society