Human amniotic membrane as a chondrocyte carrier

vehicle/substrate: In vitro study

G. Krishnamurithy,
1
P. N. Shilpa,
1
R. E. Ahmad,
2
Soah Sulaiman,
3
C. L. L. Ng,
4
T. Kamarul
1
1
Tissue Engineering Group, Department of Orthopaedic Surgery, NOCERAL, University of Malaya,
50603 Kuala Lumpur, Malaysia
2
Department of Physiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
3
Department of Obstetric and Gynaecology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
4
Department of Surgery, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Received 1 February 2011; accepted 20 May 2011
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33184
Abstract: Human amniotic membrane (HAM) is an estab-
lished biomaterial used in many clinical applications. How-
ever, its use for tissue engineering purposes has not been
fully realized. A study was therefore conducted to evaluate
the feasibility of using HAM as a chondrocyte substrate/car-
rier. HAMs were obtained from fresh human placenta and
were process to produced air dried HAM (AdHAM) and
freeze dried HAM (FdHAM). Rabbit chondrocytes were iso-
lated and expanded in vitro and seeded onto these prepara-
tions. Cell proliferation, GAG expression and GAG/cell
expression were measured at days 3, 6, 9, 12, 15, 21, and
28. These were compared to chondrocytes seeded onto
plastic surfaces. Histological analysis and scanning electron
microscopy was performed to observe cell attachment.
There was signicantly higher cell proliferation rates
observed between AdHAM (13-51%, P0.001) or FdHAM (18-
48%, p 0.001) to chondrocytes in monolayer. Similarly,
GAG and GAG/cell expressed in AdHAM (33-82%, p 0.001;
2260%, p 0.001) or FdHAM (4181%, p 0.001: 2860%,
p 0.001) were signicantly higher than monolayer cul-
tures. However, no signicant differences were observed in
the proliferation rates (p 0.576), GAG expression (p
0.476) and GAG/cell expression (p 0.135) between AdHAM
and FdHAM. The histology and scanning electron micros-
copy assessments demonstrates good chondrocyte attach-
ments on both HAMs. In conclusion, both AdHAM and
FdHAM provide superior chondrocyte proliferation, GAG
expression, and attachment than monolayer cultures making
it a potential substrate/carrier for cell based cartilage ther-
apy and transplantation. VC
2011 Wiley Periodicals, Inc. J Biomed
Mater Res Part A: 00A: 000000, 2011.
Key Words: human amniotic membrane, articular chondro-
cytes, tissue engineering, orthopaedics, biomaterials
How to cite this article: Krishnamurithy G, Shilpa PN, Ahmad RE, Sulaiman S, Ng CLL, Kamarul T. 2011. Human Amniotic
Membrane as a Chondrocyte Carrier Vehicle/Substrate: In Vitro study. J Biomed Mater Res Part A 2011:00A:000000.
INTRODUCTION
The use of cell substrates/carriers has been shown to be
benecial in cell transplantation procedures.
1
These carriers,
which are either made from natural, synthetic, or a combi-
nation of both materials provides a stable platform during
the delivery of cells in damaged sites.
2
The challenge none-
theless remains in developing an ideal material which is
bio-compatible and, cause minimal or no immune reaction.
Furthermore it is expected that this biomaterial should
undergo complete bio-degradation as soon as its function is
replaced by the transplanted or surrounding native tissues.
3
Currently more advanced engineering techniques have
reported the use of synthetic material such as PLA or PGA,
molded or designed using precision tools to produce cell
carriers/scaffolds.
4
However, due to their resilient but less
biologically compatible properties, naturally processed
materials are being favored more, for example the use of
type I collagen mesh. The use of such material has been
well established in surgery involving cartilage repair, such
as that which has been described for matrix assisted chon-
drocyte implantation (MACI).
5
There are nevertheless issues
involved in the manufacturing of these materials. First, the
costs of production have been shown to be highly exorbitant
making it less likely to be used in clinical applications. Sec-
ond, the engineering and manufacturing skills and facilities
needed to produce these materials are highly advanced and
costly, making the availability of such technologies limited
to patients attending advanced medical centers located
mainly in developed nations.
6
The need for more cost-effec-
tive and easily available material is therefore a necessity.
Correspondence to: T. Kamarul; e-mail: tkzrea@um.edu.my
Contract grant sponsor: University of Malaya Research Grant (UMRG); contract grant number: RG019/09HTM
Contract grant sponsor: Short-Term Research Grant; contract grant number: FS125/2008A
Contract grant sponsor: University of Malaya HIR-MOHE
VC 2011 WILEY PERIODICALS, INC. 1
It has been demonstrated that amniotic membrane (AM)
may be a potential candidate for this purpose.
7
AM is a
readily available, thin, highly exible material which is bio-
compatible and biodegradable. These membranes originate
from a layer, covering the extra-embryonic tissues which
consist of the chorionic plate and maternal decidua. Follow-
ing child birth, AM becomes separated from the chorionic
plate leaving behind this layer of tissue. AM appears as a
thin matrix composed of an amniotic epithelial cell (AEC)
layer, basal membrane and stromal matrix.
8,9
AM has been
used in various medical applications, such as wound dress-
ing,
10
tissue adhesion barriers,
11
and ophthalmologic sur-
gery.
12
It also exhibits anti-adhesive,
13
anti-inammatory,
14
and antimicrobial properties.
15
However, its application as a
cell carrier has only been recently realized.
1619
It has been
described that ECM components which include collagen, -
bronectin, laminin, and other proteoglycans present within
its basement membrane, provide superior cell growth.
20
These molecules form ligands with the integrin binding
receptors located on cell surfaces, which in turn provides
cell adhesion upon surface contact.
21,22
An increased
ECMcell interaction is also known to enhance cellular
expression. Hence, because these constituents duplicate the
cartilage microenvironment, it has been postulated that
HAM supports chondrocyte proliferation when implanted on
its surface thereby making it a suitable cell carrier. Despite
the sound hypothesis made with regards to the function of
HAM, this has yet to be proved in any known experimental
study. To prove this, a study was thus investigated to deter-
mine if HAM would improve cell proliferation and expres-
sion using an in vitro model as predicted.
MATERIALS AND METHODS
Preparation of air dried (AdHAM) and freeze
dried (FdHAM)
Human placentas were obtained from individuals under-
going caesarean-section. Informed consents were obtained
prior to AM harvesting. Nine subjects (n 9) aged between
25 and 35 years old were recruited for this study. They
were screened for hepatitis B and C, syphilis and human im-
munodeciency virus (HIV). The initial processing of the
amniotic membranes involved mechanical peeling (blunt
dissection) of the HAM from the chorionic layer of the pla-
centas under sterile conditions (Fig. 1). The amniotic mem-
branes were subsequently washed with sterile deionized
distilled water followed by 0.05% sodium hypocholoride
(Sigma-Aldrich, USA). HAMs were then rinsed three times
using sterile 0.9% normal saline (Sigma-Aldrich) to ensure
complete cleaning of the extracted membranes. The clean
HAMs were sectioned into two pieces to produce two differ-
ent types of HAM preparations: air dried HAM (AdHAM)
and freeze dried HAM (FdHAM). For the AdHAM prepara-
tion, the rst half of the initially processed HAM was left to
dry in a bio-safety cabinet overnight (minimum 12 h).
Another half was stored in a 20

C freezer overnight before

being transferred into a freeze drier machine (Labconco,
USA) to produce FdHAM. The air dried and freeze dried
amnions were further sectioned into 3 cm rounded shaped
scaffolds and sealed into autoclavable plastic bags. The
sealed AdHAM and FdHAM were labeled and sent for 25
kGy gamma irradiation to ensure sterilization of these
HAMs (Nuclear Agency of Malaysia).
Isolation and culture of autologous chondrocyte
Cartilage tissue were harvested from knee, hip and shoulder
joints of (n 3) New Zealand White rabbits aged 23
months old approved by the Animal Care and Use Commit-
tee, University of Malaya (Ref.no.OS/08/09/2008/DCSK).
Articular cartilage was isolated under sterile conditions. The
cartilages were washed using 1 phosphate buffer saline
(PBS) containing 100 U mL
1
of penicillin and 100 lg mL
1
of streptomycin (Gibco, Invitrogen, USA). Tissue obtained
were nely minced and washed several times in PBS (1)
and subsequently digested for 1316 h using 0.6% type II
collagenase (Gibco, Invitrogen, USA). The digested tissue
was centrifuged at 1700 rpm for 10 min, and the cell pellets
were suspended in Dulbeccos modied Eagles medium
(DMEM/F12; Gibco, Invitrogen) containing 10% of newborn
calf serum (NCS) (Gibco, Invitrogen), 100 U mL
1
of penicil-
lin and 100 lg mL
1
of streptomycin, 1% of glutamine, and
0.01% of ascorbic acid. Cell number and viability were
determined using the Trypan blue exclusion method. The
suspended cells were seeded in tissue culture asks and
placed in a humidied incubator with 5% carbon dioxide
supply.
Chondrocyte seeding on AdHAM, FdHAM,
and plastic surfaces
Both AdHAM and FdHAM were placed into 3.5-cm HydroCell
culture plates (CellSeed, Japan). Rabbit chondrocytes at pas-
sage 2 (P2) were seeded at 2 10
5
cells per-well onto the
basement membrane of the two different HAM substrates.
The same density of cells was also seeded onto the
FIGURE 1. Preparation of HAM by blunt dissection. [Color gure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
2 KRISHNAMURITHY ET AL. HUMAN AMNIOTIC MEMBRANE AS A CHONDROCYTE CARRIER VEHICLE/SUBSTRATE
uncoated surfaces of six-well tissue culture plates. The
specimens were cultivated in 3 mL of culture media
(DMEM-F12) supplemented with 10% of newborn calf se-
rum (NCS), 100 U mL
1
of penicillin and 100 lg mL
1
of
streptomycin, 1% of glutamine and 0.01% of ascorbic acid
for nourishment. Further, these specimens was placed in a
humidied incubator supplied with 5% carbon dioxide at
37

C and analyzed at different time points, that is, days 3,

6, 9, 12, 15, 21, and 28. A total of nine (n 9) samples of
AdHAM and FdHAM were used in this experiment.
Alamar blue assay for cell proliferation
A correlation between the % of alamar blue reduction and
cell numbers were carried by measuring the AB absorbance
changes and numbers obtained using haemocytometer cell
count. The 2 10
5
cell density was initially seeded in the
individual wells of a six well plate. AB was added to the
well and observed for cell proliferation at days 3, 6, 9, 12,
and 15 and cells. At these time points cells were subjected
to trypsinization followed by cell counting, which was deter-
mined by using a modied Neubauer-heamocytometer. The
standard curve of % of AB reduction against cell concentra-
tion was plotted and determined.
Cell proliferation assay was carried out based on the %
of alamar blue (AB) reduction. AB was added directly into
culture media at nal concentration of 10% and the six-well
plates were returned to the incubator for 6 h. Monolayer
cultures in six well plates were used as comparison. The
100 lL of incubated media from all treated plate was trans-
ferred in 96-wells plate. To serve as a negative control, AB
added to media without cell was used. Optical density of
the plate was measured at 570 and 600 nm using a stand-
ard micro plate reader (Plate Chameleon, Finland). A total
of nine (n 9) samples of AdHAM and FdHAM were used
in this experiment.
Sulfated glycosaminoglycan (GAG) analysis for cellular
extra-cellular expression
GAG expression was assessed on all preparation based on
the concentration of proteoglycan in cell culture media. Pro-
teoglycan in these media was determined at different time
points, that is, days 3, 6, 9, 12, 15, 21, and 28 using a blycan
scan kit (Biocolor, Northern Ireland, UK). The 100 lL of
media from all preparation was pipetted and mixed with
blyscan dye reagent (composed of 1,9-dimethyl-methylene
blue in an organic buffer) for 30 min. The glycosaminogly-
can-dye complex was collected using centrifugation method.
The dye bound to the pellet was solubilized by mixing a dis-
sociation reagent into the solution. The absorbance of the
samples was measured at a wavelength of 656 nm using a
nanophotometer (Implen, Bio-Red, Germany). Statistical
evaluation of the data was performed using statistical soft-
ware package, SPSS version 17.0. Parametric analysis was
performed using One-Way ANOVA to determine the statisti-
cal signicance of the results. Statistical signicance was set
at 95% condence interval (p < 0.05). The GAG/cell was
obtained by substracting the total GAG/well over total cell
number on days 3, 6, 12, 15, 21, and 28 for AdHAM, FdHAM
and monolayer, respectively.
Histological analysis
Hematoxylin and eosin (H&E) staining was conducted to
study the attachment of the seeded chondrocytes on
AdHAM and FdHAM, respectively. The specimens were xed
in 10% formaldehyde, embedded in parafn and then sec-
tioned at a thickness of 4 lm at day 14. The sections of
each specimen were subjected to H&E staining and
observed with the bright eld microscope (Let Letzar,
Germany).
Scanning electron microscopy (SEM)
SEM analysis was used in this study to determine the micro-
structural properties of AdHAM and FdHAM seeded with
chondrocytes, respectively. The specimens at day 14 were
xed overnight in 4% glutaraldehyde in 0.1M calcodylate
and postxed for 1 h in 1% aqueous osmium tetraoxide.
These specimens were washed three times in distilled water
before being dehydrated through a graded ethanol series
(50, 70, 80, 90, 95, 100%, acetone mixture and in pure ace-
tone). The specimens were subsequently dried at a critical
point in a critical point drier (Bal Tec, CPD030) supplement
with CO
2
. The specimens were mounted on the aluminum
stub and sputter coated with gold before being examined
using a digital scanning electron microscope (JSM 6400;
JEOL, Tokyo, Japan).
RESULTS
Cell proliferation
The percentage of AB reduction was plotted against the cell
concentrations from day 1 to 28 showed linear regression
between the two ranging from 2.19 10
5
to 4.68 10
5
cells (Fig. 2). This data were used as a reference value for
the proliferation experiment as the percentage of AB reduc-
tion was converted in cell numbers.
A signicant increase in chondrocyte proliferation was
observed between both HAMs and Monolayer cultures (p
0.001). There was signicantly higher cell proliferation rates
observed between AdHAM (13-51%, p 0.001) or FdHAM
(1848%, p 0.001) to chondrocytes in monolayer (Fig. 3).
There were no signicant differences in cell proliferation
between AdHAM and FdHAM (p 0.576).
Expression of sulfated glycosaminoglycan (GAG)
AdHAM and FdHAM showed signicant increase in total
GAG (p 0.001) as compared to monolayer cultures from
day 3 to 28 (Fig. 4).The comparison of GAG content per cell
of monolayer, AdHAM and FdHAM is shown in Figure 5.
There was no signicant difference in GAG content per cell
between than AdHAM and FdHAM (p > 0.05).
Histological analysis
The H&E histological stained slides revealed that AdHAM
maintain the hierarchy of the structure which consist of epi-
thelial cells layer, followed by basal membrane and stromal
matrix [Fig. 6(A,B)]. In the case of FdHAM, the membrane
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2011 VOL 999A, ISSUE 00 3
consists of vast range of pores [Fig. 6(C,D)] formed due to
evaporation of water crystal during freeze drying. The
nuclei of the chondrocytes, which were attached on both
AdHAM and FdHAM, appeared large, dense, ovoid and cen-
trally located within its cytoplasm.
Scanning electron microscope (SEM)
The 3D morphology of AdHAM, FdHAM, and chondrocytes
attachment on day 14 was analyzed using SEM. Based on
the topographical analysis; both preparations indicated the
presence of surface for the cells attachment. In AdHAM, the
basal lamina was clearly present and intact which formed a
continuous at and smooth layer above the brous collage-
nous stroma. On the surface of this layer, the seeded chon-
drocytes formed continuous layer of fusiform cells over the
AdHAM. ECM materials or proteins were found at the
peripheries of these cells. In FdHAM cultures, chondrocytes
appear to be highly attached within the pores form in this
material, most likely due to the larger surface area available.
Unlike that of AdHAM, cells on FdHAM formed large colo-
nies [Fig. 7(AD)].
DISCUSSION
Cell phenotype and proliferation analysis on cell-material
construct is an essential step in the evaluation of biocom-
patible products. In this study, there was signicant differ-
ence in cell proliferation and GAG expression when chon-
drocytes were implanted onto AdHAM or FdHAM as
compared to monolayer cultures. It appears that prolifera-
tion and ECM expression of chondrocytes is inuence by the
presence of biomaterial, which was also observed in studies
using other natural scaffolds.
23
This may be contributed fur-
ther by the presence of naturally occurring biological mole-
cules within the substrate resulting in increased cellmatrix
interactions.
24
Because the ECM of AM is composed of vari-
ous structural proteins such as type III and type IV collagen,
native hydrated proteoglycan and glycoproteins that resem-
ble the in vivo environment of native cartilage, cells seeded
on AM is expected to produce cell expressions that are
equal to that of natural tissues. Therefore, it is only right to
assume that the characteristic present in AM will provide
the ideal environment for the chondrocytes to thrive when
seeded onto these surfaces.
25
The increase in both cell proliferation and expression
can be explained through the cellmatrix interaction
FIGURE 2. Standard curve of % of Alamar blue reduction against cell
concentration(chondrocytes).
FIGURE 3. The rate of cell proliferation using Alamar blue assay.
[Color gure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
FIGURE 4. GAG content expressed in AdHAM and FdHAM at day 3
28. [Color gure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
FIGURE 5. GAG content /cell in AdHAM and FdHAM at days 328.
[Color gure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
4 KRISHNAMURITHY ET AL. HUMAN AMNIOTIC MEMBRANE AS A CHONDROCYTE CARRIER VEHICLE/SUBSTRATE
FIGURE 6. H&E staining of cross section of two different HAM substrates at 40: AdHAM control (A), AdHAM (B), FdHAM control (C) and
FdHAM (D). E epithelial, C seeded cells, B basement membrane. [Color gure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
FIGURE 7. SEM analysis of AdHAM and FdHAM seeded with chondrocytes. A,B-AdHAM; C,D-FdHAM, ECM !, Chondrocyte --->, Pores >.
[Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2011 VOL 999A, ISSUE 00 5
phenomenon that enhances cell signaling through direct or
indirect cell signaling mechanisms.
26
Attachment of cells
onto ECM is induced through various surface adhesion mol-
ecules (SAMs) which allows ECM molecules such as bro-
nectin, tenasin and collagen (through a number of amino
acids, for example arginine-glycine-aspartic acid-RGD pep-
tide sequence) to be attached onto integrin binding proteins
located on surfaces of cells.
27
The binding of these integrin
receptors leads to the activation of proteins associated with
the cytoskeleton, such as paxillin,
28
and other intracellular
signaling proteins, e.g., focal adhesion kinase (FAK)
29
and
MAPK signaling molecules.
30
All of which leads to increased
cellular proliferation and expression, similar to what was
observed in this study. It is however incorrect to assume
that the activation of these pathways will result in a total
summation of cellular response resulting in an overall
change in cell expression. More so when one considers that
a particular activation of a known pathway leads to a spe-
cic cell response, despite the presence of cross-talk signal-
ing which may be present during this process.
31
Mechanical
stability, which provides a consistent outside-inside signal-
ing, appears to play an important role in cell proliferation
and ECM remodeling as demonstrated in previous studies.
32
In addition, it has also been shown that both cell prolifera-
tion and ECM production is inter-related, demonstrating a
close relationship between the two.
20
The determination of
GAG production per cell was therefore important to demon-
strate the true increase in ECM production which is unaf-
fected by cell numbers. In the present study, it was found
that there was signicant difference in GAG synthesized by
chondrocytes in AdHAM and FdHAM as compared to mono-
layer despite having divided the total GAG to the number of
cells present in this culture system. In other studies, Kawa-
saki et al.
33
demonstrated similar effect when hyaluronic
acid was used in chondrocytes cultured in collagen gel. The
rate of GAG synthesis over a wide range of densities of
chondrocyte showed a reduced dependency on cell den-
sity.
34
It has been shown that cartilage-specic extracellular
matrix (ECM) components such as type II collagen and gly-
cosaminoglycan (GAG) play a critical role in regulating
expression of the chondrocytic phenotype.
35
However, the
apparent balance between the extent to which proliferation
and GAG has important implications for tissue engineering
strategies of functional repair.
36
It was evident that there was a signicant increase in
the ability of cells to produce ECM irrespective of cell num-
bers when implanted onto HAM surfaces. As far as the
authors of this article are aware, this is the rst study that
has demonstrated as such in studies involving chondrocyte
carriers. More interestingly, it is noted that an increase in
GAG per cell production was signicantly higher AdHAM or
FdHAM than monolayer. This can be explained by the
change in the material surface conformation as a result of
air drying or freeze-drying. Ice crystals formed within HAM
during the freezing stage is evaporated during the lyophili-
zation process, creating spaces which appear as pores as
demonstrated in our SEM images. Connecting pores from
one surface to the other creates communicating networks of
varying sizes. These networks provide continuous cell-to-
cell contact and signals enabling efcient cell functions and
expressions.
37
Although not as extensive as certain biomate-
rials, the three-dimensional cell culture environment pro-
vided by AdHAM and FdHAM adds the advantage of anchor-
age-independent cell growth, while maintaining the
differentiated phenotype that allows the synthesis of cell-
specic pericellular or intercellular matrix.
38
Although, one
may argue that HAM is essentially a 2D material, the images
presented in this study have demonstrated otherwise. The
freeze-drying process involved in producing FdHAM has ex-
plicitly caused changes to the topographic arrangement of
this material, which also involves the creation of pores and
networks.
In the present study, feasibility of AdHAM and FdHAM
as a substrate of chondrocytes was analyzed and evaluated
for its implications as a cell delivery vehicle. The results of
cell attachment, viability, proliferation, and GAG analysis
supports the possible role of AdHAM and FdHAM in carti-
lage tissue engineering. However, in this study, among sev-
eral ECM proteins expressed during chondrocyte prolifera-
tion only GAG content was measured. Other important
protein expressions need also be considered. In addition,
further studies need to be conducted to elucidate the gene
expression related with GAG content and mechanism behind
MAPK and FAK pathway against the upregulation of integrin
receptor.
Despite its inadequacies, this study has demonstrated
with a high degree of condence that HAM is a promising
and a potentially suitable material for use as a chondro-
cyte carrier, which not only promotes cell proliferation but
also ECM expression. Coupled with the fact that HAM has
been used for many other clinical applications thus leaving
an excellent track record as a safe biomaterial, it is more
likely that its application for tissue engineering purposes
will be forthcoming. However, study involving in vivo
model would be necessary to determine its robust poten-
tial, as the native environment for which this material is
transplanted may inuence the nal outcome. This is
because transplanting cells/materials in living organisms
may involve changes in matrix organization and conforma-
tion of the transplanted cells, which cannot be demon-
strated in an in vitro study.
CONCLUSION
The results of this study indicate that HAM could potentially
be an effective chondrocyte carrier, increasing both cell pro-
liferation and ECM expression.
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ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2011 VOL 999A, ISSUE 00 7