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TISSUE ENGINEERING CONSTRUCTS AND CELL SUBSTRATES
Evaluation of the growth and osteogenic differentiation of ASCscultured with PL and seeded on PLGA scaffolds
Abdalla Awidi • Nidaa Ababneh • Hussein Alkilani •
Bariqa Salah • Shymaa Nazzal • Maisaa Zoghool •
Maha Shomaf
Received: 11 February 2014 / Accepted: 24 October 2014 / Published online: 3 February 2015
� Springer Science+Business Media New York 2015
Abstract Scaffold serves as an important component of
tissue engineering, which facilitates cell attachment, pro-
liferation and differentiation of cultured cells. In this study
we aimed to use platelet lysates as a substitute for FBS in
culturing and proliferation of human adipose tissue-derived
stromal cells (ASCs), which constitute a promising source
for cell therapy. We characterized ASCs in the presence of
PL, and then we seeded them onto poly(lactic-co-glycolic
acid) (PLGA) scaffolds, osteogenic media was used to
induce their proliferation and osteogenic differentiation.
Gene expression analysis revealed higher expression of
osteogenic related genes, immunohistochemical staining
showed proper cell attachment, growth and collagen matrix
formation with the ability to induce vascularization. In
conclusion, expansion of ASCs in PL-supplemented med-
ium could promote cell proliferation and osteogenic dif-
ferentiation of cells seeded on PLGA scaffolds, therefore it
could be considered as a suitable and effective substitute
for FBS to be used in clinical applications.
1 Introduction
Large bone defects in human are commonly treated by
autologous bone grafting. This invasive technique is asso-
ciated with potential complications including chronic pain
and risk of infection. Tissue engineering has emerged as a
new approach in bone regeneration, which involves the
combination of osteoprogenitor cells, biomaterial scaffolds
and growth factors that promote cell growth, differentiation
and mineralized bone tissue formation, [1, 2].
The main goal of bone tissue engineering has been to
develop biodegradable materials as bone graft substitutes
for filling large bone defects. Scaffolds are artificial
matrices designed to mimic the mechanical and biological
properties of the tissue matrix [3]. These materials should
maintain adequate mechanical strength, be osteoconductive
to promote cellular interactions and tissue development,
osteoinductive to induce proliferation and differentiation of
cells into osteoblasts lineage, and degraded at a controlled
space for the formation of new bone [4]. Furthermore,
scaffolds should have high porosity and well connected
pores to provide good environment for sufficient cell
seeding density, cell–cell communications, and exchange
of nutrients and metabolic product [5]. Pore size plays an
important role, as larger pore size weakens the scaffold,
whereas smaller pore size hampers neovascularization [1].
Several types of porous scaffolds have been shown to
support in vitro bone formation by human cells, including
those made of ceramics [6], native and synthetic polymers
[7], and composite materials [8]. The use of biodegradable
polymers would be a promising material for bone
grafts, and have been extensively investigated [4].
Poly(a-hydroxy acids), including PGA, PLA, and their
copolymer PLGA, are the most popular and widely used
synthetic polymeric materials in bone tissue engineering
A. Awidi (&) � N. Ababneh � S. Nazzal � M. Zoghool
Cell Therapy Center, University of Jordan, Amman 11942,
Jordan
e-mail: [email protected]
H. Alkilani
Chemistry Department, University of Jordan, Amman, Jordan
B. Salah
Department of Plastic Surgery, Jordan University Hospital,
Amman, Jordan
M. Shomaf
Department of Pathology, Microbiology and Forensic Medicine,
University of Jordan, Amman, Jordan
123
J Mater Sci: Mater Med (2015) 26:84
DOI 10.1007/s10856-015-5404-8
[9]. PLGA was reported as the most popular polymer [10],
because of good biodegradability and biocompatibility.
PLGA and its copolymers have been widely used in bone
tissue engineering research [11, 12].
Mesenchymal stromal cells (MSCs) can be obtained
from various tissue sources, like bone marrow, adipose
tissue, umbilical cord, or placenta [13]. The cells are plastic
adherent, fibroblast-like in morphology, they have the
ability to differentiate toward osteoblasts, adipocytes and
chondrocytes in vitro, and they have a cell surface
expression of CD73, CD90, CD44 and CD105 and are
negative for CD45, CD34, CD31, CD19, CD14 [14].
Adipose tissue stem cells (ASCs) represent an attractive
source for bone tissue engineering [12], due to their
accessibility and potential for differentiation into osteo-
genic, chondrogenic, adipogenic and endothelial lineages
[15]. In previous studies, ASCs reflected features of oste-
ogenic cells after induction, including an osteoblast-like
morphology, a deposited calcified matrix, and the expres-
sion of specific genes and proteins [16, 17]. ASCs have
several advantages, compared with BMSCs, from the per-
spective of clinical applications; for example, they are
easier to obtain, carry relatively lower donor site morbidity
and are available in large numbers of stem cells at harvest.
Characteristics of human PL-cultured ASCs have been
evaluated previously, indicating enhanced proliferation rate
and a similar cell surface marker profile [17, 18]. Several
groups have reported the formation of bone-like constructs
from BMSCs and ASCs cultured on porous scaffolds
[7, 18].
Currently, there is a growing interest to avoid the use of
FBS, due to the potential of xenogenic immune reactions of
bovine pathogens and a high lot-to-lot variability that
hampers reproducibility of the results [19]. Human platelet
lysate has been shown to be an efficient replacement for
FBS [19]. Platelet granules contain many growth factors;
including platelet derived growth factor (PDGF), fibroblast
growth factor (FGF), insulin growth factor (IGF), and
transforming growth factor-b (TGFB) [20].
The purpose of this study was to evaluate the osteogenic
differentiation capacity of ASCs cultured on PLGA scaf-
folds in the presence of PL supplemented media, to esti-
mate the cell-scaffold interaction for bone regeneration and
to determine the role of PL for enhancement of the growth,
proliferation and osteogenic differentiation.
2 Materials and methods
2.1 Platelet lysate preparation (PL)
PL was obtained from different platelet apheresis collec-
tions prepared at Blood Banking unit in Jordan University
Hospital (JUH). The platelet count was performed at the
hematology unit, using automated hematology analyzer.
The collected samples were subjected to three repeated
temperature cycles, frozen at -80 �C then heated at 37 �C
and then frozen at -20 �C until future use. Platelets were
eliminated by centrifugation at 1,4009g for 10 min.
2.2 ASCs culture and seeding conditions
Lipoaspirate samples were obtained from the department of
plastic surgery (Jordan University Hospital, Amman, Jor-
dan), according to hospital guidelines after written
informed consent. Lipoaspirate samples were harvested
and washed with sterile phosphate-buffered saline (PBS)
containing 1 % antibiotics, then incubated in 0.075 % type
I collagenase containing PBS for 40 min at 37 �C with
intense stirring. Cells were filtered using cell strainer and
centrifuged at 1,2009g for 10 min to obtain the pellet.
Then the pellet was washed repeatedly with medium. After
that, the pellet was suspended in a-MEM media and cul-
tured with a seeding density of 0.18 9 106 cells/cm2 in the
presence of 5 % PL, 100 mg/ml L-glutamine, and 1 %
penicillin/streptomycin (Invitrogen) and incubated at 37 �C
with 5 % CO2.
2.3 Immunophenotypic characterization of ASCs
A panel of cell surface markers was used to evaluate their
immunophenotypic characteristics of ASCs cultured in PL-
supplemented media at passage 2. ASCs were harvested in
0.25 % Trypsin/EDTA and incubated in 1 % BSA in PBS,
and aliquots of 105 cells/100 ll were incubated with
monoclonal antibodies. Cells were washed and analyzed by
fluorescence activated cell sorter (FACS) Calibur flow
cytometer (Becton Dickson, San Jose, CA, USA) with the
Cell-Quest pro software (Becton Dickson, San Jose, CA,
USA).
2.4 Multilineage differentiation of human ASCs
Cells were seeded on six-well plates for osteogenic and
adipogenic differentiation using aMEM media supple-
mented with 5 % PL. For adipogenic differentiation, cells at
70 % confluency were stimulated with growth medium
containing 10-7 M dexamethasone, 0.5 lM isobutylmeth-
ylxanthine, and 50 lM indomethacin. On day 18, Adipo-
genic monolayer cultures were then rinsed twice with PBS,
fixed with 10 % (v/v) formalin for 10 min, washed with
distilled water, then rinsed with 60 % 2-propanol and cov-
ered with a 0.3 % Oil Red O solution. After 10 min of
incubation, cells were briefly rinsed again with 60 %
2-propanol and thoroughly in distilled water and let dried at
room temperature then visualized under inverted microscope
84 Page 2 of 9 J Mater Sci: Mater Med (2015) 26:84
123
and photographed. For osteogenic differentiation, we used
aMEM media supplemented with 5 % PL, 50 lm L-ascorbic
acid-2-phosphate and 10 mM B-Glycerophosphate with
0.1 lm dexamethasone. On day 18, the monolayers were
fixed in 70 % ethanol for 1 h at 4 �C then stained for 15 min
with Alizarin Red-S for 15 min at room temperature to check
for calcium deposition. After that, cells were washed four
times with distilled water then visualized by inverted
microscope and photographed. Finally, The presence of
alkaline phosphatase activity was detected by washing the
cells with cold PBS and fixing them in 10 % neutral formalin
buffer solution for 30 min, then the cells were stained with
naphthol AS-MX-PO4 (Sigma) solution and Fast red violet
LB salt (Sigma) for 45 min in the dark at room temperature.
Cells were washed three times with distilled water and
visualized by inverted microscope and photographed.
2.5 Preparation and characterization of PLGA scaffold
Scaffolds of PLGA were prepared by proprietary particu-
late leaching technique using chloroform as a solvent for
polymer dissolution. Commercial grade Poly (D,L-lactide-
co-glycolide) (Lakeshore BiomaterialsTM) with co-mono-
mer composition of 75:25 having an inherent viscosity (dl/
g) 0.60–0.80. Porous scaffolds were prepared by solvent
casting/particulate leaching process (SL), using a bed of
partially fused sieved sugar crystals (106–355 lm); after
chloroform evaporation at room temperature, the scaffolds
were thoroughly washed to leach the porogen. Finally the
scaffolds were sterilized with gradient ethanol. Prepared
scaffolds had 98.6 % porosity with the following dimen-
sions (diameter = 2.0 cm, thickness = 4.0 mm).
2.6 Cell seeding on PLGA scaffolds
The characterized ASCs at passage 2 were seeded on cul-
ture flasks, aMEM containing 5 % PL and 1 % penicillin/
streptomycin (Invitrogen) was used and cells were incu-
bated at 37 �C with 5 % CO2 in a humidified incubator.
The medium was changed twice weekly to wash out all
non-adherent cells. After the cells reached 80 % conflu-
ence, they were trypsinized and resuspended in aMEM.
PLGA scaffolds were prepared in circular shape. Then
characterized and sterilized by 70 % ethanol and incubated
in 10 ng/ll fibronectin for overnight, then they kept to air
dry under sterile biosafety cabinet and incubated into cul-
ture medium for 3 h. Scaffolds were distributed in a
24-well cell culture plate (Nunc). Seeding was performed
as droplets on the top of scaffold, cell suspension of
0.7 9 106 cells/scaffold was distributed and incubated for
3–4 h in humidified incubator at 37 �C with 5 % CO2 to
allow the cells to attach to the scaffold. An additional
culture medium was added to each scaffold, the medium
was changed in the next day and replaced by osteogenic
differentiation media. The cells/scaffold constructs were
cultured for 1, 4, 7, 14, and 21, 28 days under the same
culture conditions. The day 1 culture was considered as
control scaffold. Representative samples were taken at
different time points to prepare the evaluation assays.
2.7 Cell proliferation
Cell proliferation was measured for ASCs adherent to
plastic surface and for cells seeded on PLGA scaffold by
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide) assay at 1, 4, 7, 14, 21, and 28 days after
seeding. At a given culture time, 20 ll of MTT solution
(Promega, USA) was added into each sample and incu-
bated for 4 h at 37 �C. Finally, 200 ll stop solution was
added and the samples were incubated for 10 min to dis-
solve the formazan pigment. The plate was read at 570 nm
by a microplate reader, and the optical density values were
normalized to that of the culture media.
2.8 Alkalie phosphatase (ALP) assay
Osteoblast differentiation of the ASCs cultured on plastic
surface and on scaffolds was evaluated by the measurement
of ALP activity using p-nitrophenyl phosphate (PNPP)
solution as the reaction substrate, on days 1, 4, 7, 14, 21
and 28. Monolayer ASCs or cells seeded on PLGA scaffold
was incubated with 250 ll of cell lysis solution (0.2 % v/v
Triton X-100, 10 mM Tris (pH 7.0), 1 mM EDTA) then
conducted into two freeze/thaw cycles, at -70 �C for
30 min and thawing at 37 �C for 40 min. After the final
thaw, samples were crushed into small fragments with a
pipette tip to improve cell lysis and vortexed vigorously for
3–5 min. The lysate stored at -70 �C until analysis. To
determine ALP activity, the lysate was centrifuged for
10 min at 13,000 rpm. An 25 ll of cell lysate was added to
125 ll ALP substrate buffer, (1 mg/ml PNPP in 0.1 M
diethanolamine buffer, 1 % Triton X-100 and 1 mM
MgCl2 (pH 9.8)), and the mixture was incubated at 37 �C
for 30 min with shaking. The enzymatic reaction was
stopped by the addition of 0.5 M NaOH, and the product of
p-nitrophenyl (PNP) was immediately measured at 405 nm
using an ELISA plate reader.
2.9 RT-PCR for ASCs osteogenic genes expression
Osteogenic differentiated monolayer ASCs cells and cel-
lular scaffold constructs were collected on days 1, 4, 7, 14,
21 and 28 after osteogenic induction. ASCs on plastic were
trypsinized and used for RNA extraction. Each scaffold
J Mater Sci: Mater Med (2015) 26:84 Page 3 of 9 84
123
was broken in lysis buffer, and mRNA was extracted
using RNEasy Mini Kit (Qiagen) according to the man-
ufacturer’s instructions. The expression of osteopontin
(OP), alkaine phosphatase (ALP), osteonectin, collagen
type I (Col I), osteoclacin (OC), and RUNX2 were
evaluated in monolayer ASCs and cells seeded on PLGA
scaffolds, with a previously published primers (22). RNA
was extracted using RNeasy mini kit (Qiagen). One
microgram of each RNA sample was used for reverse
transcription in a final volume of 20 lL. The RNA was
mixed with 1 ll of random hexamer, heated to 65 �C for
5 min and then chilled on ice and mixed with 4 ll of 5X
reaction buffer, 20 mM of dNTPs, and 20U of RNase
inhibitor, and then mixed with 200U of M-MLV reverse
transcriptase (Promega, USA). The reaction was carried
out at 42 �C for 1 h and 70 �C for 10 min. Then, cDNA
was amplified using GoTaq� qPCR Master Mix (Pro-
mega, USA). The PCR reaction was performed in a final
volume of 25 ll, in a mixture composed of 2 ll of cDNA
template, and 1X of GoTaq� qPCR Master Mix, 10 pmol
of each primer. The reaction was started with heating at
95 �C for 5 min, and the cycles consisted of denaturation
at 94 �C for 30 s, annealing for 30 s at 57 �C, and
extension at 72 �C for 1 min, and the elongation was
performed at 72 �C for 10 min. Amounts of gene
expression were normalized to that of GAPDH, results are
reported as relative gene expression 2-DDct.
2.10 Histology, immunohistochemistry and flow
cytometry analysis
After 1, 4, 7, 14, 21 and 28 days, the PLGA scaffolds were
fixed in 10 % (v/v) buffered formaldehyde, dehydrated in
an ascending grades of ethanol, and embedded in paraffin.
For observation of cell attachment and proliferation and
collagen matrix formation, paraffin blocks were sectioned
at a 5 lm thickness and stained with hematoxylin and eosin
(H&E) and Massson’s trichrome to routine histology pro-
tocols, immunohistochemistry was performed for CD31
marker to evaluate vascularization and new blood vessels
formation. Flow cytometry analysis of CD31 (PE-conju-
gated Monoclonal Antibody) and VEGF (APC-conjugated
Monoclonal Antibody) was performed to confirm the
immunohistochemistry results.
2.11 Statistical analysis
All measurements were performed in triplicates. Statistical
analysis was performed using the GraphPad Prism 4.0c
software. Data were shown as mean ± SD (n = 3).
3 Results
3.1 Characterization of ASCs
ASCs at passage 2 were cultured in a-MEM medium sup-
plemented with 5 % PL. Then cells were characterized for
their morphology, osteogenic and adipogenic differentiation
potential, and for their immunophenotype (Figs. 1, 2). ASCs
were analyzed for their antigen expression profile using
monoclonal antibodies conjugated with fluorescein isothio-
cyanate, phycoerythrin, activated peridinin-chlorophyl-pro-
tein complex or allophycocyathin, they demonstrated the
typical panel of MSC surface markers. Cells were positive
for CD90, CD73, CD44, and CD105, and negative for
CD19, CD14, CD45, CD34, CD31, and VEGF (Fig. 2).
After 21 days of induced culture, the differentiated cells
developed mineral deposition nodules that stained positive
by Alizarin Red and clear regions of ALP activity observed
after histochemical staining which indicates the osteogenic
differentiation potential (Fig. 1a–d). The adipogenic differ-
entiation was evaluated by Oil Red staining for the detection
of lipids in the vacuoles after 21 days of cell culture in
adipogenic medium (Fig. 1e, f), We observed very robust
and earlier osteogenic differentiation in the presence of PL.
3.2 Cell viability on PLGA scaffold
We developed Poly(D,L-lactide-co-glycolide) (PLGA) scaf-
folds with high porosity and interconnectivity, both of which
being very important for tissue growth, signaling and vas-
cularization. We used PL-cultured ASCs for seeding onto
PLGA scaffolds, precoated with fibronectin. Cell prolifera-
tion in PLGA scaffold was evaluated by measuring the
number of viable cells using the MTT (3-dimethylthiazol-
2,5-diphenyltetrazolium bromide) colorimetric assay to
observe the growth stimulation of the PL in ASCs cultures.
The assay reflected the activity of a mitochondrial dehy-
drogenase to transform light yellow MTT into dark blue
formazan. The intensity of the resulting color is determined
photometrically. Figure 3a shows the proliferation status of
ASCs cultured on monolayer and on PLGA scaffolds in
different time points. PL has been shown to significantly
increase ASCs expansion in vitro; cell proliferation was
increased over the first 2 weeks of cell culture in the presence
of PL-supplemented osteogenic media with higher growth
observed in monolayer cell culture than in PLGA scaffold.
3.3 Alkaline phosphatase activity
ALP is an enzyme secreted by osteoblasts and act as one of
the markers to confirm the osteoblastic phenotype and
84 Page 4 of 9 J Mater Sci: Mater Med (2015) 26:84
123
mineralization. Therefore, it has been used as a marker that
appears early during osteoblastic differentiation. The ALP
activity of the osteoblasts cultured on monolayer and on
PLGA scaffolds had increased during the first week of
culture and reached a maximum level between 10 and
14 days in both samples and then started to decrease
gradually (Fig. 3b).
3.4 Osteoblastic gene expression
Osteoblastic gene expression was analyzed in monolayer
ASCs culture and cells on PLGA scaffolds using quanti-
tative RT-PCR and they were normalised to GAPDH
housekeeping gene. The expression of the osteogenic genes
has been observed to be significantly high in PL cultures
ba c
d e f
100 μm 100 μm 100 μm
50 μm 50 μm 50 μm
Fig. 1 Characterization of ASCs. a Morphology of MSCs isolated
from human adipose tissues. b Minerals deposition of unstained ASCs
after 21 days. c ARS Staining appears as orange-red nodules of
minerals deposition. d Alkaline phosphatase Staining of ASCs; pink
areas represent sites of enzyme activity under an inverted microscope.
e Fat droplets formation of ASCs cultured in adipogenic medium.
f Adipogenic differentiated cells stained with Oil Red O (Color figure
online)
Fig. 2 Immunophenotyping of ASCs by flow cytometry. The majority of gated cells were positive for CD73, CD44, CD90 and CD105, and
negative for CD19, CD14, CD45, CD34, CD31 and VEGF (Color figure online)
J Mater Sci: Mater Med (2015) 26:84 Page 5 of 9 84
123
(P value \ 0.001). Figure 3c–h shows the transcription
profile of bone related genes, including ALP, Col I, ON,
RUNX2, OP and OC of PL-cultured ASCs inside the
scaffold (n = 3). We had similar expression profile for
ASCs cultured as monolayer and cells on PLGA scaffolds,
with slightly increased expression of cells cultured on
PLGA scaffolds. A peak in ALP expression was observed
at day 14 (Fig. 3d) in both samples, which could support
the results of ALP activity assay (Fig. 3b). The osteogenic
medium with PL enhanced higher expression levels of
osteocalcin and RUNX2 in ASCs, especially at 14 and
21 days. Osteocalcin started to increase after 4 days of
1 4 7 14 21 28
1 4 7 14 21 28
0
5
10
15
Osteopontin
Time (Days)
Time (Days)
1 4 7 14 21 28
Time (Days)
Rel
ativ
e Ex
pres
sion
ASCs/PlasticADSCs/PLGA
0
1
2
3
4
ALP
Rel
ativ
e Ex
pres
sion
ASCs/PlasticADSCs/PLGA
0
5
10
15
Osteonectin
Rel
ativ
e Ex
pres
sion ASCs/Plastic
ASCs/PLGA
0
2
4
6
8
COL I
Time (Days)
Rel
ativ
e Ex
pres
sion
ASCs/PlasticASCs/PLGA
0
1
2
3
4Osteocalcin
Rel
ativ
e Ex
pres
sion
ASCs/PlasticASCs/PLGA
1 4 7 14 21 28
1 4 7 14 21 28
Time (Days)1 4 7 14 21 28
0
1
2
3RUNX2
Time (Days)
Rel
ativ
e Ex
pres
sion ASCs/Plastic
ASCs/PLGA
MTT
Time (Days)A
bsor
banc
e0 4 8 12 16 20 24 28
0.0
0.5
1.0
1.5ASCs/Plastic
ASCs/PLGA
a b
c d
e f
g h
Fig. 3 Evaluation of the growth
and osteogenic differentiation of
ASCs in a monolayer culture
and on PLGA scaffold. a MTT
assay for the proliferation of
human ASCs cultured on plastic
and on PLGA scaffolds, at
different time points and under
the same culture conditions.
b ALP activity. c–h mRNA
expression levels of
osteopontin, alkalie phosphatase
(ALP), osteonectin, collagen
(Col I), osteocalcin, and runt-
related transcription factor 2
(RUNX2). Results are
expressed as mean ± SD
84 Page 6 of 9 J Mater Sci: Mater Med (2015) 26:84
123
differentiation. Expression of osteonectin was observed; it
reached a higher level after 14 days of culture in osteo-
genic media then declined at the end of differentiation
(Fig. 3c–h).
3.5 Immunohistochemistry and flow cytometry for Cell
growth and attachment
Cell distribution on the scaffold was determined using HE
stain, to observe the distribution of cells into the surface and
internal pores of the scaffolds. PLGA scaffolds allowed the
adhesion and proliferation of the seeded cells over 4 weeks
culture period. H&E images show early cell attachment
after 24 h of seeding on PLGA scaffolds at lower and higher
magnification (Fig. 4A a, b). Cells growth after 21 days was
robust and started to form a matrix around the pore structure
(Fig. 4c). Figure 4d shows positive Masson Trichrome
staining of collagen matrix after 28 days of cultivation.
Serial sections of the scaffold after 21 days of culture have
been treated with CD31 immunohistochemical staining to
observe the revascularization (Fig. 4e).
Figure 4B represents the flow cytometry analysis of
angiogenesis-stimulating factors (CD31 and VEGF) for
ASCs cultured on PLGA scaffolds, at day 4 and 28 of
osteogenic induction with PL-supplemented media. Results
demonstrated that ASCs started to express CD31 and
VEGF after 28 days of induction which may indicate new
vessels formation. These results suggest that PLGA scaf-
fold supports the attachment and proliferation of cells and
collagen matrix formation.
4 Discussion
Bone is a dynamic tissue that is constantly undergoing a
process of resorption, synthesis, and remodeling. Scaffolds
serve as a cell delivery and attachment vehicles, and the PL
as a supplement rich of natural growth factors and for the
enhancement of cell proliferation activity of MSCs.
Recently, the use of PL for expression and differentiation of
stem cells has been suggested as a promising FBS substitute
[21, 22].
In our study, we evaluated the cells before their use on
PLGA scaffolds. ASCs were characterized for their mor-
phology, osteogenic and adipogenic differentiation potential
and for their immunophenotype (Figs. 2, 3). Flow cytometric
analysis revealed that ASCs had similar profile of surface
markers as stem cells. Cells were positive for CD90, CD73,
CD44, and CD105 and negative for CD19, CD14, CD45,
CD34, CD31, and VEGF (Fig. 2). We demonstrated that
ASCs expanded in a PL medium enhanced osteogenic dif-
ferentiation potential and minerals deposition as shown by
ALP cytochemical staining and Alizarin red S stain (Fig. 1).
We also observed that adipogenic differentiation of ASCs
cultured in the presence of PL as oil droplets when stained by
Oil Red O stain. Our results suggest the ability of PL to
induce ASCs toward the osteoblastic, and adipogenic lin-
eages. Thus the multipotent characteristics of ASCs, as well
as their abundance in the human body, make these cells a
potential source in tissue-engineering applications [23].
Successful induction of growth and differentiation in the
presence of PL leads us to use them for further analysis using
PLGA scaffold biomaterials.
An ideal bone graft substitute material is one that is
biodegradable and completely replaced by new bone for-
mation, mechanically stable, and highly porous with
interconnected pores. MSCs seeded onto a PLGA bioma-
terials have been used in several experimental animal
models [7, 24, 25]. A uniform distribution of MSC inside
tissue-engineered PLGA scaffolds is considered to be
essential for the in vivo osteogenesis.
To define the osteoblastic differentiation potential of
ASCs, osteogenic genes expression was studied using qRT-
PCR, on monolayer culture and PLGA scaffold loaded with
ASCs. Our results clearly revealed a potent effect of PL-
combined with osteogenic induction supplements, in
inducing an accelerated expression of the osteogenic genes.
All the genes were upregulated, as measured by qRT-PCR
with slightly increase in PLGA scaffold samples. The
elevated levels of alkaline phosphatase seen at day 4 from
seeding, the elevated levels of osteocalcin on day 7 seen on
the same matrices. The secretion of collagen I from oste-
oblasts is an important marker of normal phenotypic
development and function. For the osteoblasts seeded onto
the scaffolds, collagen I expression was evident after
14 days of cell growth on scaffold, suggesting that the
scaffold surface supporting normal phenotypic develop-
ment of the osteoblasts.
Our results by HE analysis showed that seeded ASCs
infiltrate the internal parts of the PLGA scaffold which could
provide a suitable 3D environment for adhesion, distribution,
growth and diffentiation of osteopogenitor cells (Fig. 4A a–
c). Collagen was secreted around cells and formed a matrix
(Fig. 4A d). Furthermore, immunohistochemical analysis of
PLGA scaffold loaded with ASCs exhibited positive
immunostaining with anti-CD31 (Fig. 4A e). We confirmed
that by flow cytometry for the angiogenesis-stimulating
factors (CD31 and VEGF). These results indicate active cell
growth with vascularization and new blood vessels
formation.
Our results suggest that PLGA scaffolds loaded with
ASCs and incubated in PL-supplemented osteogenic
medium has excellent osteogenic characteristics and sup-
port its use in tissue engineering to repair bone defects.
PLGA scaffolds have been shown to support the attach-
ment, proliferation and differentiation as indicated by
J Mater Sci: Mater Med (2015) 26:84 Page 7 of 9 84
123
histochemical staining and gene expression profile of bone
related genes.
5 Conclusion
In conclusion, PL could provide sufficient growth and
differentiation for ASCs grown on biomaterial scaffolds
and enhance the cell viability, identity, and potency of
ASCs without altering the phenotype of expanded cells.
PLGA scaffold has an excellent characteristics in terms of
safety and osteogenic potential, and support its potential
use in tissue engineering to repair bone defects.
Acknowledgments This work was supported by a research Grant
from the deanship of scientific research of the University of Jordan
Grant Nos. 1281 and 1442.
Conflict of interest The authors indicate no potential conflicts of
interest.
a
b
d
c
50 μm
50 μm
50 μm150 μm
50 μm
e
a
A
B
Fig. 4 A Histology and immunohistochemistry of ASCs cultured on
PLGA scaffold. a HE staining of seeded ASCs onto PLGA scaffold
after 24 h of culture at low magnification. b HE staining of seeded
ASCs on PLGA scaffold at higher magnification. c HE staining of
seeded cells after 21 days of culture in osteogenic media with
collagen matrix formation. d Masson staining of the scaffold after
28 days of cultivation. The scaffold (in pink) has been observed and
collagen matrix appears in blue. e Immunohistochemical staining of
CD31 shows cells stained positive for CD31(as shown by the arrows).
B Flow cytometry analysis of CD31 and VEGF of cells on PLGA
scaffold, at 4 days and 28 days of culture (Color figure online)
84 Page 8 of 9 J Mater Sci: Mater Med (2015) 26:84
123
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