Annals of Biomedical Engineering ( 2015)

DOI: 10.1007/s10439-015-1478-1

Physical and Biological Modification of Polycaprolactone Electrospun

Nanofiber by Panax Ginseng Extract for Bone Tissue Engineering
Application
SEYEDRAMIN PAJOUMSHARIATI,1,2 SEYEDEH KIMIA YAVARI,3 and MOHAMMAD ALI SHOKRGOZAR3
1
Department of Chemical Engineering, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin,
1983963113 Tehran, Iran; 2Chemical Engineering Department, University of South Carolina, Columbia, SC 29208, USA; and
3
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
(Received 18 April 2015; accepted 28 September 2015)

Associate Editor Dan Elson oversaw the review of this article.

Abstract—Medicinal plants as a therapeutic agent with
osteogenic properties can enhance fracture-healing process.
In this study, the osteo-inductive potential of Asian Panax
Ginseng root extract within electrospun polycaprolactone
(PCL) based nanofibers has been investigated. Scanning
electron microscopy images revealed that all nanofibers were
highly porous and beadles with average diameter ranging
from 250 to 650 nm. The incorporation of ginseng extract
improved the physical characteristics (i.e., hydrophilicity) of
PCL nanofibers, as well as the mechanical properties.
Although ginseng extract increased the degradation rate of
pure PCL nanofibers, the porous structure and morphology
of fibers did not change significantly after 42 days. It was
found that nanofibrous scaffolds containing ginseng extract
had higher proliferation (up to ~1.5 fold) compared to the
pristine PCL. The qRT-PCR analysis demonstrated the
addition of ginseng extract into PCL nanofibers induced
significant expression of osteogenic genes (Osteocalcin,
Runx-2 and Col-1) in MSCs in a concentration dependent
manner. Moreover, higher calcium content, alkaline phos-
phatase activity and higher mineralization of MSCs were
observed compared to the pristine PCL fibers. Our results
indicated the promising potential of ginseng extract as an
additive to enhance osteo-inductivity, mechanical and phys-
ical properties of PCL nanofibers for bone tissue engineering
application.
Keywords—Ginseng extract, PCL nanofiber, Osteogenic
differentiation, Bone tissue engineering.
INTRODUCTION
Polycaprolactone (PCL) is one of the most com-
monly used polymers in tissue engineered scaffolds due
Address correspondence to Seyedramin Pajoumshariati, Chemi-
cal Engineering Department, University of South Carolina,
Columbia, SC 29208, USA. Electronic mail: pajoumsh@cec.sc.edu
to its combined advantages of optimal biocompatibil-
ity, degradability, mechanical properties, structural
stability and the fact that is approved by the United
States food and drug administration (FDA) for clinical
use.16 However, application of PCL scaffolds might be
limited because of its low degradation rate (ranging
from 2 to 4 years) which might be related to its high
crystallinity,13 and its hydrophobicity.36 Moreover, the
scaffolds made from PCL do not display the favorable
mechanical properties and bioactivity2 that are essen-
tial for bone tissue constructs. Several reports have
shown that the properties of PCL based scaffolds can
be improved with blending the PCL polymer with
other macromeres.8,26,36 For example, PCL had an
accelerated degradation and improved biocompatibil-
ity and mechanical properties when it was blended with
chitosan36 Further, hydrophilic natural polymers such
as starch have been extensively utilized to improve the
biocompatibility of PCL.36
Around 25% of current prescription drugs are
derived from medicinal plants.8 Plant extracts were
medically applied mostly due to their wound healing
effects, anti-inflammation, anti-bacterial, anti-diabetic,
and anti-cancer properties.8 Further, the effect of ex-
tracts from various types of plants on bone healing has
been studied. It was reported that medicinal plants can
enhance the collagen matrix production and conse-
quently accelerate the fracture healing.26
Asian ginseng (extract of Panax ginseng root) has a
long history of medicinal application as a general
health-promoting tonic.14 There are extensive reports
which have demonstrated that Panax ginseng has
many considerable pharmacological effects on human
skin, cardiovascular, immune, endocrine, and central

 2015 Biomedical Engineering Society

 PAJOUMSHARIATI et al.
nervous systems.14 Moreover, Panax ginseng extract
promotes production of collagen in human dermal
fibroblast cells.14 Few studies indicated that ginseng
extracts have positive effects on osteogenesis10,11,19,33
and cell proliferation.33
However, despite the various reported medicinal
applications of the ginseng extract, no studies have yet
reported the potential effect of Panax ginseng extract
on bioactivity of the bone tissue engineering scaffolds.
In this study nanocomposites of PCL and ginsenoside
(the major component of ginseng9) was fabricated by
electrospinning technique and the physical and
mechanical properties of electrospun nanofibers as well
as their biocompatibility were evaluated. Furthermore,
the osteogenic differentiation of rabbit mesenchymal
stem cells (MSCs) seeded on fabricated bio-composite
scaffolds was evaluated.
MATERIALS AND METHODS
Panax Ginseng C.A. Meyer Root Extraction and
Characterization
The type and content of ginsenosides are influenced
by the type of ginseng (America, Korea, Canada and
China), extraction method and extraction solvent.34
Ginsenoside was extracted from Panax Ginseng C.A
Meyer root (collected from Medicinal Plants and
Drugs Research Institute, Shahid Beheshti University,
Iran) using the Soxhlet method.14 Briefly, 1.000 g
powder of the clean root (weighted in a cellulose car-
tridge) was extracted in Soxhlet with 95 ml of di-
chloromethane for a 60 min boiling, 15 min rinsing
and 45 min drying periods. After discarding the di-
chloromethane and residue, ginsenosides in the same
cartridge were extracted with 95 ml of 96% ethanol for
90 min boiling and 90 min rinsing periods. The ethanol
extract was evaporated under reduced pressure and the
residue was added to 25 ml water and then sonicated
for approximately 5 min. This aqueous solution was
extracted three times with 8 ml of 1-butanol in a sep-
arating funnel. The resulting butanol extracts were
freeze dried for ginsenoside determination using HPLC
as described elsewhere.17
Scaffold Fabrication
Poly (e-caprolactone) (PCL, MW: 80,000 gr/mol)
was purchased from Sigma-Aldrich. The solvent type
used and the concentration of each polymer solution
prepared for electrospinning are listed in Table 1. Each
polymer solution was mixed well by vortexing over-
night at room temperature. Each sample solution was
placed in a 2 ml standard syringe fitted with 18-G
 2015 Biomedical Engineering Society
blunted stainless steel needle at voltage of 16 kV, flow
rate of 1.5 ml/h and distance of 12 cm between needle
tip and aluminum foil wrapped collector.
Physical and Mechanical Characterization of Fibers
Each electrospun nanofiber sample was coated with
gold using a sputter coater (Technics, Hummer II,
Japan) and their morphologies were investigated by
Scanning Electron Microscopy SEM (SEM, HITACH
S4160, Japan) and imaged at an accelerating voltage of
15 kV. The diameter and mean pore size of the nano-
fibers were analyzed from SEM images by applying
analysis software (Image J, National Institutes of
Health, USA). The hydrophilic/hydrophobic proper-
ties of each nanofiber sample were investigated by
applying sessile distilled drop water contact angle
measurement using a VCA Optima Surface Analysis
system (AST products, Billerica, MA). The Fourier
Transform Infrared (FTIR) analysis of each electro-
spun nanofiber was carried out in an Avatar 380
(Thermo Nicolet, Waltham, MA) spectrometer over
the range 600–400 cm21 at a resolution of 4 cm21.
Tensile properties of the fibrous samples (1 cm 9
6 cm 9 5 lm) were determined using a tabletop tensile
tester (Instron 3345, USA) at a load cell capacity of
10 N. Test specimens were tested at a crosshead speed
of 10 mm/min and gauge length of 20 mm under
ambient conditions. The test was repeated three times
for each nanofiber scaffold.
In order to perform the degradation test under
sterile conditions, samples were kept in 1009 sterilizing
solution [containing penicillin G (10,000 U/ml)/strep-
tomycin (10,000 lg/ml) and amphotericin B (250 lg/
ml)] overnight and washed 3 times with PBS solution.
At each time point, the nanofiber samples were washed
with distilled water and then vacuum-dried at 60 C
TABLE 1. Ginsenoside types and their content in Panax
ginseng C.A Meyer root extract-derived by soxhlet method-in
this study.
Type of ginsenoside Content
Rea 0.84
Rfa 1.96
Rg1a 1.13
Rg2a 2.34
Rg3a 1.24
Rb1b 11.45
Rb2b 11.29
Rcb 9.01
Rdb 4.78
Total ginsenosides content 44.04
PD/PT 5.47
a
Protopanaxatriol (PPT).
b
Protopanaxadiol (PPD).
 Physical and Biological Modification of PCL Electrospun Nanofiber
and weighted at room temperature. The percentage of
water uptake and weight loss of each sample, were
calculated using Eqs. (1) and (2) respectively;
Water uptake ð%Þ ¼ 100  ðWa  W0 Þ=W0 ð1Þ
Weight loss ð%Þ ¼ 100  ðW0  Wt Þ=W0 ð2Þ
where W0 is the initial dry weight, Wa is the weight of
wet samples and Wt is the weight of dried samples at
the given degradation time (t). In addition, the mor-
phology of each nanofiber samples was evaluated via
SEM after 42 days of degradation test.
Proliferation and Osteogenic Differentiation of MSCs
MSCs derived from the femur of 3.5 kg skeletally
mature male New Zealand white rabbit were used in
this work. The isolation of rabbit MSCs was according
to Park et al.22 The procedure for isolation of MSCs
from rabbit bone marrow was approved by Institu-
tional Animal Care and Research Advisory Committee
of Pasteur Institute of Iran and performed following
the guidelines for experimental animals approved by
Ethics Committee of the Pasteur Institute of Iran. The
culture medium was Dulbecco’s Modified Eagle’s
Medium (DMEM) (Cellgro, Herndon, VA) supple-
mented with 10% fetal bovine serum (FBS, Seromed,
Germany), penicillin G (100 U/ml)/streptomycin
(100 lg/ml) and amphotericin B (2.5 lg/ml) (pH 7.2,
all from Gibco-BRL, Germany). Each electrospun
nanofiber sample (1 cm 9 1 cm) was sterilized as de-
scribed before and then placed in 24 well plates. Each
type of scaffold was prepared in triplicate. The cul-
tured rabbit MSCs were seeded on each scaffold at
density of 10,000 cells per well and placed in incubator
at 37 C and 5% CO2 for 4 h. After 4 h of cells
adhesion onto scaffolds, 1 ml culture medium were
added to each well and the plate was placed in incu-
bator for 14 days. For osteogenic differentiation MSC
seeded fibers were kept in osteogenic medium (DMEM
containing 1028 M dexamethasone, 0.2 mM ascorbic
acid and 10 mM b-glycerolphosphate; Sigma) for
21 days. Culture medium was changed every 3 days.
The control group was MSCs cultured in normal os-
teogenic medium in the cell culture flask.
The rabbit MSC proliferation on each electrospun
nanofiber substrates was determined by using colori-
metric 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxyme
thoxyphenyl)-2(4-sulfophenyl)-2H tetrazolium (MTS)
assay (CellTiter 96TM Aqueous One Solution Cell
Proliferation Assay). At each time point (3, 6, 9 and
14 days) the culture medium was removed and scaf-
folds were washed with PBS in order to remove de-
tached cells and then incubated in serum-free medium
at 37 C for 3 h. Absorbance of the obtained dye was
measured at 490 nm using a spectrophotometric plate
reader (FLUOstar Optima, BMG Lab Technologies,
Offenburg, Germany) and correlated to the number of
cells.
Total RNA was extracted from MSC-seeded fibers
at each time point (7, 14 and 21 days) using a Hybrid-
R extraction kit (Gene All, Korea). Genomic DNA
was eliminated by DNase I from Thermo Scientific
(Germany) according to the kit’s instruction. The
concentration and purity of RNA sample was deter-
mined by a NanoDrop spectrophotometer (ND-1000,
ThermoFisher, MA). cDNA was synthesized using a
Prime Script RT Reagent (RR037A-TaKaRa) and
underwent a heating cycle (37 C for 15 min and 85 C
for 5 s) in a Genius thermocycler (Techne, Cam-
bridge, UK). Gene expression was evaluated by real
time polymerase chain reaction (qRT-PCT) using
Power SYBR-Green (Applied Bio System). Primers
including GAPDH, Osteocalcin (OCN), Runx-2 and
col1, were designed using Primer Express software
and purchased from GeneAll (GAPDH: 5¢-CGTC
TGCCCTATCAACTTTCG-3¢, 5¢-GTTTCTCAGGC
TCCCTCT-3¢, Col1: 5¢-GCGGTGGTTACGACT
TTGGTT-3¢, 5¢-AGTGAGGAGGGTCTCAATCTG-
3¢, Runx-2: 5¢-GGAGTGGACGAGGCAAGAGT-3¢,
5¢-AGGCGGTCAGAGAACAAACTAGG-3¢, OCN:
5¢-GACACCATGAGGACCCTCTC-3¢, 5¢-GCCTG
GTAGTTGTTGTGAGC-3¢). Each reaction con-
tained 10 ll Master Mix SYBR Green, 50 nM of each
primer, 100 nM cDNA, and water in a total volume of
20 ll. Reactions were carried out in duplicates at 95 C
for 10 min (holding time), denaturized at 95 C (for
30 s) and annealing and extension at 60 C for 1 min
and 40 cycles. Amplification data was collected during
the last 15 s of annealing-extension time. For melting
curve analysis, reactions were heated for 10 min in
95 C then cooled to 60 C and finally stepwise heated
to 95 C with a ramp rate of 0.3 C. Fluorescence was
detected at each step continually. CT of each target was
normalized with GAPDH and fold change expression
calculated with 2DDCt method.
At each time point (0, 7, 14 and 21 days), cell-seeded
samples were placed in serum- free DMEM for 8 h to
remove serum proteins, followed by washing with PBS
three times and lysed with lysis buffer (10 mM Tris
supplemented with 0.2% triton in PBS). Double-
stranded DNA content, ALPase activity and calcium
content of the lysed samples were quantified with Pi-
coGreen DNA assay (Invitrogen), QuantiChrom AL-
Pase assay (Bioassay Systems) and QuantiChrom
Calcium Assay (Bioassay Systems), respectively fol-
lowing manufacturer’s instructions. Normalization
was performed for ALPase activities and calcium
 2015 Biomedical Engineering Society
 PAJOUMSHARIATI et al.

contents based on cell numbers (obtained from DNA RESULTS

contents data) of each sample at each time point.
In addition to calcium content, mineralization of
nanofibers was demonstrated by Alizarin Red Staining
(ARS). For that, cells were fixed in 4% paraformalde-
hyde and stained with Alizarin Red (Sigma-Aldrich
A5533) for 20 min at room temperature. Furthermore,
bone nodules were carefully peeled off from the base of
electrospun nanofibers and analyzed by energy-disper-
sive X-ray system (EDS, Tescan) in order to identify the
elemental composition of the nodules as well as their
calcium to phosphate (Ca/P) ratio.
Western blot analysis was performed to quantify the
protein expression of bone specific markers (osteocal-
cin and collagen type 1). Briefly, rabbit MSCs cultured
on electrospun nanofibers in each group at day 21,
were lysed with RIPA buffer containing the protease
inhibitor cocktail (Complete Mini, Roche). After
determining the total protein of lysates by BCA kit,
lysates with the same protein content (adjusted to
10 lg of total protein per each well) were run through
7.5% SDS-PAGE and transferred to nitrocellulose
membrane. Blotto solution (Santa Cruz Biotechnol-
ogy) was used as blocking solution. Blotted mem-
branes were incubated for 1 h in a mixture of primary
antibodies (including, mouse anti-rabbit Osteocalcin
(1:1000), Collagen type 1(1:1000) and b actin (1:5000)
(all from Abcam), diluted in 19 PBS, 5% dry milk,
0.1% Tween-20 and then incubated for 1 h in peroxi-
dase-conjugated secondary antibody solution, goat
anti-mouse (1:5000, Abcam). Western blotting luminol
reagent (Santa Cruz Biotechnology) was used for
visualization of antigen–antibody complexes using
Biorad ChemiDoc MP System. Imaged bands were
quantified by Image J software (NIH, Bethesda, MD).
Statistical Analysis
All the presented values are expressed as
mean ± standard deviation (SD) of the mean and each
experiment was repeated three times. Statistical dif-
ferences were determined by student’s two-tailed t test.
Ginsenosides Component of Panax Ginseng Root
Extract
Table 2 summarizes the composition of ginseng
extract derived by soxhlet extraction method in the
present study. Ginsenosides generally divided into two
categories based on their chemical structure: pro-
topanaxadiol (PD, including Rb1, Rb2, Rc, Rd, and
Rh2) and protopanaxatriol (PT, including Re, Rf,
Rg1, Rg2, and Rh1). According to our analysis, PD
group found in Panax ginseng root was about 5.5 times
more than the PT group. Moreover the highest and
lowest amounts of ginsenoside were obtained for Rb1
and Re respectively.
Physical and Mechanical Properties of Electrospun
Fibrous Scaffolds
All samples were bead-less with randomly oriented fi-
brous structure (Fig. 1). The diameter of nanofibers ranged
from 250 to 650 nm depending on electrospinning solu-
tions and conditions. The average fiber diameter for PCL/
Gin30, PCL/Gin20, PCL/Gin10 and PCL nanofibers was
442 ± 87, 507 ± 141, 537 ± 162, and 605 ± 143 nm,
respectively; with PCL/Gin30 fibers the finest and pure
PCL the thickest (Fig. 1). While PCL/Gin10 nanofibers
had a broad range of diameter distribution (Fig. 1c), other
groups showed a uniform and narrow diameter distribu-
tion (Fig. 1). Figure 1 also, illustrates that all nanofibrous
scaffolds possess highly interconnected porous network.
The mean pore size of PCL/Gin30, PCL/Gin20, PCL/
Gin10 and PCL was 860 ± 32, 970 ± 41, 1180 ± 73 and
1720 ± 65 nm, respectively.
The FTIR spectra of fibers are shown in Fig. 2. It is
illustrated that PCL-related stretching modes exist in
all nanofiber samples containing Panax ginseng extract
with relatively lower peak strengths. Figure 2 shows
that the intensity of those peaks pertained to gin-
senoside (see section ‘‘Physical and Mechanical Char-
acteristics of Scaffolds’’) changed in accordance with

TABLE 2. The electrospinning solutions of pure PCL, PCL/Gin10, PCL/Gin20 and PCL/Gin30 and their mechanical properties
including tensile stress, strain and young modulus.

Tensile Strain Young’s

Samples Solutions stress (MPa) (%) Modulus (MPa)

PCL 8% (wt/v) PCL + chloroform (40 v/v) + DMF (20 v/v) + methanol (20 v/ 2.48 134.10 18.6
v) + butanol (20 v/v)
PCL/Gin 10 8% (wt/v) PCL +chloroform (40 v/v) + DMF (20 v/v) + methanol (30 v/ 2.72 118.00 19.1
v) + ginsenoside solution (10 v/v)
PCL/Gin 20 8% (wt/v) PCL + chloroform (40 v/v) + DMF (20 v/v) + methanol (20 v/ 3.29 148.05 26.5
v) + ginsenoside solution (20 v/v)
PCL/Gin 30 8% (wt/v) PCL + chloroform (40 v/v) + DMF (20 v/v) + methanol (10 v/ 3.88 143.09 34.2
v) + ginsenoside solution (30 v/v)

 2015 Biomedical Engineering Society

 Physical and Biological Modification of PCL Electrospun Nanofiber
FIGURE 1. SEM images of electrospun nanofibers (a) PCL/Gin30, (b) PCL/Gin20, (c) PCL/Gin10 and (d) PCL, and inset histograms
of the fiber diameter distribution.
change in concentration of Panax ginseng extract for
each nanofiber scaffolds.
Figure 3 shows that with incorporation of Asian
ginseng extract, PCL/Gin10, PCL/Gin20 and PCL/
Gin30 nanofiber scaffolds became more hydrophilic
with a measured contact angles of 90 ± 2, 45 ± 3
and 23 ± 2, respectively compared to pure PCL na-
nofibers (135 ± 5).
The percentage of water uptake and weight loss of
nanofiber scaffolds soaked in PBS, at different time
points, are shown in Fig. 4. The rate of water uptake
slightly increased for all nanofiber scaffolds with the
time of incubation (Fig. 4a). As shown in Fig. 4a,
water uptake of all samples increased slightly and
reached a plateau at day 21, and then decreased slowly.
An incorporation of Panax ginseng extract increased
significantly the water uptake of PCL nanofibers. The
PCL/Gin20 sample had the highest water adsorption
at all-time points with a 15 ± 1.2% water uptake after
40 days.
In contrast to a low rate of weight loss in pure PCL
(less than 0.2% in 42 days), there was a significantly
higher weight loss for PCL samples loaded with gin-
seng extract (Fig. 4b). The percentage of weight loss
was directly correlated to the concentration of ginseng
extract within the fiber with PCL/Gin30 having the
highest weight loss (4.2% after 42 days).
Moreover the morphology and degradation of
nanofibrous scaffolds was evaluated by SEM at day 42
(Fig. 5). PCL nanofibrous matrices were able to
maintain the original fiber diameter over 42 days of
incubation with a slight decrease in pore size. In other
sample scaffolds containing ginseng extract, the fiber
became slightly swollen and there was a decrease in
pore size, but pores were still detectable after 42 days
of degradation. SEM analysis showed that PCL/Gin
nanofibers were more degradable in aqueous solution
in comparison to pure PCL nanofibers.
Figure 6 shows the stress–strain curve for nanofiber
scaffolds. It is illustrated that elongation of all fibrous
scaffolds resulted in a non-linear increase in stress
followed by a sharp drop at yield point. The Young’s
modulus of all samples was in the range of 18–35 MPa
with PCL/Gin30 having the highest young’s modulus.
The Young’s modulus of fibers increased with
increasing the ginseng extract concentration in a dose
 2015 Biomedical Engineering Society
 PAJOUMSHARIATI et al.

FIGURE 2. FTIR Spectra of PCL/Gin10 (red line), PCL/Gin20 (pink line), PCL/Gin30 (green line) and pristine PCL (blue line)
electrospun nanofibers.

FIGURE 3. Water contact angle; Photographs of water droplets on PCL (a), PCL/Gin10 (b), PCL/Gin20 (c) and PCL/Gin30 (d)
electrospun nanofibers.

dependent manner. Further, the ultimate tensile stress The tensile strength of electrospun nanofibers is sum-
value (MPa) of all ginseng loaded nanofiber scaffolds marized in Table 1. The tensile strength of the samples
was higher than that of pure PCL nanofiber scaffolds. ranged between 2.48 and 3.88 MPa and in a dose

 2015 Biomedical Engineering Society

 Physical and Biological Modification of PCL Electrospun Nanofiber
FIGURE 4. Water adsorption (a) and weight loss (b) of PCL/
Gin10, PCL/Gin20, PCL/Gin30 and pure PCL electrospun na-
nofibers after soaking (immersion) in PBS during 6 weeks
degradation study. One star indicates a statistically signifi-
cant difference (p < 0.05) between the test and PCL group at
each time point. Two stars indicate a statistically significant
difference between the test and all other groups at each time
point.
dependent manner- increased with increasing the con-
centration of ginseng extract.
Proliferation and Differentiation of MSCs on
Electrospun Scaffolds
After 9 days of cell culture, the density of rabbit
MSCs increased on all nanofiber substrates (Fig. 7a).
Results revealed that MSCs were metabolically active
on nanofiber scaffolds containing ginseng extract and
demonstrated that there was no cytotoxic effect due to
the presence of ginseng extract. The proliferation of
MSCs on PCL/Gin30 was found to be considerably
higher than PCL/Gin10 and PCL/Gin20 after 6 days
of cell culture. However, after 9 days of cell culture,
PCL/Gin20 had the highest cell proliferation among all
other nanofiber scaffolds. The proliferation of MSCs
on PCL, PCL/Gin10, PCL/Gin20 and PCL/Gin30
nanofibers increased by 3.66, 4.00, 7.66, and 6.06 folds
respectively from day 1 to 9.
The rabbit MSCs coverage on all nanofiber scaf-
folds is depicted in Fig. 7b. More specifically, the
number of MCSs attached on PCL/Gin30 and PCL/
Gin20 after 9 days was significantly higher and the
fraction of empty surface area was lower than the ones
for PCL and PCL/Gin10 nanofibers.
RT-PCR, based on SYBR Green-I, presented in
Fig. 8 showed a significant change in expression of
OCN, Col-I and Runx-2 in PCL/Gin20 and PCL/
Gin30 over PCL and PCL/Gin10 (p < 0.04) while the
expression of these genes showed no significant dif-
ference between PCL/Gin20 and PCL/Gin30 scaffolds.
The significant increase in protein expression of OCN
and Col-I for both PCL/Gin20 and PCL/Gin30 groups
compared to control was confirmed by western blot
analysis (Fig. 8i). The representative bands of OCN
(12 kDa), Col-I (130 kDa) and b actin (42 kDa) have
been shown in Fig. 8j.
DNA content of MSCs seeded on nanofibers and
cultured in osteogenic medium (Fig. 8d) showed a
slight decrease with incubation time over 21 days of
culture. ALPase activity of cells seeded on the scaffolds
increased significantly from day 7 to 14, reached a
maximum at day 14 and then decreased from day 14 to
21 (Fig. 8e). The increase in ALPase activity of MSCs
between groups, was correlated to the ginseng extract
content of scaffolds. Calcium content of MSCs seeded
on scaffolds is shown in Fig. 8f. PCL and PCL/Gin10
groups had a slight increase in calcium content from
day 7 to 21 while in other groups the calcium content
increased with a higher rate over the incubation time.
PCL/Gin30 showed a higher calcium content and
ALPase activity compared with the other groups.
Figure 8g demonstrates Alizarin Red staining
images of MSCs seeded on nanofibers at day 21. A
relatively high density of orange-red areas on the PCL/
Gin20 and PCL/Gin30 groups qualitatively confirmed
a higher calcium content (Figs. 8g3, 8g4) of the
aforementioned groups compared with the other
groups. Formation of bone nodules, three dimensional
specifically mineralized and organized structures was
observed as large dark spots only on the PCL/Gin30
group. The number density and total area of bone
nodules on the PCL/Gin30 group was 128 counts/cm2
and 2.9 ± 0.4 mm2/cm2. EDS analysis further con-
firmed that bone nodules formed on PCL/Gin30 con-
sisted of calcium (Ca) and phosphorus (P) with a Ca/P
ratio of 1.69 (Fig. 8h).
DISCUSSION
Physical and Mechanical Characteristics of Scaffolds
The obtained pore size for pristine PCL is slightly
lower than that of reported by Venugopal et al. (2–
15 lm)30 while fitted in the range of (0–10 lm) pro-
posed by Vaquette et al.29 The difference in porosity
can be explained by the difference in electrospinning
 2015 Biomedical Engineering Society
 PAJOUMSHARIATI et al.
FIGURE 5. SEM images of biodegraded PCL/Gin30 (a), PCL/Gin20 (b), PCL/Gin10 (c) and pure PCL (d) electrospun nanofibers
after 42 day of immersion in PBS.
FIGURE 6. The influence of ginseng extract at different
concentrations on the tensile behavior of nanofibers- typical
tensile stress–strain curve of PCL, PCL/Gin10, PCL/Gin20 and
PCL/Gin30 electrospun nanofibers.
conditions. Li et al.16 have shown that the increase in
fiber diameters results in an increase in mean pore size
at a certain mass per unit area. It is well established
 2015 Biomedical Engineering Society
that the fiber diameters and scaffold thickness play a
crucial role in controlling the pore size and pore size
distribution of the networks.16 Therefore, here, the
thickness of all nanofibrous scaffolds was kept con-
stant (~40 lm).
PCL nanofibers showed the characteristic carbonyl
stretching peak at 1736 cm21 (amorphous phase),
symmetric CH2 stretching peak at 2865 cm21 C–O
stretching at 1050 cm21, C–O–C stretching at
1240 cm21 (asymmetric).8 According to literature, the
presence of extra peaks at 2927 cm21 (stretching C–H
in methyl and methylene) and 1123 cm21 are the
bending vibrations of C–C–O or C–C–OH in starch.25
The stronger the relative intensity of this peak, the
higher starch content in the sample. Peaks at 1154,
1078 and 1026 cm21 which represents the bending
vibration of saccharide ring in the FTIR of PCL/
Gin10, PCL/Gin 20 and PCL/Gin30 nanofibers is at-
tributed to the presence of Asian Ginseng on the sur-
face of PCL nanofibrous scaffold.25 Based on literature
review, the FTIR spectrum for pure ginseng extract
powder shows characteristic peaks at 2923 cm21 due
 Physical and Biological Modification of PCL Electrospun Nanofiber

FIGURE 7. Proliferation of rabbit MSCs on electrospun nanofibers cultured in basal medium at day 3, 6, 9 and 14 (a); SEM images
of rabbit MSCs on PCL/Gin30 (A), PCL/Gin20 (B), PCL/Gin/10 (C) and pristine PCL (D) electrospun fibers after 9 days of cell
proliferation (b). One star indicates a statistically significant difference (p < 0.05) between the test and PCL group at each time
point. Two stars indicate a statistically significant difference between the test and all other groups at each time point.

to stretching vibration of –CH2– groups and at
1633 cm21 due to the stretching vibration of carbonyl
group.18 Peaks at 1056–1026 cm21 are characteristic
peaks to distinguish different types of Ginseng (from
Korea, America or china).18
PCL nanofibers have an extremely hydrophobic
nature which is undesirable for tissue engineering
applications especially as a bone construct. The
hydrophobicity of pristine PCL nanofibers is the major
reason for a slow degradation rate and a weak cell
adhesion and proliferation on the surface of the
fibers.13,36 The presence of a peak at 1123 cm21 in
FTIR spectra of Panax ginseng extract, which repre-
sents the C–C–OH group, can be a cause of pro-
nounced hydrophilicity of ginseng containing PCL
compared with the pristine one.13
Mechanical properties of nanofibrous sheets indi-
cated that the incorporation of Panax ginseng extract
can improve the tensile strength as well as young
modulus of PCL nano fibers in a dose dependent
manner. The Young’s modulus of cortical bone ranges
between 3.8 (GPa, for humerus) and 35.3 (GPa for
tibia)32 while this number for trabecular bone varies
between 0.1 and 0.5 GPa.31
It is worth mentioning that the tensile strength of
cortical bone ranges between 50 and 151 MPa while its
range in trabecular bone is 1–5 MPa.31 Therefore,
based on the results of Fig. 6 and Table 1, ginseng
extract loaded PCL nanofibers might be a good can-
didate for regeneration of trabecular bone. There are
different techniques for enhancing the mechanical
properties of nanofibers for bone constructs such as
using high polymer concentration, crosslinking, sheet
lamination,38 deposition of nano hydroxyapatite
(HA),7 blending with other polymers or HA.37 It was
shown that the addition of HA can improve the
mechanical properties of PCL nano fibers. However
optimum mechanical properties (modulus ~19.5 MPa)
obtained at 25% HA concentration while increasing
the level of HA resulted in decrease the modulus.37
Here we introduce Panax ginseng extract as a potential
material for improving the mechanical properties of
PCL nano fibers. Mechanical properties of PCL na-
nofibers were also improved by incorporation of other
plant extracts8 which highlight the role of plant ex-
tracts as a potential supplement for load bearing
applications.
A higher mass loss of PCL/Gin compared to PCL
nanofibers can be attributed to a higher hydrophilicity
of PCL/Gin as well as relatively fast degradation of the
ginsenoside.
The water uptake of nanofiber scaffolds depends on
several factors including hydrophobicity, pore size and
interconnectivity.1 Typically, the water uptake of a
nanofiber scaffold increases with increasing the surface
hydrophilicity, pore size and interconnectivity of the
scaffold. The addition of ginsenoside solution to the
hydrophobic PCL increases the hydrophilicity of the
fibers (see contact angle results, Fig. 3). However, as
mentioned earlier, an increase in ginsenoside content
and the subsequent decrease in pore size, might lead to
a reduction in interconnectivity, hence limiting the
water diffusion into the nanofiber scaffolds. Therefore,
the water uptake of the scaffolds initially increased
with increasing the ginsenoside concentration up to
20% and then decreased with further increasing the
ginsenoside concentration to 30%.
However, the mechanism of the degradation in
PCL/Gin nanofibers seemed to be different with that of
the other biodegradable and hydrophilic polymers

 2015 Biomedical Engineering Society

 PAJOUMSHARIATI et al.

FIGURE 8. Osteogenic differentiation of rabbit MSCs cultured on the scaffolds at days 7, 14 and 21 in the osteogenic media: gene
expression (Col1 (a), Runx-2 (b) and OCN (c)); DNA (d), ALPase activity (e) and calcium content (f); Alizarin Red staining of
deposited calcium ions on the PCL (g-1), PCL/Gin10, (g-2) PCL/Gin20, (g-3) and PCL/Gin30 (g-4) electrospun nanofibers at day 21;
elemental analysis (EDS image) of bone nodule formed on PCL/Gin30 electrospun nanofibers at day 21 (h); protein expressions of
osteocalcin and collagen type 1 for rabbit MSCs cultured on the scaffolds at day 21 in the osteogenic media relative to the b actin
expression (i); representative of western blotting bands for OCN, Col 1 and b actin (j).

including PLGA, PGA and PDLLA. The degradation
of the mentioned polymers proceeds with fiber swel-
ling, fusion and elimination of pores16; However, in
all PCL/Gin nanofibers, pores were remained
detectable even after 42 days of incubation.
MSCs Proliferation and Differentiation on Electrospun
Scaffolds
Our results indicated that the Panax ginseng extract
can improve the proliferation of MSCs.
This increase in proliferation with increasing the
ginsenoside content can partially be due to the
prominent increase in water adsorption in scaffolds (up
to 18 folds for PCL/Gin20) which can induce the better
cell adhesion and proliferation by providing better
transportation of nutrients for seeded cells.15 It is
established that cell adhesion is higher on the scaffolds
with a hydrophilic surface.5 The initial attachment of
mouse osteoblast-like cells (MC3T3-E1) increased
when the surface had a higher surface hydrophilicity.35
Moreover, cell spreading and differentiation are re-
lated to the hydrophobicity of the surface. For exam-
ple mouse osteoblasts on hydrophilic surface of plasma
treated PCL showed accelerated metabolic activity and
higher osteogenic differentiation compared to the un-
treated one.39 Here we showed that by incorporation
of Panax ginseng extract into the electrospinning
solution of PCL the hydrophilicity of fibers signifi-
cantly increased. The results of present study together

 2015 Biomedical Engineering Society

 Physical and Biological Modification of PCL Electrospun Nanofiber
with the previous study done by Jin et al.8 suggest that
incorporation of plat extracts can be an interesting
method for improving the hydrophobic nature of PCL.
Further, there are some reports on the increase of
cell proliferation by incorporating ginsenosides into
the media. For example ginsenoside RG1 increased the
proliferation of human dental pulp stem cells
(hDPSCs)33 and bone marrow stromal cell (BMSC)21
by upregulating the fibroblast growth factor-2(FGF-2)
gene and via the activation of the estrogen receptor-
mediated signaling pathway respectively. Gong et al. 6
also reported that Rg1 enhanced the proliferation of
rat osteoblasts while Rb1 did not. Moreover gin-
senoside Rd had no effect on the proliferation of
mouse osteoblastic cells (MC3T3-E1).11 Since the gin-
seng extract used in this study was a mixture of dif-
ferent types of ginsenosides, from the obtained results
it can be concluded that the ginseng extract derived by
the aforementioned method can improve the prolifer-
ation of rabbit MSCs.
To test the effect of ginseng extract loaded scaffolds
on osteoblastic gene expression and osteogenic differ-
entiation of MSCs, we evaluated the expression of three
specific genes including collagen type I (col-I)—which is
essential for extracellular matrix (ECM) formation and
mineralization in bone3—, Runt-related transcription
factor (Runx-2)—which is crucial for differentiation of
osteoblasts and bone formation4,23,28—and osteocalcin
(OCN)—which plays a role in terminal differentiation
of osteoblasts and mineralization.27,12 In addition to
transcriptional level, protein expression of OCN and
col-I was investigated to evaluate the effect of ginseng
addition to PCL nanofibers in translational level.
The sharp increase in Runx-2 gene expression
between days 7 and 14 is related to the early stage of
differentiation.28 The change in Runx-2 gene expres-
sion level among samples directly relates to the
observed change of OCN expression, since Runx-2 can
directly stimulate transcription of OCN—the most
abundant nano collagenous protein in bone ECM.4–23
The up-regulation of OCN during matrix synthesis and
mineralization,27 is curtail for later stages of osteoge-
nesis and ECM mineral formation. The obtained gene
expression profile for MSCs seeded on PCL group was
in agreement with other studies. It has been demon-
strated that PCL can increase the expression of colla-
gen 1,20 Osteocalcin20 and Runx-2.20 However the
effect of total ginseng extract on osteogenic properties
of scaffolds has not been evaluated. In other reports
though, the osteogenic properties of some components
of ginseng have been studied. For example it has been
shown that ginsenoside Rg1,34 Ginsenoside Rh2(s),10
Ginsenoside Rd11 and Panax notoginseng saponins
(PNS)19 can upregulate the OCN gene expression and
collagen-I (Col-I) gene expression have been increased
by Ginsenoside Rh2(s)10 and Ginsenoside Rd.11
Observation of bone nodules, three dimensional
specifically mineralized and organized structures with
proliferation, differentiation and mineralization stages
of MSCs simultaneously,24 can be used as a proof of
last stage of osteogenic differentiation (especially in
PCL/Gin30 scaffolds). Dark spots on the others
groups (Fig. 8) were too small (<35 lm) to be con-
sidered as bone nodules.24 The mineral composition of
the bone nodules were consistent with the previous
report40 and indicated the presence of calcium rich
minerals similar to those nodules formed in natural
bone. The improved mineralization on the PCL/Gin30
and PCL/Gin20 can be partially explained by the in-
crease in Osteocalcin and collagen expression.
The ALPase activity and calcium content of the
MSCs seeded on all groups demonstrated that the
presence of ginseng extract ameliorates the osteogenic
differentiation of MSCs (in consistent with gene
expression evaluation).
CONCLUSION
The extract of Asian Panax Ginseng root at three
different concentrations was incorporated with PCL in
order to produce bioactive PCL/ginseng extract na-
nofibers. Physical and mechanical characteristics of
PCL nanofibers were significantly improved by the
addition of ginseng extract. Cell attachment and
proliferation were improved by addition of ginseng
extract. Moreover ginseng extract enhanced the os-
teogenic differentiation of MSCs by up regulating the
gene expression of Runx-2, Col1 and OCN genes, as
confirmed by high levels of ALPase activity and cal-
cium content deposited on the surface of fibers. The
ginseng extract can potentially be a powerful supple-
ment for PCL electrospun nanofibers for bone tissue
engineering applications.
REFERENCES
1
Arabi, N., and A. Zamanian. Effect of cooling rate and
gelatin concentration on the microstructural and mechan-
ical properties of ice template gelatin scaffolds. Biotechnol.
Appl. Biochem. 60:573–579, 2013.
2
Diba, M., M. Kharaziha, M. H. Fathi, M. Gholipour-
malekabadi, and A. Samadikuchaksaraei. Preparation and
characterization of polycaprolactone/forsterite nanocom-
posite porous scaffolds designed for bone tissue regenera-
tion. Compos. Sci. Technol. 72:716–723, 2012.
3
Franceschi, R. T., B. S. Iyer, and Y. Cui. Effects of
ascorbic acid on collagen matrix formation and osteoblast
 2015 Biomedical Engineering Society
 PAJOUMSHARIATI et al.
differentiation in murine MC3T3-E1 cells. J. Bone Miner.
Res. 9:843–854, 1994.
4
Franceschi, R. T., and G. Xiao. Regulation of the osteo-
blast-specific transcription factor, Runx2: responsiveness
to multiple signal transduction pathways. J. Cell. Biochem.
88:446–454, 2003.
5
Goddard, J. M., and J. Hotchkiss. Polymer surface modi-
fication for the attachment of bioactive compounds. Prog.
Polym. Sci. 32:698–725, 2007.
6
Gong, Y.-S., J. Chen, Q.-Z. Zhang, and J.-T. Zhang. Effect
of 17b-oestradiol and ginsenoside on osteoporosis in
ovariectomised rats. J. Asian Nat. Prod. Res. 8:649–656,
2006.
7
Jaiswal, A. K., H. Chhabra, V. P. Soni, and J. R. Bellare.
Enhanced mechanical strength and biocompatibility of
electrospun polycaprolactone-gelatin scaffold with surface
deposited nano-hydroxyapatite. Mater. Sci. Eng. C
33:2376–2385, 2013.
8
Jin, G., M. P. Prabhakaran, D. Kai, S. K. Annamalai, K.
D. Arunachalam, and S. Ramakrishna. Tissue engineered
plant extracts as nanofibrous wound dressing. Biomaterials
34:724–734, 2013.
9
Kang, T. H., H. M. Park, Y.-B. Kim, H. Kim, N. Kim, J.-
H. Do, C. Kang, Y. Cho, and S. Y. Kim. Effects of red
ginseng extract on UVB irradiation-induced skin aging in
hairless mice. J. Ethnopharmacol. 123:446–451, 2009.
10
Kim, D. Y., M. S. Jung, Y. G. Park, H. D. Yuan, H. Y.
Quan, and S. H. Chung. Ginsenoside Rh2 (S) induces the
differentiation and mineralization of osteoblastic MC3T3-
E1 cells through activation of PKD and p38 MAPK
pathways. BMB Rep. 44:659–664, 2011.
11
Kim, D. Y., Y. G. Park, H.-Y. Quan, S. J. Kim, M. S.
Jung, and S. H. Chung. Ginsenoside Rd stimulates the
differentiation and mineralization of osteoblastic MC3T3-
E1 cells by activating AMP-activated protein kinase via the
BMP-2 signaling pathway. Fitoterapia 83:215–222, 2012.
12
Komori, T. Regulation of bone development and extra-
cellular matrix protein genes by RUNX2. Cell Tissue Res.
339:189–195, 2010.
13
Kweon, H., M. K. Yoo, I. K. Park, T. H. Kim, H. C. Lee,
H.-S. Lee, J.-S. Oh, T. Akaike, and C.-S. Cho. A novel
degradable polycaprolactone networks for tissue engineer-
ing. Biomaterials 24:801–808, 2003.
14
Lee, J., E. Jung, J. Lee, S. Huh, J. Kim, M. Park, J. So, Y.
Ham, K. Jung, C.-G. Hyun, Y. S. Kim, and D. Park. Panax
ginseng induces human Type I collagen synthesis through
activation of Smad signaling. J. Ethnopharmacol. 109:29–
34, 2007.
15
Lee, H., M. Yeo, S. Ahn, D. O. Kang, C. H. Jang, H.
Lee, G. M. Park, and G. H. Kim. Designed hybrid
scaffolds consisting of polycaprolactone microstrands
and electrospun collagen-nanofibers for bone tissue
regeneration. J. Biomed. Mater. Res. B Appl. Biomater.
97:263–270, 2011.
16
Li, W.-J., J. A. Cooper, Jr, R. L. Mauck, and R. S. Tuan.
Fabrication and characterization of six electrospun poly(a-
hydroxy ester)-based fibrous scaffolds for tissue engineer-
ing applications. Acta Biomater. 2:377–385, 2006.
17
Li, T., G. Mazza, A. Cottrell, and L. Gao. Ginsenosides in
roots and leaves of American ginseng. J. Agric. Food Chem.
44:717–720, 1996.
18
Li, Y.-M., S.-Q. Sun, Q. Zhou, Z. Qin, J.-X. Tao, J. Wang,
and X. Fang. Identification of American ginseng from
different regions using FT-IR and two-dimensional corre-
lation IR spectroscopy. Vib. Spectrosc. 36:227–232, 2004.
 2015 Biomedical Engineering Society
19
Li, X.-D., J.-S. Wang, B. Chang, B. Chen, C. Guo, G.-Q.
Hou, D.-Y. Huang, and S.-X. Du. Panax notoginseng sa-
ponins promotes proliferation and osteogenic differentia-
tion of rat bone marrow stromal cells. J. Ethnopharmacol.
134:268–274, 2011.
20
Lu, Z., S.-I. Roohani-Esfahani, P. C. L. Kwok, and H.
Zreiqat. Osteoblasts on rod shaped hydroxyapatite
nanoparticles incorporated PCL film provide an optimal
osteogenic niche for stem cell differentiation. Tissue Eng.
Part A 17:1651–1661, 2011.
21
Lu, X.-Z., J.-H. Wang, X. Wu, L. Zhou, L. Wang, X.-W.
Zhang, K.-J. Cao, and J. Huang. Ginsenoside Rg1 pro-
motes bone marrow stromal cells proliferation via the
activation of the estrogen receptor-mediated signaling
pathway. Acta Pharmacol. Sin. 29:1209–1214, 2008.
22
Park, H., J. S. Temenoff, Y. Tabata, A. I. Caplan, and A.
G. Mikos. Injectable biodegradable hydrogel composites
for rabbit marrow mesenchymal stem cell and growth
factor delivery for cartilage tissue engineering. Biomaterials
28:3217–3227, 2007.
23
Ryoo, H.-M., M.-H. Lee, and Y.-J. Kim. Critical molecu-
lar switches involved in BMP-2-induced osteogenic differ-
entiation of mesenchymal cells. Gene 366:51–57, 2006.
24
Schecroun, N., and C. Delloye. Bone-like nodules formed
by human bone marrow stromal cells: comparative study
and characterization. Bone 32:252–260, 2003.
25
Schulten, H. R., and F. Soldati. Identification of ginseno-
sides from Panax ginseng in fractions obtained by high-
performance liquid chromatography by field desorption
mass spectrometry, multiple internal reflection infrared
spectroscopy and thin-layer chromatography. J. Chro-
matogr. A 212:37–49, 1981.
26
Soumya, S., K. M. Sajesh, R. Jayakumar, S. V. Nair, and
K. P. Chennazhi. Development of a phytochemical scaffold
for bone tissue engineering using Cissus quadrangularis
extract. Carbohydr. Polym. 87:1787–1795, 2012.
27
Temenoff, J. S., H. Park, E. Jabbari, T. L. Sheffield, R. G.
LeBaron, C. G. Ambrose, and A. G. Mikos. In vitro os-
teogenic differentiation of marrow stromal cells encapsu-
lated in biodegradable hydrogels. J. Biomed. Mater. Res.
Part A 70:235–244, 2004.
28
Thirunavukkarasu, K., D. L. Halladay, R. R. Miles, X.
Yang, R. J. Galvin, S. Chandrasekhar, T. J. Martin, and J.
E. Onyia. The osteoblast-specific transcription factor Cbfa1
contributes to the expression of osteoprotegerin, a potent
inhibitor of osteoclast differentiation and function. J. Biol.
Chem. 275:25163–25172, 2000.
29
Vaquette, C., and J. J. Cooper-White. Increasing electro-
spun scaffold pore size with tailored collectors for im-
proved cell penetration. Acta Biomater. 7:2544–2557, 2011.
30
Venugopal, J., P. Vadgama, T. S. Kumar, and S.
Ramakrishna. Biocomposite nanofibres and osteoblasts for
bone tissue engineering. Nanotechnology 18:055101, 2007.
31
Wagonerjohnson, A. J., and B. A. Herschler. A review of
the mechanical behavior of CaP and CaP/polymer com-
posites for applications in bone replacement and repair.
Acta Biomater. 7:16–30, 2011.
32
Wang, X., J. Nyman, X. Dong, H. Leng, and M. Reyes.
Fundamental biomechanics in bone tissue engineering.
Synth. Lect. Tissue Eng. 2:1–225, 2010.
33
Wang, P., X. Wei, F. Zhang, K. Yang, C. Qu, H. Luo, and
L. He. Ginsenoside Rg1 of Panax ginseng stimulates the
proliferation, odontogenic/osteogenic differentiation and
gene expression profiles of human dental pulp stem cells.
Phytomedicine 21:177–183, 2014.
 Physical and Biological Modification of PCL Electrospun Nanofiber
34
Wang, Y., J. You, Y. Yu, C. Qu, H. Zhang, L. Ding, H.
Zhang, and X. Li. Analysis of ginsenosides in Panax gin-
seng in high pressure microwave-assisted extraction. Food
Chem. 110:161–167, 2008.
35
Wei, J., T. Igarashi, N. Okumori, T. Igarashi, T. Maetani,
B. Liu, and M. Yoshinari. Influence of surface wettability
on competitive protein adsorption and initial attachment of
osteoblasts. Biomed. Mater. 4:045002, 2009.
36
Wu, F., J. Wei, C. Liu, B. O’Neill, and Y. Ngothai.
Fabrication and properties of porous scaffold of zein/PCL
biocomposite for bone tissue engineering. Compos. B Eng.
43:2192–2197, 2012.
37
Yang, F., S. K. Both, X. Yang, X. F. Walboomers, and J.
A. Jansen. Development of an electrospun nano-apatite/
PCL composite membrane for GTR/GBR application.
Acta Biomater. 5:3295–3304, 2009.
38
Yang, S., K.-F. Leong, Z. Du, and C.-K. Chua. The design
of scaffolds for use in tissue engineering. Part I. Traditional
factors. Tissue Eng. 7:679–689, 2001.
39
Yildirim, E. D., R. Besunder, D. Pappas, F. Allen, S.
Güçeri, and W. Sun. Accelerated differentiation of osteo-
blast cells on polycaprolactone scaffolds driven by a com-
bined effect of protein coating and plasma modification.
Biofabrication 2:014109, 2010.
40
Zhang, Z.-Y., S. H. Teoh, W.-S. Chong, T.-T. Foo, Y.-C.
Chng, M. Choolani, and J. Chan. A biaxial rotating
bioreactor for the culture of fetal mesenchymal stem cells
for bone tissue engineering. Biomaterials 30:2694–2704,
2009.
 2015 Biomedical Engineering Society