Physical, morphological, and biological studies on PLA/nHA composite

nanofibrous webs containing Equisetum arvense herbal extract for

bone tissue engineering
Maliheh Khakestani,1,2 Seyed Hassan Jafari ,1 Payam Zahedi,1 Reza Bagheri,3 Reza Hajiaghaee4
1
Department of Polymer, School of Chemical Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563,
Tehran, Iran
2
Department of Chemistry, Payame Noor University, P.O. Box 19395-4697, Tehran, Iran
3
Department of Materials Science and Engineering, Sharif University of Technology, Tehran, Iran
4
Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran
Correspondence to: S. H. Jafari (E–mail: [email protected])

ABSTRACT: A series of herbal extract incorporated into poly(lactic acid) (PLA) composite nanofibrous scaffolds were successfully pre-
pared by using electrospinning technique. Equisetum arvense extract (EE) and nanohydroxyapatite (nHA) in different quantities were
loaded into PLA solution to fabricate composite nanofibrous webs under various electrospinning conditions. Uniform nanofibers were
obtained with an average diameter of 157 6 47 nm in the case of those containing the herbal extract. Characterization of the webs was
carried out by means of Fourier transform infrared (FTIR) spectroscopy, field emission-scanning electron microscopy (FE-SEM), energy-
dispersive X-ray spectroscopy (EDX), and differential scanning calorimetry (DSC) techniques. Mechanical properties, porosity, and con-
tact angle of the prepared webs were also determined. Releasing behavior was investigated in phosphate buffer solution (pH 7.2)
medium. Moreover, cell studies and osteogenic capacity were assessed in vitro using human adipose tissue-derived mesenchymal stem
cell (AT-MSC). Evaluations of cell attachment, spreading, and proliferation of AT-MSC were done by SEM observation and thiazolyl
blue (MTT) assay. Osteogenic differentiation capability of AT-MSC on the nanofibrous webs was analyzed by alkaline phosphatase activ-
ity and calcium content assay. It was found that with the addition of nHA and EE to PLA nanofibrous webs, their surface hydrophobic-
ity was reduced while the tensile strength and Young’s modulus were increased satisfactorily. Regarding the samples containing EE and
nHA, cellular adhesion was observed with flattened normal morphology. Osteogenic differentiation of AT-MSC on PLA/nHA/EE webs
showed the highest mineralization capacity after 3 weeks which, was about 1.8 and 3 times higher than that of PLA/nHA and tissue cul-
ture polystyrene as control, respectively. V
C 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45343.

KEYWORDS: biocompatibility; biodegradable; biomedical applications; biopolymers and renewable polymers; structure-property
relationships

Received 14 March 2017; accepted 29 May 2017

DOI: 10.1002/app.45343

INTRODUCTION nanofibers in tissue engineering and drug delivery systems.2,3 Nano-

fibers have special properties such as high specific surface area and
Currently, development and improvement of scaffolds in tissue
appropriate porosity, which make them promising candidate for
engineering are considered as an essential research area in regener-
scaffolds. These properties facilitate cell adhesion and growth as well
ative medicine. High porosity scaffolds have a substantial role in
as the transfer of vital substances for cell proliferation. Furthermore,
cell seeding, cell proliferation, spatial formation, and regeneration
electrospun nanofibers owing to their unique properties simplify dif-
of new tissues. An ideal scaffold not only should have suitable
fusion of biomolecules loaded into them to the surroundings and
physical and mechanical properties but also it must be biocompat-
increase the mass transfer. These features have boosted utilization of
ible and biodegradable. Furthermore, such a scaffold should
nanofibers in drug delivery applications.4
improve intercellular interactions to assist tissue regeneration.1
Electrospinning is a simple and flexible method to produce fibers Biodegradable polyesters such as poly(lactic-co-glycolic acid),
with average diameters from tens of nanometers to several microns. poly (e-caprolactone) (PCL), poly(lactic acid) (PLA), and
In recent years, this method has been extensively utilized to produce poly(glycolic acid), are considered as most common synthetic

C 2017 Wiley Periodicals, Inc.

V

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polyesters used in bone tissue engineering and drug delivery.5–7 inflammatory, and antibacterial attributes. The herb is consid-
Among them, PLA has been widely utilized in bone regenera- ered to have therapeutic effects such as a diuretic, regenerating
tion applications because of its semicrystalline nature, good bone and cartilage as well as preventing osteoporosis.24 Phyto-
strength, low toxicity, and predictable biodegradation rate. The chemical compounds in this plant consist of alkaloids, phytos-
mentioned polymers alone do not provide a proper surface for terols, tannins, triterpenoids, and phenolic compounds. Some
cell adhesion and growth due to lack of cellular recognizable flavonoids such as kaempferol, quercetin, apigenin, luteolin,
signals. This deficiency can be eliminated by using bioactive phenolic acids, and styrylpyrones are among phenolic com-
ceramics such as nanohydroxyapatite (nHA) and preparing pounds of this plant.25 This herb has minerals like silica, potas-
composite scaffolds.8 nHA is a phosphate-based ceramic that sium, manganese, magnesium, and sulfide. Since it has
has a chemical structure similar to the minerals in bone. Using considerable silica content (5%–8%), this plant has great ability
nHA in PLA enhances activity and viability of seeded cells and to absorb and use protein and facilitates collagen formation.
neutralizes the acidic products of PLA degradation by forming a The effect of EE hydromethanolic extracts in bone tissue regen-
buffer.9 It has suitable mechanical properties, osteoconductivity, eration was studied by Pereira et al., and showed the dose-
and biocompatibility. Therefore, it has been extensively used in dependent effect of the extract on cell viability and ALP activity
scaffolds of bone tissue engineering.9,10 Moreover, nHA of human bone marrow cells.19 In another work, Asgharikha-
improves surface topography, increases cell adhesion and tooni et al. reported the effectiveness of topical application of
growth, and causes calcium-bearing minerals to participate in EE ointment in wound healing and reduction of inflammation
boosting the formation of new bone tissue. nHA facilitates the and pain relieving after episiotomy.26
differentiation of mesenchymal stromal cells and osteoblast pre-
Despite unique specifications of the EE herb and particularly its
cursor cell line to osteoblast and increases activities of alkaline
high osteogenic differentiation potential, so far its application
phosphatase and osteocalcin expression.11,12
influence as a scaffold for bone tissue engineering has not been
In order to benefit from therapeutic properties of medicinal explored. Therefore, this work is aimed to explore, for the first
plants and botanicals such as their antioxidant characteristics, time, the beneficial roles of EE herbal plant and influential role
anti-inflammatory, and antibacterial attributes, etc., the extracts of nHA in improving PLA characteristics for making it a suit-
of these herbs are loaded into the drug delivery systems to make able candidate as a scaffold for bone tissue engineering. For this
use of their positive effects in vitro and in vivo via controlled purpose hydroethanolic extract of EE was loaded into electro-
delivery methods.13–15 Another research area for medicinal herbs spun PLA and PLA/nHA composite nanofibers and their mor-
is their application in bone regeneration. To improve attachment, phological, physical, and mechanical characteristics were probed
proliferation, and differentiation of bone cells, various biomole- via various techniques. Moreover, EE release behavior in phos-
cules and growth factors have been employed in bone tissue engi- phate buffer solution (PBS) was investigated. Cell viability and
neering. These will improve bone regeneration at the bone/ cellular adhesiveness were also determined by culturing of a
material interface. Generally, these materials are expensive, mesenchymal stem cell isolated from human adipose tissue (AT-
degrade easily, and have considerable limitations in widespread MSC). Moreover, osteogenic differentiation capability of AT-
applications.16 Therefore, using active ingredients of medicinal MSC on composite nanofibrous scaffolds was assessed by ALP
plants in bone regeneration studies have a promising potential as activity and calcium content assays.
alternative options. There are many pieces of evidence in in vitro
and in vivo studies that show some herb species have a positive EXPERIMENTAL
effect on bone metabolism.17–22 As a typical example Suganya Materials
et al.17 incorporated petroleum ether extract of Cissus quadrangu- PLA grade 2002 D was obtained from NatureWorks in pellet
laris (CQ) and nHA in polymeric nanofibers for bone tissue form. This grade of PLA contains both D-lactide and L-lactide
regeneration application. They exhibited that CQ and CQ-nHA isomers. Spherical nHA (KF-HAP04) with average size of 20 to
loaded PCL nanofibrous scaffolds increased the potential for pro- 40 nm and 99% purity was provided by Kinfon Pharma, China.
liferation and differentiation of human fetal osteoblast cells com- EE plant extract was gathered from north of Iran. Chloroform
pared with neat PCL nanofibrous scaffolds and also synergic (CF), dimethylformamide (DMF), and dimethyl sulfoxide
effect of CQ and nHA was studied. Zhang et al.22 investigated (DMSO) were supplied by Merck Co., Germany. All the materi-
the effect of total flavonoids of Epimedium koreanum Nakai on als were used without further purification.
proliferation and differentiation of primary osteoblasts. They
Preparation and Characterization of Plant Extract
reported inhibition of cell proliferation at most concentrations;
The hydroethanolic extract was prepared by maceration method.
however, differentiation promotion of primary osteoblasts was
In this procedure, 200 g of the plant was washed, milled, and
observed by accelerating the alkaline phosphatase (ALP) activity.
then transferred into a glass balloon. 1000 mL of extracting liq-
The potential role of some medicinal herbs on proliferation and
uid, distilled water: ethanol (1:1), was added to the balloon and
differentiation of stem cells was also studied.23 However, the
it remained for 3 days as the maceration process. The final
studies on the effect of herbal extract loading on polymeric com-
extracted solution was percolated and dried by using a rotary
posite scaffolds for bone tissue engineering applications and eval-
evaporator, operating at 50 8C under reduced pressure. The
uation of cell responses are very rare.
obtained extract was kept in sterile Petri dish and stored in a
Equisetum arvense (EE) plant, also known as horsetail, is a refrigerator at 4 8C. The concentration of total flavonoids in the
medicinal herb which has antioxidant characteristics, anti- EE was determined using spectrophotometric method.27 The

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total flavonoids content of the extract was expressed in terms of Three samples were considered for each test. The thickness of
rutin equivalent (mg of RU/g of extract). The amount of silica samples was measured by Schopper type thickness gauge OSK
was measured by atomic absorption method. (model B-12). Porosity (E) was estimated according to eq. (1),
where q0 is the density of neat PLA (1.24 g cm23). It is to be
Preparation of the Electrospinning Solutions
noted that the density of webs loaded with EE and nHA was
To prepare PLA pure solution, a weighed amount of PLA gran-
found to be close to this value.
ules was dissolved in CF at ambient temperature for 3 h, then  
DMF and DMSO (3:1) were added to the PLA/CF solution to q
E5 12 3100; (1)
give a solution concentration of 7.5% (w/v). In order to provide q0
PLA solution containing EE, first EE was dissolved in DMSO Thermal Analysis. Thermal behavior was carried out using a
and then added to the PLA/CF/DMF solution to obtain an EE- differential scanning calorimeter (DSC) (Q100, TA instrument).
contained solution with a concentration of 10% (w/w). Com- About 6 to 8 mg of samples were heated from 0 to 200 8C by a
posite nanofibrous webs containing EE was prepared by loading heating rate of 10 8C min21 under N2 atmosphere. Glass transi-
an appropriate amount of nHA into the solution. For this pur- tion temperature (Tg), cold recrystallization (Tcc), and melting
pose, different amounts of nHA were dispersed in DMF by son- temperatures (Tm) as well as cold crystallization (DHcc) and
ication for 30 min (Bandelin power 70 W) to prepare melting (DHm) enthalpies were determined from the second
polymeric solutions containing 2.5, 5, 7.5, and 10% (w/w) of heating scan. The degree of crystallinity (Xc) of the as-produced
nHA. Then each dispersed solution was added to EE/DMSO nanofibrous webs was calculated according to:
solution, stirred for 5 min, and instantly added to PLA solution
DHm2DHcc
dropwise to achieve a series of PLA/nHA/EE mixtures with pre- XC ð%Þ5 3100; (2)
determined concentrations. DH0

Electrospinning Process where DHcc and DHm were derived from the DSC thermograms,
For this purpose, the polymeric solution was inserted into a DH0 is the theoretical enthalpy of the fully crystalline polymer,
5 mL of plastic syringe and a high voltage of 15 kV was applied. for PLA the considered value is 93 J g21.28
Electrospinning set up was supplied by Nanoazma Co., Iran, the Mechanical Properties. The tensile properties of the electro-
flow rate was 0.6 mL/h, and the electrospun nanofibers were spun nanofibrous webs were determined at ambient tempera-
collected on a rotating aluminum collector kept 20 cm away ture using an Instron (model 5566, Amersham, England) with a
from the tip of the needle (23 gauge; 0.43 mm inner diameter). load cell capacity of 50 N under a crosshead speed of 5 mm/
The processing parameters were obtained by optimization of min. The thickness of samples was measured by a Schopper
preliminary experimental results. The electrospun nanofibers type thickness gauge OSK (model B-12). The webs were cut
were dried under vacuum for 48 h to evaporate any residual into rectangular specimens having dimensions of 6 3 30 mm.
solvents. Three specimens were considered for each electrospun webs.
Nanofibrous Webs Characterization The Young’s modulus (E) was evaluated between 0% and 5% of
FTIR Studies. Attenuated total reflectance Fourier transform strain. Tensile strength and elongation at break were calculated
infrared (ATR-FTIR) spectroscopic analysis of electrospun nano- based on the generated stress–strain curves of each specimen.
fibrous webs was performed using a Bruker instrument (Equi- In Vitro EE Release. The EE-loaded PLA nanofibrous webs
nox 55LS 101 series, Germany) with the resolution of 4 cm21 were immersed into 15 mL of PBS at pH 5 7.2 under gentle
(averaging 50 scans) for determination of functional groups of shaking at 37 8C. About 20 mg of EE-loaded PLA/nHA nanofi-
as-prepared nanofibrous webs. brous webs were immersed into 15 mL of PBS. At prespecified
FE-SEM Studies. The electrospun nanofibers were sputter- time intervals, 3 mL of supernatant was taken and immediately
coated with a thin layer of gold and visualized using a field replaced with 3 mL of the fresh PBS in order to keep the vol-
emission scanning electron microscope (FE-SEM; ume constant. The obtained supernatants were analyzed by a
MIRA3TESCAN-XMU, Czech Republic) with a 20,0003 magni- UV–vis spectrophotometry at a wavelength of 275 nm. The
fication. The average diameter of the electrospun nanofibers was cumulative release percentage curve was plotted versus time.
calculated by measuring 30 to 40 individual single nanofibers The cumulative amount of EE released from the samples was
within each FE-SEM micrographs using image analysis software assessed using the following equation:
Pt
(ImageJ). Cellular adhesion morphology was also investigated Mt
Cumulative EE released ð%Þ 5 t50 3100; (3)
by SEM (VEGA-TESCAN-XMU, Czech Republic) with different Mtotal
magnifications.
where Mt is the amount of EE released from the webs at time t,
Contact Angle Measurements. Hydrophilic/hydrophobic nature and Mtotal is the total amount of EE loaded into the electrospun
of the electrospun nanofibrous webs was determined by water PLA webs. The entrapment efficiency of EE in PLA nanofibrous
contact angle measurements using a G10 goniometer (Kruss, webs was about 0.68. This entrapment efficiency has been con-
Germany) at 25 8C and relative humidity of 30%. Average of sidered for calculating the reported release data.
three measurements was reported.
Cell Culture and Differentiation. AT-MSCs were cultured in
Porosity Measurements. Density (q) was estimated as mass to Dulbecco’s modified Eagle medium supplemented with 2 mM
volume ratio on 20 mm diameter discs cut out of the webs. of L-glutamine, 1% of penicillin-streptomycin, and 10% of fetal

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bovine serum. The pH of the medium was adjusted to 7.2 to

7.4 by NaHCO3 (all reagents were supplied by Gibco). Compos-
ite nanofibrous webs with and without EE were cut into circular
scaffolds with a diameter of 1.5 cm and sterilized under UV
irradiation for 40 min on each side and soaked in cell culture
medium overnight prior to cell seeding to improve protein
adsorption and cell attachment. The cells seeded on the scaf-
folds at a cell density of 2 3104 cells/well and incubated at
37 8C in 5% of CO2 for cell proliferation. For osteogenic differ-
entiation evaluation, cell-scaffold constructs were cultured in
standard culture medium (Dulbecco’s modified Eagle medium
supplemented with 10% of fetal bovine serum) which was sup-
plemented with 0.2 mM of ascorbic acid 2-phosphate, 100 nM
of dexamethasone, and 10 mM of b-glycerophosphate (all
reagents were supplied by Sigma-Aldrich, Pilsburg, The Nether-
lands). Tissue culture polystyrene (TCP) was used as a control.
In order to evaluate cell attachment on the composite nanofi-
brous scaffolds with and without EE, chemical fixation of cells Figure 1. Attenuated total reflectance Fourier transform infrared (ATR-
was performed for each sample. After 7 days of incubation, the FTIR) spectra of poly(lactic acid) (PLA), Equisetum arvense extract (EE),
nano hydroxyapatite (nHA), and PLA/nHA/EE nanofibrous webs. [Color
scaffolds were rinsed twice with PBS and afterward fixed in
figure can be viewed at wileyonlinelibrary.com]
2.5% of glutaraldehyde for 1 h. Thereafter each sample was
rinsed with distilled water and dehydrated with ethanol for 10
min. Finally, the samples were held in a vacuum chamber and Statistical Analysis
then sputter coated with a thin layer of gold for cell morphol- All data were expressed as the mean 6 standard deviation. Sta-
ogy observation by using SEM. tistical comparisons were performed by the t-test using SPSS
software. P values <0.05 were considered statistically significant
MTT Assay. The viability of cultured AT-MSCs was carried out (n 5 3).
using the colorimetric 3-(4,5-dimethyl-2-thyazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay. Regarding MTT
assay, the scaffolds put on the bottom of the 24-well plate. Cells RESULTS AND DISCUSSION
(2 3 104 cells/well) were seeded and cultured on the nanofi- Characterization of EE
brous scaffolds and TCP as a control for 1, 4, and 7 days. A The yield of solid residue after extraction and evaporation from
total of 50 mL of MTT solution was added to each well and 100 g dried plant was determined. The concentration of flavo-
incubated for 4 h. Then 200 mL of DMSO was added to each noids in the EE extracts was investigated using the spectropho-
well to terminate the reaction and dissolve purple crystals (for- tometric method with aluminum chloride. The silica content of
mazan). Then 100 mL of each plate was transferred to 96-well the EE was evaluated by atomic absorption. The results show
plate and the number of living cells was measured by using an the yield of extraction was 17.5 g per 100 g of dried plant. The
ELISA reader (Dana 3200, Iran) at 570 nm. total flavonoids and silica contents were 17.51 6 0.84 mg rutin/
ALP Activity. ALP activity was measured by p-nitrophenol assay g of EE and 564.4 mg/g of EE, respectively.
(Pars Azmun Kit, Iran) on days 7, 14, and 21. First, 200 mL of
RIPA buffer was added to cell scaffolds and TCP construct to FTIR Studies of the EE, nHA, PLA Nanofibers, and
extract total protein of cells, followed by centrifuging (14000 PLA/nHA/EE Composite Nanofibrous Webs
rpm) at 4 8C for 15 min to deposit cell debris. Then 1 mL of p- In order to evaluate the chemical compositions of EE, nHA,
nitrophenyl phosphate solution was added to each sample. The PLA nanofibers, and PLA/nHA/EE composite nanofibrous webs
absorbance was read at a wavelength of 405 nm and the activity their corresponding FTIR spectra are presented in Figure 1. The
of the enzyme was expressed as IU/L. ALP provided in the kit is results reveal the existence of various chemical constituents. The
used as a standard. wavenumber (cm21) of prominent peaks obtained from absorp-
tion spectra are described in Table I.
Calcium Content Assay. The amount of calcium minerals
deposited on scaffolds and TCP by AT-MSCs under osteogenic The characteristic peaks of EE are described as follows: the
differentiation media was evaluated using calcium content assay. absorption bands in 664 and 771 cm21 were assigned to CAH
In this method, o-cresol phthalein complexone was used as a bonds in alkenes and benzene ring, respectively. The characteris-
reagent. 250 mL of HCl (0.6 N) was added to each well to tic peak at 1064 cm21 vibration related to CAO bond existing
extract calcium and homogenized by shaking, then 20 mL of the in alcohols, ethers, carbocyclic acids, and esters. CAH bending
solution was taken and 1 mL of the reagent added to it. Cal- and stretching vibrations were seen in 1405 and 2925 cm21.
cium content was determined using the solution absorption val- Vibration at 1621 cm21 might be related to C@C in alkenes or
ues at a wavelength of 570 nm and a standard curve prepared conjugated. A broad peak in 3332 cm21 represented hydroxyl
by serial dilution of the calcium concentrations. group in alcohols, phenols containing hydrogen bonding.

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Table I. Characteristic Absorption Bands of EE, PLA, nHA, and PLA/nHA/EE

Characteristic group EE (cm21) PLA (cm21) nHA (cm21) PLA/nHA/EE (cm21)

CAH (alkenes and benzene ring) 664 703 — 701
771 754 753
CAC — 868 — 868
PO4 — — 968 —
1014
1070
CAO (phenol, alcohol, ethers, 1064 1045 — 1043
carboxylic acids, esters)
1084 1087
1126 1128
1183 1185
CAH bending vibration 1405 1379 — 1380
1451 1452
C@C (alkenes, conjugated) 1621 — — 1644
C@O — 1748 — 1752
CAH stretching vibration (alkanes) 2925 2947 — 2990
2994
OAH (absorbed water, H-bonded) 3332 3334 633 3351
3334

In FTIR spectra of PLA, absorption bands for CAH bond were vibration of alkanes were observed at 2947 and 2994 cm21. Two
observed at 703 and 754 cm21. Also, CAO vibration bands characteristic peaks of nHA were related to APO4 and AOH
were observed at 1045, 1084, 1126, and 1183 cm21. The CAH groups in 1014 and 633 cm21. Regarding EE incorporated into
bending vibration peaks were observed at 1379 and 1451 cm21. PLA/nHA composite nanofibrous webs, characteristic chemical
The maximum absorption peak of C@O characteristic group was bonds of PLA were seen with some changes. The new characteris-
seen at 1748 cm21, CAH asymmetric and symmetric stretching tic peak at 1644 cm21 was detected, this might be due to the

Figure 2. FE-SEM images (a) poly(lactic acid) (PLA), (b) PLA/Equisetum arvense extract (EE), and (c) PLA/EE/nanohydroxyapatite (nHA) nanofibers
along with their diameter size distribution.

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Table II. Physical Properties of PLA, PLA/EE, and PLA/nHA/EE Nanofi-

brous Webs

Sample Nanofibers diameter (nm) Porosity (%)

PLA 316 6 51 77.5 6 2.3
PLA/EE 157 6 47 80.0 6 3.2
PLA/EE/nHA 217 6 44 83.4 6 3.3

C@C interaction of the EE with PLA. Furthermore, the C@O

vibration peak of PLA at 1748 cm21 were shifted to 1752 cm21
in the composite nanofibers. These changes could be attributed
to the formation of hydrogen bonding between C@O functional
group of PLA and OH group of nHA.

Morphological Studies
The morphology of PLA, PLA/EE, and PLA/nHA/EE nanofibers
was observed via FE-SEM and the results are represented in Fig-
Figure 4. Contact angles of poly(lactic acid) (PLA), PLA/Equisetum
ure 2(a–c). As it is seen, the resulting morphology of the nano-
arvense extract (EE), and PLA/EE/nanohydroxyapatite (nHA) nanofibrous
fibers containing EE and nHA were appeared beadless, smooth,
webs.
and homogenous. Size measurements of nanofibers showed that
these values were decreased due to the addition of EE to PLA
observed by other reseachers.17 According to the determination
solution. This effect could be attributed to the high conductivity
of electrical conductivity of PLA and PLA/EE solution, by the
of the solution containing EE. Similar results have been
addition of EE to PLA solution, the electrical conductivity
increased strongly from 1.53 to 29.5 mS cm21. Herbal extracts
mainly contain ionic compounds and therefore their dissolution
in a solvent leads to enhancement of electrical conductivity of
the solution. The average diameters of nanofibers including
PLA, PLA/EE, and PLA/EE/nHA are reported in Table II. By EE
incorporation into polymeric solution, the nanofibers diameter
was declined. Loading nHA particles into nanofibers led to a
slight change in composite nanofibers diameter. Elemental map-
ping of calcium (Ca) and phosphorus (P) in PLA/nHA compos-
ite nanofibers and corresponding EDX spectrum as well as
related elemental analysis shown in Figure 3 confirmed presence
of nHA and its uniform dispersion within the composite
nanofibers.
Scaffolds with high porosity are essential for cell growth and
viability. The average porosity values of PLA, PLA/EE, and PLA/
EE/nHA nanofibers are shown in Table II. As it can be seen, the
porosity of PLA nanofibers was 77.5% and by incorporating EE
and nHA, it increased to 80% and 83.4%, respectively. Gener-
ally, the reduction in nanofibers diameter could tend to porosity
promotion, but in this case, with adding nHA particles porosity
increased despite an increase in fibers diameters.
Although composite nanofibers were prepared with different
percentages of nHA, just the results related to the optimum per-
centage i.e., 2.5% are reported here.
Water Contact Angle Measurements of the Nanofibrous
Samples
Hydrophilicity has a major effect on cell adhesion. Figure 4 rep-
resents contact angle results of nanofibers such as PLA, PLA/EE,
Figure 3. Elemental mapping of (a) calcium and (b) phosphorus, (c) and PLA/EE/nHA. The contact angle of PLA nanofibers due to
EDX spectrum and (d) elemental analysis of nanohydroxyapatite (nHA) its hydrophobic nature was about 1308 which decreased to 958
in poly(lactic acid) (PLA)/nHA composite nanofibers. [Color figure can by incorporation of EE and nHA. These results revealed that EE
be viewed at wileyonlinelibrary.com] and nHA due to possessing polar and hydrophilic groups, like

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Table III. Thermal Characteristics of PLA, PLA/EE, and PLA/EE/nHA Nanofibrous Webs

Sample Tg (8C) Tcc (8C) Tm (8C) DHcc (J/g) DHm (J/g) XC (%)
PLA 62.0 93.9 149.0 16.5 24.7 8.8
PLA/EE 58.2 86.6 151.7 10.3 25.5 16.3
PLA/EE/nHA 58.7 85.1 152.3 11.4 27.5 17.3

carbonyl and hydroxyl, promoted hydrophilicity, and hence, corresponding data namely Tg, Tcc, Tm, DHcc, DHm, and XC are
decreased contact angle of PLA. summarized in Table III.
Also, the DSC thermograms of various nanofibers webs are
Thermal Analysis
shown in Figure 5. The appearance of cold recrystallization exo-
The thermal characteristics of PLA, PLA/EE, and PLA/EE/nHA
thermic peak was due to very low crystallization rate of PLA. As
nanofibrous webs were obtained by the use of DSC and
seen, the Tg of PLA/EE nanofibers (58.2 8C) has been decreased.
This effect could be related to the plasticizing effect of EE. The
Tccs of PLA, PLA/EE, and PLA/EE/nHA were 93.9, 86.6, and
85.1 8C, respectively. The addition of EE and nHA to PLA
decreased the cold crystallization enthalpy and shifted Tc to
lower temperatures. This observation could be attributed to the
slow rate of freezing webs containing EE and nucleating effect
of nHA. All samples showed negligible changes in melting tem-
perature as compared to the neat PLA. However, the highest
crystallinity belonged to PLA/EE/nHA nanofibers (17.3%).
Loading EE into PLA increased its crystallinity from 8.8% to
16.3%. These findings corroborated the nucleation effect of
nHA in PLA/EE nanofibers.
Mechanical Properties
The tensile stress–strain curves of PLA, PLA/EE, and PLA/EE/
nHA nanofibers are plotted in Figure 6. Tensile strength at
yield, Young’s modulus, and strain at break are summarized in
Table IV. In composite nanofibers, the tensile strength at yield
Figure 5. Differential scanning calorimetry (DSC) thermograms of poly(- and modulus were increased from 2.7 6 0.1 MPa and 0.6 6 0.1
lactic acid) (PLA), PLA/Equisetum arvense extract (EE), and PLA/EE/ MPa to 4.5 6 0.4 MPa and 1.7 6 0.1 MPa, respectively and the
nanohydroxyapatite (nHA) nanofibers. [Color figure can be viewed at strain at break was reduced from 85.6 6 2.8% to 42.4 6 4.5% by
wileyonlinelibrary.com] adding nHA to PLA/EE nanofibers. This could be attributed to
inherent high modulus and rigidity of nHA. Incorporation of
EE into PLA, as a result of its plasticizing effect, reduced the
tensile strength and modulus of PLA and increased its strain at
break despite increasing PLA crystallinity. These results were in
agreement with similar works.17
In Vitro EE Release
Figure 7 displays cumulative release profile of EE from PLA
nanofibrous web. As can be seen, at the initial stages a plenty of
EE was released. This burst release could be related to low com-
patibility of hydroethanolic herbal extract with the hydrophobic

Table IV. Tensile Strength, Modulus, and Strain at Break of PLA, PLA/EE,
and PLA/EE/nHA Nanofibers

Tensile strength Young’s

at yield modulus Strain at
Sample (MPa) (MPa) break (%)
PLA 2.8 6 0.0 0.9 6 0.1 65.7 6 2.9
Figure 6. Stress–strain curves of poly(lactic acid) (PLA), PLA/Equisetum
PLA/EE 2.7 6 0.1 0.6 6 0.1 85.6 6 2.8
arvense extract (EE), and PLA/EE/nanohydroxyapatite (nHA) nanofibers.
[Color figure can be viewed at wileyonlinelibrary.com] PLA/EE/nHA 4.5 6 0.4 1.7 6 0.1 42.4 6 4.5

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hydrolytic degradation. The results were similar to the findings

reported on PLA and their copolymers by other researchers.29,30
Morphology and Proliferation of AT-MSC
In order to confirm cell proliferation and adhesion on scaffold
surface, SEM observation was conducted. SEM images of AT-
MSCs on PLA/nHA and PLA/EE/nHA scaffolds after day 7 of
culture are shown in Figure 8(a–f). Due to the desirable effect
of nHA on cell adhesion, proliferation and mineralization, bio-
logical studies were done on PLA composite nanofibers with
and without EE. Cell growth (matured cells) could be observed
in two composite nanofibrous scaffolds. The higher magnifica-
tion images (b, c, e, and f) showed good cell extensions and
adhesion on scaffolds. Nanofibers due to the large surface area
to the volume have a high potential for cell proliferation. As it
is evident, both scaffolds provided suitable conditions for cell
Figure 7. Cumulative release profile of Equisetum arvense extract (EE) growth but the population of AT-MSCs cells proliferated and
from poly(lactic acid) (PLA) composite nanofibrous web. migrated onto the surface of the PLA/EE/nHA nanofibrous scaf-
fold was qualitatively higher than those of the PLA/nHA
polymer and its adsorption onto the surface of nanofibers. This scaffolds.
led to EE accumulation onto the surface, and thus, to the fast MTT Assay
release rate in the release media in the early stages of the experi- To assess cell viability, measurements of cell growth were per-
ment. The release profile of EE could be categorized into three formed. Cell viability onto the surfaces of composite nanofi-
stages: about 50% of EE was quickly released in the first 3 h brous scaffolds with and without EE at days 1, 4, and 7 are
(stage 1), and then showed a release with a slower rate (stage 2) displayed in Figure 9. The cells on PLA/EE/nHA composite
followed by approximately a constant release rate (stage 3). nanofibrous scaffold compared to TCP and PLA/nHA scaffold
However, about 83% of the EE-loaded nanofibers were released have the highest viability. These results and the cell growth
within 3 weeks. The remaining of EE will be released by images demonstrated the capability of PLA/EE/nHA composite

Figure 8. Scanning electron microscopy (SEM) images of human adipose tissue-derived MSC on poly(lactic acid) (PLA)/nanohydroxyapatite (nHA)
(a–c) and PLA/Equisetum arvense extract (EE)/nHA composite nanofibers (d–f) at day 7. [Color figure can be viewed at wileyonlinelibrary.com]

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ARTICLE WILEYONLINELIBRARY.COM/APP

showed the highest mineralization capacity after 3 weeks, being

about 1.8 and 3 times higher than that of PLA/nHA and TCP
as control, respectively. Obtained results of differentiation
potential increase of AT-MSCs on composite nanofibrous scaf-
folds containing and free of EE were almost comparable to the
results of human fetal osteoblast, hFOB, cell differentiation on
PCL/CQ/nHA nanofibrous scaffolds.17 In the mentioned study
when nHA was incorporated into the scaffold containing
extract, an acceptable enhance in osteogenic potential was
observed. However, in our study the obtained notable improve-
ment in the osteogenic differentiation was due to active compo-
nents of EE such as flavonoids, which could promote the cell
differentiation. It is necessary to note that the considerable
increase in mineralization of AT-MSCs on PLA composite nano-
fibrous scaffolds was obtained while the osteogenic potential of
AT-MSCs in comparison with other mesenchymal stem cells

Figure 9. Viability of adipose tissue-derived mesenchymal stem cells (AT-

MSCs) onto the surface of poly(lactic acid) (PLA)/nanohydroxyapatite
(nHA) composite nanofibrous scaffold with and without Equisetum
arvense extract (EE) on days 1, 4, and 7 (one and two stars stand for P
value <0.05 and P value >0.1, respectively). [Color figure can be viewed
at wileyonlinelibrary.com]

nanofibrous scaffold usage in the biomedical application as scaf-

folds and drug delivery carriers.
Osteogenic Differentiation
In order to study osteogenic differentiation of AT-MSCs on
composite nanofibers and influence of EE on the cell differenti-
ation, two important key markers (ALP activity and calcium
content assay) were investigated. Figure 10(a) exhibits ALP
activity of AT-MSCs on PLA/nHA, PLA/EE/nHA composite
nanofibrous scaffolds, and TCP on days 7, 14, and 21. As can
be seen, there was no significant difference between ALP activity
of AT-MSCs on both scaffolds and TCP on day 7, but on days
14 and 21, it was higher for scaffolds as compared to TCP. AT-
MSCs on composite nanofibrous scaffold containing herbal
extract showed the highest amount of ALP activity in those
days. Due to the fact that the EE plant contains different com-
ponents including flavonoids such as kaempferol, quercetin, api-
genin, luteolin, etc., and minerals like silica, it seems these
compounds improve osteogenic differentiation capacity of AT-
MSCs. Similar observations have been reported on ALP activity
improvement. Zhang et al. reported the positive effect of flavo-
noids of Epimedium koreanum Nakai plant on the differentia-
tion of primary osteoblasts by promoting ALP activity.22 The
effect of quercetin, one of the flavonoids, on increasing osteo-
genic differentiation in human adipose stromal cells was investi-
gated by Jeong et al.31 Figure 10(b) shows mineralization of AT-
MSCs on PLA/nHA and PLA/EE/nHA composite nanofibrous
scaffolds. As expected with rising cell culture time in osteogenic
media, mineralization of AT-MSCs increased especially on scaf- Figure 10. (a) Alkaline phosphatase activity, and (b) quantitative analysis
folds containing the extract. Moreover, both PLA/nHA and for the mineralization of adipose tissue-derived mesenchymal stem cells
PLA/EE/nHA composite nanofibers promoted osteogenic differ- (AT-MSCs) on tissue culture polystyrene (TCP), poly(lactic acid) (PLA)/
entiation of AT-MSCs, a greater extent of mineral deposition nanohydroxyapatite (nHA), and composite nanofibrous scaffolds on days
was found in the PLA/EE/nHA composite nanofibers. Osteo- 7, 14, and 21 (one star stands for P value <0.05). [Color figure can be
genic differentiation of AT-MSC on PLA/EE/nHA scaffolds viewed at wileyonlinelibrary.com]

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ARTICLE WILEYONLINELIBRARY.COM/APP

sources such as human fetal mesenchymal stem cells and human 10. Liuyun, J.; Chengdong, X.; Lixin, J.; Lijuan, X. Compos. Sci.
umbilical cord mesenchymal stem cells was minimum.32 Technol. 2014, 93, 61.
In summary, cellular proliferation and mineralization were 11. Balaji Raghavendran, H. R.; Puvaneswary, B.; Talebian, S.;
indicative of cell ossification and might be due to compounds Raman Murali, M.; Vasudevaraj Naveen, S.; Krishnamurithy,
present in the extract that affected the osteogenic-related signal- G.; McKean, R.; Kamarul, T. PLoS One 2014, 9, e104389.
ing pathways. 12. Qi, H.; Ye, Z.; Ren, H.; Chen, N.; Zeng, Q.; Wu, X.; Lu, T.
Life Sci. 2016, 148, 139.
CONCLUSIONS
13. Motealleh, B.; Zahedi, P.; Rezaeian, I.; Moghimi, M.;
In this study, new PLA composite nanofibrous scaffolds contain- Abdolghaffari, A. M.; Zarandi, M. A. J. Biomed. Mater. Res.
ing herbal extract were developed and characterized. FTIR studies B 2014, 102, 977.
showed EE and nHA were successfully loaded into PLA webs. 14. Jin, G.; Prabhakaranb, M. P.; Kai, D.; Annamalai, S. K.;
Morphology of the samples was investigated by FE-SEM in which Arunachalam, K. D.; Ramakrishna, S. Biomaterials 2013, 34, 724.
a good dispersion of nHA in composite nanofibers and a signifi- 15. Nguyen, T. T. T.; Ghosh, C.; Hwang, S.-G.; Dai Tran, L.;
cant decrease in fiber diameters were observed. Thermal analysis Park, J. S. J. Mater. Sci. 2013, 48, 7125.
indicated the plasticizing effect of EE and nucleation effect of
16. Han, Q.-Q.; Du, Y.; Yang, P.-S. Future Med. Chem. 2013, 5,
nHA in nanofibers. The softening effect of EE in mechanical 1671.
assessment led to a reduction in modulus and a promotion in
strain at break values. However, in composite nanofibrous webs, 17. Suganya, S.; Venugopal, J.; Ramakrishna, S.; Lakshmi, B. S.;
Giri Dev, V. R. J. Appl. Polym. Sci. 2014, 131, DOI: 10.1002/
the addition of nHA increased tensile strength and modulus and
app.39835.
at the same time decreased strain at break values. In vitro EE
release of nanofibers in PBS showed a burst release followed by 18. Wirries, A.; Schubert, A.-K.; Zimmermann, R.; Jabari, S.;
slower release rate. Composite nanofibrous scaffold containing Ruchholtz, S.; El-Najjar, N. Int. Immunopharmacol. 2013, 15,
herbal extract showed excellent cell attachment and promoted 381.
proliferation and osteogenic differentiation of AT-MSCs by 19. Bessa Pereira, C.; Gomes, P. S.; Costa-Rodrigues, J.; Almeida
increasing cell viability, ALP activity, and mineralization content. Palmas, R.; Vieira, L.; Ferraz, M. P.; Lopes, M. A.;
Our findings indicated that EE extract has a great potential for Fernandes, M. H. Cell Prolif. 2012, 45, 386.
osteogenic differentiation of AT-MSCs and can be recommended 20. Costa-Rodrigues, J.; Carmo, S. C.; Silva, J. C.; Fernandes, M.
as a suitable candidate for bone tissue engineering application. H. R. Cell Prolif. 2012, 45, 566.
21. Wang, X.-L.; Wang, N.-L.; Zhang, Y.; Gao, H.; Pang, W.-Y.;
ACKNOWLEDGMENTS
Wong, M.-S.; Zhang, G.; Qin, L.; Yao, X.-S. Chem. Pharm.
The authors thank the Iran Nanotechnology initiative Council for Bull. 2008, 56, 46.
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M.-S.; Yao, X.-S. Phytomedicine 2008, 15, 55.
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