Khak Estan I 2017
Khak Estan I 2017
Khak Estan I 2017
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
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
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
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.
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
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
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]
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.
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indicative of cell ossification and might be due to compounds Raman Murali, M.; Vasudevaraj Naveen, S.; Krishnamurithy,
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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.;
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Wong, M.-S.; Zhang, G.; Qin, L.; Yao, X.-S. Chem. Pharm.
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partial support of this work. 22. Zhang, D.-W.; Cheng, Y.; Wang, N.-L.; Zhang, J.-C.; Yang,
M.-S.; Yao, X.-S. Phytomedicine 2008, 15, 55.
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WWW.MATERIALSVIEWS.COM 45343 (10 of 10) J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45343