Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58

Contents lists available at ScienceDirect

Journal of the Mechanical Behavior of

Biomedical Materials
journal homepage: www.elsevier.com/locate/jmbbm

Thermo-chemical modification of a natural biomembrane to induce T

mucoadhesion, pH sensitivity and anisotropic mechanical properties
⁎
Hamidreza Gharibia, Amir Abdolmalekia,b,
a
Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran
b
Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71467-13565, Islamic Republic of Iran

A R T I C LE I N FO A B S T R A C T

Keywords: In the present study due to the distinctive mechanochemical/biological characteristics of natural biomembranes,
Eggshell membrane we state the preparation, characterization and cytocompatibility of modified eggshell membrane (ESM) by citric
Citric acid acid (CA) for biomedical and pharmaceutical applications. FTIR spectroscopy and CHNS analysis demonstrated
Mucoadhesive the successful reaction of ESM with CA. Also, successful modification of the ESM was observed by the change in
Anisotropic
thermogravimetric analysis. SEM micrographs of neat ESM and ESM-CA gave further insight into membranes
Lipophilic drug delivery
morphology and revealed that aligned oriented fibrous frameworks were prepared using thermo-chemical
process. The ESM-CA displayed dense and orderly shapes with tailorable architectures to mimic the intended
tissue. Moreover, mechanical analyzes for ESM-CA indicated anisotropic mechanical properties and proved that
the ESM-CA could induce enhanced mucoadhesion, because of the existence of an enormous amounts of func-
tional groups. It was found that by modification of ESM the swelling behavior was significantly changed.
Indomethacin release from the ESM-CA showed enhanced pH sensitivity. The modified membranes have clearly
presented adequate mucoadhesion, pH sensitivity and cell viability which can be tailored for potential use in
controlled lipophilic drug delivery systems and tissue engineering.

1. Introduction
Many authors have investigated polymers with various molecular
characteristics essential for mucoadhesion. The presence of polar
functional groups increase their interaction with the mucin glycopro-
teins and favor's adhesion. At present, the most widely conducted group
of mucoadhesives are anionic, cationic and thiolated polymers
(Andrews et al., 2009; Smart, 2005; Khutoryanskiy, 2011; Laffleur,
2014).
Additionally the use of synthetic polymers may have cytotoxic ef-
fects and economically unfavourable (Dang and Leong, 2006; Sell et al.,
2010). Obviously, the development of biological mucoadhesive mem-
brane like Eggshell membrane (ESM) is of remarkable importance and
no efforts have been reported to adjust this natural membrane as
pharmaceutical release systems.
Eggshell membranes (ESM) regards to its unique structure and
chemical composition, reveal exciting features for numerous potential
applications as a biotemplate for the production of nanoparticles,
membrane for the adherence of stromal cells, a biological dressing for
burn, wound healing dressing, biomaterial scaffold in tissue en-
gineering, antibacterial/antimicrobial activities, heavy metals,
organics, dyes, sulfonates and fluorides sorbent material, the template
of biosensors, the material applicable in medicine etc (Dong et al.,
2007; Tavassoli, 1983; Maeda and Sasaki, 1982; Yi et al., 2007, 2004;
Rose and Hincke, 2009; Liu and Huang, 2011; Chen et al., 2012; Liu,
2011; Wang et al., 2010a; Song et al., 2012; Xiao and Choi, 2002; Li
et al., 2012). The ESMs have a numerous amount of bioactive con-
stituents, from which approximately 10% are types I, V and X collagens
(type I collagen is found mainly in the cores of the outer ESM fibers,
while the inner ESM core fibers contain predominantly types I and V)
and 70–75% of further proteins and glycoproteins, and due to the
presence of an enormous level of functional groups (−OH, −CO2H,
−NH2, −CHO, -SO4H, -SH, -S-S-), it is conceivable that have potential
use for mucoadhesive applications (Kaweewong et al., 2013; Arias
et al., 1997; Leach, 1982; Fernandez et al., 1997; Wong et al., 1984;
Zhao and Chi, 2009). Additionally, The ESM also reveals adequate
flexibility to spread through the mucus network, as well as bio-
compatibility, biodegradability, non-toxicity and economic efficiency
(Yi et al., 2007, 2004; Sah and Pramanik, 2014).
Here we report a new concept to engineer the ESM, which provides
two functions: mucoadhesion and controlled drug release. The abun-
dant functional groups in the ESM act as the versatile platform for extra

⁎
Corresponding author at: Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran.
E-mail addresses: abdolmaleki@cc.iut.ac.ir, amirabdolmaleki@yahoo.com (A. Abdolmaleki).

https://doi.org/10.1016/j.jmbbm.2018.07.014
Received 9 April 2018; Received in revised form 9 July 2018; Accepted 10 July 2018
Available online 11 July 2018
1751-6161/ © 2018 Elsevier Ltd. All rights reserved.
H. Gharibi, A. Abdolmaleki
chemical modification with other favorite bioactive molecules to adjust
their chemical and functional characteristics that can enormously en-
hance the worth and efficacy of these biomembranes Baláž (2014). ESM
with a pore structure (Zhou et al., 2011), high pore volume and large
surface area chosen as mucoadhesive drug delivery system after func-
tionalization with citric acid (CA). Citrate-based biomaterials (CBBs)
empower the bioengineer to target a diversity of regenerative en-
gineering and biomedical applications, owing to their adaptable ma-
terial characteristics, tailored mechanical and degradation properties,
easy synthesis reaction, simple modification, and scalability Tran et al.
(2015). CA, as an important intermediate in the Krebs cycle, and be-
cause of multifunctionality, nontoxicity and biocompatibility, is a key
and cost-effective monomer used in the design of all CBBs, which re-
veals tunable antioxidant, antimicrobial, adhesive, and fluorescent
properties (Tran et al., 2015; van Lith et al., 2014; Yang et al., 2014; Su
et al., 2014a; García-Argüelles et al., 2013; Mehdizadeh et al., 2012;
Zhang and Yang, 2013). The existence of three carboxyl groups and one
hydroxyl group in the CA chemical structure, provides opportunities for
innovation in cardiovascular, musculoskeletal and nervous systems, as
well as applications in bioimaging, drug/cell delivery, and bioadhesives
(Tran et al., 2015, 2014; Su et al., 2014b; Gyawali et al., 2010; Namazi
and Adeli, 2005; Chen et al., 2008). Herein, citric acid (CA) modified
ESM network (ESM-CA) has been prepared as drug release vehicle.
2. Experimental procedure
2.1. Materials
Citric acid, indomethacin, acetic acid, ethanol, potassium hydro-
genphosphate trihydrate, potassium dihydrogen phosphate, NaOH and
HCl were obtained from Merck and Sigma-Aldrich Chemical Co.
2.2. Membrane preparation
2.2.1. Preparation of outer ESM (O-ESM)
Before modification of the membranes, commercial hens' eggs were
cracked and evacuated. The eggshells were rinsed efficiently with
water. The outer ESM (O-ESM) was carefully detached from the shell
and peeled from the inner ESM by the air cell, cleaned with distilled
water to thoroughly eliminate the albumen from the O-ESM and
afterwards dried at room temperature. The membranes were cut out
using a cutting tool, with the size of 35 mm × 20 mm, in both latitu-
dinal (L) and hemispherical (H) directions. The obtained O-ESM, with
the size of 35 mm × 20 mm, was stored in DI water at 4 °C.
2.2.2. Preparation of CA-ESM
The O-ESMs were thermo-chemically modified by the following
procedures (Wing, 1996; Reddy and Yang, 2010). For this purpose, the
air-dried O-ESMs were dipped in solution of CA (4 g CA in 10 ml DI
water) and were heated in an oven to dehydrate at 80 °C for 24 h, then
membranes were stirred vigorously. Thereupon, all surface moisture
was wiped out and O-ESMs were covered by CA. After anchoring the CA
on the membrane surface, the oven temperature was adjusted and
thermo-chemical modification was performed at 140 °C for 3 h. Then
the reaction products (ESM-CA) were immersed in DI water to remove
CA residues. Subsequently, the membranes were suspended in acetic
acid solution (pH 2.0). Finally, the membranes were washed with DI
water 3 times and were air dried at ambient temperature (Scheme 1).
2.3. Membrane characterization
2.3.1. Structural properties
FT-IR spectra were achieved using a Jasco-680 FT-IR spectro-
photometer (Japan) with KBr pellet. Vibration bands were presented as
wavenumber (cm-1). Elemental analysis was implemented with a CHNS-
932, Leco. Thermal gravimetric analysis (TGA) was accomplished with
51
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
a STA503 win TA (Bahr-Thermoanalyse GmbH, Hüllhorst, Germany) at
a heating rate of 10 °C/min from 25 °C to 800 °C under nitrogen at-
mosphere. The morphology of the membranes was analyzed with a
scanning electron microscope (SEM, Philips, XL30) at an accelerating
voltage of 10 kV. Moreover, the average fiber diameter, surface area
porosity and total surface pore area of membranes were evaluated from
the SEM images using an image analysis software package (Image J,
National Institutes of Health, USA).
Tensile properties of the membranes in both latitudinal (L) and
hemispherical (H) directions were specified at medium temperature
(25 °C) and moist state using Testometric Universal Testing Machine
M350/500 (UK) with a load cell capacity of 10 N, according to ASTM
D882 (standards) at ambient temperature. The dimensions of the test
samples were 35 mm × 20 mm at cross-head speed of 10.0 mm/min.
The stress-strain curvatures (n = 5) were schemed and the mechanical
properties including elastic modulus, tensile strength and energy per
volume (toughness) were computed.
2.3.2. Membrane adhesion strength
To measure the adhesive properties between sample membranes (O-
ESM and ESM-CA) and a model substrate (ESM), specimens were cut
along the long axis of the shell without damaging the membranes in the
dimension of 10.00 mm × 40.00 mm. The force required to separate the
sample membranes (O-ESM and ESM-CA) from a model substratum
(ESM) was obtained using a commercial tensile tester adjusted for
bioadhesion assessments. The biological substrate was fixed to the
lower tensile jaw and was moistened with the hydration liquid (5 ml of
1% w/w mucin) for 2.5 min. The sample membrane (O-ESM or ESM-
CA) was fixed to the upper jaw grip of the tensile machine and then
situated in contact with the hydrated substrate, so that the sufficient
adhesion bonding between the membrane and substrate could be es-
tablished. After completion of preload time (an optimized preload of
25 g for 1 min (preload time)), the membrane and the substrate were
separated with a crosshead extension rate of 5.0 mm/min until a
complete detachment of the membrane-substrate bond was reached. A
force vs. distance diagram was integrated. Using digital tensile soft-
ware, the separation force and the work of adhesion (the area under-
neath the curve of force vs. distance) were evaluated to determine the
strength of detachment of the adhesive bond. All measurements were
carried out at least 3 times at ambient temperature (n = 3).
2.3.3. Studies of swelling pH-response
The swelling of the membranes in response to the changes in pH
were examined at ambient temperature by using gravimetric technique.
The membranes were dehydrated until their weight remained constant.
Then, the membranes were submerged in phosphate buffer (at pH 4.0
and 7.4) and the proliferation of weight for predetermined periods of
time was determined. Next, their excessive water surfaces were blotted
out by filter paper and then, they were quickly weighed on a micro-
balance. Water uptake was measured at different pH related to time
until the hydrated weight reached a constant value. The water uptakes
of the membranes were calculated as follows (Eq. (1)):
Wwet − Wdry
DS = × 100
Wdry (1)
where Wwet is the weight of moist membrane and Wdry is the weight of
dry network.
2.3.4. Drug loading and controlled release
Indomethacin as a model drug was incorporated into the sample
membranes by a swelling equilibrium technique. The sample mem-
branes were allowed to swell in saturated solutions of indomethacin in
ethanol-water (80: 20, vol/vol%) for 1 days at room temperature.
During this process, drug in the solvent was absorbed into the mem-
brane. After incubation, the membranes were rapidly rinsed with DI
H. Gharibi, A. Abdolmaleki
Scheme 1. Schematic procedure for preparation of ESM-CA via thermo-chemical reaction between CA and ESM.
water to remove the indomethacin that weakly adhered to the mem-
brane surface. The membranes were subsequently freeze-dried to avoid
drug migration towards the surface of membrane with quick evapora-
tion of the solvent until all water was removed. Typically the loaded
pharmaceutical is liable to migrate toward the membrane surface in the
procedure of drying. This movement of drug, however, was hindered by
reduction temperature under − 35 °C in the freeze-thawing process. To
accomplish the process of drug loading, one milliliter of each remaining
solution was diluted to 25 ml and the absorbance was measured by UV
spectrophotometry (UV/Vis/NIR spectrophotometer, JASCO V-570) at
318.00 nm at predetermined time intervals. The amount of drug in the
supernatant solutions was specified and determined using the extinc-
tion coefficient obtained from an appropriate calibration curve (Fig.
S1). The adsorbed amount of indomethacin into each membrane was
evaluated by the difference between the initial drug value and the final
remaining drug amount in the solution.
Indomethacin release experiments were implemented in constant-
temperature phosphate buffer saline (PBS) (pH 2.0, 7.4 and 10.0)
(which simulates physiological conditions e.g. gastrointestinal, body
fluid and wound environments) and stirred gently. The membrane was
held in the 50 ml release media using a Teflon tape. At predetermined
time periods, samples of media (3 ml) were withdrawn and returned
back to the release media in order to maintain the same volume of
solution. The concentration of indomethacin was calculated from the
absorbance at 318.00 nm by UV spectrophotometer (UV/Vis/NIR
spectrophotometer, JASCO V-570) at specific time points. Each drug
release experiment was repeated three times and final results were
obtained as an average by previously established calibration curve.
The drug loading efficacy and cumulative in vitro release efficacy
were measured according to the following equations (Eqs. (2) and (3)):
Mads
Drug loading efficiency(%) = ×100
M∞
Mrel
Drug release efficiency(%) = ×100
M∞
where Mads, M∞ and Mrel are the mass of drug adsorbed in the O-ESM
and CA-ESM at time t during the loading procedure, entrapped in the
sample membranes at equilibrium state and released from drug-loaded
membranes at time t throughout the release progress, respectively.
2.3.5. MTT assay
The in vitro cytotoxicity evaluation of membranes was carried out
by direct-contact assay with MG-63 cell line from the National Cell
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
Bank of Iran at the Pasteur Institute. Before cell seeding, the membranes
were sterilized with ethanol (30 min), UV ray (2 h), and culture medium
(overnight at 37 °C). The complete growth medium was Dulbecco's
Modified Eagle Medium (DMEM-low, Bioidea, Iran) supplemented with
10% (v/v) fetal bovine serum (FBS) (Bioidea, Iran) and 1% (v/v)
streptomycin/penicillin (Bioidea, Iran). MG-63 cells were subcultured
from stock culture by trypsinization and subsequently seeded in a 96-
well plate at a density of 10,000 cells/well. Then, previously sterilized
membranes were allowed to keep in contact with the cells at 37 °C in a
humidified incubator containing 5% CO2 for 1 and 2 days. In parallel,
cells seeded on a tissue culture plate (TCP) used as the control group
(n = 3 per group). After being cultured for 24 h and 48 h at 37 °C, the
cytotoxicity was quantitatively measured by MTT assay, which evalu-
ates the metabolic reduction of 3-(4,5-dimethylthiazolyl-2)-2,5-di-
phenyl tetrazolium bromide (MTT) to formazan by viable cell.
Therefore, the degree of the reduction of MTT can reveal the level of
cell metabolism.
2.3.6. Statistical analysis
Statistical analysis were undertaken three times separately with
triplicates and results were represented using one-way ANOVA analyses
and stated as mean values ± standard deviation (SD). To assess a
statistically remarkable variance between groups, the Tukey's post-hoc
test using Graph Pad Prism Software (V.6) with a probability of
P < 0.05 was then investigated to be statistically significant.
3. Results and discussion
3.1. Preparation and characterization of membranes
The ESM fibers have consisted mainly of a collagen-rich core and a
glycoprotein rich mantle containing lysine-derived cross-links. This
(2)
membrane has the potential for further modifications due to the pre-
sence of an enormous amount of hydrophilic functional groups and
(3) large variety of amino acids (Proline, Glutamic acid, Glycine,
Hydroxyproline, Aspartic acid, Arginine, Histidine, Cysteine, Lysine,
Tyrosine, Phenylalanine and etc.) (Baláž, 2014; Sah and Rath, 2016;
Nakano et al., 2003; Harris et al., 1980).
As revealed in Scheme 1, the existence of three carboxyl groups and
one hydroxyl group in CA furnish key advantages for biomaterial
modification and consequently, provide the crucial functionality for
cross-linking of biopolymer chains in quite simple and cost-effective
catalyst-free thermal postpolymerization or polycondensation reaction
to synthesize a homogeneous cross-linked membrane of hydrolysable
52
H. Gharibi, A. Abdolmaleki
Fig. 1. FT-IR spectra of ESM and ESM-CA.
ester and amide linkages Menzel et al. (2013).
In the current research, the ESM–CA was prepared by thermo-che-
mical process. The ESM contains amine, hydroxyl, carboxyl, amide and
other hydrophilic superficial functional groups which fasten the CA
molecules to the meshwork filament surface by hydrogen bond inter-
actions where modification can take place. As the temperature was
enhanced, the neighboring carboxyl groups on CA dehydrated to pro-
duce a cyclical anhydride, and consequently citric anhydrides could
react with free amine and hydroxyl groups in the ESM to form amide
and ester linkages and the chains were gradually cross-linked by CA
(Scheme 1) (Coma et al., 2003; Anand et al., 2013).
To confirm the reaction between citric acid and ESM throughout the
development of ESM-CA by thermo-chemical modification, FTIR spec-
troscopy was utilized to assess the chemical bond transformation of
parallel neat ESM and ESM-CA membranes synthesized according to the
above-mentioned procedure.
The FTIR spectra of neat ESM and ESM-CA are depicted in Fig. 1.
The presence of characteristic peaks in the ESM are demonstrated in
Fig. 1, which shows considerable peaks at 3488 cm-1 (associate to the
O–H and N–H stretching type), 3064 cm-1, 2940 cm-1, and 2865 cm-1
(correspond to the asymmetric stretching vibrations of the C–H bonds
present in =C–H and =CH2 groups), 1628 cm-1 (owing to the C˭O
stretch of amides), 1522 cm-1 (CN stretching / NH bending forms), and
1237 cm-1 (CN stretching/NH bending types), which the last three
peaks can be specified to the amide I, amide II, and amide III vibrations
of the glycoprotein strands of the fibers, respectively. The peaks at
1449 cm-1, 1130 cm-1 and 536 cm-1 can be attributed to the stretching
modes of C˭C, C–O and C-S bonds, respectively (Dong et al., 2007;
Baláž, 2014; Lee et al., 2010). The appearance of strong shoulder cen-
tered at 1731 cm-1 and a peak at 1184 cm-1, which were present in ESM-
CA spectrum and clearly absent in the neat ESM spectrum, are char-
acteristics of C˭O stretching vibration of ester/acid moieties and the C-
O stretching groups of ester respectively, confirmed the formation of
ester bonds, and the shift of the amide I (due to amidation) and the O–H
and N–H stretching (due to the introduction of –COOH groups) modes
from 1628 cm-1 to 1650 cm-1 and 3488 cm-1 to 3416 cm-1 for ESM-CA
sample can be assigned to amide groups formed by cross-linking of the
amino moieties of the ESM fibers with CA (Gyawali et al., 2010;
Tsutsumi et al., 2004). Furthermore, the relative absorption intensities
of the main bands for ESM-CA such as amide I, amide II, and C-H bands
increased after the modification and crosslinking of ESM with CA. All
the above evidence demonstrating the successful amidation and ester-
ification of ESM with CA through crosslinking by a thermo-chemical
reaction.
In addition, the modification of ESM with CA was verified by the
elemental analysis results of the as-prepared neat ESM and ESM-CA
dehydrated samples (Table 1). ESM has around 14.30% N, 46.15% C
53
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
Table 1
Elemental analysis data of ESM and ESM-CA.
Membrane N (%) H (%) C (%) S (%)
ESM 14.30 6.00 46.15 3.65
ESM-CA 13.50 6.44 47.71 3.27
Fig. 2. TGA curves of ESM and ESM-CA.
and 6.00% H in its organic matter. After modification, the carbon and
hydrogen contents increased to 47.71% and 6.44%, respectively and
nitrogen content decreased to 13.50%, indicating that most of the
amino and hydroxyl functional groups are participated in the thermo-
chemical modification process of CA in the preparation procedure.
Also, successful modification of the ESM was also observed by the
change in thermogravimetric analysis (TGA). Thermal properties of
neat ESM and ESM-CA were determined using TGA and DTG.
Specimens were heated from 25 to 800 °C at a rate of 10 °C/min under
nitrogen atmosphere. The TG curve of ESM and ESM-CA are given in
Fig. 2, from which it can be understood that the ESM shows a multi-step
thermal decomposition pattern starting very early (around 53 °C) (Yi
et al., 2004; Baláž, 2014). The primary stage of the TG curve could be
referred to the thermal denaturation of collagen and the loss of bound
water which is completed around 130 °C. The second step of decom-
position occurs in the range between 260 °C and 500 °C, which mainly
might be a result of the thermal degradation of collagen and glycan
chains and the products were composed of small molecular segments
Molavian et al. (2017). Finally, the slow continuous decrease of weight
loss at 500–500 °C is ascribed to the decomposition of the ESM back-
bone and final membrane matrix. The major weight loss region for both
neat ESM and ESM-CA occurs in the second degradation step, which is
attributed to the decomposition of the membranes proteinous fibers and
the ester/amide bonds. Moreover, in the second decomposition step,
ESM-CA begins to lose weight with more intensity at about 200 °C,
which is earlier than ESM (which begins at about 260 °C). This is also
clearly indicated in the derivative thermogravimetric (DTG) curve in
Fig. S2 where the rate of mass change in the second step in degradation
of ESM-CA occurs earlier, thus the thermo-chemical modification and
introduction of new ester/amide bonds as the function of CA changed
the bonding among the molecules, so that the thermal stability of the
ESM-CA decreased in the second stage. As reported, the thermal de-
composition of citric acid is followed by dehydration and decarbox-
ylation processes resulting in the creation of methyl maleic anhydride
and the change in thermal stability between the ESM and ESM-CA
maybe related to compositional and morphological changes during the
thermos-chemical membrane modification Wyrzykowski et al. (2011).
The weight residues at 800 °C for both specimens are about 22%. This
specifies that the products have no major changes in terms of thermal
properties.
H. Gharibi, A. Abdolmaleki Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58

Fig. 3. Membranes morphologies: SEM images of a) neat ESM and b) ESM-CA; SEM images of a′) neat ESM and b′) ESM-CA which were modified with the ImageJ
software.

3.2. Morphological properties increased temperature enhances chain mobility and facilitates re-
ordering of chains. Secondly, when modification occurs, some dense
The illustrative microstructure images of the neat ESM as well as charge centers are formed in protein and also, the hydrogen bond be-
ESM-CA fibrous networks are presented in Fig. 3. The porosity of the tween chains is increased due to the introduction of the CA moieties
membranes was also studied in detail within this work and analyzed by Molavian et al. (2017). Therefore, the introduction of these charged
the ImageJ software (Fig. 3a′-b′). The O-ESM (Fig. 3a) revealed the centers lead to electrostatic attraction and increase protein chains
random fibrous morphology with nearly interconnected pore networks alignment. For ESM-CA (Fig. 3b), addition of the CA groups increased
that has the potential for biomedical application Sah and Rath (2016). polar interactions (hydrogen bonding) between the chains. Further-
The decrease of the porosity and surface area of the fibers from the neat more, increase of temperature during the modification rearranged the
membrane to the modified membrane of the ESM was confirmed (Fig. 3 protein chains and a more regular morphology was obtained for the
and Table 2) and the average fiber sizes about 2.30 ± 0.37 µm were ESM-CA. Moreover, morphological properties of the neat and modified
observed. After modification of the ESM by CA using the thermos- fiber mats reveal both randomly orientated and well-aligned fiber
chemical method, the pore size of ESM significantly decreased and the patterns with fiber diameters that are desirable for neurite and
density of the fibers increased dramatically, depending on the thermo- Schwann cell outgrowth and proliferation (Wang et al., 2010b; Silva
chemical process. In the case of ESM-CA (Fig. 3b), the thermo-chemical et al., 2004).
modification of the fibers reduced the average pore size, pore surface
area and pore surface porosity of ESM from 6.34 ± 0.72 µm to
3.52 ± 0.33 µm, 159.26 ± 7.83 µm2 to 46.19 ± 3.48 µm2 and 3.3. Mechanical properties
12.83 ± 2.26% to 5.42 ± 0.84%, respectively. Moreover, the or-
ientation of fibers significantly changed confirming that the membrane Desirable mechanical properties would be expected for application
morphology changed during the ESM modification. In this condition, of engineering scaffolds. Fig. S3 shows the illustrative tensile stress–-
the fiber orientation of ESM significantly enhanced, whereas the pore strain (σ–e) plots of the neat O-ESM and resultant ESM-CA in order to
dimension noticeably diminished (P < 0.05) from 6.34 ± 0.72 µm to evaluate the elastic modulus, ultimate tensile strength and tensile
less than 3.52 ± 0.33 µm which may have critical role in the me- toughness from stress-strain curves according to the ASTM D882. Fur-
chanical and biological features of the membrane. This is because of ther, the σmax and emax are listed in Table 3. Representative mechanical
partial crosslinking between the chains forming fibrils of the membrane behavior of the ESM was comparable to other biologic systems (e.g.
fiber and denaturation of the twisted molecular chains by denaturation tendons, and DNA molecules) as a consequence of the entropic and
of collagen and glycoprotein and the change of membrane structural enthalpic (The linear region of the stress–strain curve) contributions of
properties altered the membrane features drastically (Table 2). Two
main factors are responsible for denaturation of proteins: firstly, Table 3
Mechanical properties of ESM and ESM-CA cut in Hemispherical (H) and
Latitudinal (L) directions.
Table 2
Pore average size (D), Pore surface area (A) & Porosity (P) of ESM and ESM-CA. Membrane UTS (N/mm2) Strain (%) E-Modulus Work up to
(N/mm2) break (N mm)
2
Membrane A (μm ) D (μm) P (%)
ESM (H) 1.67 ± 0.28 38.41 ± 10.15 5.57 ± 0.90 5.98 ± 1.08
ESM 159.26 ± 7.83 6.34 ± 0.72 12.83 ± 2.26 ESM (L) 1.54 ± 0.23 54.65 ± 13.65 4.71 ± 0.47 6.31 ± 0.92
ESM-CA 46.19 ± 3.48 3.52 ± 0.33 5.42 ± 0.84 ESM-CA (H) 2.67 ± 0.34 63.42 ± 12.38 7.74 ± 0.64 7.18 ± 0.79
ESM-CA (L) 1.25 ± 0.16 48.07 ± 8.26 3.22 ± 0.31 4.62 ± 0.88
All data were obtained by the ImageJ software.

54
H. Gharibi, A. Abdolmaleki
the molecules present in their structure Torres et al. (2013). Further-
more, there was not a significant difference between the mechanical
properties of the neat ESM in latitudinal and hemispherical directions
Torres et al. (2010). However, there was a meaningful difference be-
tween the mechanical strength of the modified fibrous membrane
(ESM-CA) in latitudinal and hemispherical directions. These trends
specified that the mechanical behaviors of the various membranes were
severely controlled by the alignment of fibers and introduction of polar
groups (CA moieties). The special relationship between the fiber or-
ientation, loading direction and the mechanical properties of the shell
membrane in both latitudinal (L) and hemispherical (H) directions was
discovered because of changes in fiber orientation and membrane
porosity as previously revealed. The result is a complex distribution of
anisotropic mechanical properties. It was observed that the σmax and
emax values of the neat O-ESM in latitudinal (L) and hemispherical (H)
directions were 1.54 ± 0.23 MPa - 54.65 ± 13.65% and
1.67 ± 0.28 MPa - 38.41 ± 10.15%, respectively, as compared with
1.25 ± 0.16 MPa - 48.07 ± 8.26% of the ESM-CA (L) and
2.67 ± 0.34 MPa - 63.42 ± 12.38% of the ESM-CA (H). Although the
mechanical properties of the membranes such as ultimate tensile
strength (UTS), young moduli (E) and elongation percentage, in
hemispherical directions were higher than latitudinal direction, that is
because of the increase in the cross-linking between the chain, the chain
orientation and fiber density in hemispherical direction of the mem-
branes by thermo-chemical modification, while the mechanical prop-
erties of the neat O-ESM and ESM-CA membranes in the latitudinal
direction noticeably declined due to non-uniform and randomly or-
iented distribution of the neat ESM fibers in both direction and low
orientation of the ESM-CA fibers in the latitudinal direction. The tensile
strength of the aligned fibers (ESM-CA (H)) was higher
(2.67 ± 0.34 MPa) than that of randomly oriented fibers
(1.67 ± 0.28 MPa in ESM (H)) that is attributed to the presence of the
citrate groups (enthalpic deformation) and the level of orientation of
fibrous proteins in modified membrane (entropic deformation). These
results were consistent with other reports that the mechanical char-
acteristics of fiber scaffolds are highly dependent on the alignment of
the fibers (Table 3) (Zhou et al., 2013; Yin et al., 2010).
According to SEM images of the scaffolds at Fig. 3, modification of
ESM matrix through thermo-chemical process, due to the partial
crosslinking between the chains forming fibrils of the membrane fiber,
enhancement of polar interactions (hydrogen bonding) between the
chains and denaturation of the twisted molecular chains, resulted in the
reduction of the average pore size, pore surface area and porosity of
ESM which enhanced the stiffness of the porous membranes versus
mechanical load. Besides, chain cross-linking through amidation and
esterification of ESM with CA acted as sacrificial linkages which partly
separated to dissipate energies and led to the significant enhancement
of the mechanical strength by energy dissipating mechanism (Gong
et al., 2003; Lin et al., 2015).
3.4. Adhesive properties
ESMs were engaged as a substitute substrate to mimic animal mu-
cosa in the assessment of in vitro maximal detachment force (Fdet) and
total work of adhesion (Wadh) by which mucoadhesion mechanism
takes place by the contact (wetting) step followed by the consolidation
period (the formation of the adhesive interactions).
Table 4 and Fig. 4 reports the result of adhesion tests for neat and
Table 4
Mucoadhesion strength data of ESM and ESM-CA.
Membrane Detachment force (N) Work of adhesion (N mm)
ESM 0.022 ± 0.004 0.083 ± 0.013
ESM-CA 0.034 ± 0.006 0.156 ± 0.026
55
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
Fig. 4. Force versus extension curves for detachment of ESM and ESM-CA from
substrate.
modified ESMs which depend on the chemical and physical properties
of the network. ESM as a natural source of collagen, glucosamine,
chondroitin, and hyaluronic acid is a potential biomaterial for mu-
coadhesion and cell adhesion applications (Baláž, 2014), particularly
provides anchorage to cell adhesion via surface receptors and cell ad-
hesive RGD sites. Based on the propensity of the Arginine-Glycine-As-
partate (RGD), a tripeptide glycoprotein motif revealed to be cell ad-
hesion peptide sequence which is also present in the ESM, it may
promote adhesion when anchored on the surface of substrates (Sah and
Rath, 2016; Hersel et al., 2003).
In particular, high amount of charge, polar and hydrophobic amino
acids in ESM and specific characteristics of the side chain amino acids,
provides it with its distinctive role in adhesive protein structure. The
charged amino acid residues comprise lysine (+), arginine (+), as-
partate (-) and glutamate (-), which cause the crucial mechanism of
mucoadhesion at the molecular level through the establishment of
electrostatic bonds. In fact, positively charged amino acids could in-
teract with negatively charged sialic acid (pKa 2.6) and sulphate re-
sidues of mucin glycoprotein (Nakano et al., 2003). Polar amino acids
including serine, threonine, asparagine, glutamine, histidine and tyr-
osine, promote adhesion to the biological substrates through polar in-
teractions and hydrogen bond formation. The hydrophobic amino acids
include alanine, valine, leucine, isoleucine, proline, phenylalanine,
tryptophane, cysteine and methionine which act as indirect bonds due
to the assembly of non-polar groups to diminish the increase in entropy.
Further, the hydroxyl, amino and sulphate groups are also likely to
participate in the development of the mucoadhesive junctions through
hydrogen bonds and/or charge–charge interactions depending on the
environmental pH (Smart, 2005; Khutoryanskiy, 2011).
In addition, the presence of a significant amount of the bridging
structure (cysteine-rich ESM proteins, higher cysteine content (10%))
and repetitive patterns in ESM proteins equip strength, self-assembling
arrangement and adhesive functions to the ESM due to the existence of
molecular interactions between repetitive collagen motifs and disulfide
linkages found in cysteine-rich subdomains Kodali et al. (2011). Thus,
ESM is introduced as a new generation of membrane which being able
to form covalent bonds with the mucus gel layer through thiol/disulfide
exchange reactions, and/or simple oxidation process (Leitner et al.,
2003; Trapani et al., 2014). Inter- and/or intrachain disulfide bonds are
occurred between cysteine-rich subdomains of ESM proteins and mucus
glycoproteins which are expected to be responsible for the enhanced
mucoadhesive properties.
ESM-CA showed superior mucoadhesion values than neat ESM; in
fact, the detachment force increased until a value of 0.034 ± 0.006 N.
This behavior can be described with the presence of a more amount of
charge centers (carboxyl groups charge density) which influences the
extent of ionic interactions with the mucin macromolecules and con-
sequently, with a greater presence of secondary polar interactions
(Smart, 2005; Patel et al., 2003.
H. Gharibi, A. Abdolmaleki
Fig. 5. Swelling behavior of ESM and ESM-CA at different simulated pHs.
3.5. Swelling
In order to explore the stability of membranes over time, water
absorption and swelling of ESM and ESM-CA were measured in aqueous
PBS solution at pH 4.2 and 7.4 at room temperature. Here, variations in
membrane weight at different times were measured in order to assess
membranes stability by dry mass membranes at 80 °C for 12 h. Due to
the different properties of the ESM-CA from the unmodified ESM, this
membrane also behaved differently in swelling capacity. The ESM-CA
and neat ESM absorption curves in PBS solution are shown in Fig. 5 and
DS was measured in accordance with Eq. (1). As can be observed, the
initial rate of DS sharply enhanced (except for ESM-CA at pH 4.2) and
then began to level off. The ESM-CA had a higher water absorption than
the unmodified ESM at pH 7.4, which this behavior was due to the
presence of hydrophilic carboxyl group derived from modification with
citric acid in ESM-CA. When the ESM-CA membranes were soaked into
PBS solution, the carboxyl groups lose their acidic protons and convert
to carboxylate groups. These negatively charged carboxylate groups
repel each other in the membrane network, consequently membrane
starts to swell. Simultaneously, increased electrostatic repulsion in the
meshwork reinforced the osmotic force difference. Theoretically, the
higher variation of the osmotic pressure, lead to the superior molecular
penetration rate inside the network. Moreover, as the more water mo-
lecules penetrated into the membrane to overcome the osmotic pressure
inside the network, the swelling rate reduced due to chains mobility
restriction, and finally reached equilibrium state. Furthermore, the
ESM-CA membrane showed less swelling in the acidic medium, which
may be due to the protonation of carboxylate groups (–COO-) to form
carboxyl groups (–COOH), and consequently this phenomenon de-
creased the negative charge of the networks, weakened the electrostatic
repulsive force among anionic groups, enhanced intramolecular and
intermolecular hydrogen bonds and finally hindered the network from
swelling (Chiou et al., 2013; Xu et al., 2015; Kim et al., 1999). Besides,
this phenomenon can be ascribed to the reaction of the amine groups
with the carboxylate group of citric acid (amidation of ESM with CA)
through crosslinking by thermo-chemical reaction, meanwhile with the
reduction of amine groups, the protonation capacity of amine groups
which caused the repulsive forces (by creating ammonium-positive
ions) decreased in the membrane, then the swelling of the membrane
diminished even further El-Sherbiny et al. (2005).
3.6. Drug release
Several investigates have revealed that indomethacin can be effec-
tive in colon cancer and inflammatory syndromes treatments which
works by inhibiting both isomers of cyclooxygenase (COX), tumor ne-
crosis factor-α (TNF-α) and interleukin 6 (IL6) (Ettarh et al., 2010;
Quidville et al., 2004; Hawcroft et al., 2002). Besides, indomethacin
can suppress cancer cell migration through inhibition of endothelial
growth factor (EGF)-mediated Ca2+ signals Guo et al. (2013). However,
indomethacin as a non-steroidal anti-inflammatory drug (NSAIDS) has
56
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
a lot of adverse effects on gastrointestinal tract and belongs to class II
drugs whose solubility is too low. Owing to these challenges and with
an increasing number of lipophilic drugs under development (Chorny
et al., 2002; Tao et al., 2009), modified fibrous ESM because of their
high surface to volume ratio, biocompatibility and the presence of high
amount of charge, polar and hydrophobic amino acids might be a
promising candidate to enhance the bioavailability and clinical efficacy
of lipophilic drugs (such as indomethacin).
The drug loading efficiency of ESM-CA and neat ESM (according to
the Eq. (2)) are shown in Fig. S4 at room temperature for therapeutic
purposes. As can be realized, loading curves revealed a characteristic
sustained mode, which is related to the membranes swelling and mo-
lecular properties. Over the sequence of 4 h, indomethacin loading was
increased sharply due to the rapid swelling of membranes, which pro-
vided conditions for rapid drug loading and permeation. Over time, the
absorption capacity of the drug was reduced to a balanced state. Hy-
drogen bonds and intermolecular π–π stacking interactions played role
in junction of drug molecules to the membranes network firmly.
pH-sensitive release behaviors of indomethacin were evaluated at
37 °C with change in pH. In this aparatus, the key issue for adjusting the
released drug is the swelling of the network. Indomethacin release
decreased significantly for ESM-CA at pH 2.0 compared with pH 7.4
and pH 10.0 (Fig. 6). Therefore, modified membrane can protect the
drug from harsh environments, e.g. the low pH in the stomach (˂3) to
develop controlled release formulations for oral administration. At pH
2.0, ESM-CA acidic groups are in the form of carboxylic acid (acidic
medium (pH 2.0) less than its pKa) and strong hydrogen bonds are
established among chains, which diminish the migration of solvent into
the membrane and consequently decrease the release of drug. However,
after the membrane was placed at pH 7.4, definitely, carboxyl groups in
the ESM-CA network converted to negatively-charged carboxylate
groups (deprotonation of carboxyl groups in a medium of pH degree
over its pKa), and the resulting carboxylate groups repelled one another
and the network initiated to swell, resulting in the drastic increase of
drug permeation at this pH, then this membrane has the potential for
drug delivery in intestinal tract (nearly neutral pH) (Cuggino et al.,
2014; Cinay et al., 2017). At pH 10.0, due to the hydrolysis of the ester
linkages, alteration of the network three-dimensional structure and the
destruction of salt bridges in proteins, the drug release enhanced sig-
nificantly. At pH 2.0, the release amounts from neat ESM and ESM-CA
were different, and ESM-CA presented lower values owing to re-
markably lower swelling in comparison with the neat ESM. Also, it is
investigated that the release rate from networks is approximately in-
dependent of the composition of the membranes at pH 7.4. Accordingly,
ESM-CA is an optimal membrane to progress the bioavailability of li-
pophilic drugs, vaccines and proteins. Further this system has great
potential for the wound care and gastro-resistant release dosage forms.
Fig. 6. Release kinetics at 37 °C from drug loaded ESM and ESM-CA at different
simulated pHs.
H. Gharibi, A. Abdolmaleki
Fig. 7. MTT assay, formazan absorbance was indicated as a measure of cell
viability from MG-63 seeded on ESM and ESM-CA after 1 and 2 days of culture.
3.7. MTT assay
To assess the cell viability, MTT assay (specifies the active mi-
tochondrial enzymes present in a cell that can reduce MTT) was used to
verify the ability of the membranes to support cell proliferation. In this
study, cell survival assay was performed at 24 and 48 h interval after
cell culture (Fig. 7). The ability of the neat ESM and ESM-CA matrices to
support cell survival and proliferation showed that these membranes
exhibited a high biocompatibility (greater than control) (Fig. 7). It is
evident that the percentage of cell survival for ESM on the first day
increased from 110% to about 165% on the second day (compared to
the MG-63 cell control as 100%), which is due to the presence of rich
source of collagen, hyaluronic acid and other glycoproteins that found
numerous biomedical applications including improvement of suppor-
tive arrangement for tissue regeneration and matrix biomineralization
of osteoblasts, then the key components of ESM play a crucial role in
the bioengineering of live tissues (Baláž, 2014; Sah and Rath, 2016).
Also RGD motifs of ESM proteins, revealed to be cell adhesion, and
calcium binding peptide sequence, promote osteoblast adhesion and
proliferation, then minimize the risk of immune reactivity and regulate
physiological and pathological processes Sah and Rath (2016). Besides,
topology and interwoven morphology of the neat ESM surface may
afford a physical support to anchor the cells and play great role on
adhesion and proliferation of the cells Yi et al. (2004).
We next seeded MG-63 cells on ESM-CA autoclaved, and compared
with neat ESM. As specified by Fig. 7, the cell viability of MG-63 cells
cultured on modified membrane is more than that cultured on neat ESM
on the second day which may be due to the structural or compositional
modifications accomplished by thermo-chemical treatment. As dis-
cussed previously, thermo-chemical process, due to the denaturation of
the twisted molecular chains, enhancement of hydrophilic moieties and
introduction of the CA as bioactive intermediate that plays a role in the
Krebs cell cycle, may favors proliferation of cells on the modified net-
work and furnishes suitable biological signals/space to the cells for
growth, expansion and repair (Tran et al., 2015; Griesser et al., 1994;
Daw et al., 1998). However, the results with ESM-CA, proposes that the
biocompatibility of ESM-CA revealed in cell culture maybe devoted to
its aligned fiber morphology which afford favorable topographical
features, regardless of the compositional properties (Tavassoli, 1983;
Furth et al., 2007).
4. Conclusion
ESM mimics the intrinsic mechanism of concealed mucus glyco-
proteins, which reveals adhesion ability. The results demonstrated that
thermo-chemical process of ESM modification significantly affects ar-
chitecture, mechanical, adhesive and cellular behaviors. Morphological
characterization of the neat and modified fiber networks reveal both
57
Journal of the Mechanical Behavior of Biomedical Materials 87 (2018) 50–58
randomly orientated and well-aligned fiber mats that are favorable for
tissue engineering application. Additionally, the synergistic enhance-
ment in pH sensitivity is made possible by the modification of economic
efficient biomembrane (ESM) with selected bioactive agent (CA) for
therapeutic treatment and mucoadhesive drug delivery system. Further,
MTT assay specify that novel ESM-CA scaffolds could enhance the
proliferation of MG-63 cells. We assume that this approach can be de-
veloped to design scaffolds for the healing of complex tissues with
spatial adjustment of mechanical and biological properties.
Acknowledgements
We thankfully acknowledge the partial financial funding from the
Research Affairs Division Isfahan University of Technology (IUT),
Isfahan. Further partial financial support of National Elite Foundation
(NEF) and Center of Excellency in Green Chemistry (IUT) is also
gratefully acknowledged.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jmbbm.2018.07.014.
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