Materials Science & Engineering C 86 (2018) 70–82

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Tuning biological properties of poly (vinyl alcohol) with amino acids and T
studying its influence on osteoblastic cell adhesion
⁎
S. Mohanapriya, V. Raj
Advanced Materials Research Laboratory, Department of Chemistry, Periyar University, Salem 636 011, India

A R T I C L E I N F O A B S T R A C T

Keywords: A new class of bio composite materials for orthopedic applications were prepared by blending polyvinyl alcohol
Amino acid (PVA) with certain amino acids (AA) such as glycine, lysine and phenyl alanine followed by in-situ cross-linking
Citric acid cross-linking with citric acid (CT). CT cross-linked PVA offers sufficient chemical, thermal, and morphological stability by
Bio composite films establishing intermolecular interaction. These biocomposite films were characterized for mechanical and
Hydrophilic domains
thermal behaviors as well as analyzed through SEM, TGA and AFM studies. Zwitterionic characteristics of AA
create cell-adhesive hydrophilic domains that act as potential cell-adhesion sites and facilitate osteointegration.
Biocompatibility studies proved that PVA-AA/CT is non-toxic and exhibits good anti-bacterial activity. In-vitro
bioactivity test and cell adhesion results predict that presence of AA is advantageous to enhance apatite growth
and promote cell-substrate binding through modulating cellular activity of PVA polymer. Hydrophilicity of AA
zwitterions significantly facilitates cell-substrate binding and CA cross-linking helps osteoblastic integration of
PVA-AA/CT bio composite films. The rational design of microstructure with zwitterionic-hydrophilic domains is
a key to enhance cell-substrate interaction of PVA-AAs/CT bio composites.

1. Introduction
Joint and articular cartilage injuries are the most frequent difficul-
ties suffered by many people leading to progressive cartilage tissue loss,
further exposing the bony ends, leaving them without protection. This
finally deteriorates into most common arthritis-osteoarthritis [1]. Total
joint replacement is commonly performed using ceramics, composites
and/or metallic materials (titanium, chromium, etc.), which are both
stiffer than cartilage and do not have lubrication, shock absorption, and
deformation properties of native cartilage. Enormous research efforts
have been put forward in last few decades to develop numerous
ceramic-metal, bio-ceramics and glass-ceramic base materials for bone
replacement in the view of growing demand for bone grafts [2,3]. Major
shortcomings correlated with these materials are as follows: (a) metal
implants get corrode with time and become deficient in bioactivity, (b)
inert ceramics also loss bioactivity, (c) bioactive ceramics and bio
glasses do not go with mechanical properties of bone [4,5]. Polymeric
materials are viable alternatives due to the ease of fabrication, flex-
ibility and biocompatible nature besides their wide range of mechan-
ical, electrical, chemical and thermal behaviors when combined with
different materials as composites. Additional advantages of polymer
and polymer composites are that they are non-magnetic and are radio
transparent for X-ray radiography and MRI scans [6].
Poly vinyl alcohol (PVA) is one of the first synthetic polymers,
which is applied as artificial cartilage. In orthopedics, PVA implants
have been commonly used in meniscus and cartilage replacements as it
possesses several useful properties, including permeability, hydro-
philicity and low frictional function [7,8]. Beyerlein et al. implanted
PVA hydrogel in white rabbits for up to 52 weeks as an artificial ar-
ticular cartilage replacement resulting in low inflammatory responses
and high in vivo biocompatibility [8]. Maiotti et al. studied the effec-
tiveness of these PVA hydrogel implants in 18 patients with a mean age
of 56 over a period of 2 years and demonstrated their better perfor-
mance compared to autograft or allograft tissue donor transplantation
[9]. However, use of PVA in the orthopedic surgery field has been
thought to be limited because of its low mechanical strength, poor os-
teointegration, low antibacterial activity and durability. Studies on
biological response of PVA hydrogels implanted into canine knee joints
as an artificial osteochondral composite material reveal that PVA hy-
drogel composite replacement with titanium fiber mesh caused negli-
gible damage to articular cartilage and meniscus in comparison with
hard-implant materials [10]. PVA based gelatin/hydroxyapatite scaf-
folds were proven to be biocompatible cartilage scaffolds for tissue
engineering applications [11]. Lack of antibacterial action of implanted
materials pose major problems as infectious complications of open
surgery continue to be a significant factor contributing to patient

⁎
Corresponding author.
E-mail addresses: priyaechem@gmail.com (S. Mohanapriya), vraj@periyaruniversity.ac.in (V. Raj).

https://doi.org/10.1016/j.msec.2018.01.006
Received 3 May 2017; Received in revised form 13 October 2017; Accepted 28 January 2018
Available online 01 February 2018
0928-4931/ © 2018 Elsevier B.V. All rights reserved.
S. Mohanapriya, V. Raj
morbidity and poor healing outcomes [12]. The ensuing infection of
bone by bacteria (osteomyelitis) is characterized by levels of in-
flammation and destruction of viable bone tissue. Often infection be-
comes chronic, resulting in increased rates of surgical revisions, non-
union and extremity amputation [13]. As a whole, an ideal implant
replacement for cartilage would mimic its structure, mechanical prop-
erties and should have good biocompatibility and antibacterial activity.
Following this line of investigation, in order to fine tune surface
characteristics as well as to instigate both osteointegration and anti-
bacterial activity, PVA was combined with amino acids (AAs). AA are
advantageous compared to other compounds since they originate in the
blood stream basically due to protein metabolism and they move freely
through blood circulation in all tissues of the body of vertebrates. Many
research works have been done on blending biomolecules such as an-
tibody and antibiotics with biopolymers to enhance antibacterial ac-
tivity [14–16]. But they behave as affinity probes only for specific pa-
thogens through hydrogen bonding which might be ineffective to other
bacteria. Huang et al. synthesized amine-functionalized magnetic na-
noparticles that could effectively capture both Gram-positive and Gram-
negative bacteria through nonselective electrostatic interaction and
hydrophobic interaction [17]. It is also established that PLA membranes
modified with alkaline amino acids chitosan derivatives (e.g. arginine
and lysine-g-chitosan) achieved the most efficient cell attachment and
growth of chondrocytes [18]. Also it has been proved that peptide se-
quence Lys-Arg-Ser-Arg that selectively influences heparan sulfate
mediated osteoblast adhesion mechanisms [19]. All these studies es-
tablish that biological functions of materials could be tuned with amino
acids. Based on these investigations we have modified PVA with three
different amino acids namely glycine (GL), lysine (LY) and phenyl
alanine (PA). Since reactive hydroxyl groups are present in PVA
structure, it is capable of forming hydrogen bonds readily with AAs due
to its polar nature. It is demonstrated in this study that introduction of
AA moieties to the backbone of PVA has been shown to improve the
physicochemical properties through existence of interesting synergy
between PVA and AA as well as some beneficial biomedical properties
such as antibacterial activity. Over and above biocompatibility of PVA
is enhanced without altering already well-established safety profile of
PVA. The influences of material dosage, solution pH, ionic strength,
competitive anions, as well as natural organic matter (NOM) on the
bacteria capture were systematically studied.
Many researchers have cross-linked PVA by thermal and chemical
methods (using cross linkers like glutaraldehyde, glyoxal etc.) but most
of the chemical cross-linkers are toxic. So, in the present study citric
acid (CT) is used as a cross-linker, which is shown to establish proper
proportion between hydrophilic and hydrophobic domains of polymer.
CT is a cheap, non-toxic polycarboxylic acid comprises both carboxylic
acid and hydroxyl groups, hence could effectively involve in hydrogen
bonding and crosslink with PVA through esterification reaction thereby
ascertain proper arrangement of micro-domains within PVA resulting in
good mechanical properties.
Eventually, purpose of this investigation is to illustrate strategies for
rational design/selection of AAs and elucidating its influence on os-
teoblastic cell-adhesion properties. Since cell-adhesion is a central
progression that must occur before subsequent osteoblast (an ancho-
rage-dependent cell) functions (such as proliferation, migration, pro-
duction, and deposition of mineralized matrix) can take place, proac-
tive biomaterials should be designed to support and enhance osteoblast
adhesion. Also this study aims to prove that CT cross-linking provides
adequate thermal and mechanical stabilities and ascertain proper hy-
drophilic domains for cell attachment. The ability of hydrophilic group
to interact with water molecules and the hydrophobic group to be
connected the surface of the material will reduce the surface tension
arising from the contact of a solid material with a solution therefore
impose mechanical stability. Designed bio composite films are expected
to have an optimal mechanical performance and a controllable de-
gradation rate combined with eminent bioactivity and this will be of
71
Materials Science & Engineering C 86 (2018) 70–82
great importance for bone remodeling and growth. Hence the present
study proves that modification of PVA with AA followed by CA cross-
linking constitute appropriate bio composite film that is suitable to act
as a substitute for bone. To the best of our knowledge this is the first
report wherein CA cross-linked PVA-AA bio composite films is sub-
jected for cell studies.
2. Experimental
Poly vinyl alcohol (PVA), (99.7% hydrolyzed, M.W. 115,000) was
obtained from Loba Chemie, India. Glycine (GL), lysine (LY) and phenyl
alanine (PA) were procured from Acros organics. Citric acid (CT) was
obtained from SRL chemicals. Sulfuric acid (98%) was obtained from
S.D. Fine Chemicals, India. All the chemicals were used as-received.
Deionized (DI) water (18.4 MΩ cm) from Millipore was used during the
experiments.
2.1. Bio composite film preparation
PVA-AA bio composite films were prepared by solution-casting
technique. In brief, 30 mL of 4 wt% PVA solution was prepared by
dissolving the required amount of PVA in water at 60o C followed by
mechanical stirring until a clear solution was obtained. Similarly, 20 mL
of 10 wt% AA in relation to PVA was dissolved in aqueous medium at
room temperature followed by stirring until a homogeneous solution
was obtained. Both the solutions were mixed and further stirred for 2 h
to form a compatible blend. The composition of AAs was varied from
5 wt% to 20 wt% in relation to PVA and optimized to 10 wt% with re-
spect to mechanical stability. 10 wt% of CT in relation to PVA was
added as a cross-linker followed by the addition of a drop of sulfuric
acid to catalyze the reaction. The mixture was allowed to stir at 60 °C
for 1 h to complete the cross-linking reaction. The viscous solution was
cast on a flat Plexiglass plate to from a membrane by evaporating the
solvent at room temperature. Pristine PVA film was prepared in a si-
milar manner without the addition of AAs. After evaporation of solvent,
the films were dried at room temperature and the thickness of it was
~120 μm.
2.2. Characterization of fabricated films
2.2.1. Physicochemical characterization
Micro structural and morphological characterizations of prepared
films were carried out using Scanning Electron Microscope (SEM)
(Hitachi, Japan, S-3400N). Gold film of thickness < 100 nm was sput-
tered on PVA or PVA-AA bio composite film (thickness in the range of
40–60 nm approximately) surfaces using a JEOL Fine Coat Ion Sputter-
JFC-1100 Unit, prior to their examination under SEM. Elemental
composition of the films were analyzed by Energy dispersive X-ray
spectroscopy (EDS) along with elemental mapping. Universal testing
machine (UTM) (Model AGS-J, Shimadzu) with an operating head-load
of 10 kN was used to study the mechanical properties of the mem-
branes. The test samples were prepared in the form of dumb-bell shaped
object as per ASTM D-882 standards. The films were then placed in the
sample holder of the machine. The film was stretched at a cross-head
speed of 1 mm/min and its tensile strength was estimated using equa-
tion
Maximum load
Tensile Strength =
Cross sectional area (1)
Swelling behavior of the films under investigation (size
5 cm × 6 cm) were soaked in SBF controlled at 40 °C for 24 h.
Dimensional changes in length (L) and breadth (B) were calculated by
using the following equations
(L 2 − L1)
L (%) =
L1 (2)
S. Mohanapriya, V. Raj
(B2 − B1)
B (%) =
B1 (3)
where L1 and B1 are length and breadth of the sample before soaking in
de-ionized water and L2 and B2 are corresponding parameters measured
after soaking for 24 h.
Thermo-gravimetric analysis of all the membranes were carried out
using a SDT Q600 V8.2 TGA/DTA instrument in the temperature range
between 273 K and 1073 K at a heating rate of 5 K/min with nitrogen
flushed at 200 mL/min. Fourier Transform infrared (FTIR) spectra (400
Perkin Elmer infrared spectrometer) in the wave number range of
400–4000 cm−1 was used to investigate the functional groups present
in the films. The surface topography of the coated samples was analyzed
by atomic force microscopy (AFM) and the images were obtained using
a Multimode Scanning probe microscope (NTMDT, NTEGR Aprima,
Russia) operating in the semi contact mode.
Average contact angle and surface wetting energy measurements
were conducted on bio composite films using a Surface Electro Optics
Model Phoenix-300 unit by sessile drop method. By automatic pipette
water drop of volume (8.0 ± 0.2) μL was deposited on the film surface
and the consequently contact angle was measured. Estimation of sur-
face energy was also based on the measurement with the SEE System.
The measurement was carried out at room temperature. Triplicate
measurements carried out on different parts of the same membrane
sample for accurate results.
2.3. In vitro assays
2.3.1. Anti-bacterial studies
Anti-bacterial activity of fabricated PVA and PVA-AA bio composite
films are tested by viable cell count method against Staphylococcus
aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia and
Pseudomonas aeruginosa as model bacteria. Viable count method mea-
sures bacterial growth. For this study, 108 colony forming units (CFU)
of respective bacteria were grown in 10 mL nutrient broth supplement
with film discs (20 mm dia). The bacterial viability was determined by
measuring their O.D values using UV–Vis spectrophotometer at 600 nm.
2.3.2. Apatite growth
In order to investigate the formation of apatite on PVA film and
different PVA-AA bio composite films they were soaked in 15 mL SBF
solution made according to a method adopted by Kokubo [20]. SBF was
prepared by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl,
K2HPO4.3H2O, MgCl2. 6H2O, CaCl2 and Na2SO4 using de-ionized water
and buffering at pH 7.4 with tris-hydroxymethylaminomethane
((CH2OH)3CNH2) and 1.0 mol/L HCl, at 37 °C as shown in Table 1 and
solution was refreshed every other day. After soaking in SBF for 7 and
14 days, samples were taken out and rinsed with de-ionized water.
2.3.3. Cytotoxicity studies
The proliferative potential of MG-63 osteoblast-like cells on PVA
film and different PVA-AA bio composite films were assessed by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
[21]. Uniform number of cells (2.5 × 104 cells per well) in Modified
Eagle's Medium supplemented with 100 μ/mL penicillin, 100 μg/mL
streptomycin, 30 μg/mL gentamycin and 2.5 μg/mL amphotericin B
with 10% fetal bovine serum was seeded onto PVA films as well as PVA-
AA bio composite films and allowed to proliferate for 24 h. After
Table 1
Ionic concentration of SBF.
Na+ K+ Mg2+ Ca2+
SBF 142.0 5.0 1.5 2.5
Blood plasma 142.0 5.0 1.5 2.5
72
Materials Science & Engineering C 86 (2018) 70–82
incubation, the cells were treated with MTT (0.5 mg mL−1) for 3 h. The
formazan complex formed was solubilized with DMSO (dimethyl
sulphoxide) and the absorption was measured at 570 nm with a re-
ference wavelength of 630 nm, in a micro plate reader (Bio-Rad). The
percentage of cell viability was calculated using the optical density of
the control and treated cells.
2.3.4. Cell adhesion assay
Based on physicochemical analyses and in vitro analysis cell adhe-
sion of PVA-AA bio composite films was determined as it is more ap-
propriate for implant applications. PVA film and PVA-AA bio composite
films were placed onto 24-well tissue culture plates containing a sus-
pension of MG-63 osteoblast-like cells (5 × 104 cells per well) in
Modified Eagle's Medium supplemented with 100 μg/mL penicillin,
100 μg/mL streptomycin, 30 μg/mL gentamycin and 2.5 μg/mL am-
photericin B with 10% fetal bovine serum. The cells were incubated for
4 h at 37 °C in a humidified atmosphere of 5% CO2. After incubation the
wells were washed gently with warm PBS 3–5 times to remove the non-
adherent cells. The adhered cells were then fixed with 96% ethanol for
10 min and stained with 0.1% crystal violet for 30 min. Excess dye was
removed by washing the wells thoroughly with de-ionized water. The
adhered cells were then imaged using Leica microscope, Germany [22].
3. Results and discussion
3.1. Physicochemical characterization
UV analysis of precursor polymer solutions supply evidence for the
formation of PVA-AA compatible blend and also proves existence of
intermolecular interaction between PVA and AA. Fig. 1 represents UV
spectra of pristine PVA, LY and PVA-LY is precursor solutions. PVA and
LY solutions exhibit a shoulder-like band at 250 nm and 195 nm re-
spectively. When LY is blended with PVA, absorption peak of PVA-LY
shifted towards lower wavelength substantiating the presence of in-
termolecular bonding and successful blending of the components.
Cross-linking of PVA chains with citric acid and blending of PVA with
AAs is also validated by analysis of FTIR spectra of non-cross-linked
PVA, cross-linked PVA, PVA-GL, PVA-PA and PVA-LY as shown in
Fig. 2. Both intermolecular and intra molecular hydrogen bonds are
possibly present in PVA due to high hydrophilic forces. Non-cross-
linked PVA presents a broad band centered at 3500 cm−1 related to
both inter-molecular hydrogen bonding and eOH stretching vibration
of PVA. Characteristic vibration of semi-crystalline PVA appeared at
1143 cm−1 and that of CeO vibration emerged at 1110 cm−1. In con-
trast, cross-linked PVA spectra illustrates a distinct absorption band at
1078 cm−1 that might be ascribed to CeO ester groups, indicating
cross-linking reaction through esterification is accomplished between
CT and PVA [23]. Also it is to be noted that intensity of OeH stretching
vibration peak is decreased after cross-linking reaction which ulti-
mately proves that eOH groups of PVA are consumed for esterification.
These results reveal that, due to presence of lower number of eOH
groups hydrogen bonding becomes weaker in case of cross-linked PVA.
As well, the CeO stretching at approximately 1100 cm−1 in FTIR
spectrum of non-cross-linked PVA is replaced by a broader absorption
band (from 1000 to 1140 cm−1), which can be attributed to the ester
(CeO) bands formed by the cross-linking reaction of PVA with citric
acid. From these results it is clear that PVA polymer chains are cross-
linked by CT.
Cl− HCO3− HPO4− SO42− pH
147.8 4.2 1.0 0.5 7.4
103.8 27.0 1.0 0.5 7.2–7.4
S. Mohanapriya, V. Raj Materials Science & Engineering C 86 (2018) 70–82

30 5.5
1.4 Tensile strength
Elongation-at-Break
1.2

Tensile Strength (MPa)

Elongation -at-Break (mm)

5.0
1.0 28
Absorbance (A.U)

0.8
4.5
0.6 26
(i)
0.4
4.0
(iii)
0.2
24
(ii)
0.0
3.5
-0.2 PVA/CT PVA-GL/CT PVA-PA/CT PVA-LY/CT
100 200 300 400 500 600 700 Fig. 4. Tensile strength and % Elongation-at-Break associated with PVA film and PVA-
Wavelength (nm) AA/CT bio composite films.

Fig. 1. UV–Visible absorption spectra (i) PVA blend (ii) LY and (c) PVA-LY solutions.
Table 2
Dimensional stability of PVA film and PVA-AA/CT bio composites.

Type of film Dimensional change (%)

(v)
Length Breadth

PVA/CT 26.9 26.9

(iv) PVA-GL/CT bio composite 23.8 23.8
PVA-PA/CT bio composite 21.7 21.89
Intensity(A.U)

PVA-LY/CT bio composite 18.3 18.3

(iii)

(ii) Molecular interaction between PVA and AA was studied by com-

paring cross-linked PVA-CT spectrum with that of PVA-AA/CT. It is
(i) evident from Fig. 2 that PVA-AA/CT spectra exhibit combined features

1078 cm-1
of both PVA-CT and AA in which few peaks appear newly and other
2995 cm-1 peaks appear with a shift in their position. Spectral properties of PVA/
CT are strongly influenced by the interaction of AA with the polymer
backbone. Distinct absorption of AA due to presence of amino and

3500 cm-1
carboxylic groups occurred in all PVA-AA/CT bio composites demon-
strating successful blending of PVA with AA. Although, spectral char-
500 1000 1500 2000 2500 3000 3500 4000 acteristics depend upon nature of AA, all the three PVA-AA/CT bio
composite films present a broad band in the region from 2600 to
Wavenumber (cm-1) 3400 cm−1 centered at around 3000 cm−1. Both symmetric and
Fig. 2. FTIR spectra of (i) PVA Cross-linked with CA (ii) Non-cross linked PVA film (iii) asymmetric vibrations of amino groups emerge at around 1550 cm−1
PVA- GL/CT (iv) PVA-PA/CA and (v) PVA-LY/CT bio composite films. and 1650 cm−1 respectively. In modified samples, these peaks overlap
with broad bands of the stretching and deformation modes of car-
boxylic and amino groups of AA as evident from the spectra. These
100 spectral changes clearly point out the presence of interfacial interac-
tions between PVA with both AA and CA.
90

80 3.2. Thermo-mechanical stabilities of membranes

70
Weight (%)

Fig. 3 shows typical TG plots PVA/CT film, PVA/CT-GL, PVA/CT-PA

60 and PVA/CT-LY bio composite films. These films show analogous
thermal behavior with three-phase degradation involving the processes
50
of thermal dehydration, thermal degradation and thermal decomposi-
40 tion [24]. Initial weight-loss between 303 K and 473 K is caused by
(iii) evaporation of absorbed water molecules from PVA/CT film, PVA/CT-
30 (iv) GL, PVA/CT-PA and PVA/CT-LY bio composite films. It is noteworthy
20 that weight-loss associated with PVA/CA-AA bio composite film is
(ii)
(i) higher than that of PVA/CT, designating the fact that PVA/CT-AA bio
10
300 400 500 600 700 800 900 1000 composite film are highly hydrophilic. Decomposition of the main
Temperature (K) polymeric PVA chains and linkage created during CT cross-linking to-
gether constitute second stage weight-loss at 473–673 K. The third stage
Fig. 3. TGA plots (i) Pristine PVA/CT film (ii) PVA-GL/CT (iii) PVA-PA/CA and (iv)PVA- weight loss occurring between 723 K and 1073 K is due to the decom-
LY/CT bio composite films.
position of main chains of PVA. It is clear from the thermal studies that

73
S. Mohanapriya, V. Raj
bio composite films are thermally stable in physiological environment
after implantation.
The mechanical properties of all films under study were determined
by its tensile strength and elongation-at-break as represented in Fig. 4.
It is obvious from the data that blending AA marginally improved the
tensile strength and resulting PVA-AA/CT bio composite membranes
possess good stability. Apparently, elongation-at-break is fairly de-
creased because of obstructed segmental mobility of PVA polymer
chains. Presence of AA, constitutes additional hydrogen bonds which
perform as reinforcing units preventing the easy movement of PVA
polymer chains. Hence, hydrogen bonding and electrostatic interactions
existing among PVA polymeric chains and AA/CT give rise to good
mechanical stability to the bio composites. Among different PVA-AA/
74
Materials Science & Engineering C 86 (2018) 70–82
Fig. 5. SEM morphologies of (i) PVA film and (ii) PVA-LY/
CT bio composite film.
Fig. 6. SEM-EDS elemental mapping of PVA-LY/CT bio
composite film by SEM: (i) original picture, (ii) elemental
analysis, (iii) C k mapping, (iv) O k mapping, (v) N k
mapping.
CT bio composites under study, PVA-LY/CT bio composite film displays
maximum tensile strength along with good elongation characteristics,
which is basically ascribed due to the strong interaction between LY
and PVA.
These results throw light on the fact that existence of an interfacial
interaction among PVA, CT and AA components steadily influences
mechanical behavior of bio composite films. CT comprising multi-car-
boxyl groups reinforces the intermolecular binding by introducing
covalent bonds that supplement natural intermolecular hydrogen bonds
so as to improve the mechanical properties and water resistibility [25].
PVA-AA/CT bio composites possess advantages of both suppleness of a
polymer and adequate mechanical stability that make them con-
structive to be utilized as an orthopedic implant.
S. Mohanapriya, V. Raj Materials Science & Engineering C 86 (2018) 70–82

Fig. 7. 2D AFM images of (i) PVA film and

(ii) PVA-AA/CT bio composite film. 3D AFM
images of (iii) PVA film and (iv) PVA-AA/CT
bio composite film.

Table 3 AA under study, an additional basic amino group is present in LY. Due
Surface wetting properties of PVA film and PVA-AA/CT bio composites. to presence of two positively charged amino groups, LY actively in-
volved in the interfacial bonding and an additional amino group in LY is
Type of film Surface properties
responsible for increased stability that takes full advantage of PVA
Average contact angle Surface wetting energy polymeric voids to efficiently form closely-packed arrangement. Ac-
(degree) (mNm−1) cordingly, hydrophilicity associated with bio composites shows more
affinity towards water which in turn may impact membrane properties.
PVA/CT 71 1.19
PVA-GL/CT bio 63 1.35 These outcomes are in good line with mechanical stability results.
composite FE-SEM images of PVA film and PVA-LY/CT bio composite film are
PVA-PA/CT bio 61 1.47 as presented in Fig. 5. It is clearly visible from the figure that the PVA-
composite LY/CT bio composite membrane possesses homogeneous morphology
PVA-LY/CT bio 58 1.52
with no phase separation, implying that the AAs uniformly blended
composite
with PVA through favorable electrostatic and hydrogen bonding inter-
actions. PVA exhibits a smooth surface structure.
Table 2 provides data related to dimensional changes associated The density distribution of elements is EDS mapped for both PVA
with PVA film and different PVA-AA/CT bio composites after soaking in film and PVA-LY/CT bio composite film as shown in Fig. 6. SEM image
SBF for 24 h. It is obvious from the data that blending PVA with AA of sample under investigation is provided in Fig. 6a and the corre-
could decrease dimensional changes in PVA polymer matrix. In- sponding EDS data are represented in Fig. 6b. It is inferred from these
corporation of AA brings PVA polymer chains closer therefore PVA-AA figures that carbon and oxygen are main constituents present in PVA
comprise more closely packed network as compared to PVA. Besides, film but in addition to these elements, PVA-LY/CT bio composite film
the interactions between PVA-AA and CT such as ionic cross-linking also comprises N that provides evidence for blending of components. It
(electrostatic forces) and hydrogen-bonding bridges, contribute to the should be noted that N is distributed evenly on the entire surface of
control of membrane swelling without a decrease in flexibility. These PVA-LY/CT bio composite film. It is to be pointed out here that in-
hydrogen bonds offer well-connected hydrophilic domains by bringing corporation of AA generates hydrophilic domains which are essential
the proton-conducting groups closer. Consequently PVA-AA polymeric for cell-substrate interaction. Existence of interfacial interaction be-
network is stronger and undergoes less degree of deformation. PVA-LY/ tween AA molecules and PVA as well as homogenous blending AA into
CT bio composite experiences least degree of dimensional changes in- polymeric matrix is evident from these results.
dicating that PVA-LY/CT hold higher stability. Differences in the be- It is well known that surface roughness and morphology play an
havior of bio composite films could be possibly due to the diverse important role in biological responses of biomaterial surfaces. Fig. 7a
chemical structure of AAs. In spite of similar zwitterionic structure of all and b display 2D topographical view of PVA film and PVA-LY/CT bio

75
S. Mohanapriya, V. Raj
composite film respectively. The related 3D images are provided in
Fig. 7c and Fig. 7d. RMS value of roughness factor is provided along
with the respective image. Similar surface topography is observed for
both films under study; however roughness factor of PVA-LY/CT bio
composite film is higher than that of PVA film. It is apparent from that
Sa value (average height) increases when PVA is combined with AA,
which is favorable for enhanced cell-substrate interaction. Earlier stu-
dies prove that extent of osteointegration process greatly depends on
Ssk, Sdq and Sa values [26]. Higher roughness factor for PVA-LY/CT as
compared to PVA film elucidates morphological changes caused due to
the presence of LY. Based on this, it could be predictable that blending
AA brings the polymer chains closer and as a consequence, surface with
higher roughness is created. For example, RMS value associated with
PVA film is low consequently a smooth and homogenous surface is
observed. As envisaged by a recently developed biomechanical model,
higher Sdq associated with surface results in larger interfacial retention
strength with bone cells and accordingly facilitates osteointegration
[27]. The results are in accordance with our earlier studies wherein
TiO2-SiO2 bio ceramic coatings on Ti alloy and PVA/TiO2 nano-
composites are investigated as bone implant models [28].
Potential surface energy effects on bone cell mineralization may be
a very important consideration in orthopedic implants [29]. Previous
reports demonstrate that high surface energy (hydrophilic) surfaces not
only supported homogeneous spatial cell growth and mineral deposi-
tion but also increased the quantity (mineralized area) and quality (the
mineral-to-matrix ratio) of cell mineralization, relative to low surface
energy (hydrophobic) surfaces [30,31]. Since, cell adhesion onto dif-
ferent polymeric surface was governed by surface wettability of poly-
meric substrate, contact angle and surface wetting properties of bio
composite films are evaluated and data are provided in Table 3. Cell
adhesion is in general better on hydrophilic surfaces. Enhanced surface
76
Materials Science & Engineering C 86 (2018) 70–82
Fig. 8. SEM images of apatite formed on the samples
dipped in SBF for 7 days: (i) PVA film (ii) PVA-AA/CT bio
composite. After 14 days (c) PVA film (d) PVA-AA/CT bio
composite.
hydrophilicity of bio composite films in relation to pristine PVA film is
due to the presence of amino and carboxylic acid groups [32,33].
Subsequent increase of surface wetting energy is related to possible
reorientation and rearrangement of water molecules on the polymeric
surface. It is well-known that changes in the physicochemical proper-
ties, which influence the hydrophilicity of polymer surface, will mod-
ulate cell attachment and proliferation. Therefore, increased wettability
enhances interaction between implant surfaces and the biologic en-
vironment [34,35]. Surface wettability is affected not only by surface
chemistry but also by topography parameters such as roughness and
micro-texture, these results are in congruent with AFM studies. It can be
concluded that surface energy and resultant wettability significantly
affect short-term anchorage-dependent cell functions.
3.3. In-vitro assays
3.3.1. Hydroxyapatite formation in SBF
Bone-like apatite plays a central role in formation; growth and
maintenance of
tissue-biomaterial integrate and keeps durability of biomaterials
during implantation. The apatite inducing ability of PVA-AA/CT sam-
ples were evaluated by inspecting their representative SEM images
obtained by immersing these samples in SBF for 7 and 14 days re-
spectively as shown in Fig. 8. 7 days after immersing in SBF, numerous
white tiny HAp grains are visible on PVA film as shown in Fig. 8a. HAp
growth is medium and barely distributed over PVA-GL/CT surface. By
contrast, PVA-LY/CT bio composite film displays fine network-like
structures with nano-pores of size 100 nm (shown in Fig.8b). Here the
surface coverage of HAp grains is higher than that of PVA film. The
growth and surface coverage of the HAp grains presents in the order
PVA-GL/CT < PVA-PA/CT < PVA-LY/CT. Based on the results it
S. Mohanapriya, V. Raj
could be inferred that nature of AA exhibit a profound influence both
on the mechanism and kinetics of HAp nucleation. The results are in
agreement with an earlier study on role of AA on regulating HAp crystal
growth wherein AA immobilized on PVA matrix become HAp nuclea-
tion site and regulate orientation of HAp crystals [36]. Depending on
the nature of AA, growth rate and surface coverage also differ. Also it is
illustrated that bio mineralization usually occurs through specific or
selective interaction between inorganic moieties and the organic tem-
plates on the surface of macromolecular frameworks, cell walls or lipid
membranes [37]. Many researchers have reported that biocompatible
polymers rich in negatively charged groups have the potential of in-
ducing nucleation of the calcium phosphate phase under simulated
physiological conditions [38,39]. In the present case, presence of AA
comprising amino and carboxyl groups stimulates the apatite forma-
tion. As a result, rate of HAp growth is favored over PVA-AA/CT bio
composite films rather than PVA film. The observed results also sub-
stantiate AFM results. Similar growth pattern is retained by bio com-
posite films under study after 14 days of immersion in SBF. However
growth of HAp grains is higher after 14 days of immersion for all the
films under study and they show a ring worm like structure with reg-
ularly arranged Nano flakes. The results reveal that growth of HAp
grains is depressed over PVA-GL/CT bio composite film compared to
PVA-PA/CT and PVA-LY/CT film as shown in Fig. 8d. Nevertheless,
PVA-LY/CT bio composite film displays a regular densely-packed HAp
77
Materials Science & Engineering C 86 (2018) 70–82
Fig. 9. Anti-bacterial activity of PVA film and
different PVA-AA/CT bio composite films
against (a) S. aureus (b) B. subtilis (c) E. coli (d)
K. pneumoniae (e) P. aeruginosa.
nanostructure that covers nearly the whole surface. The results are
depicted in Fig. 8e. Difference in the HAp growth kinetics over PVA-
AA/CT bio composite films is attributed to chemical composition of AA.
Nonetheless, morphological characteristics of apatite grown on bio
composite films are nearly identical as indicated by Fig. 8e and f re-
spectively.
3.4. Anti-bacterial assay
Bone-implants with good antibacterial activity are desirable to cir-
cumvent material deterioration after implantation. Hence materials
with self-antibacterial ability are advantageous. Since B. subtilis and E.
coli commonly cause implant-associated infections, PVA and PVA-AA/
CT films are tested for antibacterial action with these bacteria. Fig. 9
displays typical anti-bacterial test results of PVA and PVA-AA/CA bio
composite films by viable count method. Previous studies have revealed
that amino groups of AAs are nonselective ligands to interact with both
Gram-positive and Gram-negative bacteria via the electrostatic attrac-
tion [40,41]. Based on this merit, we have compared the antibacterial
activity of PVA and PVA-AA/CT bio composite films against Gram-po-
sitive (B. subtilis) and Gram-negative bacteria (E. coli) with initial cell
concentration of 1.5 × 107 CFU mL−1.
Pristine PVA film could not capture bacteria efficiently against both
gram positive and gram negative bacteria. Specifically, the capture
S. Mohanapriya, V. Raj
efficiencies for B. subtilis and E. coli by bare PVA/CT film were only 14%
and 15%, respectively. Although the capture efficiencies increased in
the case of GL blended PVA film, the cell removal efficiencies for B.
subtilis and E. coli by PVA-GL/CT film increased to 33% and 30%, re-
spectively. Notably, the capture efficiency of PVA-LY/CT bio composite
film increased very fast and almost 35% of the bacterial cells are re-
moved. The capture efficiency of PVA-PA/CA bio composite is mod-
erate and their efficiency is 28%. Specifically, all AA modified PVA bio
composite films could capture more number of bacteria for both cell
types compared to pristine PVA/CT films. This observation showed that
modification of PVA film with AA could greatly increase the bacteria
capture efficiencies of both B. subtilis and E. coli used in this study. This
may be due to the presence of positively charged free eNH3+ group
(belong to zwitter ion) present in AAs that can bind tightly to the
components of bacterial cell walls, resulting in pore formation in cell
walls, severe leakage of cell constituents and eventually the cell death
[42]. Among all three bio composites under study, PVA-LY/CT film
shows higher activity. It may be due to the presence of two amino
groups that strongly interact with bacterial cell walls. Likewise better
antibacterial activity is shown by PVA-GL/CT and PVA-LY/CT bio
78
Materials Science & Engineering C 86 (2018) 70–82
Fig. 10. Optical images of cells cultured on various samples
for a period of 1 day: (i) Control (ii) PVA film (iii) PVA-GL/
CT (iv) PVA-PA/CT and (v) PVA-LY/CT bio composite
films. After 3 days (vi) PVA film (vii) PVA-GL/CT (viii)
PVA-PA/CT and (ix) PVA-LY/CT bio composite films. After
5 days, (x) PVA film (xi) PVA-GL/CT (xii) PVA-PA/CT and
(xiii) PVA-LY/CT bio composite films. (xiv) Cell viability of
osteoblast cells expressed as a percentage after 1, 3 and
5 days incubation.
composite comprising aliphatic AA as active component in comparison
with PVA-PA/CT bio composite incorporated with aromatic AA. Ob-
served difference in the activity may be possible due to the presence of
methyl group with electron donating nature that may enhance accu-
mulation of positive charge over amino group which in turn helps to
destruct the bacterial cell wall. By contrast, PA encompassing phenyl
group with electron withdrawing characteristics tend to diminish po-
sitive charge accumulated over amino group. As a result bacterial
capturing efficiency of PVA-PA/CT is lower. Presence of both AA and
CT mitigate adverse effects associated with foreign body infection.
3.5. Cell culture studies
3.5.1. Cytocompatibility
New biopolymer derivatives must always be tested thoroughly for
their potential toxic effects after application. Following this line, the
measurement of cytotoxicity in L929 cell type is a good starting point to
ensure their safety. Their application in pharmaceutical and tissue en-
gineering fields is an exciting opportunity to improve our arsenal of
biomaterials used for development of safe, efficient and innovative
S. Mohanapriya, V. Raj
therapies. The cytotoxicity of PVA film and PVA-GL/CT, PVA-PA/CT and
PVA-LY/CT bio composites were evaluated in terms of cell proliferation
and viability at 1, 3 and 5 days with respect to control. The respective
optical microscopy morphologies of MG-63 osteoblast-like cell lines cul-
tured on various samples are presented in Fig. 10 (i-ix) and the corre-
sponding MTT data is shown in Fig. 10(x). All PVA-AA/CT bio composites
under study show no cytotoxicity against the normal cells indicating that
incorporation of AA has little affected the toxicity of PVA. It is worth
mentioning that CA cross-linking introduces no toxicity however glutar-
aldehyde is generally used as a cross-linker for PVA that introduces
toxicity. PVA/CT film exhibits 45%, 62% and 74% viability after 1, 3 and
5 days respectively. The cell viability of PVA-GL/CT bio composite film
after 1, 3 and 5 days are found to be 55%, 72% and 79%. In the case of LY
modified PVA film cell viability is higher compared to the previous one
and their viability values are 82%, 84% and 89% obtained after 1, 3 and
5 days respectively. Among the three AA modified PVA films, PVA-LY/CT
film shows higher cell viability than any other. This may be due to the
presence of more number of positive charges on the surface compared to
others. In our case, AAs are incorporated into the PVA backbone, and they
are relatively short, their cytotoxicity is significantly lowered because of
the presence of PVA backbone.
Newly synthesized biomaterials with an ability to simulate and in-
itiate biomimetic processes have been used for applications in artificial
bones and in tissue engineering. The surface property of the material is
essential for cell attachment and proliferation, tissue growth and nu-
trient passage. Here in the present case, the biocompatibility of the AA
modified PVA films were examined by cultured L929 cells on various
samples. Cells on the PVA-GL/CT film were more spread and larger in
number compared to pristine PVA film. It is obvious that the presence
of AA blended with PVA significantly improved the cellular responses.
While in the case of PVA-LY/CT bio composite film, more number of
cells appeared than the former and it also shows some elongation. Less
number of cells was spread out on PVA-PA/CT film compared to the
two aliphatic amino acids modified PVA bio composite films namely
PVA-LY/CT and PVA-GL/CT. However, all PVA-AA/CT bio composite
films exhibit cell spreading higher than the pristine PVA film.
79
Materials Science & Engineering C 86 (2018) 70–82
Fig. 11. Fluorescence images of osteoblast cells adhered on
(i) Control (ii) PVA film and (iii) PVA-LY/CT bio composite
film (iv) % Cell viability associated with (A) Control (B)
PVA film and (C) PVA-LY/CT bio composite film.
3.5.2. Cell adhesion studies
Cellular phenomenological behavior of MG63 cell lines over PVA-LY/
CT bio composite film is shown in Fig. 11 and corresponding data are
presented in Fig. 12. It is inferred from figures that presence of LY is
beneficial for initial cell attachment. The plausible reason may be that LY
produces more positive charge on PVA film surface due to the presence of
two amino groups. Gao et al. have reported that existence of positive
charges would enhance the cell-biomaterial interaction, which is a pro-
mising way to improve cell adhesion and proliferation [43]. Furthermore,
final product would have potential capacity to form polyelectrolyte
complexes, which are of high practical relevance in biomedical applica-
tions. Scheme 1 depicts preferential adhesion of osteoblasts over PVA-AA/
CT bio composite surface rather than pristine PVA surface. The results
obtained in the present case are relevant with earlier findings [44,45]. As
well, better dimensional stability of PVA-AA/CT bio composite film pre-
vents release of toxic ions, effectively promoting the cell attachment. This
study elucidate that incorporation of AA into polymer matrix clearly
improves thermo-mechanical properties and dimensional stability. As a
consequence, addition of AA into PVA produces a positive impact on
biocompatible properties. Hence, bio artificial advanced polymeric ma-
terial with better bio compatibility and other biological functions is rea-
lized by using AA bio molecule as an additive. Biocompatibility is a sig-
nificant parameter for use as a medical device component material,
positive biocompatibility test results indicate that fabricated bio compo-
site films could be a potential alternate for metal based implants [46]. AA
modified PVA possess a great potential for application in these biomedical
fields, whereby a combination of the properties of PVA and those be-
longing to different amino acid moieties could produce materials with
synergetic properties.
3.5.3. Cell morphology studies
Structural morphology of cells envisioned through SEM for pure
PVA/CT film and PVA-LY/CT bio composite film are presented in
Fig. 12. Difference in the cellular activity and cell-material interaction
could be clearly visible from these images wherein morphology of os-
teoblasts seeded on PVA-LY/CT bio composite film exhibit extensive
S. Mohanapriya, V. Raj
spreading and better extracellular matrices. In contrast, cells grown on
pure PVA/CT film display a primarily flat appearance. Underlying
reason behind this behavior is that there exist a synergy between PVA
and AA, which stimulates cell functions such as cell attachment and
proliferation and synthesis of proteins. Hence, PVA-LY/CT showed
markedly higher development of cytoplasmic process, these results
80
Materials Science & Engineering C 86 (2018) 70–82
Fig. 12. Scanning electron microscopic images of osteo-
blast cells adhered on pure PVA film and PVA-LY/CT bio
composite film under different magnifications. (i), (iii) and
(v) are related to pure PVA film. (ii), (iv) and (vi) are re-
lated to PVA-LY/CT bio composite film.
Scheme 1. Preferential adhesion of Osteoblast over PVA-
AA/CT biocomposite film surface comprising zwitterion
hydrophilic domains.
substantiate growth of osteoblasts over PVA-AA/CT bio composite film.
4. Conclusions
The present study proposes a specific strategy to tune biological
properties of PVA and constitutes an example of rational design/
S. Mohanapriya, V. Raj
selection of bioactive molecules. Results confirm that osteoblast adhe-
sion to substrates can be controlled selectively and significantly by in-
corporation of amino acids that elucidates criteria and strategies for the
design of proactive orthopedic implant biomaterial. These results
documented that citric acid could successfully accomplish cross-linking
of PVA polymer chains, also enhance hydrogen bonding network
among PVA and AA molecules. Incorporation of the AA bio molecules
into PVA strongly influences the physicochemical and biological prop-
erties of PVA. Developed films exhibited high thermo-mechanical sta-
bilities with good antibacterial activity along with excellent dimen-
sional stability. Antibacterial activity is enhanced by the presence of
amino acid and citric acid. In vitro studies carried out with PVA film
and PVA-AA/CT bio composite film proved better in vitro bioactivity of
later elucidating the role of bipolar amino acid molecules towards fa-
voring cell viability which is attributed due to improved surface
chemistry, roughness and cell adhesion activity. Zwitterionic molecules
create hydrophilic nano-domains into PVA that play as potential active
sites for cell attachment, therefore pseudopods of osteoblasts desired to
anchor over bio composite films. In addition to this, cross-linking PVA-
AA bio composite with citric acid substantially improves the membrane
properties through balancing hydrophobic-hydrophilic microstates. To
end on a high note, this study establishes a biologically-tuned polymer
film fabrication strategy that enhances bone repair and orthopedic
implant integration application.
Acknowledgments
One of the authors S. Mohanapriya is grateful to University Grants
Commission (UGC), Government of India, for providing fund under the
scheme of ‘UGC-Dr. D. S. Kothari Post-Doctoral Fellowship’. (Ref: No.
Award Letter-No. F.4-2/2006 (BSR)/CH/14-15/0102 dated 5-5-2015).
M. Gowdhami, Research scholar and Prof. R. Balagurunathan, Head of
the Department, Department of Microbiology are gratefully acknowl-
edged for their assistance in carrying out antibacterial studies.
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