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 Materials Letters 186 (2017) 109–112

Contents lists available at ScienceDirect

Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Enhanced mechanical strength and sustained drug release of gelatin/

keratin scaffolds
crossmark
⁎
Preethi Ramadossa, K. Thanigai Arula,b, , J. Ramana Ramyaa, M. Rigana Begamc,
V. Sarath Chandraa,g, E. Manikandand,e,f,⁎⁎
a
Crystal Growth Centre, Anna University, Chennai 600025, India
b
Dept. of Physics, Nano Functional Materials Technology Centre and Materials Science Research Centre, Indian Institute of Technology Madras (IIT-M),
Chennai 600036, Tamil Nadu, India
c
Dept. of Physics, Indira Gandhi College of Engineering & Technology For Women (IGCET), Kancheepuram High Road, Chengalpattu, Athur 603101, Tamil
Nadu, India
d
Dept. of Physics, TUCAS Campus, Thiruvalluvar University, Thennangur 604408, Serkkadu, Vellore, Tamil Nadu, India
e
UNESCO UNISA Africa Chair in Nanosciences & Nanotechnology, College of Graduate Studies, University of South Africa, Muckleneuk Ridge, PO Box 392,
Pretoria, South Africa
f
Nanosciences African Network (NANO-AFNET), iThemba LABS-National Research Foundation, 1Old Faure Road, Somerset West 7129, PO Box: 722, Cape
Town, South Africa
g
Photonic Nanomaterials Laboratory, Dept of Chemistry & Dept of Carbon Materials, Chosun University, 309, Pilmun-daero, Dong-gu, Gwangju, 501-759,
South Korea.

A R T I C L E I N F O A BS T RAC T

Keywords: Three dimensional natural polymer composites with highly interconnected pores of gelatin/keratin (GK) and
Biomaterials gelatin/silk (GS) prepared by freeze-drying technique. FTIR analysis confirmed the functional group of GS and
Keratin GK. GK possesses superior mechanical strength and sustained release of drug. Both GK and GS are
Polymers hemocompatible in nature. Hence, in comparison with GS, GK is a potential candidate for sustained drug
Surface
release and could be availed as a natural bandage material. Therefore, GK is a green, non-toxic material to be
Composite materials
Mechanical
employed in biomedical field.
Drug delivery

1. Introduction create a favorable three dimensional matrix that permits cellular

The structures are called scaffolds, which play a pivotal role in vivo
to recapitulate its environment and allowing cells to influence the own
microenvironments. To accomplish the goal of tissue reconstruction
and scaffolds must meet the specific prerequisites. High porosity and
an adequate pore size are necessary to facilitate cell seeding and
diffusion throughout the whole structure. Keratins are a group of
cysteine-rich structural proteins formed in the epithelial cells of higher
vertebrates that exhibit high mechanical strength due to a large
number of disulfide bonds [1]. The extracted keratin proteins possess
an intrinsic ability to self-assemble and polymerize into a porous and
fibrous scaffold. Keratin derived from wool and human hair possess cell
binding motifs, such as leucine-aspartic acid-valine (LDV) and gluta-
mic acid-aspartic acid-serine (EDS) binding residues, which are cap-
able of supporting cellular attachment [2]. Together, these attributes
infiltration, attachment and proliferation [3].
Silk fibroin (SF), is a natural macromolecule spun by the silkworm
(Bombyx mori), and has drawn the interest of scientists of various
fields for a longer time. It is a mechanically resilient biomaterial that
offers a full range of mechanical and operational properties for
biomedical applications [3,4]. Silk fibroin (SF) fibers are a form of
protein, which consists of 18 amino acids, such as glycine, alanine,
serine, etc. Application of three-dimensional scaffolds provides in-
creased surface area, support of large cell mass and capable of shaping
particular structures [5]. Gelatin is a derivative of collagen and is the
major component of skin, bones and connective tissues and show signs
of antigenicity [6,7]. It shows interesting biological characteristics since
it contains arginine-glycine-aspartic acid (RGD) like sequence which
promotes cell adherence [8]. In the present study, we report on gelatin/
keratin (GK) and gelatin/silk (GS) prepared by freeze-drying technique

⁎
Corresponding author at: Crystal Growth Centre, Anna University, Chennai 600025, India.
⁎⁎
Corresponding author at: Dept. of Physics, TUCAS Campus, Thiruvalluvar University, Thennangur 604408 Serkkadu, Vellore, Tamil Nadu, India
E-mail addresses: thanigaiarul.k@gmail.com (K. Thanigai Arul), maniphysics@gmail.com (E. Manikandan).

http://dx.doi.org/10.1016/j.matlet.2016.09.095
Received 29 April 2016; Received in revised form 30 August 2016; Accepted 24 September 2016
Available online 26 September 2016
0167-577X/ © 2016 Elsevier B.V. All rights reserved.
P. Ramadoss et al.
for physicochemical, mechanical, hemocompatible and drug release
were carried out.
2. Materials and methods
Scaffolds were fabricated by a mixture of gelatin and silk Fibroin
(GS) and a mixture of gelatin and keratin (GK). Unpolished Bombyx
mori silk was obtained and degummed by boiling the silk in 0.02 M
Na2CO3 and rinsed thoroughly with distilled water and dried. It is then
dissolved in 200 ml of a solution containing a mixture of CaCl2: H2O:
ethanol in the ratio 1:8:2 respectively at 60 °C for approximately for
12 h. The resultant salt/silk solution is then dialyzed against water with
a cellulose membrane [12,000 Da molecular weight cut off (MWCO)]
for a week. 150 ml of silk fibroin solution was obtained from 5g of silk.
It was then centrifuged for 40 min at 9000 rpm to remove the
impurities. 3.6 g of gelatin was mixed with 10% Keratin (w/w) using
distilled water as a solvent for preparing GK. Similarly, 3.6g of gelatin
was mixed with 12% (w/w) of silk Fibroin by distilled water as a solvent
in order to fabricate GS. The mixture was allowed to stir at 60 °C for 6 h
and then cooled at 25 °C followed by pre-freezing at −25 °C for 24 h.
The freeze dried scaffolds were then cross linked with 0.4% gluter-
aldehyde [9] at room temperature for 2 h and then the scaffolds were
washed before being lyophilized again.
The silk fibroin once separated from the sericin is very hydrophilic
and bonding with water molecules. Silk fibroin along with gelatin were
prepared and with loading of drug for examining the drug release. For
turning the secondary structure for more stable, gluteraldehyde was
cross-linked for better mechanical strength and stability to the blended
silk fibroin scaffolds especially for tissue engineering and controlled
release of drug [9]. The above mixture was carried out at 60 °C in order
to allow the β sheet formation for maintaining the stability [10].
The cross-linked composites were loaded with Diclofenac Sodium
drug for its anti-inflammatory action. Since the scaffolds are fabricated
to be implanted as skin graft material, inflammation is likely to occur
upon implantation; hence the scaffolds were tested for the drug
delivery efficiency for the purpose. The scaffolds were weighed 50 mg
each and soaked in 3 ml of Diclofenac sodium drug for 4 h and dried at
37 °C for 48 h. The quantity of drug absorbed (75 mg/ml) into the
scaffolds was determined by variation in concentration of the drug,
before and after loading. It was measured with the help of UV
spectrophotometer (UV-3900, Hitachi High-Tech).
The in vitro drug release of diclofenac was carried out by immersing
the scaffolds in 4 ml of saline solution and incubating at 37 ± 0.1 °C.
The medium was withdrawn at various time intervals and replenished
with fresh medium. All experiments were carried out in triplicates. The
drug release was measured by the optical density using a UV spectro-
photometer (U-3900 Spectrophotometer, Hitachi High-Tech) at
280 nm for Diclofenac.
Scanning electron microscope (Carl Zeiss MA 15/EVO 18) was used
for surface morphology of the composites. Imagej software was
employed to investigate pore size. FTIR analysis was carried out using
a model FTIR-6300 in ATR mode. Young's modulus was analyzed with
the help of Instron 3369 with a load cell of 100 N. The samples were
placed in the tester and loads of different magnitudes (0-1N) were
applied at gradual increment. Hemolysis was analyzed with the help of
UV-Spectrometer.
3. Results and discussion
FTIR spectra of the GS and GK composite scaffolds were shown in
the Fig. 1a–b. The peak at 1738 cm−1 attribute the stretching vibration
of (C˭O) O of the silk fibron [11]. The broad peak at 3074 cm−1
corresponds to the N-H stretching mode of the gelatin. The peaks at
1621 and 1628 cm−1 are the amide I of the β-sheet whereas, the band
at 1508 cm−1 indicates the amide II of the β-sheet molecular con-
formation of the silk fibron. The peaks at 1370 cm−1 and 1443 cm−1
110
Materials Letters 186 (2017) 109–112
attribute to the bending vibration of the CH2 and CH3 of the β sheets of
the silk fibron [12]. The peaks at 1645 cm−1 was due to the C˭O
stretching of amide I, 1548 cm−1 was the N-H deformation of amide II
and 1240 cm−1 was the C-N stretching vibration of the amide III of the
gelatin in the structure respectively [13]. The peak at 1217 cm−1 was
responsible for the Amide III-β sheets of the silk fibron. The formation
of the peak at 1611 cm−1 corresponds to the increase β sheet structure
and the band at 1668 cm−1 indicates the loss of random coiling of the
silk structure during the process of polymerization [14]. In keratin-
gelatin composite scaffold (Fig. 2b), the characteristic peak of amide A
was observed at 3292 cm−1 region. The region at 1659 cm−1 was due to
the stretching vibration of the C-O of the amide I and the peak at
1528 cm−1 correspond to the C-H stretching and N-H bending of the
amide II region. The region around 1220–1300 cm−1 assigned to the
amide III. The peak at 2935 cm−1 attributed to the stretching vibra-
tions of CH2. The band at 1450 cm−1 was due to the -COOH stretching
of carboxyl groups. The peaks at 1080 cm−1 and 1030 cm−1 are C-O
stretching vibrations of carboxyl groups [14]...
The pore size of GS is 13 ± 11 µm; however, for GK, the pore size
enhanced up to 366 ± 49 µm (Fig. 2a-b) could be due to the remarkable
self assembling nature of keratin molecules and also it attached with
gelatin to form larger pore sizes. A highly porous scaffold will provide
sufficient space for cell growth and the average size of human cells lies
within the range 2–120 µm [15].
High Young's modulus of GK (Fig. 2c) could be due to keratin
makes one salt bridge with gelatin which could be the reason behind its
outstanding mechanical strength compared to GS. It is found that the
addition of keratin to gelatin decreases flexibility of the scaffold. It is
necessary that the scaffold possesses more strength along with good
elasticity when being used as a skin substitute [16,17].
Swelling behavior of the two composites was as shown in Fig. 3a.
GS contains high proportion of hydrophilic amino acids which attracts
water molecules. Gelatin and silk is found to increase the interaction
with water molecules [18]. Due to the reason GS seems to have higher
swelling behavior when compared to that of GK..
Hemocompatibility carried out on the composites (Fig. 3b) to
evaluate the disposition of the polymer composite to interact with
blood cells. Hemolysis of GS and GK scaffolds was 3.8% and 4.1%
respectively which are less than 5% indicates highly hemocompatible.
Gui-Bo et al. studied poly L-lactic acid (PLA)/Silk Fibroin (SF), PLA/
SF-gelatin (70:30), PLA/SF-gelatin (50:50) scaffolds and their corre-
sponding hemolysis were 4.5%, 3.2%, and 3.1% which were all less
than 5% [19]. As the composites engrafted into the skin to react with
the blood cells. Both the composites showed high hemocompatible
behavior. Percentage of hemolysis is calculated utilizing the formula
[20].
OD for the test sample − OD for the negative control
Hemolysis(%)=
OD for the positive control − OD for the negative control
The calculated percentages of hemolysis for all the samples were
compared with ASTM standard.
1. Highly hemocompatible ( < 5% hemolysis)
2. hemocompatible (within 10% hemolysis) and
3. non- hemocompatible ( > 20% hemolysis)
Release percentage of the drug was taken for different time intervals
of 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 42 h and 72 h (Fig. 3c). It was
found that GK composite shows a rather sustained release when
compared to GS. This can be ascribed to the fact that keratin is more
hydrophobic when compared to gelatin. Less permeable the scaffolds
for water that is responsible for a lesser amount of the drug release.
Since, diclofenac is a water soluble drug and gets easily released o the
system. Hence, the keratin based scaffold revealed sustained drug
release compared to silk based scaffold [21–24].
P. Ramadoss et al. Materials Letters 186 (2017) 109–112

Fig. 1. (a) FTIR spectra of GS composite scaffold, (b) FTIR spectra of GK composite scaffold.

Fig. 2. (a) SEM analysis of GS composite scaffold, (b) SEM analysis of GK composite scaffold and (c) Young's modulus of composite scaffolds.

Fig. 3. (a) Swelling behavior of the GS and GK composites scaffolds, (b) Hemolysis of the GS and GK composites scaffolds and (c) Drug release of the GS and GK composites scaffolds.

111
P. Ramadoss et al.
4. Conclusions
GK and GS three dimensional scaffolds were successfully prepared
by freeze drying technique. The functional groups of GK and GS were
confirmed by FTIR. The addition of keratin to gelatin has provided a
better mechanical strength and sustained drug release. The SEM
images of GK composite shown higher porosity (366 ± 49 µm) when
compared to GS scaffold. Both composite scaffolds were hemocompa-
tible in behavior. Therefore from overall studies, GK scaffold is an
excellent material for would healing natural bandage and to be used in
biomedical field.
Acknowledgement
Ms. Preethi Ramadoss gratefully acknowledges Dr. S. Narayana
Kalkura for providing all the facilities to carry out the research work. I
thank Prof. S. Subramanian, Dept. of Textile Technology for providing
the silk that was required for the research and Dr. V.R. Giridev, Mr.
Shiva Shankar and Dr. Senthil Ram of the Dept. of Textile Technology,
Anna University, Chennai for UV spectrophotometer and contact angle
characterization of the composites.
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