International Journal of Polymeric Materials and

Polymeric Biomaterials

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Biopolymer film fabrication for skin mimetic tissue

regenerative wound dressing applications

Logesh Kumar Sellappan, Sanmugam Anandhavelu, Mukesh Doble,

Govindaraj Perumal, Ji-Hoon Jeon, Dhanasekaran Vikraman & Hyun-Seok
Kim

To cite this article: Logesh Kumar Sellappan, Sanmugam Anandhavelu, Mukesh Doble,
Govindaraj Perumal, Ji-Hoon Jeon, Dhanasekaran Vikraman & Hyun-Seok Kim (2022)
Biopolymer film fabrication for skin mimetic tissue regenerative wound dressing applications,
International Journal of Polymeric Materials and Polymeric Biomaterials, 71:3, 196-207, DOI:
10.1080/00914037.2020.1817019

To link to this article: https://doi.org/10.1080/00914037.2020.1817019

View supplementary material Published online: 20 Sep 2020.

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INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
2022, VOL. 71, NO. 3, 196–207
https://doi.org/10.1080/00914037.2020.1817019

Biopolymer film fabrication for skin mimetic tissue regenerative wound dressing
applications
Logesh Kumar Sellappana , Sanmugam Anandhavelub , Mukesh Doblec, Govindaraj Perumalc,
Ji-Hoon Jeond, Dhanasekaran Vikramand , and Hyun-Seok Kimd
a
Department of Biomedical Engineering, Dr. N. G. P. Institute of Technology, Coimbatore, Tamil Nadu, India; bDepartment of Chemistry, Vel
Tech Multi Tech Engineering College, Chennai, Tamil Nadu, India; cDepartment of Biotechnology, Bhupat and Jyoti Mehta School of
Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India; dDivision of Electronics and Electrical Engineering, Dongguk
University-Seoul, Seoul, Republic of Korea

ABSTRACT ARTICLE HISTORY

A biopolymer film serving as a wound-healing substitute was fabricated using keratin and fibrin Received 9 June 2020
(extracted from a goat hoof and blood, respectively) via modified Shindai techniques. The import- Accepted 26 August 2020
ance of keratin and fibrin along with gelatin and mupirocin for the fabrication of a biofilm by solv-
KEYWORDS
ent casting method is shown in this work. In vitro microscopic images of the fibroblast NIH 3T3
Biopolymer; biowaste; fibrin;
cell lines revealed the biocompatibility, cell viability, cell adhesion, and proliferation of biopolymer keratin; Teflon;
film in the wound site. The low-cost and biowaste-source-extracted keratin and fibrin blended bio- wound dressing
polymer film supports the wound-healing process in humans.

GRAPHICAL ABSTRACT

1. Introduction will occur, thereby leading to a poor quality of life[4].

During the wound-healing process, the human body gener-
ates an auto-response for the restoration of skin to damaged
tissues[1–3]. In the case of severe wounds that ensue either
from clinical injuries, traumatic, or long-standing diseases,
external therapeutic treatment is necessary. This treatment is
avoided, however, if bacterial septicity and medical burden
Wound healing is a multidisciplinary process of the epider-
mal layer that maintains cellular and biochemical func-
tions[5]. Owing to the lack of protein, closing the extra cell
matrix is difficult during the wound medicinal process.
Naturally generated biopolymers (e.g., gelatin, chitosan,
keratin, collagen, silky fibroin, fibrin, albumin) have been

CONTACT Prof. Hyun-Seok Kim hyunseokk@dongguk.edu Division of Electronics and Electrical Engineering, Dongguk University-Seoul, Seoul 04620,
Republic of Korea.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
extensively investigated as biomaterials for providing pro-
teins to wound sites[6,7]. The keratin polymer is structured
with disulfide bonds, which provide high resistance to the
tissue matrix[7,8]. The goat-extracted keratin matrix protein
is associated with cysteine, which mainly provides the nat-
ural mechanical stability, adhesive property, biodegradability,
cell adhesion, and cellular proliferation[9,10].
Gelatin is a tetrahedron of collagen that is important for
the biodegradability of tissue. The structure shows good
optical properties and helps in the creation of a flexible film
with good optical properties[11]. In addition, gelatin is easily
soluble in water at 45–65
C and blended with copolymers.
Fibrin helps to develop a major plasma protein that removes
leakage during the developmental form of a moist environ-
ment and a matrix layer, which helps the cell migration on
the site[12]. Moreover, fibrin helps to develop the connective
granulating tissue[13]. Mupirocin is a tropical antibiotic that
prevents pathogens from penetrating the deep skin and epi-
dermis layer on the site of the wound. In this context, some
films using chitosan and gelatin blend a polymer mixture of
fibrin and chitosan with a crosslinking agent of polyethylene
glycol as the coating material[14,15]. The simple solvent cast-
ing route is an appropriate route to fabricate thin biofilm
for biomedical applications[16]. In pharmaceutical technol-
ogy, solvent casting methodology is broadly used to increase
drug suspension while the involvement of hydrophobic mol-
ecules[17,18]. For the biofilms preparation, various stabilizing
agents were used for solvent casting. Among glycerol as a
plasticizer, helps to deteriorate the intermolecular interaction
along polymer chains thereby improve the flexibility and
chain mobility of the prepared biofilms[17,19]. In addition, it
provisions to diminish the brittleness and shrinkage proper-
ties during the usage and storage. The inclusion of tetrathy-
lorthosilicate supports to improve the surface properties and
durability of the prepared film[20]. Recent studied models
have revealed the progressive characteristics of physical
properties such as surface binding sites, degradation, swel-
ling, thermal stability, crystalline behavior, and porosity of
biofilm in the drug release nature capability to
wounded sites[21,22].
In earlier, keratin and fibrin were derived from the
bovine biological waste by freeze-drying method and used
for skin tissue regeneration by Singaravelu et al.[23]. In this
study, an interesting alternative is developed using the
extracted keratin (from the goat’s hoof) and fibrin (from the
goat’s blood) blended with gelatin and mupirocin to produce
a new biofilm using a simple solvent casting method[18,24].
For the comparison, the biopolymer film was prepared with-
out and with mupirocin drug along with blends of keratin,
fibrin, and gelatin. The physical properties of the biopolymer
film were investigated via scanning electron microscopy
(SEM), X-ray diffraction (XRD), Fourier transform infrared
spectroscopy (FTIR), and thermogravimetric analysis (TGA).
The medicinal capability of our prepared biopolymer films
was determined through a swelling study, drug release ana-
lysis, and enzymatic degradation. Furthermore, the wound-
healing property of our biopolymer films was determined
through in vitro viability tests performed against an NIH
197
3T3 fibroblast cell line by MTT assay. The antimicrobial
properties of the biopolymer film against gram-positive
(Staphylococcus aureus and Bacillus subtilis) gram-negative
(Pseudomonas aeruginosa and Escherichia coli) pathogens
were analyzed via the well-diffusion method. From this
study, an easy-to-skin mimetic film was evaluated with a
long-term drug and subsequently released for the acceler-
ation of the wound rate.
2. Materials and methods
2.1. Extraction of keratin and fibrin
The keratin was extracted from goat’s hoof by using a
Soxhlet apparatus (Aldrich-Z556203), as reported in previ-
ous studies[24,25]. In addition, the keratin extract was centri-
fuged and then subjected to a 60-h dialysis process against
deionized water. Pure keratin was obtained by releasing the
dialyzed solution in a rotary vacuum evaporator to eliminate
the excess water from the keratin solution. Afterward, the
extracted keratin solution was stored in the refrigerator at
15
C for the experiment. The yield of keratin was 80% from
goat hoof and their molecular weight was 43 kDa with low
dispersity which included 12–15% cysteine residues.
The fibrin was extracted from physiologically clotted
goat’s blood, in accordance with Babu et al.[26], and then
separated from the blood by washing under streaming water
to eliminate unwanted debris from the blood clot.
Subsequently, the fibrin was sensed with 0.5 M sodium acet-
ate and 30% hydrogen peroxide solutions, lyophilized from
the resultant product to remove water, and then crushed
using a laboratory blender.
2.2. Preparation of KFG and KFGD biopolymer film
The keratin-fibrin-gelatin (KFG) biopolymer film was pre-
pared by dissolving 5% of gelatin powder in 100 mL of water
and stirring for 20 min at room temperature. Various stoi-
chiometric proportions (1:1:1, 1:1:2, and 1:1:3) of the keratin
and fibrin solutions were then blended with gelatin using a
magnetic stirrer at room temperature. Subsequently, 2% (w/
w) glycerol and 3% (w/w) tetrathylorthosilicate were blended
by dropwise at 55–65
C for 30 mins with KFG homogenous
solution to improve the stability of KFG biofilm. Afterward,
the homogenized mixture was poured into a 4 cm  8 cm
Teflon sheet by solvent casting method, allowed the solvent
evaporation at room temperature for 48 h and then endorsed
to dry at 60
C for 1 h. The keratin–fibrin–gelatin–mupirocin
(drug) (KFGD)-based biopolymer biofilm was arranged by
mixing 40 mg of mupirocin with KFG 100 mL solution, in
accordance with the above methodology.
2.3. Characterization details
The prepared KFG and KFGD biopolymer films were char-
acterized via various techniques, including FTIR (Nicolet 20
DXB FTIR spectrometer) performed over a range of
4000–400 cm1. The crystallinity and thermal stability of the
198 L. K. S ET AL.
biofilms were validated via X-ray diffractometer (X’Pert
PRO PANalytical) and TGA (Universal V4.4A TA instru-
ment), respectively. Furthermore, the surface properties and
mechanical properties of the films were evaluated via SEM
(JEOL JSM-5200) and tensile tests (Universal Testing
Machine, Instron Model 1405).
2.4. Swelling study analysis
As-prepared biopolymer films were submerged in a phos-
phate buffer solution (PBS pH 7.4) at room temperature up
to the film attained equilibrium. The equilibrium–swelling
ratio (Esw) was calculated as follows[27]:
Ww  Wd
Esw ð%Þ ¼  100 (1)
Wd
where Ww and Wd are the wet and dry weights of the bio-
polymer film, respectively. After the swelling experiment,
the sample was dehydrated overnight at 37
C and then used
for mechanical property analysis.
2.5. Drug release experiments
The mupirocin drug discharge by the KFGD biopolymer
film was identified at 37
C using a Franz-type cell. The
receiver component was equipped with PBS (pH-7.4) under
1 h of constant stirring. Afterward, the buffer from the
receiver part was extracted and centrifuged to remove the
supernatant solution. The mupirocin drug content ratio was
determined from the residue by gauging the absorbance
(Shimadzu ultraviolet (UV) spectrophotometer at
218 nm)[28]. The percentage of drug out from the KFGD
biopolymer film was estimated as follows:
Qp
E¼  100 (2)
Qt
where Qt is the total quantity of drug blended to the film,
Qp is the drug released quantity, and E is the drug release
percentage of from KFGD.
2.6. Enzymatic degradation
The biological stability and degradation rate of KFG and
KFGD biopolymer films were evaluated by using collagenase
enzyme (Sigma #C0773). The triplicate of known weight of
each prepared sample was taken and endorsed to dry in air
at 37
C. Enzyme solutions were prepared using PBS (pH
7.4). Afterward, all the prepared samples were exposed with
the collagenase enzyme (100 units/mL) at 37
C (pH 7.4) in
an incubator. Subsequently, the sensed samples were
detached from the incubator after 24 h, dehydrated via blot-
ting, and dried at room temperature in air for 24 h. The
amount of sample degradation was calculated gravimetric
weight loss[29].
2.7. In vitro MTT assay
The cell cytocompatibility of biopolymer films was deter-
mined in accordance with a previously reported proced-
ure[30]. In the 96-well tissue culture polystyrene (TCP) plate,
Swiss mouse NIH 3T3 fibroblast cells (density: 1  104 cells/
mL) were cultivated on both KFG and KFGD films contain-
ing DMEM media accompanied with 10% fetal bovine
serum (FBS) and 1% antibiotic–antimicotic mixture. The
cells were then incubated at 37
C with 5% CO2. The culture
media was substituted with 0.5 mg/mL of MTT solution
after 24 h and 72 h, and further nurtured for 3 h at 37
C.
Consequently, MTT solution was replaced and blended the
DMSO to liquefy the formazan crystals. The absorbance
behavior was validated using a multimode plate reader at
570 nm (Enspire, Perkin Elmer, USA). The cell viability % of
each sample was measured using the formula of absorbance
of test/absorbance of control 100.
The NIH 3T3 cell linkage and propagation were deter-
mined using fluorescein isothiocyanate (FITC) stain (Medox
Biotech, India), in accordance with a previously reported
method[31]. The cells were cultured (using the previously
mentioned procedure) and incubated for 12 h with DMEM
media. Subsequently, the cultured cell-well media were
replaced with sample extracts (except control wells), which
were prepared using the same media, incubated further for
24 h, and then removed. These media were then washed
with PBS, and 4% paraformaldehyde was added after wash-
ing. After 30 min, the media were then washed repeatedly
with PBS. The fixed cell layers were permeabilized by adding
Triton X 100 solution for 10 min and washed with PBS.
Afterward, the cells were stained by fluorescein isothiocyan-
ate and observed with a fluorescence microscope
(Olympus, Japan).
2.8. In vitro scratch test
The wound-healing performance of fibroblast NIH 3T3 cells
with various samples were appraised using acridine orange
exposure[30,31]. As-prepared biopolymer films were sterilized
via UV treatment for 30 min on each side. The cells (dens-
ity: 1  105 cells/well) were then cultured in a six-well TCP
and nurtured in a moistened atmosphere of 5% CO2 at
37
C for 24 h to form a cell monolayer. Afterward, media
were removed from all the wells and an artificial injury was
created on the cell monolayer using a 20 mL sterile pipette
tip. The isolated cells from the scratch region were carefully
removed by washing with PBS. The wells were added to
sample extracts containing media and incubated at 37
C.
Subsequently, the scratched-region cell migration or wound-
healing capacity was analyzed by capturing images at prede-
termined time intervals (0 and 24 h). At time 0, images were
captured of the wells without the addition of the fluorescent
dye. After 24 h, images were captured of the complete cell
migration between the scratched regions by the cells stained
with acridine orange live cell stain. Images of the control
sample and test sample scratched regions were captured by
a fluorescence microscope.
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS 199

Figure 1. Schematic showing extraction and fabrication of KFGD biopolymer film.

Table 1. Tensile strength analysis of biopolymer films.

Presence of Mean extension at
Sample mupirocin Mean maximum Mean elongation Mean Young’s Mean tensile maximum
ratio (K:F:G) drug (40 mg) load (N) break (%) modulus (MPa) strength (MPa) load (mm)
1:1:1 No 26.74 10.91 0.74 5.27 4.95
1:1:2 32.80 6.37 1.46 7.15 3.05
1:1:3 38.46 3.83 1.95 9.48 1.80
1:1:3 Yes 36.07 2.96 1.68 8.13 1.51

2.9. Antibacterial study at 37
C for 24 h and the diameters of inhibition zones were
measured in mm. The triplicated results mean values are
Microbial cells were acquired from Himedia Laboratories
used in the discussion.
Pvt. Limited, Mumbai. The KFG and KFGD biopolymer
films were evaluated against the gram of positive bacteria (S.
aureus (ATCC 11632) and B. subtilis (ATCC 11774)) and
the gram of negative bacteria (P. aeruginosa (ATCC 2799) 2.10. Statistical analysis
and E. coli (ATCC 10536)) using the well diffusion All the results were validated from the triplicated experiment
method[32]. The mentioned microorganisms were patterned outputs and their mean values are given with standard deviation
to each of the Muller-Hinton Agar (MHA) plates and cul-
(n ¼ 3). Acquired all the results were appraised with GraphPad
tured for 24 h at 37
C. The prepared biofilm disks were
Prism Software (version 6.01). The post hoc test with analysis of
positioned onto the surface of the inoculated MHA plates
variance (ANOVA) was carried out to identify the significance
and surged down to made widespread contact with the agar
of the particular differences between the pairs of means. All the
surface. Then, MHA plates were nurtured in a vertical site
experimental values were statistically significant with p˂0.05.
200 L. K. S ET AL.
Figure 2. Photograph and SEM image (a) KFG-3 and (b) KFGD biopolymer films.
3. Results and discussion
The biopolymer KFG films were prepared using natural pro-
tein polymer of keratin from the goat’s hoof waste and
fibrin from the goat’s blood with gelatin. The mupirocin
drug was blended with KFG to prepare KFGD biopolymer
film. The preparation scheme is provided in Figure 1.
3.1. Physical properties of biopolymer films
The different ratios of blended KFG and KFGD films are
given in Table 1. As the table displays, the Young’s modu-
lus and tensile strength of the prepared biopolymer films
increase with increasing concentration of gelatin. The low-
est elongation break (3.61%) and the highest tensile
strength (9.48 MPa) were obtained for the 1:1:3 biopolymer
film. Therefore, the 1:1:3 ratio was selected for preparing
the mupirocin-incorporated biopolymer film. After incor-
porating the mupirocin drug, the tensile strength of the
film decreases slightly to 8.13 MPa (with the elongation
break at 2.96%), which is significant for the wound-heal-
ing process.
Different ratios using blended KFG films SEM images
are provided in the supporting information Figure S1 in
Supplementary Material. The observed smooth surface for
KFG with 1:1:3 than the other ratios and physical
properties are made them to use for further analysis and
mupirocin blended biofilm preparation. Figure 2a–b
shows a photograph and SEM micrograph of the KFG
with 1:1:3 (further denoted as KFG-3) and KFGD biopoly-
mers, respectively. The KFGD film shows a highly smooth
surface, owing to the disulfide bonds of keratin interact-
ing with co-polymers as well as with the evenly distrib-
uted mupirocin[33,34]. The FTIR spectra of the films (see
Figure 3) reveal several trends. For the KFG-3 biofilm
(Figure 3a), the peak occurring at 3350–3450 cm1 corre-
sponds to N–H band of keratin[33,34] . The characteristic
peaks (keratin and fibrin) occur at 1544 cm1 and
1450 cm1 and are attributed to amide I and II, respect-
ively[35–38]. C–H and C¼O stretching of acetyl groups of
KFG comprising peaks at 2924 cm1 and 1652 cm1 ,
respectively[24,39]. For the KFGD film, the characteristic
peaks of keratin and fibrin are slightly shifted. The major
interaction peak of KFGD, which occurs at 1236 cm1, is
attributed to amide III[40] . In addition, C¼O bending and
asymmetric and symmetric S–O stretching peaks show at
1166 cm  11,082 cm1 and 1042 cm1 , respectively, for
KFGD[41] . The stabilized higher molecular weight poly-
mers are merged with mupirocin, resulting in lower
molecular weight compounds. This leads to significant
interaction of the disulfide bond between protein poly-
mers and the drug in a defined structure of the
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
Figure 3. (a) Fourier transform infrared spectra, (b) X-ray diffraction patterns, and (c) thermogravimetric spectra of KFG-3 and KFGD biopolymer films.
biopolymer film, and is beneficial for wound-healing
applications. Further FTIR band details are given in Table
S1 in Supplementary Material.
Figure 3b shows the XRD patterns of the KFG-3 and
KFGD biopolymer films. Peaks related to the a-helix struc-
ture and b-sheet structure of keratin occur at 11
and
19–20
, respectively (K1-K3)[12]. The amorphous structure
of the gelatin shows the broad hump at around 17–23

(G1)[42]. In addition, the fibrin characteristics peaks occur at
26–33
[43]. For KFGD, the additional peaks result from the
addition of the mupirocin drug (M1-M5) to the biopolymer
film (see Table S2 in Supplementary Material for further
XRD peak information)[4,7,44].
The thermal characteristics of the biopolymer film are
determined via TGA[45]. Figure 3c shows the thermograms
of KFG-3 and KFGD. For the KFG-3 film, the initial weight
loss (15%) at 260
C and the second weight loss (76%) at
480
C result from the water evaporation from keratin and
the decomposition of gelatin, respectively. For the KFGD
film, the initial weight loss (10%) and second weight loss
(50%) occur at 210
C and 400
C, respectively. The thermal
stability increases considerably, owing to the addition of
mupirocin along with the KFG complex.
201
3.2. Swelling, drug release, and enzymatic degradation
performance
Moreover, the swelling study of biopolymer film helps to
determine the swelling nature of the film against body flu-
ids. Figure 4a shows the swelling nature of the KFG-3 and
KFGD biopolymer film as a function of time. From this
viewpoint, the concentration of gelatin is directly propor-
tional to the degree of swelling and the hydrophilic property
of gelatin in the biofilm improves the swelling degree[16].
Pictures obtained at different times during the swelling
study performed on the KFGD biofilm are provided in
Figure S2 in Supplementary Material.
In vitro drug release property of the KFGD biofilm is
determined using PBS (pH-7.4) with Franz diffusion cells.
As shown in Figure 4b, 15%, 20%, 40%, and 70% of
the drug from the biofilm are released in 1, 10, 50, and
120 h, respectively. The initial 15% drug release promotes
formation of the wound surface from debris and 20% at
10 h clearly indicates the proper binding of drug with the
biopolymer film. From the observed drug delivery results,
the KFGD biofilm is sustainably drug released up to 130 h
and, hence, represents an ideal wound-healing substitute[28].
202 L. K. S ET AL.
Figure 4. (a) Swelling study of KFG-3 and KFGD biopolymer films. (b) Drug release study of KFGD biopolymer film. (c) Degradation study of KFG-3 and KFGD bio-
polymer films.
The stability of the biopolymer film and its degradation
rate was measured versus the collagenase enzyme
(Figure 4c). The degradation of KFG-3 and KFGD biopoly-
mer film occurs at 60.9% and 57.8%, respectively. Owing to
the incorporation of mupirocin in KFGD, the degradation
rate decreases considerably. The observed degradation study
results lend further support to the use of our biopolymer
film for wound healing[29].
3.3. In vitro studies on NIH 3T3 cell lines
The in vitro cell viability of Swiss mouse fibroblast NIH 3T3
cells at different times (e.g., 12, 24, and 72 h) on KFG-3 and
KFGD films is shown in Figure 5a. The cell viability percen-
tages are 80%, 84% and 88% in KFGD and 78%, 81% and
83% in KFG-3 biofilm for 12 , 24, and 72 h, respectively.
Compared with the control sample (well without biofilm),
spectra exposed the more active cells on both KFG-3 and
KFGD biofilms, whereas the cell viability on the KFGD bio-
film is higher than that of KFG-3 biofilm. In addition,
Figure 5b shows the fluorescence microscope images of
fibroblast cell adhesion and proliferation after 12, 24, and
72 h, where the viable cells are stained with a green color.
The cell adhesion and proliferation associated with the
KFGD film are greater than those of the KFG-3 film, owing
to mupirocin. Moreover, the amount of keratin released
from the blended polymers reveals the excellent cell to cell
interaction. The controlled release of mupirocin induces
rapid tissue regeneration at the wound site, without induc-
ing any cytotoxicity to the cells[15]. The biologically derived
KFGD film shows very good cellular attachment and the cell
proliferation on the film-surface cell lines is higher than that
on the KFG-3 film-surface cell line. Thus, keratin shows
strong cellular activities with respect to the effect of fibrous
fibrin on cell adhesion and the hydrophobic nature of gel-
atin on cell binding motifs[23,46].
3.4. In vitro wound-healing performance
To investigate the molecular mechanism and characterize
the effects of KFG-3 and KFGD on fibroblasts, we first
observe the tissue proliferation and cell migration of the
NIH 3T3 Swiss mouse fibroblasts shown in Figure 6. These
results indicate that keratin enhances cell propagation and
the cell voyage rate during the wound-healing performance.
We also observed the activation of fibrin and the mupirocin
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS 203

Figure 5. (a) In vitro cell % cell viability at different time intervals. (b) Fluroscence images of in vitro cell linkage and propagation on KFG-3 and KFGD biopolymer
films at different time intervals for NIH 3T3 fibroblast cells.

Figure 6. In vitro cell migration of fibroblast NIH 3T3 cells on KFG-3 and KFGD biopolymer films at 12 and 24 h (Scratch test region between the yellow lines).
204 L. K. S ET AL.

Figure 7. Antibacterial study of biopolymer film (a) Staphylococcus aureus, (b) Pseudomonas aeruginosa, (c) Bacillus subtilis and (d) Escherichia coli. (e) Zone of inhib-
ition ratio against the different pathogens.
 INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS
drug in mouse fibroblasts. Moreover, we use acridine orange
(AO) live-cell stain to quantitatively define the number of
viable cells in each duration. The increased contribution of
protein in biofilm induces fibroblast cell migration[29,47,48].
The cell migration is observed at an artificial wound cre-
ated in the 3T3 fibroblast cell lines after 12 and 24 h. Both
of the KFG-3 and KFGD films show 100% wound healing,
but the cell migration occurring for the wounded area
(between the yellow lined region) associated with KFGD is
higher than that occurring for the KFG-3 film. The percent-
age of area of cell migrations of 25% and 60% in KFG and
55% and 80% in KFGD for 12 and 24 h, respectively, are
occurred in the wound scratch test. The high efficiency of
cell migration in KFGD results from the strong reaction of
the mupirocin drug with protein polymers. These results
show that the cell migration leads to complete closure of the
wounded part, which synergizes the fibroblast cell growth
rate and promotes rapid cell migration at the wound
scratch. Therefore, the results suggest that the KFGD bio-
polymer film can enhance fibroblast cell migration as a
wound-healing accelerator in tissue engineering applications.
3.5. Antibacterial study
Figure 7 shows the biopolymer film zone of inhibition activ-
ity. The antibacterial activity of keratin, gelatin, and fibrin
has been well-established from previous studies[36]. The
antibacterial properties of blended system comprising the
KFG-3 and KFGD biopolymer film are studied versus gram-
positive (S. aureus (Figure 7a) and B. subtilis (Figure 7b))
and gram-negative (P. aeruginosa (Figure 7c) and E. coli
(Figure 7d)) pathogens[48,49]. Figure 7e shows the zone of
inhibition variations of KFG and KFGD with different
pathogens. The better zone of inhibition values of 25, 22,
27, and 32 mm for KFGD are observed compared with 14,
11, 7, and 30 mm for KFG-3. The improved inhibition zone
shows for KFGD biofilms than KFG-3 which proves the
importance of drug inclusion with KFG.
4. Conclusions
A novel source of biowaste materials for the extraction of
keratin and fibrin is reported in this paper. The extracted
keratin and fibrin were successfully blended with gelatin to
form a KFG biopolymer film, whereas KFG was combined
with mupirocin drug for organization of the KFGD com-
plex. The thermal resistance, mechanical strength, surface
properties, and inhibition against Gram-negative and Gram-
positive bacteria of the KFGD film were superior to those of
the KFG film. Moreover, compared with its KFG counter-
part, the KFGD film showed superior cell adhesion and cell
migration symptoms of NIH 3T3 fibroblast cells. The
observed results suggest that the simple and biological waste
generated by the prepared structure of KFGD would be a
fruitful wound-healing recipe that is suitable for
in vivo studies.
205
Acknowledgments
LKS extends his gratitude to his institute (Dr. N. G. P. Institute of
Technology, Coimbatore) for the Biomedical Research Lab facility to
carry out this work.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the Mid-career Researcher Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Science and ICT [grant number 2019R1A2C2086747]
and the Department of Biotechnology (DBT) and the Government of
India for providing International Travel Grant [grant number DBT/
CTEP/02/201601450 (Dated on 25.11.2016) to LKS].
ORCID
Logesh Kumar Sellappan http://orcid.org/0000-0002-5794-3056
Sanmugam Anandhavelu http://orcid.org/0000-0002-7191-3056
Dhanasekaran Vikraman http://orcid.org/0000-0001-8991-3604
Hyun-Seok Kim http://orcid.org/0000-0003-1127-5766
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