Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsp20

Fabrication and characterization of novel bacterial

cellulose/alginate/gelatin biocomposite film

Nadda Chiaoprakobkij, Sutasinee Seetabhawang, Neeracha Sanchavanakit

& Muenduen Phisalaphong

To cite this article: Nadda Chiaoprakobkij, Sutasinee Seetabhawang, Neeracha Sanchavanakit

& Muenduen Phisalaphong (2019) Fabrication and characterization of novel bacterial cellulose/
alginate/gelatin biocomposite film, Journal of Biomaterials Science, Polymer Edition, 30:11,
961-982, DOI: 10.1080/09205063.2019.1613292

To link to this article: https://doi.org/10.1080/09205063.2019.1613292

Accepted author version posted online: 02

May 2019.
Published online: 21 May 2019.

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JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION
2019, VOL. 30, NO. 11, 961–982
https://doi.org/10.1080/09205063.2019.1613292

Fabrication and characterization of novel bacterial

cellulose/alginate/gelatin biocomposite film
Nadda Chiaoprakobkija, Sutasinee Seetabhawangb, Neeracha Sanchavanakitc
and Muenduen Phisalaphongb
a
Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University, Bangkok,
Thailand; bDepartment of Chemical Engineering, Faculty of Engineering, Chulalongkorn University,
Bangkok, Thailand; cResearch Unit of Mineralized Tissues, Department of Anatomy, Faculty of
Dentistry, Chulalongkorn University, Bangkok, Thailand

ABSTRACT ARTICLE HISTORY

Hydrogels from bacterial, algal, and animal cells—bacterial cellu- Received 13 February 2019
lose (BC), alginate, and gelatin, respectively—were combined to Accepted 27 April 2019
fabricate a biocomposite film (BCAGG) via an eco-friendly casting
KEYWORDS
technique. In addition, glycerol was added as a plasticizer to
bacterial cellulose; alginate;
improve the elasticity and water absorption capacity of the film. gelatin; glycerol; composite;
In this study, BC pellicles were simply deconstructed into fibrils human keratinocyte
suspension and then reconstructed into films with a supplement
of alginate, gelatin and glycerol. The physical appearance of fabri-
cated films resembled native BC but possessed improved ductility,
enhanced flexibility, higher water uptake ability and better bio-
compatibility. The film was found to resist tearing under suture
pullout strength in a hydrated state. In vitro cytotoxicity tests
showed that the film was cytocompatible. A cell study using a
human keratinocyte culture demonstrated enhanced cell adhe-
sion, spreading, and proliferation on the BCAGG film compared
with BC/alginate film. The BCAGG film therefore has significant
potential for use in biomedical applications, particularly in dermal
treatment, skin tissue regeneration, and wound healing.

1. Introduction
Polysaccharide-based films are considered potentially promising materials for biomed-
ical applications because they are nontoxic and biocompatible, and their degradation
products are also nontoxic. Currently, many experimental and clinical studies are
being carried out to develop biopolymeric films for wound dressing and dermal treat-
ment. Bacterial cellulose (BC) exhibits many advantageous properties including high
water uptake capacity, high crystallinity, and an ultrafine nanofibril network structure
[1]. BC also presents great mechanical stability, which is essential for biomedical
applications. Alginate derived from brown algae is also used as one of base

CONTACT Muenduen Phisalaphong muenduen.p@chula.ac.th Department of Chemical Engineering,

Chulalongkorn University, PhayaThai Road, Phatumwan, Bangkok 10330, Thailand.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/tbsp.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
962 N. CHIAOPRAKOBKIJ ET AL.

biomaterials because of its adhesiveness, gelling, transparency, biocompatibility, and

rapid ionic gelation properties with divalent cations, which are useful for biofabrica-
tion strategies [2]. The factor that may limit the potential use of BC and alginate in
biomedical application is the absence of RGD cell-binding peptide. This limitation
can be overcome by incorporating protein molecules. Such modification could further
enhance biocompatibility to meet the specific requirements for biomedical applica-
tions. Recent advances in the modification of alginate based hydrogel have been
studied, including photo thermal activity, catalysis, target drug delivery, drug degrad-
ation, removal of aquatic pollutant and bio-sensing. Alginate hydrogel integrated with
nano-particles could increase hydrogel mechanical properties, modulus and adsorp-
tion behavior [3]. Wr oblewska-Krepsztul et al. [4] summarized the recent advances
emphasizing the usage of alginate for biomedical and pharmaceutical applications.
Gelatin is derived from the hydrolysis of native collagen containing RGD peptides
making it suitable for tissue engineering applications. However, despite showing
excellent biocompatibility, gelatin has poor mechanical properties. Previously, in situ
modification of BC was carried out simply by adding gelatin to the fermentation
medium resulting in films that were denser and had a lower crystallinity index than
native BC [5,6]. However, the restrictive microbial fermentation conditions limit the
introduction of a greater variety of additives. Ex situ modification of BC was per-
formed after the BC had been formed by impregnating the polymer matrix with dif-
ferent materials to modify the films [7,8]. Wang et al. [9] modified BC by
crosslinking gelatin with BC. Pure BC films were individually immersed in aqueous
gelatin solutions. The results showed that the composite provided better support for
NIH/3T3 fibroblasts than unmodified BC. Although ex situ modification through
absorption is simpler and more versatile, it is also limited by the penetration of the
additive molecules and the thickness of the BC pellicles. Yan et al. [10] developed
alginate/chitosan/gelatin composite scaffold. The BC nanacrystals (BCNs) obtained
from sulfuric acid hydrolysis of BC were used as reinforcing agents. The developed
scaffolds were fabricated by the combined method involving internal gelation and
layer-by-layer electrostatic assembly. The resulting scaffolds exhibited good porous
structure and excellent biocompatibility with osteoblast MC3T3-E1 cells. Chen et al.
[11] proposed an alternative method to improve the mechanical properties and stabil-
ity of gelatin by using chitin nanofibers as reinforcing agents. The developed films
showed a uniform structure without aggregation and good mechanical properties.
Highly transparent and excellent mechanical properties of gelatin films reinforced by
deacetylated chitin nanofibers by immersion method were successfully developed
[12]. Chen et al. [13] used a simple NaOH treatment to form double network hybrid
hydrogel of cellulose nanofiber/polyacrylamide. The hydrogels showed excellent com-
pressive stress with 15-fold higher than the pure polyacrylamide gel. This unique
design and promising properties of the hydrogel was useful for cartilage or other soft
tissue engineering. Functionalization of polymeric membranes is one of the key
approaches to tailor polymers for biomedical applications. Pandele et al. [14] success-
fully developed osteoconductive membranes from cellulose acetate. The membranes
were functionalized with resveratrol via facile covalent immobilization. The function-
alized membranes were found to significantly increase ALP activity and ECM
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 963

mineralization of MC3T3-E1 cells. In addition, incorporation of nanomaterials (such

as iron oxide, manganese peroxidase, carbon nanotubes, nano crytalline hydroxyapatite,
and magnetic nanoparticles could produce robust hydrogel with good mechanical proper-
ties for advanced applications [15]. Novel hydrogel composed of alginate/gelatin incorpo-
rated cellulose nanocrystals was reported for the cartilage tissue engineering [16]. The
developed hydrogel showed the strain and strength similar to that of natural cartilage.
Glycerol has been used as a plasticizer in biopolymer based films owing to its
water-solubility, polarity, low molecular weight, and it being non-volatile [17].
Gelatin films plasticized with glycerol showed increased flexibility, hydrophilicity, and
workability without major alteration of the mechanical strength [18,19]. The plasti-
cizer affects the strength of the interactions between macromolecules increasing the
distance between them and reducing the internal forces which give more flexibility to
the films.
Handling infectious medical waste is one of the factors that needed to be
addressed. Non-biodegradable medical wastes are generally disposed by incineration
at high temperatures, which causes of environmental pollution problems.
Biodegradable materials can be allowed to decompose in soil into CO2 and biomass,
which are considered much better for the environment [20]. In addition, for conveni-
ent uses, biomedical materials for clinical applications are commonly desired to be
dried, packed, sterilized and stored properly. Never-dried BC in a hydrogel state
would create a favorable environment for microbial growths that could affect its shelf
life. BC in dried form is convenient to handle, lightweight and can extend shelf life.
However, the re-swelling and water holding capacity of dried BC films were markedly
decreased due to their densely packed structures after the drying process. Polymer
blending is a simple method for improving polymer properties. The addition of
hydrogel or water-drawing molecules into BC composites could enhance water
adsorption capacity, while disintegrated BC can function as both a matrix and
reinforcement in the composite materials [5, 21]. So far, there is no report of the
blends of mechanically-disintegrated BC, alginate, gelatin and glycerol. This study
focused on the fabrication and the characterizations of these hybrid bio-composite
films. A novel bio-composite film composed of mechanically-disintegrated BC, algin-
ate and gelatin plasticized with glycerol was fabricated via an eco-friendly casting
technique to combine the synergistic beneficial aspects of hydrogels from bacteria,
algae, and animal cells, i.e. BC, alginate and gelatin, respectively. The physical, chem-
ical, and mechanical properties of the film were then determined. Cell studies using
human keratinocytes (HaCat) were carried out to evaluate the cytotoxicity and bio-
compatibility of the film.

2. Materials and methods

2.1. Materials and reagents
BC pellicles (size 1 cm
1 cm
1 cm) were kindly supplied by Pramote Thamarat
from the Institute of Research and Development of Food Product, Kasetsart
University (Bangkok, Thailand). The BC pellicles were purified with 1 wt. % NaOH
and were rinsed with DI water until the pH was 7.0. Gelatin (G) (180 bloom, type A)
964 N. CHIAOPRAKOBKIJ ET AL.

Figure 1. Schematic fabrication process of novel BC biocomposite films.

was purchased from Sigma-Aldrich, USA. Alginate was purchased from Acros,
Belgium. Calcium chloride and glycerol were purchased from Ajax Finechem Pty.
Ltd., Australia.

2.2. Fabrication of composite films with/without glycerol

For the polymer blend system, a 2% (w/v) solution of alginate was prepared in DI
water at room temperature. A 15% (w/v) solution of gelatin was prepared by stirring
gelatin powder in DI water at 60  C until gelatin was completely dissolved. Purified
BC pellicles were homogenized by using a blender at 4500 rpm for 10 min at
room temperature. After the homogenization, the BC slurry with 1.5 wt.% solid
contents of disintegrated BC were obtained. The films of BC and BC-alginate (BCA)
were fabricated from the BC slurry and a blend of the BC slurry mixed with alginate
solution at a weight ratio of 60/40. Films of BC-alginate- gelatin (BCAG) were made
from a blend of the BC slurry, alginate solution, and gelatin solution at a weight ratio
of 60:20:20. Films of BC-alginate-gelatin supplemented with glycerol (BCAGG) were
fabricated from a blend of the BC, alginate, and gelatin at a weight ratio of 60:20:20,
added with glycerol at 2 g/10 g of gelatin solution. All mixtures were thoroughly
mixed for 6 h and then the homogeneous mixtures were placed in a 14.5 cm diameter
sterile Petri-dish (40 g/plate), and air-dried at room temperature for 24 h. After that,
all dried films were cross-linked in a 1% (w/v) solution of calcium chloride (CaCl2)
for 1 h. The films were then rinsed with DI water and dried at room temperature.
The films were carefully peeled off from the plates and were stored in a vacuum des-
iccator until further use. The procedure for the fabrication of composite films is
shown in Figure 1.

2.3. Characterization
2.3.1. Film thickness
The thickness of films was the average values measured using a 547-321 Mitutoyo
thickness gauge (Mitutoyo Corporation, Japan) at 10 different points of each sample.
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 965

2.3.2. FT-IR analysis

For Fourier transform infrared (FT-IR) analysis, a small amount of powder sample
(1 mg) obtained by grinding of dried sample in a mortar was mixed with KBr and
then the mixed powder was ground into a homogenous fine powder. The mixture
was poured into a pellet press. After that, the pellet was popped out and placed in a
holder for FT-IR analysis. FT-IR spectra of the samples were obtained using a
Spectrum One FT-IR Spectrometer (PerkinElmer, USA) within the range of
4000–400 cm1 at a scanning resolution of 2 cm1.

2.3.3. Water absorption capacity

Water absorption capacity (WAC) was determined by immersing the weighed sam-
ples in DI water at room temperature until they were equilibrated. The swollen sam-
ples were then removed from the water and excess water at the surface of the
samples was blotted out with soft papers. The weight of wet sample was then
reweighed. All testing was carried out in six duplicate. Water content was determined
by gravimetric method and calculated using the following equation:

ðWsWdÞ
%WAC ¼ x 100
Wd

Where Ws and Wd are denoted as the weight of hydrate and dry sample,
respectively.

2.3.4. Mechanical properties

Mechanical properties were measured in terms of tensile strength and elongation at
break (%). Tensile strength is the maximum stress that the material withstands before
it breaks. Elongation at break, or fracture strain is the ratio between increased length
and initial length after breakage of the tested specimen. The tensile strength and
elongation at break of dried and re-swollen films were measured by an INSTRON
5567 Universal Testing Machine (INSTRON, USA) equipped with a 1.0 kN load cell.
The samples were cut into strip-shaped specimens with a width of 10 mm and a
length of 10 cm. The test conditions in this assay followed ASTM D882, the standard
test method for tensile properties of thin films. The tensile strength and elongation at
break were reported as average values determined from at least six specimens.

2.3.5. Morphology
Scanning Electron Microscope (SEM) was used for observation of morphology of the
specimen. Cell-free films were cut into small pieces and fixed to sample mounts for
surface observation. The samples were coated with a thin layer of gold by sputter
coating. For cross- sectional observation, cell-free films were prepared by direct sim-
ple freeze fracture technique. Films were dipped in liquid nitrogen for approximately
5 min to ensure completely frozen. Samples were removed from liquid nitrogen and
rapidly snapped with forceps. The fractures were fixed to sample mounts and coated
with a thin layer of gold for cross-sectional observation. For the observation of cell
morphology on films, the film cultured with cells was washed with PBS and fixed in
2.5% v/v glutaraldehyde. After that, the film was dehydrated by ethanol dehydration
966 N. CHIAOPRAKOBKIJ ET AL.

series (30, 50, 70, 90, 100% ethanol) and critical point drying. Then, the film was
sputter-coated with gold and the surface morphology of films was observed. The
samples were sputtered with gold in a Balzers-SCD-040 sputter coater (Balzers,
Liechtenstein). Morphology of the samples was observed using a JSM-5410LV scan-
ning electron microscope (JOEL, Japan) operated at an acceleration voltage of 15 kV.

2.3.6. Oxygen permeation ability

The oxygen transmission rate (OTR) of the films was determined with an oxygen
permeation analyzer (Model 8000, Illinois Instruments, Johnsburg, IL) at the Thai
Packaging Centre, Thailand Institute of Scientific and Technological Research
(Bangkok, Thailand). The test condition followed ASTM D-3985. The determination
of OTR was done at 23  C and 0% relative humidity. The sample was held in such a
manner that it separated two sides of test chamber. One side was exposed to a nitro-
gen atmosphere, while the other side was exposed to an oxygen atmosphere. Testing
was completed when the concentration of oxygen in the nitrogen side was constant.

2.3.7. Surface wettability

Surface wettability of the films was evaluated as static water contact angle using an
OCA 20 contact angle analyzer (Dataphysics, Germany). A droplet of deionized water
(2 lL) was placed over the film surfaces at constant temperature (25  C). The angle
between the baseline of the drop and the tangent at the drop boundary was analyzed
by SCA20 software. Measurements were performed at 10 different locations for
each sample.

2.3.8. Size distribution, zeta potential measurement

Wet BC particle size distribution from defibrillated BC was conducted by laser dif-
fraction using a Mastersizer 3000 Laser Particle Size Analyzer (Malvern, UK) with
a Malvern Mastersizer 3000 software, under the conditions of material refractive
index 1.42, dispersant refractive index 1.33, 25  C, 2500 rpm motor speed and
0.5% (w/v) of suspensions. The results were reported as the average of triplicate
measurements.
The zeta potential of BC fibril dispersion at pH 7 was determined by dynamic
scattering (DLS) using a Zetasizer Nano-ZS instrument (Malvern, UK). Samples were
diluted to a concentration of about 0.05% (w/w) in Milli-Q water. The samples were
then placed in an electrophoresis chamber. The detection was conducted with a 4mW
He-Ne laser at 633 nm. All measurements were performed at 25  C. The zeta potential
was reported as the average of triplicate measurements.

2.3.9. Gel permeation chromatography (GPC)

Molecular weight and polydispersity of disintegrated BC were characterized by per-
meation chromatography (GPC) technique (Waters, USA), equipped with Waters
2414 RI detector and TSK gel columns using DI water as mobile phase. The operat-
ing conditions are: 30  C; flow rate of 1 ml/min; injection volume of 20 mL and the
standard curve was made with pullulan polysaccharide standards.
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 967

2.3.10. X-Ray diffraction

X-ray diffraction (XRD) patterns of the samples were determined with a D8 Discover
X-Ray diffractometer (Bruker, Germany). The operation conditions were performed
under power 40 kV and 30 mA. The diffraction angle ranged from 10 to 40 at a
scan speed of 3 /min. The crystallinity of the samples was calculated by Topas pro-
gram (version 4.2) using the following equation:

Crystalline area
100

Degree of crystallinity ð%Þ ¼
Total area ðCrystalline area þ Amorphous areaÞ

2.3.11. In vitro tests

Human keratinocyte (HaCat) and mouse fibroblast (L929) cell lines were used for in
vitro cell culture model as a direct contact test. Cells were expanded in the growth
medium composed of Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin and
0.25 mg/ml amphotericin B. Cells were grown in a humidified incubator at 37  C with
5% CO2. The medium was refreshed every 2–3 days. Cells were grown to 90% conflu-
ence and subcultured using trypsin–EDTA (Sigma Aldrich, Switzerland).
In order to study the viability of each cell type, 5
104 cells were seeded on each
film placed in 24-well plates. Glass coverslip were used as a control. The cultures
were cultivated in an incubator at 37  C with 5% CO2. The attached cell number was
evaluated by MTT assay as previously reported [21] at 24 and 48 h after seeding. The
optical densities were measured by a Multiskan EX microplate reader (Thermo
Scientific, USA) at wavelength of 570 nm.
In order to observe cell morphology on BCA and BCAGG, cells were seeded onto
the films at 5 x 104 cells/well. Cells were allowed to proliferate for 48 hrs. Films con-
taining cells were washed with PBS and fixed in 2.5 vol % fixative solution. The sam-
ples were dehydrated in serial dilutions of ethanol afterwards, and dried in an
automated Leica EM-CPD300 critical point dryer (Leica Microsystems, Austria).
Then, the samples were sputter-coated with gold and observed under a JSM-5410
Scanning Electron Microscope (JEOL, Japan).

2.3.12. Film handling characteristics

In order to test the film handling characteristics, the selected films underwent con-
ventional surgical instrumentations by using, 24 mm surgical needle, tweezers and 3.0
silk suture. All studies were assessed under wet condition in sterile normal saline for
24 h at 37  C.

2.4. Statistical analysis

All results were reported as mean ± standard deviation. Statistical analysis was
performed using Minitab 17 software (Minitab Inc., USA). The level of statistical sig-
nificance for all tests was set at P-value < 0.05.
968 N. CHIAOPRAKOBKIJ ET AL.

Figure 2. FT-IR spectra of (A) BC, (B) Alginate, (C) Gelatin, (D) BCA (60/40), (E) BCAG (60/20/20)
and (F) BCAGG (60/20/20 with glycerol).

3. Results and discussion

3.1. FT-IR analysis
Figure 2 showed the FT-IR spectra of BC, BCA, BCAG, and BCAGG compared with
those of BC, alginate, and gelatin. The BC spectrum showed bands attributed to –OH
groups at 3392 cm1, H-O-H bending of adsorbed water at 1647 cm1 and C-O-C
stretching at 1061 cm1, similar to those reported for BC [22]. For alginate, bands at
3411 cm1, 1607 cm1 and 1423 cm1 were detected, which were attributed to –OH
stretching, COO- asymmetric stretching, and COO- symmetric stretching, respectively
[23]; whereas a band at 820 cm1 was characteristic of mannuronic acid residues. In
the presence of alginate, the bands of H-O-H bending and C-O-C stretching for BCA
shifted to 1611 cm1 and 1026 cm1. The resulting shifts were similar to those of the
FTIR-Spectra for BC-alginate matrix [24]. In addition, the FTIR of BCA showed a
peak around 819 cm1, which was assigned to the characteristic absorption bands of
alginate [25]. The shifts were attributed to intermolecular interactions between cellu-
lose and alginate [26]. Gelatin showed bands at 3408 cm1 (NH-stretching),
1643 cm1 (amide I), 1535 cm1 (amide II), and 1239 cm1 (amide III). For BCAG,
the broad peak at 3400 cm1 indicated the –OH bond from the interaction between
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 969

BC, alginate, and gelatin [16]. BCAG showed bands at 1613 cm1 and 1059 cm1,
which as compared with BCA were shifted toward a higher wave number and had
lower band intensities. With the supplement of gelatin, the decreased intensity
implied decreased intramolecular bonding [27]. The band of asymmetric vibration of
–COO at 1613 cm1 and C-C stretching at 1059 cm1 are ionic binding sites. The
spectra of BCAG showed lower intensities suggesting a weaker –OH binding vibra-
tion or weaker Ca2þ binding to alginate [28]. The amino group peaks in gelatin were
not clearly visible in BCAG. This was a result of the formation of complexes between
carboxyl groups in the polysaccharides and amino groups in the protein [29]. For
BCAGG, the H–O–H bending was shifted to 1650 cm1, the COO- stretching bands
were shifted to 1431 cm1 and characteristic peaks of gelatin appeared at 1541 cm1.
The presence of glycerol intensified the characteristic gelatin bands in BCAGG. It was
previously suggested that C ¼ O and N–H bands could form intermolecular hydrogen
bonds with the –OH of glycerol [30]. The shifts in the BCAGG spectrum indicated
more intermolecular hydrogen bonding interaction between BC, alginate, gelatin, and
glycerol generated as a consequence of more available –OH groups. The formation of
complexes between BC, alginate, gelatin, and glycerol was due to multiple interac-
tions. It is thought that the anionic polysaccharide macromolecules, glycerol, and the
cationic amino groups of gelatin were tangled up via electrostatic interaction [31,32].
Our results were similar to the previous works of alginate/BC nanocrystals/collagen
composite hydrogel. It was reported that the peak of hydroxyl stretching in the
spectrum of modified hydrogel was shifted and broad, indicating the formation of
the intermolecular hydrogen bonding among composite blend [10]. The peak of
carboxyl asymmetric stretching vibration in the spectrum of the composites also
shifted and became broader compared to the alginate spectrum, which implied the
formation of polyelectrolyte complex [33]. The result of this study also corre-
sponded to the FT-IR spectra of carboxymethy cellulose/alginate/chitosan composite
films reported by Lan et al. [34]. It was suggested that hydrogen bonds increased
the degree of polarization of chemical bonds. Physical crosslinking between
hydroxyl groups of cellulose and hydroxyl groups of alginate consumed small
amounts of hydroxyl groups [35].

3.2. Water absorption capacity (WAC)

The densities of dried BC, BCA, BCAG and BCAGG films were 1.2 ± 0.2, 0.7 ± 0.2,
0.9 ± 0.2 and 1.1 ± 0.1 g/cm3, respectively. The water absorption capacity (WAC) of
the films in DI water is shown in Figure 3. The results indicated that the WAC values
increased significantly with the addition of gelatin and glycerol. The increase of WAC
for the BCAG and BCAGG films was due to the highly hydrophilic nature of gelatin.
Similar results have previously been reported [7, 23]. It was found that the water
uptake ability of alginate/gelatin blend fibers increased with gelatin content. By cross-
linking alginate with Ca2þ, in which the COO- in the alginate binds to the Ca2þ, the
alginate chains become less available to bind with H2O molecules, resulting in lower
hydrophilicity [36]. Gelatin is strongly hydrophilic and more hydrophilic than cal-
cium alginate. The hygroscopic character of glycerol also enhanced the WAC of the
970 N. CHIAOPRAKOBKIJ ET AL.

Figure 3. Water absorption capacity of BC, BCA, BCAG and BCAGG.

films. The hydroxyl groups of glycerol had a strong affinity for water molecules lead-
ing to a higher WAC [37]. The addition of gelatin and glycerol therefore enhanced
WAC of the films.

3.3. Mechanical properties

The tensile strength (TS) and elongation at break (EB) of the homogenized BC, BCA,
BCAG, and BCAGG in both the dry and wet states are shown in Figure 4(A and B),
respectively. The formation of an alginate matrix cross-linked with Ca2þ cations con-
tributed to improving the mechanical properties of the BCA, BCAG, and BCAGG
films. The addition of glycerol led to ductile behavior, resulting in a high EB in wet
conditions for BCAGG. Glycerol improved the film flexibility by increasing the inter-
molecular spacing and reducing the internal hydrogen bonding between polymer
chains making the modified films soft, flexible, and stretchable. Similar findings were
also found by Zhang et al. [38] with the study of cocoon sericin films plasticized with
glycerol. Lim et al. [39] reported that the TS and EB of plasticized gelatin films were
affected by relative humidity (RH). As RH increased, TS decreased while EB
increased. It was suggested that water behaved as a plasticizer. The effect of RH was
enhanced on plasticized films because both the water and plasticizer were capable of
plasticizing the polymers. This behavior was also observed in other films [40,41]. The
combination of cellulose nanocrystals (CNCs) and plasticizer was previously reported
to have a positive effect on EB [42,43]. CNCs presented a high surface area that was
polar and could interact strongly with a polar plasticizer (such as glycerol). This study
showed that composite films developed from homogenized BC also exhibited a plasti-
cizer effect on the mechanical properties, similar to that reported for CNCs films.
The fabricated films of BCA, BCAG and BCAGG exhibited favorable mechanical
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 971

Figure 4. Mechanical properties of BC, BCA, BCAG and BCAGG: (A) Tensile strength and (B) Percent
elongation at break.

properties for skin regeneration. Despite good water uptake, BCAG had relatively
lower mechanical properties compared to those of BCA and BCAGG. Therefore, only
BCA and BCAGG composite films were selected for the further studies.

3.4. SEM analysis

SEM overview images of surface morphology are shown in Figure 5. All films exhib-
ited good structural integrity and homogeneous morphology without phase separ-
ation. The BCAGG surface appeared rougher than the BCA surface. Ghasemlou et al.
[44] found that the roughness of soybean polysaccharide films increased with the
presence of plasticizers owing to the hydrophilicity of glycerol. They concluded that
the rough surface of the films could provide sites for water binding during moisture
absorption. A similar effect was previously reported [45,46]. Flocculation and coales-
cence during the drying process in film formation induced changes in the film struc-
ture [47]. The cross-sectional images acquired by SEM in Figure 5(F and G) show the
more compact structure of BCA compared with that of BCAGG. Some cavities were
observed for BCAGG (Figure 5(G)). Partial water and glycerol evaporation during
sample preparation for SEM analysis might also have influenced the film struc-
tures [40].
972 N. CHIAOPRAKOBKIJ ET AL.

Figure 5. SEM images of the films in dry state, surfaces views: (A) BC, (B) alginate, (C) gelatin, (D)
BCA, (E) BCAGG and cross-sectional views: (F) BCA, (G) BCAGG.

3.5. Oxygen permeability

The oxygen permeability of BCAGG was 1.1 ± 0.1 cm3/m2/day, which was slightly
higher than that of BCA (0.9 ± 0.0 cm3/m2/day). Plasticizers reduce intermolecular
forces and increase the mobility of polymer chains; therefore, the diffusion coefficients
for gas or water have been shown to rise with increasing plasticizer concentration [48].
The oxygen permeability of BCA and BCAGG was considerably close to the polyureth-
ane commercial dressing, Tegaderm. Sirvio et al. [49] reported that oxygen permeability
of Tegaderm was classified as a medium permeable dressing was around 2.7 cm3/m2/
day. According to their clinical trials, the epithelization of shallow wounds on pigs
under Tegaderm was significantly faster as compared to that on high permeable
silicone dressing (oxygen transmission rate >200cm3/m2/day). It was suggested that
high oxygen permeable dressing could lead to the loss of oxygen. Oxygen levels might
fall short for normal epidermal cells needed and led to delay wound healing [49,50].

3.6. Surface wettability

The water contact angles of the BC, BCA, and BCAGG films were 54.6 ± 3.4 , 63.4
± 2.9 , and 49.5 ± 5.6 , respectively (Figure 6(A)). The hydrophilicity of polymeric
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 973

Figure 6. (A) Water contact angle of films, (B) size dispersion of BC and BCAGG.

films is an important factor in their promotion of cell adhesion and proliferation

[51]. BCA was less hydrophilic compared with BC as calcium alginate from the cross-
linking of alginate and Ca2þ reduced the alginate hydrophilicity [23]. The addition of
gelatin and glycerol resulted in a decrease in the water contact angle indicating
enhanced surface wettability. The enhanced hydrophilicity of BCAGG was attributed
to the relatively hydrophilic gelatin and glycerol molecules. The optimal water contact
angle of material for protein and cell adhesion should be in the range of 48-62
which was considered as moderately hydrophilic surfaces [52]. Therefore, the water
contact angles of BC, BCA, and BCAGG films were in the optimal range.

3.7. Size dispersion

The size distribution of BC fibrils in the BC slurry was broad (2–300 mm), as shown
in Figure 6(B). Peak multiplicity was found for neat homogenized BC dispersion
974 N. CHIAOPRAKOBKIJ ET AL.

indicating nano-sized fractions of BC. However, only one peak was found for the
BCAGG blend. It was revealed that the initial BC fibril distribution peak was not
detected after being supplemented with alginate, gelatin, and glycerol. Aqueous dis-
persions of neat homogenized BC interacted through physical entanglement and
hydrogen bonds with small fragments of BC fibrils dispersing more freely in water.
The explanation for this observation is related to the gel-like behavior of the mixed
system. The movement of the BC nano-fibrils was impeded and stabilized in the
blend and the functional groups of the biopolymer chains in the blend system could
be joined together to create more complex and stable structures. In this study, the
size distribution of the blend system of BCAGG was uniform and the nano-sized
fragments decreased.

3.8. Zeta potential

The surface charge and stability of the suspension were evaluated by zeta potential.
Guidelines classify dispersions with zeta potential values of ±0–10, ±10–20, ±20–30,
and > ±30 mV as indications of being highly unstable, relatively stable, moderately
stable, and highly stable, respectively [53]. Typically, zeta potential values of
±30 mV reflect excellent stability of a colloidal suspension. The greater the
magnitude of the zeta potential (positive or negative), the stronger the repulsion
were, leading to a more stable suspension. The zeta potential value of aqueous sus-
pension of disintegrated BC was around -2.65 ± 1.1 mV (Figure 6(C)). This exhibited
strong aggregation. Zeta potential values for the BCA and BCAGG blends were
65.9 ± 2.8 and 55.9 ± 3.4 mV, respectively (Figure 6(D and E)). These results sup-
port the high stability of the blends, which showed no precipitation or aggregation.
The BCA slurry mixture had a higher negative zeta potential due to the contribu-
tion of repulsive force between carboxyl and hydroxyl groups of polyanionic
alginate and BC. The lower zeta potential value of the BCAGG blend was related to
the cationic molecules of gelatin. Oppositely charged macromolecules colloid could
self-assemble via electrostatic interaction [54]. Lau et al. [55] reported that gelatin
type A, which is positively charged at pH 7, could electrostatically interact with the
negatively charged carboxylic groups of manuronic and guluronic acid units
in alginate.

3.9 Gel permeation chromatography (GPC)

The average molecular weight (MW) of disintegrated BC was found to be in the
range of 83,000-194,000 Da with the polydispersity index 1.01-1.03. Mn, Mw and Mz
were 191,661, 193,757 and 195,616, respectively. The MW of disintegrated BC in
this study was considerably lower compared to those reported by Choi et al. [56], in
which the average MW of BC produced in bubble column bioreactor fell in the
range of 1,765,000 – 4,235,000 Da with the polydispersity index 1.4-1.5. The culture
conditions and disintegration method, therefore, significantly affected MW of
BC fiber.
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 975

Figure 7. XRD patterns of BC, BCA and BCAGG.

3.10. X-ray diffraction analysis

X-ray diffraction patterns of BC and BC composites are shown in Figure 7. The
crystallinities of BC, BCA and BCAGG films were 68.8%, 67.7% and 71.2%, respect-
ively. Three characteristic peaks corresponding to the crystalline structure of typical
native BC were observed in all samples [57]. For BCA film, the intensities of the
characteristic crystal peaks were lower than BC film. The addition of alginate is
expected to reduce the crystallinity of the composite film as a result of the amorphous
alginate structure that showed no crystal peaks [58]. Blend films with alginate were
less crystalline, which could be attributed to the amorphous alginate disrupting the
regular arrangement of the crystalline molecules. An increase in the diffraction inten-
sities was observed for BCAGG. Fan et al. [23] reported that gelatin increased the dif-
fraction intensities of blend films owing to the crystallinity of the close packing of
flexible gelatin molecules. The presence of glycerol has been shown to enhance the
macromolecular mobility, allowing the formation of microcrystals and recrystalliza-
tion [59]. Glycerol induced the cellulose nanocrystals to form an ordered structure,
thus crystallization in the composite films was promoted [60]. An increase in the
number of highly mobile molecules led to an increase in the crystallinity of the films
[61]. It is therefore suggested that the addition of gelatin and glycerol raised the crys-
tallinity of the BCAGG film.

3.11. In vitro tests

Assessment of cell viability was performed to determine the in vitro biocompatibility
of the films, which also gave an indication of the potential in vivo behavior. The com-
parative non-viable cell numbers on the BCA and BCAGG films are shown in Figure
8. More viable cells were found for both HaCat and L929 after continual exposure to
a BCAGG surface after 24 and 48 h. Cell proliferation on BCAGG tended to increase
over time. The morphology of HaCat and L929 at 48 h after seeding on the samples
is depicted in Figure 9. There was relatively better attachment and spreading of L929
and HaCat cells on BCAGG as compared to that on BCA (Figure 9(A–D)). The result
976 N. CHIAOPRAKOBKIJ ET AL.

Figure 8. Relative cells number (%) of BCA (䊏) and BCAGG (䊏) at 24 h, 48 h after seeding: (A)
L929 and (B) HaCat.

of the cell study revealed successful and satisfactory HaCat and L929 proliferation
on BCAGG.
Keratinocytes are found in the outermost visible layer of the skin, which is called
the epidermis. Their primary function is to protect the body from pollutants, heat,
UV radiation, and moisture loss. Direct observation revealed that HaCat cells grew to
higher densities on BCAGG compared with BCA. HaCat were found to form clusters
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 977

Figure 9. SEM images of L929 and HaCat on BCA and BCAGG at 48 h after seeding.

on BCA. Previously, a lower degree of spreading and looser cell adhesion was
observed in HaCat cultured on BC surface [62]. It was also reported that HaCat were
unevenly dispersed and showed limited adhesion across the scaffold with the addition
of alginate [63]. It was suggested that BC and alginate did not promote cell adhesion
owing to the relatively poor cell-material interaction [64]. Similar observation was
reported by Loh et al. [65] on BC/acrylic acid hydrogel, where HaCat and dermal
fibroblasts were grown with a round shape. In this study, SEM images demonstrated
that HaCat were evenly spread over BCAGG surfaces which produced a uniform
monolayer. The morphology of HaCat on BCAGG was found to be more extended
than that on BCA. Gelatin retains the crucial RGD sequence from collagen, facilitat-
ing high levels of cell adhesion. On the other hand, glycerol was reported for induc-
ing growth of myoblasts within scaffolds [66]. Modification of silk fibroin films with
glycerol also induced corneal endothelial initial adhesion and proliferation rate [67].
Therefore, the improved HaCat adhesion, spreading and proliferation on BCAGG
surface could be a result of the synergistic effect of glycerol as a plasticizer and the
gelatin component. In addition, the addition of glycerol also enhanced the flexibility
and conformity of the films.

3.12. Film handling characteristics

In this study, disintegrated BC-based films supplemented with alginate, gelatin, and
glycerol were fabricated without an organic solvent via a simple, eco-friendly and
978 N. CHIAOPRAKOBKIJ ET AL.

Figure 10. Photographs of (A) BCAGG (adhered to pig skin) under surgical tweezers and suture
needle implementation. (B) Flexibility and suturability of hydrated BCAGG.

inexpensive casting method. The BCAGG composites fulfilled a number of handling

requirement characteristics for biomedical applications. The combination of gelatin
and glycerol helped in tailoring the physical, chemical, and biological properties of
the films. The BCAGG film exhibited better flexibility, water absorption capacity, and
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 979

oxygen permeability than the other films. The BCAGG film was more ductile in both
the dry and wet states and could mimic the elastic properties of skin. In addition, the
film could help maintain moisture and prevent water evaporation. The BCAGG film
was shown to provide viable support for both L929 and HaCat cells by promoting
cell adhesion and proliferation. The BCAGG film was also easy to handle with surgi-
cal tweezers and withstood suture needle penetration (as shown in Figure 10). The
BCAGG film therefore offers significant potential for biomedical applications.

4. Conclusions
In this study disintegrated BC-based films supplemented with alginate, gelatin, and
glycerol were fabricated without organic solvent via a simple, eco-friendly and inex-
pensive casting method. The BCAGG composites fulfilled a number of handling
requirement characteristics for biomedical applications. The combination of gelatin
and glycerol helped in tailoring the physical, chemical, and biological properties of
the films. The BCAGG film exhibited better flexibility, water absorption capacity, and
oxygen permeability than the other films. The BCAGG film was more ductile in both
the dry and wet states and could mimic the elastic properties of skin. In addition, the
film could help maintain moisture and prevent water evaporation. The BCAGG film
was shown to provide a viable support for both L929 and HaCat cells by promoting
cell adhesion and proliferation. The BCAGG film was also easy to handle with surgi-
cal tweezers and withstood suture needle penetration. The BCAGG film therefore
offers significant potential for biomedical applications.

Disclosure statement
The authors declare no conflict of interest.

Funding
This research was supported by The 100th Anniversary Chulalongkorn University Fund for
Doctoral Scholarship, The 90th Anniversary of Chulalongkorn University Fund
(Ratchadaphiseksomphot Endowment Fund) and The Graduate School,
Chulalongkorn University.

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