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Abstract
Bacterial cellulose (BC) is a polysaccharide known as a suitable matrix for
proper wound healing. To improve this ability, BC was functionalized with silk
sericin (SS) that has cytoprotective and mitogenic effects. The composites
obtained by solution impregnation were stabilized by hydrogen bonds, and SS
could be released in a controlled manner. The constructs were highly porous with
interconnected pores allowing for high water uptake that varied with the SS
concentration used for sample preparation. While SS did not disrupt the stability
of the BC network, soluble SS diffusing from the composites did not influence
keratinocyte growth but enhanced fibroblast proliferation, which would further
optimize the wound healing process and improve extracellular matrix production,
accelerating healing. Further, improved cell viability was observed upon the
composites. Because of their attractive structure and properties, these BC−SS
biomaterials represent potential candidates not only for wound dressing
applications but also for tissue engineering.
INTRODUCTION
Wound dressing is a biomaterial designed to create a suitable
microenvironment for cell adhesion and proper proliferation in order to restore the
physiological and structural properties of wounded skin. It controls cell behavior
by regulating physical, chemical, and biological signals through its surface
microarchitecture. Whether used as temporary coverage or meant for chronic
applications, wound dressings are of capital importance especially for the
treatment of large wounds and may be critical if not life-saving in certain cases
such as extensive burns. In fact, according to the World Health Organization, over
300 000 people die of burn injuries, making skin wounds a major burden both
socially and financially. This high rate is due in part to poor skin healing ability,
additionally to the limitations of current treatments, mostly the organ donor
limitation. Therefore, despite the numerous wound dressing methods available to
date, there is still a pressing need for improved ones, as the former only meet
part of the requirements for an ideal wound dressing. The ideal wound dressing
as currently described represents an effective barrier to micro-organisms, which
absorbs wound exudate and allows gas exchange, while keeping the necessary
moisture at the wound interface without toxicity, allergenic responses, and can
be removed from the wound with minimum pain. This should be made from
readily available and affordable materials that require minimum processing. In
this context, nanofibrous materials, which have attracted significant interest as
scaffolds in current tissue engineering strategies aiming for the repair of various
tissues including skin, have shown to improve biocompatibility. Becaise of their
high porosity, they enable cell adhesion, growth, and differentiation while further
allowing for the delivery of bioactive molecules.
In recent years, bacterial cellulose (BC), a natural polysaccharide, has
shown great potential as a biomaterial for tissue engineering applications
including skin tissue repair. Because of its nanofibrillar structure, BC represents
an adequate matrix for optimal wound healing. In fact, similarity with collagenous
fibers facilitates the interaction between cells and BC fibers, improving its overall
biocompatibility. It is biodegradable and offers good mechanical properties and
high hydrophilicity, all of which contribute to its success in tissue engineering.
Conversely, silk sericin (SS) is a natural hydrophilic protein and a
byproduct of the silk industry that biomedical properties have been well
elucidated. It has cytoprotective and mitogenic effects on mammalian cells with
regenerative abilities on mammalian tissues; particularly, its positive effects on
fibroblasts and keratinocytes, major cell types in skin, has made it very attractive
for the development of skin tissue repair materials. In addition to its potential for
enhancing collagen production and accelerating wound healing, SS has shown
antimicrobial properties necessary for a wound dressing. Nevertheless, due to its
amorphous nature, SS only forms fragile materials unsuitable for biomedical
applications. Interestingly, it has polar side chains with diverse functional groups
(amine, hydroxyl, carboxyl groups) that allow interaction with other compounds
through blending, crosslinking, or copolymerization to yield improved
biomaterials.
This study aims at developing an improved wound dressing material which
would accelerate wound healing, with BC as the niche for optimal wound healing,
and SS as the bioactive molecule that regulates cell behavior and accelerates the
healing process thereby reducing scar formation.
EXPERIMENTAL SECTION
Materials. Bombyx mori cocoons from Ningbo Industrial Co., Ltd. (China)
were used. The bacterial strain Acetobacter xylinum (ATCC53582) was
purchased from the American Type Culture Collection (ATCC), while the NIH-
3T3 and HaCaT cell lines were obtained from China Infrastructure of Cell Line
Resources. Yeast extract and peptone were provided from Beijing Shuangxuan
Microbe Culture Medium Products Factory (China) with all other reagents of
chemical grade except for poly(ethylene glycol) (PEG, Biosharp, China)
purchased from Sigma. Dulbecco modified eagle medium (DMEM), fetal bovine
serum (FBS), and trypsin-EDTA (TE) were products of Gibco.
Penicillin/streptomycin mixture was obtained from Beyotime (China). Phosphate-
buffered saline (PBS) solution was supplied from Hyclone, MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide) powder from Sigma, and
alamarBlue from Thermo Fisher Scientific. Fluorescein isothiocyanate labeled
phalloidin (FITC-Phalloidin) was provided from Sigma, Hoechst 33342 from
Invitrogen (Life Technologies), formaldehyde from Solarbio (Beijing, China), and
triton X-100 from Amresco. The BCA (bicinchoninic acid) kit was purchased from
Pierce, Thermoscientific.
Preparation of Bacterial Cellulose Films. BC was produced in Hestrin &
Schramm (HS) medium from Acetobacter xylinum grown in static cultures at 30
°C. The HS medium was composed as follows: citric acid (0.15%, w/v), disodium
phosphate (0.27%, w/v), yeast extract (0.5%, w/v), peptone (0.5%, w/v), and
glucose (2%, w/v). The films were allowed to grow to a thickness of 2−3 mm after
which they were harvested and subjected to cleaning steps to remove the
bacteria and the adsorbed HS medium. They were washed for 3 days in distilled
water, then boiled in NaOH (1 wt %) for 40−45 min, and subsequently washed in
purified water to neutralize the pH. The clean BC membranes were autoclaved in
purified water and stored at 4 °C for further use. For different experimental
purposes, various BC sizes were obtained from different sizes culture flasks or
polystyrene cell culture vessels.
Extraction of Silk Sericin from Bombyx mori Cocoons and
Preparation of BC−SS Composites. SS was extracted from Bombyx mori
cocoons by a method described by Lee et al. with slight modification. The
cocoons were cut into small pieces and boiled under pressure at 120 °C for 1 h
in purified water (1 g of cocoon/10 mL water). Fibroin fibers were removed from
the resulting soup by filtration followed by centrifugation. SS solution was then
concentrated by dialysis against PEG solution and lyophilized. The powder was
stored at −20 °C until use. The absorbance profile of the extract between 700 and
190 nm was scanned by UV spectroscopy (UV-1600 PC spectrophotometer,
MAPADA, coupled to “Mwave − Professional 2” software), from a solution sample
constituted as described further in this section.
BC−SS composites were prepared by solution impregnation as depicted
in Figure 1. To obtain SS solution, SS powder was resuspended in purified water
and dissolved by boiling for 20 min.30 The solution was further filtered through
Minisart high flow syringe filters (pore size 0.2 μm, Sartorius Stedim) for the
removal of particulate impurities. Sterile filters were used when sterilization was
required. Different constructs (BC-SS1:24, BC-SS2:24, BC-SS3:24) were thus
fabricated by varying SS concentration (1, 2, and 3% (w/v), respectively).
STATISTICAL ANALYSIS
For comparison among groups, One-way Analysis of Variance (ANOVA)
test was used with Tukey’s Multiple Comparison test for the posthoc pairwise
comparisons in OriginPro 8 software. Statistical significance was obtained at P <
0.05.
RESULTS AND DISCUSSION
Characteristics of Hot Water-Extracted SS. For the purpose of this
study, SS was extracted from Bombyx mori silk cocoons in water, by high
pressure and temperature technique (Figure 1). Contrary to the enzymatic and
chemical extractions, the hot water extraction produces purer SS protein devoid
of impurities and byproducts that may be derived from sample processing when
using enzymes or chemicals. Additionally, this method has the advantage of
yielding higher methionine and cysteine contents in SS, two amino acids that
support the most important aspects of wound healing, that is, cell growth and
collagen production. On the basis of the ability of proteinaceous compounds to
absorb UV light due to peptide bonds (absorb between 215 and 230 nm) and
aromatic amino acids (absorb between 260 and 290 nm), UV spectroscopy was
employed to characterize the SS extract. Two absorption peaks at 276 and 216
nm were distinguished in the absorbance profile of the sample (Figure 2b),
corresponding to aromatic amino acids and peptide bonds, respectively. Similar
to previous reports, peptide bonds presented the highest absorbance, confirming
the minor representation of aromatic amino acids in SS. Interestingly, the
absorption peak for aromatic amino acids was registered at a wavelength very
close to that reported for a commercial SS sample (275.4 nm), ensuring the good
quality of the SS sample.
Figure 2. Sample characterization by FTIR (a) and UV spectrophotometry (b) and release profile
of SS from the composites scaffolds (c). n = 3.
Figure 4. Physical properties of the samples. (a) = TGA, (b) = water uptake ability; (c) = Young’s
modulus; (d) = stress−strain curve by tensile testing. n = 3.
Because physical stability is one of the leading criteria in the choice of a
material for biomedical applications particularly tissue engineering, the scaffolds
were further subjected to mechanical testing, namely, tensile testing. The
stress−strain curves and the Young’s modulus of the samples are shown in
Figure 4d,c, respectively. Unsurprisingly, BC exhibited the highest mechanical
properties, which were subsequently slightly altered upon SS incorporation.
Because of its amorphous nature, SS is a material devoid of mechanical strength
often leading to the decrease of the overall mechanical strength of the composite
after blending with polymers of higher mechanical stability such as BC, as
reported for SS-collagen composite films. Nevertheless, the Young’s modulus of
BC samples was found not significantly higher when compared with the
composites’ (Figure 4c).
Swelling Ability. Figure 4b represents the profiles of water uptake ability
measured from the scaffolds in distilled water. Similar trends were observed in all
samples, with a rapid swelling after 6 h and a decreasing swelling ratio with higher
SS content, as reported for previously developed SS-carboxymethyl cellulose
composites. As suggested by the FTIR spectra, with increased protein content,
the incorporation of SS between the molecular chains of BC leads to a weakening
of the hydrogen bonds within the network, diminishing the ability of the latter to
lock water molecules. Moreover, as discussed in the previous section, increased
SS amounts result in decreased sample porosity, restricting the free space
available for water uptake. Hence, by varying the SS concentration, different
levels of swelling ratio can be obtained, which would be interesting for the
treatment of wounds with different exudate levels. Overall, all samples
demonstrated high swelling abilities, as the smallest swelling ratio (22.92 in BC-
SS3:24 samples) represented more than 20-fold the weight of the dry samples
and remained several folds higher than the values reported elsewhere for
potential material candidates for biomedical and tissue engineering applications.
Thus, consistently with the SEM results, all scaffolds remained highly porous after
incorporation of SS, allowing for the high swelling observed in water.
Effects of BC−SS Composites on Cell Behavior. To test the
biocompatibility of the samples, NIH-3T3 fibroblast cell line was cultured in the
samples extracts, using normal culture media as control (Figure 5a). Fibroblasts
are key players in wound healing. Aside from the production of extracellular
matrix proteins (mainly collagen necessary for wound closure), they coordinate
the phases of wound healing by producing cytokines and growth factors. In our
study, in all samples no cytotoxicity was detected at either D1 or D3. Furthermore,
the extracts obtained from the composites significantly enhanced the cell viability
when compared with the control at D1. Similarly, higher cell viability was observed
for the cells cultured in composites’ extracts at D3 in comparison with those
cultured in normal culture media with a significant increase for the BCSS3:24
samples. Importantly, the cell viability with the composites’ extracts was
enhanced relatively to BC at D1 with a very significant difference at D3. A dose-
dependent effect was observed, based on the concentration of SS solution used
for composite preparation. Additionally, there was no significant difference
between the effects of BC extracts and normal cell culture media. These data
thus suggest that the positive effect of BC−SS samples’ extracts on fibroblast cell
viability resulted from soluble SS released from the scaffolds, which promoted
cell proliferation without inducing any cytotoxicity. Hence, beneficial behaviors
may be expected from these composites both in in vitro and in vivo applications,
as active SS molecules would diffuse into the milieu, assisting cell growth thanks
to sericin’ s cytoprotective and mitogenic abilities. When the extracts were tested
on keratinocytes (Figure 5c), higher cell viability was observed with the
composites relatively to pristine BC at D1, which was found statistically
insignificant except for the cells cultured in extract from BC-SS3:24 samples.
Moreover, at D3 no significant difference was noted between scaffolds. Hence,
in accordance with the research by Akturket al., SS did not have a significant
effect upon keratinocyte cell growth. However, interestingly, the cell viability with
all scaffolds’ extracts was found significantly higher as compared to that with
normal cell culture media, indicating a good biocompatibility.
Figure 5. In vitro biocompatibility of the constructs. (a,c) Viability of fibroblasts and keratinocytes
cultured in samples’ extracts, respectively: MTT assay. (b,d) Viability of fibroblasts and
keratinocytes cultured onto the scaffolds, respectively: alamarBlue assay. (*) Comparison
between scaffolds and control (PS) for the same time-point; (#) comparison of the composites’
extracts with BC extract for the same time-point; (+) comparison with D3, for scaffolds. (***) P <
0.001; (**) P < 0.01; (*) P < 0.05; (###) P < 0.001; (##) P < 0.01; (#) P < 0.05. n = 3.
Figure 6. Confocal imaging of NIH-3T3 cells cultured onto the scaffolds (10 000 cells each) for 3
days. (Green, actin filaments; blue: nuclei, scale bar, 100 μm).
Figure 7. Confocal imaging of HaCaT cells cultured onto the scaffolds (10 000
cells each) for 7 days. (Green, actin filaments; blue, nuclei; scale bar, 100 μm).
CONCLUSIONS
In this study, bacterial cellulose−silk sericin composite biomaterials were
successfully obtained by solution impregnation method without cross-linking. A
good compatibility was observed between BC and SS, as the latter integrated
perfectly the BC network, covering the random fibers and maintaining the native
BC’s nonwoven fiber structure that is attractive for wound dressing application.
Consequently, no significant alteration was observed in the thermal and
mechanical stability of the BC network upon SS incorporation. Within the
composites, the two macromolecules were stabilized by hydrogen bonding, as
shown by the FTIR spectra. All structures were highly porous and presented
interconnected pores necessary for gas exchange and absorbing of wound
exudate. Besides, SS has the ability to enhance oxygen permeability, which is an
important factor for proper wound healing. These structures may also be good
candidates for tissue engineering applications, as they would enable easy
nutrient and metabolites circulation, enhancing the overall cell viability. The
swelling ability of the constructs varied with the SS content, with high capacity for
water absorption in all scaffolds. Thus, they may be used for different types of
wounds according to their exudate level. The SS release assay confirmed the
stability of the samples, as SS effectively remained in the structures while being
released in a SS content (initial SS concentration used for the preparation of the
composite)- dependent manner. Thereby, the released SS whose amount can be
controlled would interact with cells and exercise its benefial biological effects,
mainly its mitogenic effect, thus accelerating the healing process and resulting in
reduced scar formation. This was further confirmed in vitro, as extract solutions
from the composites significantly enhanced the viability of fibroblast cells, in
comparison with both BC and normal cell culture media. However, the viability of
keratinocytes was not significantly influenced by the presence of SS. AlamarBlue
assay performed on cells cultured directly onto the scaffolds indicated no
difference between pristine BC and BC−SS composites, while inferring the need
for longer time periods to effectively allow fibroblast cell profileration upon the
constructs in the experimental conditions employed in this work. Overall, while
further in vivo investigations are required to fully validate these materials, it can
be concluded that they represent potential candidates for wound healing and
tissue engineering applications.
ABBREVIATIONS
BC: bacterial cellulose; SS: silk sericin; HS medium: Hestrin and Schramm
medium; PEG: poly(ethylene glycol); SEM: scanning electron microscope; FTIR:
Fourier transform infrared; PBS: phosphate-buffered saline; TGA:
thermogravimetric analysis; BCA: bicinchoninic acid; BET: Brunauer−
Emmett−Teller; Ws: swollen weight; Wd: dry weight; ATCC: American Type
Culture Collection; DMEM: Dulbecco’s Modified Eagle Medium; FBS: fetal bovine
serum; EDTA: ethylene diamine tetra acetic acid; MTT: 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl tetrazolium bromide; DMSO: dimethyl sulfoxide; OD: optical
density; PS: polystyrene cell culture plate; CS: coverslips; FITC: Fluorescein
isothiocyanate.