Advances in Tissue Engineering and Regenerative Medicine
Mini Review
Open Access
In Vitro wound healing model: effects of chitosan films
loaded with gentamicin and silver sulfadiazine on the
wound filling rate
Abstract
Volume 2 Issue 3 - 2017
Currently, animals use in research experiments has been widely questioned by Animal
Protect Institutions and Organizations. Moreover, preliminary tests and scientific
validation of research hypothesis are required before in vivo tests approval. Thus,
animal testing could take a long time. For all these reasons, the development of an in
vitro test that could simulate and/or replace animal research would be very interesting.
The aim of this work was the development of an in vitro wound healing model for
evaluation of the healing potential of drug-loaded cross-linked chitosan films. MG63, a cell line derived from human osteosarcoma, was used as cell line model. The
monolayer was cultured and when 100% confluence was reached, a wound was
created using a scraper. The healing process was evaluated for 21 days and cells were
stained after 0, 7, 14 and 21 days of incubation. Wounds were treated with chitosan
film (CH), chitosan-bisulfite blocked diisocyanate cross-linked film (CH-X), chitosan
cross-linked film loaded with gentamicin (CH-X-GE) and chitosan cross-linked film
loaded with silver sulfadiazine (CH-X-SS). The effects of chitosan (CH), bisulfite
blocked diisocyanate (BBDI), gentamincin (GE) and silver sulfadiazine (SS) on the
wound healing response were microscopically analyzed by the wound filling rate.
CH and CH-X films did not show deleterious effects on cell growth compared to the
positive control (no film) during the studied period. CH-X-GE demonstrated a reduced
wound filling rate at the first week due to the burst release of gentamicin at this period.
CH-X-SS showed reduced wound filling rates during all period studied probably due
to silver cytotoxicity and its accumulation by the cells. Finally, in vitro wound healing
rates were compared to in vivo results reported in the Literature. Similar behavior was
observed, suggesting that the proposed in vitro wound healing model could potentially
replace preliminary in vivo studies.
Maria Gabriela Nogueira Campos,1,3 Henry
Ralph Rawls,2 Lucia Helena Innocentini Mei,3
Neera Satsangi4
1
Federal University of Alfenas, Brazil
Comprehensive Dentistry, University of Texas Health Science
Center at San Antonio, USA
3
Department of Materials Engineering and Bioprocess, State
University of Campinas, Brazil
4
Innovative Research Solutions, Inc., USA
2
Correspondence: Maria Gabriela Nogueira Campos, Federal
University of Alfenas, Institute of Science and Technology,
Rodovia José Aurélio Vilela, 11999, Poços de Caldas/MG, Brazil,
Tel +55-35-3697-4600, Fax + 55-35-3697-4602,
Email nogueiracamp@gmail.com
Received: April 27, 2017 | Published: May 16, 2017
Keywords: in vitro model, wound healing, wound dressing, chitosan, gentamicin,
silver sulfadiazine.
CH, chitosan film; CH-X, chitosan cross-linked film; BBDI, bisulfite blocked diisocyanate; GE, gentamicin;
SS, silver sulfadiazine; CH-X-GE, chitosan cross-linked film loaded
with gentamicin; CH-X-SS, chitosan cross-linked film loaded with
silver sulfadiazine
Abbreviations:
Introduction
Animal testing or animal research refers to the use of animals in
research experiments. Worldwide, about 100 million animals are used
annually and either killed during the experiments or subsequently
euthanized.1 Animal research is mainly carried out inside universities,
medical schools and pharmaceutical companies, although some
commercial facilities provide animal-testing services to industry.2
Groups that supports animal research affirm that it has played a
vital role in every major medical advance and that many major
developments, such as penicillin (mice), organ transplant (dogs), and
poliomyelitis vaccine (mice, monkeys) involved animal researches.3
However, the topic is controversial. Opponent groups argue that
animal testing is unnecessary, poor scientific practice, poorly
regulated, that the costs outweigh the benefits, or that animals have an
intrinsic right not to be used for experimentation.4 Nevertheless, most
scientists and governments agree that animal testing should cause as
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little suffering to animals as possible, and that animal tests should
only be performed where necessary, respecting the 3R’s principle:
Reducing, Replacement and Refinement of the use of animals in
research.5 In this context, the development of an in vitro test that could
replace preliminary animals testing or serve as pre-screening before in
vivo experiments is an interesting, current and crucial research field.
Wound healing is a complex process that involves five phases
(hemostasis, inflammation, cellular migration and proliferation,
protein synthesis and wound contraction, and remodeling), but only
three major phases are effectively considered due to overlap of phases.6
Inflammatory, proliferative, and remodeling phases are associated with
considerable complexity that involves soluble mediators, extracellular
matrix formation, and parenchymal cell migration.7 Nevertheless, the
primary goal of wound healing is timely wound closure, which is
directly related to the wound filling rate.6,8 Additional help might be
provide for wound healing in order to accelerate wound closure and/
or to improve the regenerated tissue quality. Several approaches on
wound healing treatment are currently available, such as grafts (auto,
allo and xenografts), use of donor keratinocytes, cultured epithelial
autografts, delivery of growth factors and other molecules, use of stem
cells and wound dressings.6,7 Wound dressings are most commonly
used in wounds treatment due to the limitations of grafts (donor site,
Adv Tissue Eng Regen Med Open Access. 2017;2(3):186‒189
186
© 2017 Campos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and build upon your work non-commercially.
In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on
the wound filling rate
surgical complications, rejection, graft contraction and stability of
the graft) and autologous cell culture (the time required to culture
and prepare sheets of cells for grafting limit its use).6 In addition
to its general functions, such as covering and protecting the wound
against infection, avoiding wound dehydration and allowing woundenvironment gas/fluid exchanges, an ideal wound dressing should
also serve as a matrix for cell adhesion and proliferation, as well as
a vehicle to delivery growth factors and antibiotics.6–9 There are a
number of wound dressing based on polymeric materials available on
market, and chitosan is the most used polymer for this application.6
Chitosan is a biopolymer derived from chitin, the second most
abundant polysaccharides found in nature.10 Due to its interesting
biological properties such as biocompatibility, biodegradability,
antibacterial activity and bio-adhesion, it has been widely studied
and applied as a biomaterial in the biomedical area.10,11 Biomedical
applications of chitosan involve implants, scaffolds for tissue
engineering, skin substitutes and drug delivery vehicles.12–14 To
increase the timeframe and consistency of kinetics of drug delivery
from chitosan vehicles, crosslinking might be necessary.15 Several
chemical agents are available for the crosslinking of chitosan:
glutaraldehyde, genipin, epichlorohydrin, sulfuric acid, etc.; but
glutaraldehyde is the molecule most commonly used as chitosan
cross-linker. On the other hand, there are concerns over the toxicity of
residual glutaraldehyde, which may compromise the biocompatibility
of chitosan delivery system.15 A water-soluble bisulfite blocked
diisocyanate has been recently prepared and used as crosslinking
agent for chitosan.16 This bisulfite blocked diisocyanate is devoid of
any toxic effects of the relative diisocyanate and preferably reacts
with amine groups of chitosan by urea linkage.15,16
Therefore, in this present work, we evaluated the healing potential
of chitosan and chitosan-blocked hexamethylene diisocyanate crosslinked films loaded with gentamicin and silver sulfadiazine on an in
vitro wound healing model. For the in vitro model, human MG-63
cells, a cell line derived from an osteosarcoma, were seeded in sixwell tissue culture plates (Corning Co, USA). The seeded cells were
incubated in α-MEM (Minimum Essential Media Alpha Medium) with
5% fetal bovine serum (FBS) and humidity atmosphere of 5% CO2
and 95% air at 37˚C. After cells reached confluence, wounds were
created across the surface of each well (Figure 1A) and the media were
replaced. The wounds were then microscopically examined to ensure
that cellular and extra-cellular materials were completely removed
from the wound sites (Figure 1B). At this moment, the chitosan-based
films, previously prepared by solvent evaporation technique and
sterilized using UV light (Table 1), were placed in each of the wells,
and the positive control was a well with no film. The experiment was
carried out on duplicates. The culture medium was replaced at every
two days in order to provide essentials nutrients for cellular growth.
The wound filling rate was observed for three weeks (21 days). After
0, 7, 14 and 21days of incubation, films were removed from the wells
and cells were stained with 20ml of T-blue stain for 2 minutes at 37˚C.
After staining, all media were removed and cells were washed with
1ml of 0.1M phosphate buffer solution (PBS) pH 7.4. Then, PBS was
removed and 1 ml of formalin was added. Cells were kept at 4˚C for
24hours and, after this procedure, formalin was replaced for ethanol
70%. At this moment, the wound filling rate was analyzed using a
microscope with photography apparatus. The wound filling rate was
qualitatively determined by the amount of cells that grown in each
wound site at each analyzed period (Figure 2). According to Figure 2,
CH and CH-X films did not show deleterious effects on cell growth
Copyright:
©2017 Campos et al.
187
during the studied period if compared to the positive control (no film),
suggesting that both films are biocompatible and BBDI are non-toxic
to MG-63 cells. In fact, no significant difference was observed for the
wound filling rate of these films, but stimulation on cells growth by
CH at day 7 might be suggested. Ueno et al.17 reported that chitosan
stimulated migration and proliferation of fibroblast and collagen
production in a dog wound healing model. Moreover, wound healing
experiments using mouse as model have shown that the application
of chitosan hydrogel onto an open wound induced significant wound
contraction and accelerated wound healing.18 Xu et al.19 reported the
biocompatibility of a genipin-chitosan hydrogel on a rabbit model.
In addition, glycerol phosphate-chitosan hydrogel was assessed in
experimental osteochondral joint defects in horses and did not cause
relevant clinical effects, inflammatory response or toxic effects in the
joints.20 Chitosan films were also evaluated on wound repair of horse
distal limb and they had not interfered on healing time in the equine
model,21 suggesting compatible results of in vivo wound model and
this reported in vitro wound model. On the other hand, the gentamicinloaded crosslinked chitosan film CH-X-GE showed inferior wound
filling rate when compared to positive control in the first week.
Lin et al.22 reported that application of 2% gentamicin eye drops in
porcine cornea significantly disturbed the corneal epithelial healing
rate. Cooper et al.23 investigated the cytotoxic effects of gentamincin
on human fibroblasts and keratinocytes, which play an important
role in wound healing, and found profound effects of gentamicin on
these cells. Bertolaso et al.24 reported the biochemical mechanism
underlying gentamicin cytotoxicity in OC-k3 cells. Although
gentamicin ototoxicity and neuphrotoxicity have been extensively
studied, the dose-dependent deleterious effects of gentamicin have
not been well established yet.25,26 Hancock et al.27 studied the retinal
toxicity of gentamicin in vivo and in vitro and reported reversible
toxic effects in short-term exposure to gentamicin, while irreversible
retinal damages were observed after prolonged gentamicin treatment,
suggesting besides dose-dependent, toxicity of gentamicin is also
time-dependent. After 14 and 21 days of incubation, CH-X-GE film
deleterious effects on wound filling were not observed anymore in our
in vitro wound healing model (CH-X-GE wound filling rate is similar
to positive control, CH and CH-X films). This can be attributed to
the decrease on gentamicin concentration in the media, since burst
release happened in the first week and then minimal amount of
gentamicin was controlled released by CH-X-GE films during the
following two weeks.28 Again, the proposed in vitro wound healing
model showed analogous results to those found in vivo, suggesting
a dose and time-dependent effect of gentamicin on the wound filling
rate. Contrary, silver sulfadiazine-loaded crosslinked chitosan film
CH-X-SS showed reduced wound filling rate during all the analyzed
period. Silver cytotoxicity id related to its accumulation in the body.29
Thus, the decrease on the amount released by the CH-X-SS trough
the weeks does not reduce the cytotoxicity observed at 7, 14 and 21
days of incubation. Lee et al.30 also reported the cytotoxic effect of
silver sulfadiazine on HaCaT cells and the delayed epithelialization
in an animal model. In addition, Mi et al.31 observed a marked
inhibition of 3T3 fibroblasts growth caused by 1% silver sulfadiazine
cream treatment. According to authors, fibroblasts inhibition was
significantly reduced by the use of silver sulfadiazine incorporated
to an asymmetric chitosan membrane.31 Poon et al.32 studied the
cytotoxic effects of silver on keratinocytes and fibroblasts in vitro.
According to their results, silver is highly toxic to both keratinocytes
and fibroblasts, which play an important role in wound healing
process. Moreover, the cytotoxic dosage for skin cells was found to
Citation: Campos MGN, Rawls HR, Mei LH, et al. In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on the
wound filling rate. Adv Tissue Eng Regen Med Open Access. 2017;2(3):186‒189. DOI: 10.15406/atroa.2017.02.00031
Copyright:
©2017 Campos et al.
In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on
the wound filling rate
be similar to that for bacteria, suggesting the use of silver in wound
treatments should be carefully evaluated since it could delay wound
healing instead of accelerate it.32 Several in vivo studies using human
and animal burn wounds report the side effects of silver sulfadiazine
topical treatment,33–35 which corroborate with the results found in our
in vitro wound healing model.
188
Table 1 Preparation of chitosan-based films for in vitro wound healing
response evaluation.
Sample
Film preparation/composition
CH
In a 250mL flask, 1.5g of high molecular weight chitosan,
>75% deacetylated (Sigma-Aldrich, USA) was dissolved in
100ml of 1.0% acetic acid solution. After total dissolution,
the resulted solution was cast in the molds and the
films were obtained by solvent evaporation at room
temperature.
CH-X
In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15
was added to 100ml of 1.5% chitosan solution and heated
up to 40˚C under stirring for 24 h. The cross-linked
solution was cast in the molds and the films were obtained
by solvent evaporation at room temperature.
CH-X-GE
In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15
was added to 100 ml of 1.5% chitosan solution and heated
up to 40˚C under stirring for crosslinking. After 24h, 1.0%
gentamicin sulfate (w/v) was added and the solution was
homogenized under stirring for another 24h. Then, the
solution was cast in the molds and the films were obtained
by solvent evaporation at room temperature.
CH-X-SS
In a 250mL flask, 0.8g of blocked bisulfite diisocyanate15
was added to 100ml of 1.5% chitosan solution and heated
up to 40˚C under stirring for crosslinking. After 24h, 5.0%
silver sulfadiazine (w/v) were added and the solution was
homogenized under stirring for another 24h. Then, the
solution was cast in the molds and the films were obtained
by solvent evaporation at room temperature.
A. Cells being removed by a scraper in order to create a wound
*All films were sterilized by exposure to UV light for 2
hours each side.
CH, chitosan film; CH-X, chitosan cross-linked film; CH-X-GE, chitosan crosslinked film loaded with gentamicin; CH-X-SS, chitosan cross-linked film loaded
with silver sulfadiazine.
B. In vitro wound created after cell removal using a scraper (day 0). Cells
are stained, while the wound site is not.
Figure 1 In vitro wound healing model apparatus.
Conclusion
In vitro wound healing model results have shown to be consistent to
those obtained in animal studies reported in the Literature. Therefore,
it could potentially replace preliminary animal testing and/or serve as
pre-screening before in vivo experiments, collaborating to the 3R’s
principle of reduction, replacement and refinement of animal tests.
Acknowledgements
The authors thank CAPES and CNPq for financial support.
Conflict of interest
The author declares no conflict of interest.
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Citation: Campos MGN, Rawls HR, Mei LH, et al. In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on the
wound filling rate. Adv Tissue Eng Regen Med Open Access. 2017;2(3):186‒189. DOI: 10.15406/atroa.2017.02.00031
In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on
the wound filling rate
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©2017 Campos et al.
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Citation: Campos MGN, Rawls HR, Mei LH, et al. In Vitro wound healing model: effects of chitosan films loaded with gentamicin and silver sulfadiazine on the
wound filling rate. Adv Tissue Eng Regen Med Open Access. 2017;2(3):186‒189. DOI: 10.15406/atroa.2017.02.00031