Open Journal of Stomatology, 2013, 3, 22-30 OJST
http://dx.doi.org/10.4236/ojst.2013.39A004 Published Online December 2013 (http://www.scirp.org/journal/ojst/)
Insights into chitosan based gels as functional restorative
biomaterials prototypes: In vitro approach
Victoria Tamara Perchyonok1, Shengmiao Zhang2, Nicolaas J. Basson3, Sias Grobler3,
Theunis Oberholzer1, Ward Massey1
1School of Dentistry and Oral Health, Griffith University, Gold Coast, Australia
2School of Material Science and Engineering, East China University of Science and Technology, Shanghai, China
3Oral and Dental Research Institute, Faculty of Dentistry, University of the Western Cape, Cape Town, South Africa
Received 7 September 2013; revised 23 October 2013; accepted 11 November 2013
Copyright © 2013 Victoria Tamara Perchyonok et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
Restorative materials in the new era aim to be “bio-
active” and long-lasting. The purpose of the study
was to design and evaluate novel chitosan hydrogels
containing melatonin and/or propolis (antioxidant
containing material), nystatin (antifungal), naproxen
(pain relieve medication) and combinations thereof
(chitosan-H-melatonin, chitosan-H-melatonin-na-
proxen, chitosan-H-propolis, chitosan-H-propolis-
naproxen, chitosan-H-naproxen-propolis-melatonin,
Chitosan/Propolis/Nystatin, Chitosan/Melatonin/Pro-
polis/Nystatin, Chitosan/Propolis/BSA/Nystatin, Chi-
tosan/Melatonin/BSA/Nystatin) as functional additive
prototypes for further development of “dual function
restorative materials”, to determine their effect on
the dentin bond strength of a composite, to evaluate
stability of the encapsulated antioxidants as well as
evaluate antimicrobial properties of the selected
group of “designer” functional materials. Materials and
Methods: The above mentioned hydrogels were pre-
pared by dispersion of the corresponding component
in glycerol and acetic acid with the addition of chito-
san gelling agent. The surface morphology (SEM),
drug-polymer solid state interaction (FT-IR spectro-
scopy), released behaviours (physiological pH and
also in acidic conditions), stability of the therapeutic
agent-antioxidant-chitosan and the effect of the hy-
drogels on the shear bond strength of dentin were
also evaluated. Results: The release of naproxen con-
fers the added benefit of synergistic action of a func-
tional therapeutic delivery when comparing the newly
designed chitosan-based hydrogel restorative materials
to the commercially available products alone. Neither
the release of naproxen or the antioxidant stability
was affected by storage over a 6-month period. The hy -
drogel formulations have a uniform distribution of
drug content, homogenous texture and yellow colour
(SEM study). All chitosan dentin treated hydrogels
gave significantly (P < 0.05; non-parametric ANOVA
test) higher shear bond values (P < 0.05) than dentin
treated or not treated with phosphoric acid. Conclu-
sion: The added benefits of the chitosan treated hy-
drogels involved a positive influence on the naproxen
release as well as increased dentin bond strength as
well as demonstrating good antimicrobial properties
and enhanced antioxidant stability. The therapeutic
polymer approach described here has a potential to
provide clinical benefit, through the use of “designer”
adhesive restorative materials with the desired prop-
erties.
Keywords: Therapeutic Polymers; Adhesives; Chitosan,
Hydrogels; Propolis; Melatonin; Naproxen;
Antimicrobial; Dentin Bonding; Antioxidants; Bioactive
1. INTRODUCTION
The field of “functional designer dental materials for
restorative treatment” is currently a hot topic in dentistry.
The properties such as mechanical, physical and bonding
properties have been greatly improved and modified and
now represent a significant arsenal available to a front
row researchers as well as dental practitioners as many of
recent products on the market exhibit excellent/accept-
able clinical performance [1,2]. Accordingly, it is pro-
posed that innovation of restorative materials in the new
era could be directed toward a new dimension: develop-
ment of materials with “bio-active functions” to provide

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 23
therapeutic effects. The one bio-active function proposed
for restorative materials, antibacterial activity can be
highlighted for the restorative treatment of caries [3].
The acid-etch technique, introduced by Buonocore in
1955, was seminal and opened the doors to the possibili-
ties of achieving a bond to natural tooth substrates with
artificial acrylic-based restoratives [4]. Whilst bonding to
enamel has changed little since its inception more than
half a century ago, bonding to dentin has proved far more
elusive, undergoing enormous changes. A major ad-
vancement for achieving a sustainable bond to dentin
was the introduction of the total-etch technique in the
late seventies [5,6]. The SE primers not only simplified
bonding to dentin, but also eliminated the clinical errors
associated with this exacting procedure. Bio-adhesive
polymers appear to be particularly attractive for the de-
velopment of alternative etch-free dentin bonding system
with an added advantage of additional therapeutic deliv-
ery systems to improve intra-dental administration of
therapeutic and prophylactic agents, if necessary [7].
Chitosan, which is a biologically safe biopolymer, has
been proposed as a bio-adhesive polymer and are of con-
tinuous interest due to their unique properties and flexi-
bility in a broad range of oral applications as reported
recently [7,8].
Null Hypothesis
The purpose of the study was firstly to design and evalu-
ate (surface morphology, stability, swelling, release be-
haviour) novel chitosan hydrogels containing propolis
(anti-oxidant containing material and natural antimicro-
bial agent), melatonin (antioxidant), naproxen (pain re-
lieve medication and free radical scavengers) and com-
binations thereof (chitosan-H-propolis, chitosan-H-pro-
polis-naproxen, chitosan-H-naproxen, chitosan-H-napro-
xen-nystatin, chitosan-H-melatonin-naproxen and chito-
san-H-melatonin) as functional additive prototypes for fur-
ther development of “dual function restorative materials”
such as the designer material which is able to improve
the adhesion to the tooth structure as well as provide
additional therapeutic property. The ability to control
bacteria would be advantageous to eliminate the risk of
further demineralization and cavitation, since dental car-
ies is an infectious disease and eradication of cariogenic
bacteria is the important principle. Secondly it is to de-
termine their effect on the dentin bond strength of a
composite and thirdly to evaluate some antimicrobial
properties of this functional biomaterials.
2. MATERIALS AND METHODS
Propolis (Aurora Pharmaceuticals, Australia), melatonin
(Now Food, Australia) nystatin, bovine serum albumin,
naproxen (Sigma, USA) were used as received. Chitosan
(Aldrich, Australia), glycerol (Sigma, USA), glacial ace-
tic acid (E. Merck, Germany) were used as received. The
degree of deacetylation of typical commercial chitosan
used in this study is 87%. Chitosan with molecular
weight 2.5 × 103 KD was used in the study. The isoelec-
tric point is 4.0 - 5.0.
2.1. Preparation of Various Naproxen
Containing Hydrogels
The naproxen containing gel was prepared by dispersion
of naproxen powder 0.3 gm in glycerol (5% w/w) using a
mortar and a pestle. Ten milliliters of glacial acetic acid
(3% w/w) was then added with continuous mixing and
finally chitosan polymer was spread on the surface of the
dispersion and mixed well to form the required gel. The
strength of the prepared gel (10 gm) is 0.3 g of naproxen
in each gm of the base. Naproxen gel had been prepared
with three different concentrations of chitosan gelling
agent (5%, 6% and 7% w/w). The summary of the newly
prepared materials is highlighted in Table 1.
Where P is propolis as additive, Nap is naproxen as
selective additive, M is melatonin as selective additive.
PB = propolis: BSA (1:1), MB = melatonin: BSA (1:1)
Hydrogels containing chitosan in different % are synthe-
sized and characterized.
2.1.1. Determination of Gel pH
One gram of the prepared gels was accurately weighed
and dispersed in 10 ml of purified water. The pH of the
dispersions was measured using pH meter (HANNA in-
struments, HI8417, Portugal).
2.1.2. Morpho l og y of the Ge l s
The samples were prepared by freezing in liquid nitrogen
for 10 min, and then were freezing-dried for 24 h. The
prepared samples were fractured in liquid nitrogen using
a razor blade. The fractured samples were attached to
metal stubs, and sputter coated with gold under vacuum
for SEM. The interior and the surface morphology were
observed in scanning electron microscope (SEM, Hitachi
S4800, Japan).
2.1.3. FTIR of the Gels
The samples were freeze-dried for 72 h, then the FTIR
spectral of the sample was recorded on an Infrared Spec-
trophotometer (Nicolet 5700). The IR-spectra of the pel-
lets were recorded from 400 - 4000 cm−1 taking air as a
reference.
2.1.4. Gel Stability
Stability of the gel formulations was also investigated.
The organoleptic properties (color, odor), pH, drug con-
ent, and release profiles of the gels stored at 20˚C were t

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 24
Table 1. Gel formulation prepared in the study.
Gel formulation Chitosan (w/w%) Naproxen (w/w%) or
Nystatin (w/w%) Melatonin (w/w%) Propolis (w/w%) pH
Chitosan-H Gel-1 5 0 0 0 5.20
Chitosan-H1 Gel-2 6 0 0 0 5.70
Chitosan-H2 Gel-3 7 0 0 0 5.17
Chitosan-H-Nap1 Gel-4 5 1 0 0 5.25
Chitosan-H-Nap2 Gel-5 6 1 0 0 5.65
Chitosan-H-Nap3 Gel-6 7 1 0 0 5.84
Chitosan-H-A1P1 Gel-7 5 1 0 1 5.24
Chitosan-H-M1N1 Gel-8 5 1 1 0 5.13
Chitosan-H-M2N2 Gel-9 5 1 1 0 5.20
Chitosan-H-M1A1N1 Gel-10 5 1 1 1 5.00
Chitosan-H-P Gel-11 5 1 (Nystatin) 0 1 5.34
Chitosan-H-M Gel-12 5 1 Nystatin 1 0 5.62
Chitosan-H-PBN Gel-13 5 1 (Nystatin) 1 propolis:BSA 0 5.78
Chitosan-H-MBN Gel-14 5 1 (Nystatin) 0 1: 1 melatonin:BSA6.21
examined on days (0, 15, and 30).
2.1.5. Studies of Equilibrium Swelling in the
Alternative Drug Delivery Systems
The known weight naproxen-containing dry gels and
naproxen-containing dry gels were immersed in pH 4.0,
pH 9.0 buffer solutions, respectively, and kept at 25˚C
for 48 h until equilibrium of swelling had been reached.
The swollen gels were taken out and immediately
weighed with microbalance after the excess of water
lying on the surfaces was absorbed with a filter paper.
The equilibrium swelling ratio (SR) was calculated using
the following equation:

SR 100%
sdd
WWW
where Ws and Wd are the weights of the gels at the equi-
librium swelling state and at the dry state, respectively
[9]. Experiments were repeated 6 times for each gel
specimen and mean value was obtained.
2.1.6. In Vitro Study of Napr o xen Release Profi l e
The release study was carried out with USP dissolution
apparatus type 1, Copley U.K., slightly modified in order
to overcome the small volume of the dissolution medium,
by using 100 ml beakers instead of the jars. The basket
of the dissolution apparatus (2.5 cm in diameter) was
filled with 1 gm of naproxen gel on a filter paper. The
basket was immersed to about 1 cm of its surface in 50
ml of phosphate buffer pH 6.8, at 37˚C ± 0.5˚C and 100
rpm [10]. Samples (2 ml) were collected at 0.25, 1, 2, 3,
4, 5, 6, 7 and 8 hours [11] and were analyzed spectro-
photometrically by U.V. Spectrophotometer (Cintra 5,
GBC Scientific equipment, Australia) at The UV-vis
absorption spectrum of naproxen in water is typical of a
2-substituted naphthalene compound, presenting a three
band system centred around 220 nm, 240 - 280 nm and
310 - 330 nm [12]. Each sample was replaced by the
same volume of phosphate buffer pH 6.8 to maintain its
constant volume and sink condition [13].
2.1.7. Cupric Ions ( Cu2+) Reducing Power and
Antioxidant Strength Assay and Stability
Measure for Microencapsulation
In order to further measure the reducing ability of nega-
tive control (35% hydrogen peroxide solution and
CuSO4), melatonin, chitosan:melatonin (1:1), propolis
and propolis:chitosan (1:1) the cupric ions (Cu2+) re-
ducing power capacity was used with slight modification
[14]. Briefly, 250 μL of 37.5% hydrogen peroxide solu-
tion and CuSO4 and 250 μL CH3COONH4 buffer solu-
tion (100 mmol/L, pH 7.0) were added to a test vial con-
taining a negative control (35% hydrogen peroxide solu-
tion and CuSO4), melatonin and propolis sample as well
as chitosan complexes of the melatonin and propolis
(250 μL). Then, the total volume was adjusted with the
buffer to 2 mL and mixed vigorously. Absorbance
against a buffer blank was measured at 568 nm at 20
minutes intervals for the total time of 2 hours. Increased

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 25
absorbance of Cu+ complex in the reaction mixture indi-
cates increased reduction capability. Trolox (water solu-
ble vitamin E) was used as the positive controls. The
results of the investigation are summarised in the graph 2.
Absorbance was measured using POLARstar Omega
Multifunction Microplate Reader (BMG LABTECH,
Spectral range: 220 - 850 nm). 24 well plates used in the
investigations are Corning Incorporated Castar 3524, 24
well cell culture cluster flat bottom with lid, Non-pyro-
genic, Polystyrene, sterile plates (Corning Incorporated
Corning, NY, 14831, USA).
Further studies were conducted to evaluate and quan-
tify the antioxidant potential of melatonin and melatonin:
chitosan, propolis and propolis:chitosan for the purpose
of determining the stability of their activity and also cor-
relating the micro-encapsulating influence of the chito-
san on stability and efficacy of corresponding antioxi-
dants.
2.1.8. In Vitro Antifungal Activity of the Newly
Prepared Hydrogels
A type strain of Candida albicans strain NCPF 3153 was
obtained from the Health Protection Agency Culture
Collections, Salisbury, UK. The yeast was sub-cultured
and maintained on Sabouraud dextrose agar. The effec-
tiveness of the prepared nystatin gel against Candida
albicans was measured using the standard Kirby-Bauer
agar diffusion method. Five to six mm deep Muller-
Hinton agar plates were inoculated by streaking a stan-
dardized inoculum suspension containing 107 - 108 col-
ony forming units with a standard throat cotton swab.
Hydrogels were dissolved 50 mg/100 µl in sterile dis-
tilled water and 10 µl were used to impregnated 6 mm
diameter paper disks. The paper discs were placed on the
Muller-Hinton agar medium and incubated at 37˚C for
24 hours. The effectiveness of the prepared gel was
compared with chitosan gel containing 0% of nystatin
and an antibiotic sensitivity disc (Mast Laboratories,
Merseyside UL) containing 100 I.U. of nystatin per disc.
The diameter of the zones of growth inhibition was
measured with a caliper. Each type of the samples was
tested in triplicate.
2.1.9. Shear Bond Strength Tests for Dentin Bonding
Extracted non-carious, intact, human molars stored in
water containing a few crystals of thymol at 4˚C were
used within two months. Samples were checked before
use for any damage caused by their removal. The roots of
the teeth were removed with a separating disc and the
occlusal enamel removed by grounding wet on 60-grit
silicon carbide (SiC) paper. The teeth were embedded in
PVC (Consjit Tubing, SA PVC, JHB, RSA) pipe con-
tainers with cold cure acrylic resin so that the grounded
occlusal surfaces projected well above the resin. The 10
mm length pipes were put on a glass surface with one
end blocked by the glass and the embedding done
through the open end. Immediately after embedding the
occlusal surfaces were ground wet with 180-grit fol-
lowed by 600-grit SiC on a polishing machine to expose
the superficial dentin. The samples were washed under a
stream of tap water. A standardized zig (Ultradent ISO
A2-70) with an internal diameter of 2.5 mm and height
of 3 mm was used to shape the composite resin stud
(SDR, Dentsply, CA, USA, Batch number 1105000609,
Exp 2013-04). Two of these studs were then bonded to
the polished dentin surface of each tooth via the bonding
agent XP bond (Dentsply, New York, USA), as sug-
gested by the manufacturer. The bonding agent con-
tained: carboxylic acid modified dimethacrylate (TCB
resin), phosphoric acid modified acrylate resin (PENTA),
urethane dimetacrylate (UDMA), triethyleneglycol di-
methacrylate (TEGDMA), 2-hydroxyethylmethacrylate
(HEMA), butylated benzenediol (stabilizer), ethyl-4-di-
methylaminobenzoate), camphorquinone, functionalized
amorphous silica, t-butanol.
In this way were 80 teeth samples (each containing 2
studs) prepared and divided into 10 groups of 8 each,
A-T (Table 2 ) and stored in a solution of artificial saliva.
These groups were then treated as outlined in Table 2.
After 24 hours one stud of each tooth was tested for
shear bond strength and the other one after 3 months. An
Instron Universal Testing Machine (Griffith University,
G12) at a crosshead speed of 0.5 mm/minute was used to
test the de-bonding strength. All data tests were analysed
using the non-parametric ANOVA test.
Table 2. Groups tested (8 teeth per groups).
Group A37% of phosphoric acid + primer +
Bonding immediately (negative control)
Group BSelf-etching primer + Bonding
immediately (positive control)
Group CGel1 + primer + Bonding immediately
Group DGel2 + primer + Bonding immediately
Group EGel3 + primer + Bonding immediately
Group FGel4 + primer + Bonding immediately
Group KGel5 + primer + Bonding immediately
Group LGel6 + primer + Bonding immediately
Group MGel7 + primer + Bonding immediately
Group NGel8 + primer + Bonding immediately
Group OGel9 + primer + Bonding immediately
Group PGel10 + primer + Bonding immediately

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 26
3. RESULTS 3.2. Studies of Equilibrium Swelling in
Naproxen-Chitosan Gels (Gel 1-10)
3.1. The Characterization of Naproxen-Chitosan
Gels (Gel-4-Gel-10) The hydrogels remain in the cylindrical form after swell-
ing. Compared with dry state hydrogels, the swollen state
hydrogel volume displays significant increases and are
summarized in Figure 2.
The SEM images were obtained to characterize the mi-
crostructure of the freeze-dried naproxen composite gels
and are presented in Figure 1. It could be seen that the
gels displayed a homogeneously pore structure. It was
thought that the micro-porous structure of the gels could
lead to high internal surface areas with low diffusional
resistance in the gels. The surfaces of the gels were also
presented (Figure 1). The “skin” of the gels can be seen,
and the collapse of the surface pores may be due to
freeze-drying process.
Equilibrium swelling ratio (SR) of hydrogels exerts an
influence on their release rates. The reduction in equilib-
rium swelling capacity is due to the formation of a tight
network structure in high content. Environmental pH
value has a large effect on the swelling behavior of these
gels. From Figure 2, it is clear that the SR value in-
creases with the increase of pH. Such pH dependent
properties of the hydrogels come from the polyelectro-
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 1. SEM photographs of interior morphology of the selected gels under investigation for (a) Gel-4, (b) Gel-5, (c) Gel-6, (d)
el-7, (e) Gel-8, (f) Gel-9, (g) Gel-10,(h) Gel-12, (i) Gel-13. G

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 27
Figure 2. Water uptake degree of the gels Gel-1.
lyte nature of chitosan segments in the hydrogel network.
Namely, when the pH value of the buffer solution (pH
9.0) was far higher than the isoelectric point (PI) of GEL
(PI 4.0 - 5.0), the carboxyl groups were de-protonized to
carry negative charges, which made molecular chains
repulsed to each other. The network became looser and it
was easy for the water molecules to diffuse into the
cross-linked network. According to above results, we
believed that the naproxen results release mechanism
could result from the superposition of various effects,
such as swelling property of hydrogels, the solubility of
the drug and erosion property of matrix; it is not neces-
sarily based on a single factor.
3.3. FTIR Investigations of the Gels 4-10
Drug-polymer solid state interaction was further inves-
tigated through FT-IR spectroscopy. Some representative
FT-IR spectra of NAP-CS, NAP-CS and NAP-CS-An-
tioxidant combinations in the C=O stretching region of
NAP (1800 - 1600 cm−1) are shown in Figure 3. Spectra
of the physical mixtures were the weighted average of
those of the single components. No appreciable modifi-
cations in the characteristic quartet of NAP frequency
bands was observed by comparing the spectra of the
physical mixtures with CS with those of corresponding
co-ground systems. On the contrary a reduction of inten-
sity together with a shift towards lower frequencies for
the NAP carbonyl band was observed in co-ground sys-
tems with Antioxidant, attributable to a variation in the
hydrogen bond pattern due to a NAP-Antioxidant inter-
action [15].
3.4. Shear Bond Strengths
Figure 4 gives the shear bond strength values (MPa)
after 24 hours.
Mean shear bond strength values and difference be-
tween the groups are summarized in Figure 4 for bond-
ing to dentin after 24 hours. In general there was an in-
crease in bond strength of the dentin treated with the
antioxidant containing hydrogels compared to the bond
strength of the conventionally bonded teeth. An increase
in the shear bond strength was also previously reported
Figure 3. FT-IR spectra from the top down are: Chitosan:
naproxen, Chitosan:naproxen:propolis, Chitosan:melatonin:na-
proxen and Chitosan:naproxen:melatonin:propolis, respectively.
Figure 4. Shear bond strength of hydrogels after 24 hours of
bonding to dentin.
[16] for chitosan-H, chitosan-propolis, chitosan-napro-
xen and chitosan-naproxen-propolis and chitosan:napro-
xen:melatonin. Interestingly the increase in bond strength
was also observed in the groups of hydrogen peroxide
exposed samples suggesting that there additional benefits
associated with chitosan:antioxidant system are in need
of further investigations [16].
The results of this study suggest that the optimum re-
sults for the strengthening of dentin can be achieved
throughout the immediate treatment with antioxidant:
chitosan with the increase of dentin bond strength. Also,
impressively an almost immediately after the corre-
sponding gel treatment and proceeding with bonding
procedures is recommended with the significant increase
in bond strength. The results of this study suggest that
the optimum results for the increased enamel dentin bond
strength can be achieved through out the immediate
treatment with gels. The additional advantage of the sys-
tem may suggest that, antioxidant release from chitosan
gel depends on the physical network structure (open cell
like structure) as well as pH properties and flexibilities of
the material. Antioxidant release occurs through the
pores of the low polymer concentration while chitosan

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 28
concentration increment resulted in more cross-linking of
the network structure; consequently slower antioxidant
release from the gel base was achieved and therefor
weaker adhesive properties of the materials such as
Gel-1 in case of groups [17].
It was shown by others and us earlier, that the swelling
properties and antioxidant release from gels were in-
creased under acidic conditions due to the protonation of
the primary amino group on chitosan [18]. Chain relaxa-
tion due to protonation of amino groups leads to a faster
hydrogen bond dissociation and efficient solvent diffu-
sion. Thus, the appreciable increase in water uptake at
lower pH values can be attributed to the high porosity of
the gels, which seems to govern the diffusion of the sol-
vent in the gel matrix, and thus, the release of the anti-
oxidant from the gel [19]. The additional benefit of using
chitosan:antioxidant system as a bonding/pre-bonding to
enamel and dentin system lies in its ability to show fa-
vourable immediate results in terms of bonding effec-
tiveness as well as the durability of resin-dentin bonds
for a prolonged time (up to 6 months) [ref]. It is well
documented that the hydrostatic pulpal pressure, the den-
tinal fluid flow and the increased dentinal wetness in
vital dentin can affect the intimate interaction of certain
enamel and dentin adhesives with dentinal tissue. There-
for the newly developed chitosan:antioxidant systems
might at least be able to address the shortfalls in the cur-
rent perspectives for improving bond durability through
understanding factors affecting the long-term bonding
performance of modern adhesives and addresses the cur-
rent perspectives for improving bond durability.
3.5. In Vitro Release of Naproxen from
Naproxen-Chitosan Gels (Gel-4,
Gel7-Gel-10)
The in vitro release of naproxen from chitosan gels was
carried out using USP dissolution apparatus type I as
previously described [20]. The release of naproxen from
chitosan gel 5% was studied with naproxen concentra-
tions (1% w/w) and gels containing corresponding anti-
oxidants, as shown in Figure 5. The principal mecha-
nism of such interactions is the formation of hydrogen
bonds involving amino group and carboxyl group of
naproxen [21,22]. Also it becomes apparent that the in-
fluence of chemical structures of antioxidants such as
propolis and melatonin has significantly improved the
release of naproxen from the hydrogels. The mechanism
of this interaction is currently under investigations in our
laboratories.
3.6. Stability of Antioxidants in the Chitosan
Hydrogels during Storage
Stability of various conventional antioxidants in the
Figure 5. The effect additive such as antioxidants (propolis
and/or melatonin) on naproxen release from naproxen:chitosan
gels (Gel-4, Gel-7, Gel-8, Gel-9, Gel-110) in phosphate buffer
pH 6.8.
Figure 6. Antioxidant capacity measured at 450 nm using the
previously described spectrophotometric assay to asses the
hydrogels and corresponding ingredients antioxidant capacity
after 24 hours under storage under ambient temperature condi-
tion. Antioxidant capacity was measured during the first 2
hours of exposure.
Figure 7. Antioxidant capacity measured at 450 nm using the
previously described spectrophotometric assay to asses the
hydrogels and corresponding ingredients antioxidant capacity
after 6-month under storage under ambient temperature condi-
tion. Antioxidant capacity was measured during the first 2
hours of exposure.

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 29
newly designed drug delivery system during storage is an
important factor to determine whether chitosan-coated
nano-size delivery vehicle can protect various conven-
tional antioxidants. So the stability of the microencapsu-
lated antioxidants has been measured by UV absorbance.
Stabilities of microencapsulated antioxidants have been
compared after 24 hours (Figure 6) and after 6-month
(Figure 7) of storage at 24˚C, the stability of antioxi-
dant-molecular carrier vehicle was over 95%. This indi-
cates that antioxidants have been protected by the mo-
lecular carrier via positive host:guest supra-molecular
interaction. Important to note that performance of the
antioxidant was enhanced by the presence of the chitosan,
which is a very interesting point in itself as the syner-
gism in increased stability and lower concentration of the
active antioxidant with the same or even higher antioxi-
dant capacity can lead to a development of broad range
to novel functional restorative materials.
3.7. Investigations into Stability of Antioxidants
in the Chitosan Hydrogels during Storage
Stability of various conventional antioxidants in the
newly designed drug delivery system during storage is an
important factor to determine whether chitosan-coated
nano-size delivery vehicle can protect various conven-
tional antioxidants. So the stability of the microencapsu-
lated antioxidants has been measured by UV absorbance.
Stabilities of microencapsulated antioxidants have been
compared and after 6 months of storage at 24˚C, the sta-
bility of antioxidant-molecular carrier vehicle was not
significantly diminished as indicated in Figures 5 and 6.
This observation suggests that the antioxidant had been
protected by the molecular carrier. Important to note that
performance of the antioxidants such as propolis, mela-
tonin, chitosan:propolis, chitosan:melatonin, chitosan:
propolis:melatonin:naproxen was enhanced by the pres-
ence of the chitosan, which is a very interesting point in
itself as the synergism in increased stability and lower
concentration of the active antioxidant with the same or
even higher antioxidant capacity can lead to a develop-
ment of broad range to novel functional drug delivery
systems and dual action restorative materials.
3.8. In Vitro Microbiological Study
Candida albicans was not susceptible to the blank chito-
san gel containing no nystatin (no zone of inhibition was
observed). The selected preparations containing Chito-
san/Propolis/Nystatin, Chitosan/Melatonin/Propolis/Nys-
tatin, Chitosan/Propolis/BSA/Nystatin, Chitosan/Mela-
tonin/BSA/Nystatin and the Nystatin antibiotic sensitive-
ity disc containing 100 IU of Nystatin all give clear inhi-
bitions zones of different diameters. The Candida albi-
cans growth inhibition zones were 26.5 ± 0.5 mm (aver-
age ± STD) for the nystatin antibiotic sensitivity disc, 9.5
± 0.8 mm for the Chitosan/Propolis/Nystatin, 8.6 ± 0.7
mm for the Chitosan/Melatonin/Propolis/Nystatin, 14.5 ±
1.3 mm for the Chitosan/Propolis/BSA/Nystatin, 10.6 ±
0.5 mm for the Chitosan/Melatonin/BSA/Nystatin and
10.0 ± 0.0 mm for the Chitosan/Nystatin formulations
respectively. All the test samples give a smaller inhibit-
tion zone than the nystatin antibiotic control disc. This
indicates that release of the nystatin from the formula-
tions were inhibited to some extent, which is in agree-
ment with the results obtained from the nystatin release
experiments where it was observed that after 8 hours
only about 50% of the nystatin was released from the
formulations [23]. This slow release of the nystatin from
the gel will be a beneficiary effect that will enable a sus-
tainable release over time [24].
4. CONCLUSION
We have developed and evaluated novel functionalized
biomaterials, which have high loading efficiency, thus
they can be used for carriers for proteinaceous drugs as
well as display certain degree of defence mechanism for
a free radical damage and increased antimicrobial prop-
erties of the novel functional drug delivery systems. The
added benefits of the unique functionality of the hy-
drogels involve increased dentin adhesive bond strengths
(after 24 h), positive influence on the naproxen release,
additional benefits of the antioxidant stability of func-
tional “designer” restorative material. This approach
highlights the importance of innovative development of
functional dental restorative material to have bioactive
and bonding properties suitable to be used in dentin and
enamel as well as show the beneficial preventative and
therapeutic properties.
5. ACKNOWLEDGEMENTS
This study is financially supported by the DDF fund of the South Afri-
can Dental Association. VTP would like to thank Dr Vanessa Reher for
support through out the challenging journey. VTP would like to ac-
knowledge partial financial support by the School of Dentistry and Oral
Health, Griffith University for the travel research fellowship.
REFERENCES
[1] Buffet-Bataillon, S., Tattevin, P., Bonnaure-Mallet, M.
and Jolivet-Gougeon, A. (2012) Emergence of resistance
to antibacterial agents: The role of quaternary ammonium
compounds—A critical review. International Journal of
Antimicrobial Agents, 39, 381-389.
http://dx.doi.org/10.1016/j.ijantimicag.2012.01.011
[2] Imazato, S., Torii, M., Tsuchitani, Y., McCabe, J.F. and
Russell, R.R.B. (1994) Incorporation of bacterial inhibit-
tor into resin composite. Journal of Dental Research, 73,
1437-1443.

V. T. Perchyonok et al. / Open Journal of Stomatology 3 (2013) 22-30 30
[3] Imazato, S., Kinomoto, Y., Tarumi, H., Torii, M., Russell,
R.R.B. and McCabe, J.F. (1997) Incorporation of anti-
bacterial monomer MDPB into dentin primer. Journal of
Dental Research, 76, 768-772.
http://dx.doi.org/10.1177/00220345970760030901
[4] Buonocore, M.G. (1955) A simple method of increasing
the adhesion of acrylic filling materials to enamel sur-
faces. Journal of Dental Research, 34, 849-853.
http://dx.doi.org/10.1177/00220345550340060801
[5] Chandler, H.H., Bowen, R.L., Paffenbarger, G.C. and
Mullineaux, A.L. (1970) Clinical investigation of a ra-
diopaque composite restorative material. The Journal of
the American Dental Association, 81, 935-940.
[6] Dorminey, J.C., Dunn, W.J. and Taloumis, L.J. (2003)
Shear bond strength of orthodontic brackets bonded with
a modified 1-step etchant-and-primer technique. Ameri-
can Journal of Orthodontics & Dentofacial Orthopedics,
124, 410-413.
http://dx.doi.org/10.1016/S0889-5406(03)00404-9
[7] Perchyonok, V.T., Zhang, S. and Oberholzer, T. (2013)
Protective effect of conventional antioxidant (β-carotene,
resveratrol and vitamin E) in chitosan-containing hydro-
gels against oxidative stress and reversal of DNA double
stranded breaks induced by common dental composites:
In-vitro model. Open Nanoscience Journal, 7, 1-7.
http://dx.doi.org/10.2174/1874140101307010001
[8] Perchyonok, V.T., Zhang, S. and Oberholzer, T. (2012)
Alternative chitosan based drug delivery system to fight
oral mucositis: Synergy of conventional and bioactives
towards the optimal solution. Current Nanoscience, 8,
541-547. http://dx.doi.org/10.2174/157341312801784320
[9] Martin, A. (1993) Physical pharmacy. Lea and Febiger,
Philadelphia, London, 496-497.
[10] Chen, S., Liu, M., Jin, S. and Wang, B. (2008) Prepara-
tion of ionic-crosslinked chitosan-based gel beads and
effect of reaction conditions on drug release behaviors.
International Journal of Pharmaceutics, 349, 180-187.
http://dx.doi.org/10.1016/j.ijpharm.2007.08.029
[11] Shin, S.C. and Kim, J.Y. (2000) Enhanced permeation of
triamcinolone acetonide through the buccal mucosa. Euro-
pean Journal of Pharmaceutics and Biopharmaceutics,
50, 217-220.
http://dx.doi.org/10.1016/S0939-6411(00)00101-6
[12] Hamilton-Miller, J.M. (1973) The effect of pH and of
temperature on the stability and bioactivity of nystatin
and amphotericin B. Journal of Pharmacy and Pharma-
cology, 25, 401-407.
h ttp ://dx. doi.org/10.1111/ j.2042-7158.1973.tb10035.x
[13] Perchyonok, V.T., Zhang, S., Grobler, S.R. and Oberhol-
zer, T.G. (2013) Insights into and relative effect of chito-
san-H, chitosan-H-propolis, chitosan-H-propolis-nystatin
and chitosan-H-nystatin on dentine bond strength. Euro-
pean Journal of General Dentistry.
[14] Akala, E.O., Kopecková, P. and Kopecek, J. (1998) No-
vel pH-sensitive hydrogels with adjustable swelling ki-
netics. Biomaterials, 19, 1037-1047.
http://dx.doi.org/10.1016/S0142-9612(98)00023-4
[15] Mura, P., Zerrouk, N., Mennini, N., Maestrelli, F. and
Chemtob, C. (2003) Development and characterization of
naproxen-chitosan solid systems with improved drug dis-
solution properties. European Journal of Pharmaceutical
Sciences, 19, 67-75.
http://dx.doi.org/10.1016/S0928-0987(03)00068-X
[16] De Munck, J., Van Landuyt, J.K., Peumans, M., Poitevin,
A., Lambrechts, P., Braem, M. and Van Meerbeek, B.
(2005) A critical review of the durability of adhesion to
tooth tissue: Methods and results. Journal of Dental Re-
search, 84, 118-132.
http://dx.doi.org/10.1177/154405910508400204
[17] Gan, Q., Wang, T., Cochrane, C. and McCarron, P. (2005)
Modulation of surface charge, particle size and morpho-
logical properties of chitosan-TPP nanoparticles intended
for gene delivery. Colloids and Surfaces B: Biointerfaces,
44, 65-73.
http://dx.doi.org/10.1016/j.colsurfb.2005.06.001
[18] Govender, T., Riley, T., Ehtezazi, T., Garnett, M.C., Stol-
nik, S., Illum, L., et al. (2000) Defining the drug incor-
poration properties of PLA-PEG nanoparticles. Interna-
tional Journal of Pharmaceutics, 199, 95-110.
http://dx.doi.org/10.1016/S0378-5173(00)00375-6
[19] Rokhade, A.P., Shelke, N.B., Patil, S.A. and Aminabhavi,
T.M. (2007) Novel interpenetrating polymer network mi-
crospheres of chitosan and methylcellulose for controlled
release of theophylline. Carbohydrate Polymers, 69, 678-
687. http://dx.doi.org/10.1016/j.carbpol.2007.02.008
[20] Li, X., Li, L., Wang, X., Ren, Y., Zhou, T. and Lu, W.
(2012) Application of model-based methods to character-
ize exenatide-loaded double-walled microspheres: In vivo
release, pharmacokinetic/pharmacodynamic model, and
in vitro and in vivo correlation. Journal of Pharmaceuti-
cal Sciences, 101, 3946-3961.
http://dx.doi.org/10.1002/jps.23236
[21] Tahara, K., Sakai, T., Yamamoto, H., Takeuchi, H., Hira-
shima, N. and Kawashima, Y. (2009) Improved cellular
uptake of chitosan-modified PLGA nanospheres by A549
cells. International Journal of Pharmaceutics, 382, 198-
204. http://dx.doi.org/10.1016/j.ijpharm.2009.07.023
[22] Tahara, K., Sakai, T., Yamamoto, H., Takeuchi, H. and
Kawashima, Y. (2008) Establishing chitosan coated PLGA
nanosphere platform loaded with wide variety of nucleic
acid by complexation with cationic compound for gene
delivery. International Journal of Pharmaceutics, 354,
210-216. http://dx.doi.org/10.1016/j.ijpharm.2007.11.002
[23] Perchyonok, V.T., Zhang, S.M. and Oberholzer, T. (2012)
Towards development of novel chitosan based drug de-
livery prototypes: Devices for targeted delivery drug the-
rapy at the molecular level in aqueous media. Current
Organic Chemistry, 16, 2437-2439.
http://dx.doi.org/10.2174/138527212803520155
[24] Perchyonok, V.T., Zhang, S.M. and Oberholzer, T. (2013)
Chitosan and gelatin based prototype delivery systems for
the treatment of oral mucositis: From material to per-
formance in vitro. Current Drug Delivery, 10, 144-150.
http://dx.doi.org/10.2174/1567201811310010020