Chitosan nanocarriers could be used in future
dental applications 9 such as in dentin pulp
regeneration procedures, 10-11 in bone regeneration
techniques, 12 in endodontics 13-15 or in periodontal
therapy. 12, 16 Moreover, chitosan containing NPs
were able to transport antitumour substances to
different cancer cell lines, 17-19 including oral cancer
cells. 18-19
Despite numerous scientific reports regarding
organic nanomaterials in medicine, more expe-
riments are needed in order to asses the effects
of organic NPs on the oral mucosa. It has been
shown that the interactions between NPs and
cells depends on the cell type, as well as on the
size and surface charge of NPs. 20 NPs behave
completely differently depending on their surface
coverings and size, while the concentration and the
exposure time to such NPs makes them cytotoxic
or biocompatible. Moreover, the oral mucosa
is composed of a variety of cells with different
properties which may react differently to the same
NPs. The pathologic conditions can also modify
the response of human oral cells to NPs, due to
changes in cell physiological status.
The aim of our study is to determine the uptake
and effect of chitosan covered poly-lacto-co-
glycolic NPs (PLGAChi NPs) on the cells found
in the oral cavity, in normal and pathological
conditions. NPs were tested on normal human oral
keratinocytes (NOKs) and human dental pulp cells
(DPCs), harvested from healthy human donors,
as well as on POE9i cell line used as a model for
precancerous oral keratinocytes. In the attempt to
create a stronger resemblance to the natural 3D
structure of the oral mucosal tissue, the PLGAChi
NPs were also exposed to 3D organotypic (OT)
oral mucosa tissues grown in vitro. The PLGAChi
NPs tested in our study were previously fabricated
and characterized by Navarro et al. 21- 22
2. Materials and methods
2.1. Cell culture
NOKs, DPCs and normal oral fibroblasts (NOFs)
were primary cells isolated from clinically healthy
adult volunteers (n=5). Samples of gingival
mucosa showing no sign of clinical inflammation at
collection time were used to generate NOKs and
NOFs. The protocol for the isolation of NOKs has
previously been described by Costea et al. 23 DPCs
were isolated following a protocol adapted from
from Ishkitiev et al. 24 and Lee et al. 25
POE9i cells are dysplastic, premalignant human
immortalized oral keratinocytes. NOKs and POE9i
keratinocytes were grown in Keratinocyte Serum-
Free Growth Medium ( KSFM) (from Sigma-Aldrich,
St. Louis, MO) medium supplemented with 1 ng/
mL epithelial growth factor, 25 μg/mL bovine
pituitary extract, 20 μg/mL l-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin and 0.25 μg/
mL amphotericin B (all supplements were aquired
from InVitrogen, Massachusetts, USA ).
DPCs and NOFs were grown in DMEM medium
(Sigma St Louis, Missouri) containing 10 % fetal
Stomatology Edu Journal
bovine serum, 20 μg/mL l-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin, and 0.25 μg/
mL amphotericin B (all supplements were aquired
from InVitrogen, Massachusetts, USA). The pro-
tocol for growing normal human organotypics
(OTs) was previously described by Costea et al. 23
The multilayered epithelium was elaborated
using NOKs grown on top of collagen matrices
populated with NOFs.
2.2. Viability Test
NOKs and POE9i cells were cultured in 6 well
plates (150.000 cells/well) with 3.5 mL of culture
medium. Cells were allowed to set for 24h into
the incubator at 37°C and supplemented with
5% CO 2 . Afterwards, the media was removed
and the cells were washed with PBS. Then 3.5
mL media containing PLGAChi NPs at the tested
concentrations was added in each well: 5 μg/mL,
20 μg/mL and 200 μg/mL. The viability was counted
with trypan blue and an automatic cell counter
(Sigma-Aldrich, St. Louis, MO). The counting was
done in triplicates for every cell culture well.
2.3. Exposure of cells to PLGAChi NPs
The cells were seeded in two-well glass chambers
(Thermo Fisher Scientific; Nunc™ Lab-Tek™)
at a density of 75.000 cells/well. Every cell type
was incubated with 1.5 mL of their own culture
medium. The cells were kept for 48 h at 37°C till
they became 70 % – 80 % confluent. Afterwards,
the media were removed and washed twice with
PBS. NOKs, DPCs and POE9i cells were exposed
for 12h and 24h at the following concentrations of
fluorescein marked PLGAChi NPs: 20 μg/mL and
200 μg/mL. 1.5 mL of media containing PLGAChi
NPs at the mentioned concentrations was placed
in every chamber slide: 20 μg/mL and 200 μg/
mL. The solutions thus prepared were rotated for
30 minutes before exposure. The glass chambers
were placed in the incubator in a humidified
atmosphere at 37°C and supplemented with 5 %
CO2 for 12 h or 24 h. At the end of the exposure
time, the cells were washed three times with PBS
in order to remove unattached particles, followed
by fixation and staining. The controls were run in
duplicate in each experiment and were placed into
the incubator for one day.
The organotypic cultures were exposed to NPs
after a total period of 10 days of coculture. The
OTs were exposed to 200 μg/mL PLGAChi NP and
let into the incubator for 24h. At the end of the
exposure time, the OTs were washed three times
with PBS in order to remove unattached particles,
followed by fixation and staining.
Imaging and image analysis was performed using
an optical microscope.
The fluorescent NPs uptaken by the cells were
visualized by flourescence microscopy ( AxioIma-
ger.M2 with ApoTome.2). The cells were mounted
in Vectashield mounting medium with DAPI for
nuclear staining and were visualized at a Zeiss
up-right Axio Imager microscope with ApoTome
slider module, using the 403 or 603 oil immersion
objective lens.
CHITOSAN MODIFIED POLY(LACTIC-CO-GLYCOLIC) ACID NANOPARTICLES INTERACTION WITH
NORMAL, PRECANCEROUS KERATINOCYTES AND DENTAL PULP CELLS
17