Key words:
Cuttlefish bone powder - cytotoxicity - osteoblast cell culture - proliferation
INTRODUCTION
Cuttlefish bone (CB) is a natural biomaterial source from the chamber of the cuttlefish
that can be ground into a powder. CB is a brittle structure found in all members of
the cephalopod family and is a chambered, gas-filled shell used to control floating.[1] A gas and liquid mixture osmotically regulates the pressure inside the CB.[2]
[3] The main chemical CB components are 87.3%–91.75% calcium carbonate and chitin. In
addition, CB also contains trace amounts of silicon, aluminum, titanium, manganese,
barium, and copper.[4] CB is a traditional Chinese medicine that is effectively used in treating gastritis
and frequently used as a hemostatic agent after tooth extraction or rhinoplasty.[4] Moreover, the synthesis of Hydroxyapatite (HAp) from natural biomaterials, such
as eggshell,[5] coral,[6] and CB[7] has been reported.
Materials that enhance bone regeneration have a wide range of potential clinical applications,
from treating nonunion fractures to spinal fusion. The use of porous material scaffolds
with bioceramic and polymer components to support bone cell and tissue growth is a
popular research topic. Current challenges include engineering materials that can
match the mechanical and biological properties of the bone tissue matrix and support
the vascularization of large tissue constructs.[8] The most common biomaterials used for bone tissue engineering include Hap, titanium,
alumina, and polymers.[9] Battistella et al.[10] revealed that cell proliferation in a three-dimensional (3D) CB scaffold increased.
They also suggested investigating the use of dynamic culture to improve cell proliferation
and differentiation. The mechanical properties of natural bone are of interest in
bone tissue engineering. A scaffold should have a highly porous matrix for transporting
nutrients, oxygen, and metabolic products.[11] In addition, cuttlefish bone powder (CBP) was added to paste or gel dentifrices
or used directly with a toothbrush to clean the teeth and improve oral hygiene.[12]
[13] We would like to investigate the biological properties of CB available in Thailand.
In the present study, the biocompatibility and effect of CBP on MC3T3-E1 osteoblast
cell proliferation were evaluated in vitro.
MATERIALS AND METHODS
Cuttlefish bone powder preparation
The bone inside the cuttlefish (from the Southern part of Thailand) was removed and
cut in the middle into small pieces (1 cm × 1 cm × 0.5 cm) [Figure 1]. The CB was rinsed with deionized water, then boiled 10 min for getting rid of the
odor and microorganisms. To desorb any impurities, the CB was dried at 103°C–105°C
for 24 h and cooled in a desiccator at room temperature. The CB was crushed, pulverized,
and sieved (Pass 80 mesh) into a 150–250 µm particle powder. The CB powder (CBP) was
used as the test material in the experiments.
Figure 1: (a) Cuttlefish bone is the hard tissue in cuttlefish that functions in floatation.
(b) Natural cuttlefish bone
Test materials
The CBP had a maximum 8% moisture and a pH range of 6.0–8.0. The minimum powder fineness
passed through a No. 80 sieve with 75% efficiency for sterilization before cell culture
experiment. The CBP (200 mg) was mixed with 1 ml of Dulbecco’s modified Eagle’s medium
(DMEM, Invitrogen, CA, USA) for a 20% (w/v) solution. The solution was incubated at
37°C in a 5% CO2 atmosphere for 24 h, per ISO 10993-12.[14] The CBP stock solution was centrifuged at 3500 rpm for 10 min, and the supernatant
was diluted into 0.5, 1, 5, 25, 50, 100, or 200 µg/ml solutions.
We used 3 cm2 polyurethane/2 ml of DMEM (Hatano Research Institute, Food and Drug
Safety Center, Kanagawa, Japan) as a positive control per ISO 10993-5.[15] The polyurethane films were sterilized by soaking in 70% alcohol for 1 min, washed
in normal saline for 1 min, and left to dry. The dry films were immersed in DMEM and
incubated at 37°C in a 5% CO2 atmosphere for 24 h before testing.
Thermanox® Coverslips (NUNC™ Naperville, IL, USA) (6 cm2/2 ml of media) served as a negative
control per ISO 10993-5.[15] Thermanox® Coverslips were cut into small pieces, soaked in DMEM, and incubated in a 5% CO2 atmosphere at 37°C for 24 h before testing.
Cell culture procedure
The cells used in this experiment were a continuous cell line, MC3T3-E1Subclone 4
Strain C57BL/B mouse osteoblast-like cell line (ATCC® CRL-2593™, USA). The cells were
maintained in DMEM containing 10% fetal calf serum, 200 μg/ml penicillin G, 200 μg/ml
streptomycin, and 2 μg/ml fungizone at 37°C in a humidified 5% CO2 atmosphere. The medium was changed every other day. When the cells reached confluence,
they were detached using 0.2% (w/v) trypsin and transferred to new culture flasks.
Cytotoxicity evaluation
At 80% confluency, the cells were trypsinized and plated in 96-well culture plates
(1 × 104 cells/well). Each well contained 100 µl of cell suspension, and the plates
were incubated for 24 h at 37°C in a 5% CO2 atmosphere. After 24 h, the media was removed from each well. Subsequently, 100 µl
of eluent from the 0.5, 1, 5, 25, 50, 100, or 200 µg/ml CBP solutions or the positive/negative
control was placed into the 96-well culture plates (8 wells/test material). After
incubation for 24 h at 37°C in a 5% CO2 atmosphere, cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The experiments were repeated in triplicate.
The mean optical density of the blank control group was set at 100% viability. The
results for the experimental, positive control, and negative control groups were normalized
to the blank control group. Statistical analysis was performed using the nonparametric
Mann–Whitney test (P<0.05). The relative cell count ratio was calculated from the following formula:
Where O.D.570e is the mean optical density of the 100% extracts of the test sample, O.D.570c is the mean optical density of the control, and O.D.570b is the mean optical density of the blanks.
Cell proliferation evaluation
MC3T3 cells were cultured in α-Minimum Eagle’s Medium containing 10% fetal bovine
serum (FBS), 100 U/ml penicillin G, and 100 µg/ml streptomycin at 37°C in a humidified
5% CO2 atmosphere. The cells were treated with 0.25% trypsin for 5 min at 37°C and diluted
with α- MEM containing 10% FBS to a concentration of 1 × 105 cells/ml. The cells (2
× 103 cells/well) were seeded in seven 96 -well culture plates (100 µl/well) and incubated
at 37°C in a humidified 5% CO2 atmosphere for 24 h. The cells were treated with 0.5, 25, or 100 μg/ml of CBP solution
and the media control group (10 wells/concentration/duration) for 1, 3, 5, 7, 10,
14, and 16 days. The MTT assay was used to determine cell proliferation at each concentration
at each time point.
The percentage of cell proliferation of the three experimental groups was calculated
using the mean optical density from 7 to 10 days this means the values from 7 to 10
days and was expressed as a percentage of the control values.
Statistical analysis
Statistical analysis was performed using SPSS-18.0 software (SPSS Inc., IL, USA).
The results are presented as the mean ± standard deviation. Statistical analysis was
performed using Student’s t-test. A value of P<0.05 was considered to be statistically significant.
RESULTS
We determined the effect of CBP on MC3T3-E1 cell viability as percentage of cell viability
[Figure 2].] CBP was not toxic to the MC3T3-E1 cells at any tested concentration. The percentage
of cell viability in the 0.5–200 µg/ml CBP groups dose dependently decreased from
107.52% ± 11.03% to 92.48% ± 5.60%, however, these differences were not significantly
different from each other or the negative control group (P > 0.05), while the positive control group showed a significant 5–6–fold reduction
compared with the other groups (P < 0.05). The results of the cell proliferation evaluation showed that cell proliferation
in the CBP groups peaked at 14 days and decreased at 16 days [Figure 3], with the 0.5, 25, and 100 µg/ml CB groups at 16 days demonstrating 123.19% ± 10.07%,
126.02% ± 15.69%, and 133.33% ± 11.74% proliferation, respectively, which was significantly
higher compared with the media control [Figure 4].
Figure 2: Cell viability percentages in the CBP, negative control, and positive control groups.
Different superscript letters signify a significant difference between groups (P<0.05)
Figure 3: Cell proliferation as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide optical density results from 1–16 days
Figure 4: Percentage of cell proliferation in the 0.5, 25, and 100 g/ml cuttlefish bone groups
at 16 days. *Indicates a significant difference between the control group (P<0.05)
DISCUSSION
Tissue engineering in dentistry is a multidisciplinary field. The purpose of tissue
engineering is to repair, maintain, or enhance tissue and organ regeneration. Promoting
the organization of cells in a 3D architecture directs the growth and formation of
the desired tissue. Bone has a low capacity for self-repair due to its limited vascular
supply and low rate of chondrocyte mitosis. Currently, osteoconductive porous biodegradable
materials are used in tissue engineering for bone repair. Once the bone healing is
accomplished, the newly formed tissue undergoes physiologic bone remodeling, which
involves the coordinated action of osteoblasts and osteoclasts.[16]
Calcium phosphate-based materials can be used as a biomaterial for tissue engineering.
Hap, Ca10(PO4)6(OH)2, is widely used as a bone substitute.[17] The composition of Hap is the same as the mineral constituents in hard tissue, i.e.,
bone and teeth. Hap has several beneficial properties; it is nontoxic, osteoconductive,
and biocompatible. Natural Hap biomaterials, such as coral and eggshell, have been
recommended as the materials of choice in bone tissue engineering.[5] CB is a Hap material that has been used in dentistry for bone repair. There are
two benefits of CB Hap in bone tissue engineering: the main component of CB is aragonite
(CaCO3) that has been converted into Hap and it also has a porous structure and bone-like
architecture.[18]
Recently, Hap with a porous morphology has been used as a scaffold. The scaffold is
placed in the bone defect area. This porous scaffold is beneficial for bone defect
repair due to its effectiveness in cell attachment, differentiation, and proliferation,
generating bone healing.[19] Polycaprolactone is a polymer commonly added to a Hap scaffold to increase its mechanical
properties.[20]
CB has been proposed as a suitable material for bone tissue scaffold. We selected
MC3T3-E1 as the target cells in our experiment because they are cell line and have
the capacity to differentiate into osteoblasts. MC3T3-E1 has been established from
a C57BL/6 mouse calvaria and selected on the basis of high alkaline phosphatase activity
in the resting state. Our study revealed the same biocompatibility with MC3T3-E1 osteoblast
cell line as shown in other studies.[10]
[18] None of the CBP concentrations we evaluated were cytotoxic to the MC3T3-E1 cell
line. The percentage of cell viability was rather high and similar to that of the
negative control, while that of the positive control group was significantly fold
lower.
We evaluated cell proliferation using three levels of CBP concentration; low (0.5
μg/ml), middle (25 μg/ ml), and high (100 μg/ml) along with media controls. We found
that the exponential phase of cells treated under these conditions was between 7 and
10 days. The percentage of cell proliferation was calculated from the optical density
in the exponential phase of the CB and control groups. The low-, medium-, and high-CB
groups demonstrated percent cell proliferation of 123.17%, 124.02%, and 133.33%, respectively,
and were significantly higher from the media control group at 16 days. Our results
correspond with those of Kim et al.,[21] where PCL/CB-Hap scaffold implantation generated significantly higher new bone formation.
Yildirim et al.[22] found that the mineral composition of CB was compatible with human bone tissue and
suggested its use as a scaffold. In addition, Kannan et al.[23] estimated that a CB channel size of 100 × 200 μm would be beneficial for bone ingrowth.
Moreover, CB and shrimp shell-derived chitosan displayed good biocompatibility and
supported cell attachment and growth.[24]
A study reported that raw CB contains 0.05 ppm mercury, 0.52 ppm copper, 2.42 ppm
zinc, 0.39 ppm lead, and 0.07 ppm cadmium and is not cytotoxic in vitro.[25] Zreiqat et al.[26] suggested that implant surfaces coated with Mg2+ promote optimal osteogenesis and
lead to the maintenance of nature and healthy bone.
The Mg2+ ion has an important role in integrins binding to their respective ligands.
Integrins transduce signals from the extracellular environment to the interior of
the cell or vice versa for cellular migration, adhesion, proliferation, and differentiation.[27] The Mg2+ present in CB is a likely reason for the cell proliferation observed in
our study.
CONCLUSION
The current study revealed that 0.5–200 g/ml CBP were biocompatible with the MC3T3-E1
cell line and that 0.5–100 g/ml CBP induced a high percentage of cell proliferation
compared with control. These findings suggest that CB has the potential to improve
in vivo bone defect healing by increasing cell proliferation.
Financial support and sponsorship
Nil.
