Keywords
adipose-derived mesenchymal stem cells - bovine teeth - scaffold - tissue engineering
Introduction
The tissue engineering technique incorporating the use of cells and growth factors
combined with scaffold for periodontal tissue regeneration is currently gaining popularity.
This technique utilizes biocompatible scaffold seeds with growth factor, stem cells,
or both that are implanted into a site to stimulate the reformation or repair of the
missing tissues.[1]
[2] Bovine teeth have been the most widely employed substitutes for the human variety
in dental research, with their use increasing over the past 30 years.[3]
[4] As bone graft material, bovine teeth possess osteoinductive and osteoconductive
properties responsible for the construction of new bones. The term “osteoinduction”
signifies that the grafted material is chemotactic to undifferentiated osteoprogenitor
cells in the host and induces differentiation into osteoblasts. Osteoconduction is
defined as a process that permits the growth of osteogenic cells from exposed bone
surface into the adjacent graft material.[5]
[6]
Bovine teeth are predominantly composed of inorganic material (70%), organic material
(20%), and water (10%). The inorganic component is largely hydroxyapatite and organic
content consisting of collagen type I and growth factor.[7]
[8]
[9] The purpose of scaffolds is to support cell attachment and migration, while also
providing growth factors to support tissue and bone formation. The combination of
scaffold and stem cells for bone growth is synergic in character. The self-renewal
abilities of stem cells and their capability to differentiate into multiple cell lineages
render them promising candidates for cell-based tissue engineering. Adipose-tissue
derived from adult stem cells is most commonly used for periodontal regeneration.[10]
Adipose-derived mesenchymal stem cells (ADMSCs) can be easily isolated, providing
an enormous number of stem cells that are vital for tissue engineering and stem cells-based
therapies.[11]
[12] The International Society for Cellular Therapy set three minimum criteria for the
definition of MSCs: plastic-adherence; expression of CD73, CD90, and CD105; and the
absence of CD45, CD14, CD19, human leukocyte antigen - DR isotype (HLA-DR) expressions
and their trilineage differentiation potential into osteoblasts, chondrocytes, adipocytes.[13]
[14] Research into bovine teeth implantation in the calvarial defects of rats showed
an increase in bone density after 6 weeks. Other research conducted by George et al
state that umbilical mesenchymal stem cells demonstrate a tendency to differentiate
and proliferate after binding to the tooth surface in vitro.[15]
[16]
The purpose of this study is to analyze the osteogenic potential and biocompatibility
test of bovine teeth scaffold seeded with ADMSCs in vitro.
Materials and Methods
This study received ethical clearance (number 637-KE) from the Animal Care and Use
Committee Faculty, Veterinary University of Airlangga, Surabaya, Indonesia. Three,
4-week-old, male Wistar rat subjects were sacrificed by euthanasia. Isolation of the
ADMSCs of these subjects was performed by washing adipose tissue with saline phosphates
containing 10% antimycotic–antibiotic agent. The adipose tissue was cut into pieces
and immersed in a 0.2% collagenase type I (Worthington, Lakewood, New Jersey, United
States) solution with the addition of Dulbecco's phosphate buffer saline (STEMCELL
Technologies, Nucleos, Singapore) and agitated slowly for 40 minutes at 37°C. The
tissue was filtered using a 10 µm mesh filter (Pluriselect; Leipzig, DE) before being
centrifuged at 1,250 rpm for 4 minutes, with the supernatant subsequently discarded.[17]
Isolation and Culture of Adipose-Derived Mesenchymal Stem Cells ADMSCs
MSCs were cultured with α-modified minimum essential medium eagle (αMEM) (Gibco, Roskilde
Denmark) plus 15% fetal bovine serum (Biowest; Missouri, United States), 2 mM of L-glutamine)
(Gibco), 100 mg/mL streptomycin (Gibco), 2.5 μg/mL fungizone) (Gibco), and 100 IU/mL
penicillin (Gibco) before being incubated at 37°C with 5% CO2. The cells were grown in six wells on a tissue culture plate at a concentration of
107 in each well. The medium was changed on the 7th day and every 3 days thereafter.
Observation of the cells was performed using an inverted microscope (80× magnification).[13]
[14]
[15]
[16]
[17]
[18]
Characterization ADMSCs by Immunocytochemical Staining and Flow Cytometry
A single cell subjected to a process of trypsinization was centrifuged, fixed with
formaldehyde (TCI, Chuo-ku, Tokyo), and washed with PBS. FITC anti-rat CD105 monoclonal
antibody (BioLegend, San Diego, California, United States) and FITC anti-rat CD45
monoclonal antibody (BioLegend) were mixed into the sample and incubated at 37°C for
45 minutes. Several drops of 50% glycerin (TCI, Chuo-ku, Tokyo) were then applied
to glass objects and observed under a fluorescent microscope. During the trypsinization
process, the single cell was centrifuged. Thereafter, it was fixed in formaldehyde
solution and closed with BSA. Cells were added to CD105 primary antibody and CD45
FITC. The fixed cells were analyzed by means of a FACSCalibur flow cytometer (Becton
Dickinson, New Jersey, United States).[19]
Culture on Osteogenic Medium
MSC culture was separated using trypsin solution (Sigma, Singapore) and poured into
a 24-well microplate (SPL, Gyeonggi-do, Korea) filled with osteogenic medium that
contained αMEM medium (Gibco) with the supplementary addition of 50 μg/mL of L-ascorbic
acid (TCI, Chuo-ku, Tokyo), 10–8 M of dexamethasone (TCI, Chuo-ku, Tokyo and 10 mM
of b-glycerophosphate (TCI, Chuo-ku, Tokyo) at a density of 2 × 105 and incubated at 37°C. The medium was replaced every 3 days for 21 days. After that
period, the cells were fixed with 10% formaldehyde (TCI, Chuo-ku, Tokyo), rinsed with
water to facilitate examination of the osteogenic differentiation, and, finally, stained
with Alizarin red (Sigma, Singapore).[20]
Preparation of Bovine Teeth
Preparation of Bovine Teeth
Bovine teeth were cleaned with peroxidase 3% for 1 week. The crowns and roots were
subsequently separated by means of a bone cutter and rongeur forceps. Powder was produced
by processing the teeth comprised of dentin and cementum tissue with a bone miller.
The resulting products were filtered until the desired amount was obtained. The demineralization
process was performed using a bone mineral removal method involving immersion in 1%
hydrochloric acid (Merck, NJ, United States) for a day and thorough rinsing before
being dried. Prior to storage, the protein content of the tooth was frozen (freeze
dried = lyophilization). Particles 355 to 710 μm in size were produced and sterilized
in Batan, Jakarta, Indonesia.[21] The bovine teeth particle shape was examined with an SEM at a magnification of 75×
and 1,500× (Electron Microscope Laboratory, Faculty of Medicine, Universitas Airlangga
Surabaya, East Java, Indonesia) before being scanned using micro-computed tomography
(micro-CT) to observe the particle shape in three-dimensional (3D) (Faculty of Mathematics
and Natural Science, Institut Teknologi Bandung West Java, Indonesia).
Micro-computed Tomography-based Investigation
The specimens were positioned in a horizontally rotating micro-CT scanning device
holder (Bruker Micro-CT Sky-Scan 1173, High Energy Micro-CT, FMIPA ITB) before being
scanned using a source X-ray voltage of 40 kV. The source current was 130 mA with
an exposure time of 500 ms using a 1.0-mm aluminum filter. The sample was scanned
through a 180°rotation with a rotation step of 0.2°. During image acquisition, 10
frames were averaged and the scanning process required approximately 2 hours per sample.
By using a camera binning of 1 × 1, the projection images produced were 2,240 × 2,240
in dimension. The projection images were reconstructed as 8-bit gray scale images
with isotropic spatial resolution of 5.7 μm/pixel. A pre-reconstruction process, a
ring artifact correction of 10, and beam hardening correction of 20% were applied
to enhance the reconstructed image quality.[23]
Toxicity Testing
A toxicity test (MTT assay) of bovine teeth scaffold in relation to the cell culture
of ADMSCs was performed using the following procedure. Bovine teeth scaffolds were
immersed in μMEM (Gibco) for 24 hours. The pellets were cultured in ninety-six 5 ×
104 cells/well and incubated for 24 hours at 37°C at 5% CO2 concentration. After 80% growth and incubation for 20 hours at 37°C and 5% CO2, 200 μL supernatant from bovine teeth scaffold and 25 μL MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (Biotium, Fremont, California, United States) were added to each well and
incubated for 4 hours at 37°C. They were subsequently observed under an inverted microscope.
The color change in the wells was observed with an Elisa Reader at a wavelength of
595 nm.[22]
[23]
Seeding of ADMSCs on Bovine Teeth Scaffold
Bovine teeth scaffolds were immersed in αMEM (Gibco) for 1 day, before the medium
was removed and replaced with a new one. After 1 day, 5 mg of bovine teeth scaffold
was inserted into 96-well culture, to which a medium suspension of 2 × 106 cells was added and incubated for an hour at a temperature of 37°C and 5% CO2. More medium was added to each 1.3 mL/well and incubated at a temperature of 37°C
and 5% CO2. The tube was periodically agitated to ensure even cell distribution within the bovine
teeth scaffold suspension. SEM observation was conducted after 1 day of seeding.[24]
Statistical Analysis
All data were expressed as the mean and standard deviation (SD). The statistical analysis
was performed using one-way analysis of variance. p-Value < 0.05 was considered statistically significant.
Results
In this research, bovine teeth scaffold seeded with ADMSCs was obtained. The characteristics
of bovine teeth scaffold and the viability rate of the ADMSCs was examined using MTT
assays. The ADMSC culture of the subjects is shown in [Fig. 1]. ADMSCs have the ability to adhere to culture plastic flasks, while they appeared,
morphologically, as spindle-shaped cells both as scattered individuals and in small
colonies. With regard to the expression of mesenchymal stem cell markers CD45 and
CD105, based on observation under a fluorescence microscope ([Fig. 2]), it transpired that CD105 expression is much stronger than that of CD45. Characterization
of MSCs with flowcytometry showed that the cells cultured were MSCs with a marked
subpopulation of cells expressing more CD105 than CD45.
Fig. 1 Culture of adipose-derived mesenchymal stem cells (ADMSCs) showing fibroblast-like
morphology and adherence to the plate: (A) Culture containing ADMSCs 1 day after isolation (passage 1) and (B) 15 days after isolation (passage 4) (inverted microscope at 200× magnification).
Fig. 2 The immunocytochemistry result showed that (A) adipose-derived mesenchymal stem cells are strongly expressed in CD105 and (B) weakly expressed in CD45 (immunofluorescence microscope at 200× magnification).
The proportion of subpopulations of MSCs cells expressing CD105 was more dominant
at 99.63%, while that expressing CD45 accounted for only 0.37% ([Fig. 3]). The effect of the bovine teeth scaffold on the mineralization ability of ADMSCs
and the deposition of calcium and phosphate was confirmed by Alizarin red staining
([Fig. 4]). The morphological shape of bovine scaffold was observed in 3D using micro-CT with
a particle size of 355 to 710 μm. [Fig. 5B] shows the pore shape of the bovine teeth scaffold particles. Particle thickness
distribution as estimated by means of 3D micro-CT had an average of ~96 μm, while
the average particle size as determined during the process ([Fig. 5]) was 500 µm. The particle size (2D) can be estimated by calculating the area and
perimeter of the previously clustered objects using a watershed process ([Fig. 6]). From the results of the study, the largest cell viability was obtained at a concentration
of 10% with a mean ± SD (97.08 + 12.67), concentration of 50% with mean + SD (88.58
± 12.38), and concentration of 100% with mean ± SD (76.64 + 5.7 6) where p < 0.05 ([Fig. 7]). The attachment and proliferation of cells on bovine teeth scaffold were demonstrated
within 1, 12, and 24 hours of incubation. After 24 hours in culture, the number of
cells in the bovine teeth scaffold was greater than that at 1 and 12 hours (p < 0.05) ([Fig. 8]).
Fig. 3 Examination of flow cytometry indicating that the majority of mesenchymal stem cells
subpopulations expressed more CD105 at ~99.63% (UL), while the minority subpopulations
expressed around 0.37% (LL) of CD45.
Fig. 4 Optical microscopy pictures showing the cultures stained with alizarin red. (A) Control cultured in a medium for 21 days. (B) Differentiation of adipose-derived mesenchymal stem cells as shown by positive alizarin
red staining in osteogenic medium for 21 days (inverted microscope at 200× magnification).
Fig. 5 (A) Bovine teeth scaffold with various particle sizes from 355 to 710 µm (micro-computed
tomography photographs). (B) Scanning electron microcopy photographs of bovine teeth scaffold visible pores.
Fig. 6 Two-dimensional particle size distribution measured by the calculation of the major
diameter. The majority of bovine teeth scaffold particles are 182 to 364 μm (purple).
Fig. 7 Cell viability of the bovine teeth scaffold.
Fig. 8 Scanning electron microcopy micrographs showed adipose-derived mesenchymal stem cells
seeding with bovine teeth scaffold at various time intervals: (A) 1, (B) 12, and (C) 24 hours of cells seeding DDM scaffold (×5,000 magnification). The arrow-marked
region is the attachment point.
Discussion
The growth of ADMSCs can be subcultured up to 9 to 10 passages, after which the cells
will degenerate.[25] ADMSCs are shown to be 80% confluent after three passages and form multilayers after
20× confluence.[26]
[27] Periodontal tissue regeneration has also been successfully demonstrated in the experimental
subject using ADMSCs.[28] Characterization of ADMSCs was confirmed MSCs by means of CD105 positive and CD45
negative. CD105 is a homodimer membrane glycoprotein associated with human vascular
endothelium. CD45 is expressed in hematopoietic progenitors, the small vessel endothelium
of many varieties of tissue, and represents a subset of bone marrow stromal cells.[29]
The osteogenic differentiation of adipose-derived stem cells was initiated after as
few as 4 days in the form of small nodules in isolated regions characterized by spindle-shaped
cells at their perimeters. Electron microscope transmission of the osteogenic cells
on day 21 of osteogenic culture showed nearly intact osteogenic progenitor cells.
These are characterized by the cytoplasm that is abundant in the mitochondria, rough
endoplasmic reticulum, oval-shaped mineralized calcium deposits, and the considerable
numbers of collagen fibrils that are distinctive features of osteoblast differentiated
cells.[30] Another study posited that osteogenic differentiation requires at least 28 to 38
days at which point calcified extracellular matrix are observable after staining with
alizarin red.[31] The results of these experiments indicate that bovine teeth scaffold plays a crucial
role in stimulating ADMSCs mineralization potential.[32]
Small pore size facilitates osteoblast cell proliferation, while lower porosity helps
osteogenic differentiation in vitro. The structure of the newly formed bone correlated
with the scaffold pore size where smaller pores support more trabeculae formation.[33] There are some factors that must be considered when designing a scaffold, such as
pore size, as this will affect the nutrition diffusion and migration of cells throughout
the scaffold, while also influencing the formation of a vascular network within the
scaffold and integration with the vascular system of the surrounding tissues. Previous
studies posited that collagen combined with demineralized bone powder with a particle
size of 250 to 500 μm is suitable for osteoblast differentiation environments and
potentially for bone tissue engineering.[34]
[35]
An MTT assay is employed to evaluate the material cytotoxicity in tissue engineering
and can indicate cell growth and proliferation. These results prove that bovine teeth
scaffolds are not toxic and may be attached to and proliferate in the ADMSCs.[36] The HA content of bovine teeth helps improve the osteoconduction process. Inorganic
materials, including HA, have also been used to promote periodontal regeneration.
Hydroxyapatite can not only eliminate the morbidity of the donor site but also lead
to granular migration and incomplete resorption.[37]
[38] Previous studies have suggested that HA scaffold seeded with mesenchymal stem cells
from bone marrow can improve the osteogenesis process in vivo.[39] Cells were attached and spread into the pores of the scaffold, being visibly attached
to the scaffold approximately 1 hour after seeding (data not shown). This result indicates
that a bovine teeth scaffold is suitable for cell environments and supports cell growth
and proliferation in addition to cell differentiation.[40]
[41]
Conclusion
The scaffold from bovine teeth is biocompatible and accelerates osteogenic differentiation
of ADMSC.
