| dc.description.abstract | Objective Adipose-derived mesenchymal stem cells (ADMSCs) have great potential for regenerative medicine. These have been combined with biomaterials such as
Bovine teeth that are preferred as a periodontal regeneration material. The main purpose of this study is to evaluate and analyze a biocompatibility test and osteogenic
differentiation of bovine teeth scaffold seeded with ADMSCs in vitro.
Materials and Methods A true experimental study with post-test only group design
was conducted. Random sampling and Lameshow’s formula were used to determine
the sample. The scaffold, obtained from bovine teeth as the bone graft material, was
analyzed using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, and its attachment was evaluated by scanning electron microcopy (SEM) and
micro-computed tomography with ADMSCs. ADMDSCs attachment present in the
bovine teeth scaffold was assessed using SEM at 1-hour, 12-hour, and 24-hour intervals.
Statistical Analysis Analysis of variance was used to analyze the MTT assay results
(p < 0.05) based on normality and homogeneity test (p > 0.05).
Results The highest viability of cells (97.08%) was found at a concentration of 10%
by means of an MTT test (p < 0.05). The results of three-dimensional bovine teeth
Abstract
Keywords
► adipose-derived mesenchymal stem cells
► bovine teeth
► scaffold
► tissue engineering
Osteogenic Differentiation Bovine Teeth Scaffold Combined Adipose Mesenchymal Stem Cells Sari et al. 207
European Journal of Dentistry Vol. 13 No. 2/2019
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-weekold, 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).13-18
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
scaffold showed the average particle size to be 500 µm. ADMSCs cell attachment to
the scaffold bovine teeth showed a significant increase in the number of cells attached
after 24 hours compared with those at 1 and 12 hours. Alizarin red staining showed
an increase in ADMSC osteogenic differentiation after it was combined with bovine
teeth scaffold. | en_US |
