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1.  Rabbit maxillary sinus augmentation model with simultaneous implant placement: differential responses to the graft materials 
Purpose
This study was performed to establish an experimental rabbit model for single-stage maxillary sinus augmentation with simultaneous implant placement.
Methods
Twelve mature New Zealand white rabbits were used for the experiments. The rabbit maxillary sinuses were divided into 3 groups according to sinus augmentation materials: blood clot (BC), autogenous bone (AB), and bovine-derived hydroxyapatite (BHA). Small titanium implants were simultaneously placed in the animals during the sinus augmentation procedure. The rabbits were sacrificed 4 and 8 weeks after surgery and were observed histologically. Histomorphometric analyses using image analysis software were also performed to evaluate the parameters related to bone regeneration and implant-bone integration.
Results
The BC group showed an evident collapse of the sinus membrane and limited new bone formation around the original sinus floor at 4 and 8 weeks. In the AB group, the sinus membrane was well retained above the implant apex, and new bone formation was significant at both examination periods. The BHA group also showed retention of the elevated sinus membrane above the screw apex and evident new bone formation at both points in time. The total area of the mineral component (TMA) in the area of interest and the bone-to-implant contact did not show any significant differences among all the groups. In the AB group, the TMA had significantly decreased from 4 to 8 weeks.
Conclusions
Within the limits of this study, the rabbit sinus model showed satisfactory results in the comparison of different grafting conditions in single-stage sinus floor elevation with simultaneous implant placement. We found that the rabbit model was useful for maxillary sinus augmentation with simultaneous implant placement.
doi:10.5051/jpis.2012.42.6.204
PMCID: PMC3543935  PMID: 23346463
Animal models; Bone substitutes; Dental implants; Guided tissue regeneration; Sinus floor augmentation
2.  Effects of immunosuppressants, FK506 and cyclosporin A, on the osteogenic differentiation of rat mesenchymal stem cells 
Purpose
The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs).
Methods
The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7.
Results
Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control.
Conclusions
These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.
doi:10.5051/jpis.2012.42.3.73
PMCID: PMC3394998  PMID: 22803008
Cell differentiation; Cyclosporin A; FK506; Immunosuppressive agents; Mesenchymal stem cells
3.  The evaluation of the correlation between histomorphometric analysis and micro-computed tomography analysis in AdBMP-2 induced bone regeneration in rat calvarial defects 
Purpose
Micro-computed tomography (micro-CT) has been widely used in the evaluation of regenerated bone tissue but the reliability of micro-CT has not yet been established. This study evaluated the correlation between histomorphometric analysis and micro-CT analysis in performing new bone formation measurement.
Methods
Critical-size calvarial defects were created using a 8 mm trephine bur in a total of 24 Sprague-Dawley rats, and collagen gel mixed with autogenous rat bone marrow stromal cells (BMSCs) or autogenous rat BMSCs transduced by adenovirus containing bone morphogenic protein-2 (BMP-2) genes was loaded into the defect site. In the control group, collagen gel alone was loaded into the defect. After 2 and 4 weeks, the animals were euthanized and calvaria containing defects were harvested. Micro-CT analysis and histomorphometric analysis of each sample were accomplished and the statistical evaluation about the correlation between both analyses was performed.
Results
New bone formation of the BMP-2 group was greater than that of the other groups at 2 and 4 weeks in both histomorphometric analysis and micro-CT analysis (P=0.026, P=0.034). Histomorphometric analysis of representative sections showed similar results to histomorphometric analysis with a mean value of 3 sections. Measurement of new bone formation was highly correlated between histomorphometric analysis and micro-CT analysis, especially at the low lower threshold level at 2 weeks (adjusted r2=0.907, P<0.001). New bone formation of the BMP-2 group analyzed by micro-CT tended to decline sharply with an increasing lower threshold level, and it was statistically significant (P<0.001).
Conclusions
Both histomorphometric analysis and micro-CT analysis were valid methods for measurement of the new bone in rat calvarial defects and the ability to detect the new bone in micro-CT analysis was highly influenced by the threshold level in the BMP-2 group at early stage.
doi:10.5051/jpis.2011.41.5.218
PMCID: PMC3213232  PMID: 22087412
Bone morphogenic protein-2; Gene therapy; Histology; X-Ray microtomography
4.  Response of osteoblast-like cells cultured on zirconia to bone morphogenetic protein-2 
Purpose
The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2.
Methods
MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days.
Results
At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7.
Conclusions
The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.
doi:10.5051/jpis.2011.41.5.227
PMCID: PMC3213233  PMID: 22087413
Bone morphogenetic protein-2; Cell differentiation; Cell proliferation; Zirconium oxide
5.  Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow 
Purpose
The aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells.
Methods
We employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis.
Results
We identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs.
Conclusions
This study demonstrated the genome-wide gene expression patterns of STRO-1+ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells.
doi:10.5051/jpis.2011.41.4.192
PMCID: PMC3175499  PMID: 21954424
Adult stem cells; Gene expression profiling; Mesenchymal stem cells; Microarray analysis; mRNA
6.  Initial adhesion of bone marrow stromal cells to various bone graft substitutes 
Purpose
The aim of this study is to determine whether certain biomaterials have the potential to support cell attachment. After seeding bone marrow stromal cells onto the biomaterials, we investigated their responses to each material in vitro.
Methods
Rat bone marrow derived stromal cells were used. The biomaterials were deproteinized bovine bone mineral (DBBM), DBBM coated with fibronectin (FN), synthetic hydroxyapatite (HA), HA coated with FN, HA coated with β-tricalcium phosphate (TCP), and pure β-TCP. With confocal laser scanning microscopy, actin filaments and vinculin were observed after 6, 12, and 24 hours of cell seeding. The morphological features of cells on each biomaterial were observed using scanning electron microscopy at day 1 and 7.
Results
The cells on HA/FN and HA spread widely and showed better defined actin cytoskeletons than those on the other biomaterials. At the initial phase, FN seemed to have a favorable effect on cell adhesion. In DBBM, very few cells adhered to the surface.
Conclusions
Within the limitations of this study, we can conclude that in contrast with DBBM not supporting cell attachment, HA provided a more favorable environment with respect to cell attachment.
doi:10.5051/jpis.2011.41.2.67
PMCID: PMC3087077  PMID: 21556256
Bone substitutes; Cell adhesion; Fibronectins; Stem cells
7.  Immunomodulatory effect of canine periodontal ligament stem cells on allogenic and xenogenic peripheral blood mononuclear cells 
Purpose
The aim of this study was to investigate the immunomodulatory effects of canine periodontal ligament stem cells on allogenic and xenogenic immune cells in vitro.
Methods
Mixed cell cultures consisting of canine stem cells (periodontal ligament stem cells and bone marrow stem cells) and allogenic canine/xenogenic human peripheral blood mononuclear cells (PBMCs) were established following the addition of phytohemagglutinin. The proliferation of PBMCs was evaluated using the MTS assay. The cell division of PBMCs was analyzed using the CFSE assay. The apoptosis of PBMCs was assessed using the trypan blue uptake method.
Results
Periodontal ligament stem cells and bone marrow stem cells inhibited the proliferation of allogenic and xenogenic PBMCs. Both periodontal ligament stem cells and bone marrow stem cells suppressed the cell division of PBMCs despite the existence of a mitogen. No significant differences in the percentages of apoptotic PBMCs were found among the groups.
Conclusions
Canine periodontal ligament stem cells have an immunomodulatory effect on allogenic and xenogenic PBMCs. This effect is not a product of apoptosis of PBMCs but is caused by the inhibition of cell division of PBMCs.
doi:10.5051/jpis.2010.40.6.265
PMCID: PMC3021166  PMID: 21246016
Allogenic; Immunomodulation; Periodontal ligaments; Stem cell; Xenogenic

Results 1-7 (7)