• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.73-80
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• 2012

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.

• International Journal of Oral Biology
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• v.36 no.4
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• The Journal of Korean Academy of Prosthodontics
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• v.43 no.6
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• pp.751-763
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• 2005

Statement of problem. To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. The surface quality of the implant depends on the chemical, physical, mechanical and topographical properties of the surface. The different properties will interact with each other and a change in thickness of the oxide layer may also result in a change in surface energy, the surface topography and surface, chemical composition. However, there is limited the comprehensive study with regard to changed surface and biologic behavior of osteoblast by anodization. Purpose of study. The aim of this study was to analyze the characteristics of an oxide layer formed and to evaluate the cellular biologic behaviors on titanium by anodic oxidation (anodization) by cellular proliferation, differentiation, ECM formation and gene expression. And the phospholipase activity was measured on the anodized surface as preliminary study to understand how surface properties of Ti implant are transduced into downstream cellular events. Methods and Materials. The surface of a commercially pure titanium(Grade 2) was modified by anodic oxidation. The group 1 samples had a machined surface and other three experimental specimens were anodized under a constant voltage of 270 V(Group 2), 350 V(Group 3), and 450 V(Group 4). The specimen characteristics were inspected using the following five categories; the surface morphology, the surface roughness, the thickness of oxide layer, the crystallinity, and the chemical composition of the oxide layer. Cell numbers were taken as a marker for cell proliferation. While the expression of alkaline phosphatase and Runx2 (Cbfa1) was used as early differentiation marker for osteoblast. The type I collagen production was determined, which constitutes the main structural protein of the extracellular matrix. Phospholipase $A_2$ and D activity were detected. Results. (1) The anodized titanium had a porous oxide layer, and there was increase in both the size and number of pores with increasing anodizing voltage. (2) With increasing voltage, the surface roughness and thickness of the oxide film increased significantly (p<0.01), the $TiO_2$phase changed from anatase to rutile. During the anodic oxidization, Ca and P ions were more incorporated into the oxide layer. (3) The in vitro cell responses of the specimen were also dependant on the oxidation conditions. With increasing voltage, the ALP activity, type I collagen production, and Cbfa 1 gene expression increased significantly (p<0.01), while the cell proliferation decreased. (4) In preliminary study on the relation of surface property and phospholipase, PLD activity was increased but $PLA_2$ activity did not changed according to applied voltage. Conclusion. The anodized titanium shows improved surface characteristics than the machined titanium. The surface properties acquired by anodization appear to give rise more mature osteoblast characteristics and might result in increased bone growth, and contribute to the achievement of a tight fixation. The precise mechanism of surface property signaling is not known, may be related to phospholipase D.

• BMB Reports
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• v.49 no.10
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• pp.548-553
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• 2016

Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering to repair articular cartilage defects. However, hypertrophy of chondrocytes derived from MSCs might hinder the stabilization of hyaline cartilage. Thus, it is very important to find a suitable way to maintain the chondrogenic phenotype of chondrocytes. It has been reported that cordycepin has anti-inflammatory and anti-tumor functions. However, the role of cordycepin in chondrocyte hypertrophy remains unclear. Therefore, the objective of this study was to determine the effect of cordycepin on chondrogenesis and chondrocyte hypertrophy in MSCs and ATDC5 cells. Cordycepin upregulated chondrogenic markers including Sox9 and collagen type II while down-regulated hypertrophic markers including Runx2 and collagen type X. Further exploration showed that cordycepin promoted chondrogenesis through inhibiting Nrf2 while activating BMP signaling. Besides, cordycepin suppressed chondrocyte hypertrophy through PI3K/Bapx1 pathway and Notch signaling. Our results indicated cordycepin had the potential to maintain chondrocyte phenotype and reconstruct engineered cartilage.

• Journal of Web Engineering
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• v.14 no.17
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• pp.3604-3622
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• 2022

The fruit of Hippophae rhamnoides has been widely used for medicinal purposes because of its anti-inflammatory, antioxidant, antiplatelet, and antimicrobial effects. Since there are no clear reports on the therapeutic efficacy of H. rhamnoides in osteoporosis, this study aimed to confirm the potential use of H. rhamnoides for the treatment of osteoporosis through its osteogenic differentiation-promoting effect in ovariectomized mice. Through an in vitro study, we compared the effects of the EtOH extract of H. rhamnoides fruits (EHRF) on the differentiation of C3H10T1/2, a mouse mesenchymal stem cell line, into osteoblasts based on alkaline phosphatase (ALP) staining and the relative expression of osteogenesis-related mRNAs. The EHRF significantly stimulated the differentiation of mesenchymal stem cells into osteoblasts and showed 7.5 times (^* p < 0.05) higher osteogenesis than in the untreated control. A solvent fractionation process of EHRF showed that the hexane-soluble fraction (HRH) showed 10.4 times (^** p < 0.01) higher osteogenesis than in the untreated control. Among the subfractions derived from the active HRH by preparative HPLC fractionation, HRHF4 showed 7.5 times (^* p < 0.05) higher osteogenesis than in the untreated naïve cells, and HRH and HRHF4 fractions showed 22.6 times (^*** p < 0.001) stronger osteogenesis activity than in the negative control. Osteoporosis was induced by excision of both ovaries in 9-week-old female ICR mice for in vivo analysis, and two active fractions, HRH and HRHF4, were administered orally for three months. During the oral administration period, body weight was measured weekly, and bone mineral density (BMD) and body fat density were measured simultaneously using a DEXA machine once a month. In particular, during the in vivo study, the average BMD of the ovariectomized group decreased by 0.0009 g/cm², whereas the average BMD of the HRH intake group increased by 0.0033 g/cm² (^* p < 0.05) and that of the HRHF4 intake group increased by 0.0059 g/cm² (^** p < 0.01). The HRH and HRHF4 intake groups significantly recovered the mRNA and protein expression of osteogenic genes, including ALP, Osteopontin, Runx2, and Osterix, in the osteoporosis mouse tibia. These findings suggest that the active fractions of H. rhamnoides fruit significantly promoted osteoblast differentiation in mesenchymal stem cells and increased osteogenic gene expression, resulting in an improvement in bone mineral density in the osteoporosis mouse model. Taken together, H. rhamnoides fruits are promising candidates for the prevention and treatment of osteoporosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.3
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• pp.214-220
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• 2001

The clinical application of the three-dimensional radiographic technique had been limited to standard Broadbent-Bolton cephalometer with biplanar stereoradiography. We developed a new method for compensating the error of head position in ordinary non-biplanar cephalostat. It became to possible to use the three dimensional cephalogram commonly in clinical bases. 1. The method of methemetical compensation of head positioning error in non-biplanar condition was evaluated with dry skull. The error of the method of first and the second trial was $0.46{\pm}1.21$, $0.33{\pm}0.90mm$, which means the error of the head positioning correction in conventional cephalogram was within clinical acceptance. 2. The reproducibility of this system for clinical application was 0.54 mm ($-2.99{\sim}2.26mm$) which defines the absolute mean difference of the first and second trial. Compare to the The landmark identification error $1.2{\pm}1.6mm$, the error of the measurement was within the range of landmark identification error. The result indicates the adequate clinical accuracy of the computation of three-dimensional coordinates by compensation of the error of the head position in ordinary non-biplanar cephalostat.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.