• Journal of the korean academy of Pediatric Dentistry
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• v.42 no.4
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• pp.312-318
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• 2015

The aim of this study was to investigate the possibility of the supernumerary teeth for the stem cell source in dentistry. The Real Time Quantitative Reverse Transcription Polymerase Chain Reaction (Real Time qRT-PCR) method was used to evaluate the differentiation toward the odontoblast of the supernumerary dental pulp stem cells (sDPSCs). Supernumerary dental pulp stem cells were obtained from 3 children (2 males and 1 female, age 7 to 9) diagnosed that the eruption of permanent teeth was disturbed by supernumerary teeth. The common genes for odontoblasts are alkaline phosphatase (ALP), osteocalcin (OC), osteonectin (ON), dentin matrix acidic phosphoprotein 1 (DMP-1), dentin sialophosphoprotein (DSPP). The sDPSCs were treated for 0 days, 8 days and 14 days with additives and then Real Time qRT-PCR was performed in intervals of 0 days, 8 days and 14 days. The alizarin-red solution staining was performed to visualize the stained color for the degree of calcification at 7 days, 14 days, 21 days and 28 days after treating additives to the sDPSCs. From the result of the Real Time qRT-PCR, the manifestation exhibit maximum value at 8 days after additive treatment and shifted to a decrease trend at 14 days. Alizarin-red solution staining exhibit light results at 7 days after staining and generalized dark result at 14 days. Consequently, in studies with sDPSCs, appropriate treatment time of additives for Real Time qRT-PCR is 8 days. Also, a suitable period of Alizarin-red solution staining is 14 days.

• Journal of Nutrition and Health
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• v.51 no.5
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• pp.379-385
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• 2018

Purpose: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to $15{\mu}M$ $ZnCl_2$ (Zn- or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. Results: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. Conclusion: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.175-181
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• 2012

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% $CO_2$ atmosphere at $38.5^{\circ}C$. The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-${\gamma}2$, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.11 no.9
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• pp.3358-3365
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• 2010

This study was carried out to investigate the effect of Sorbus commixta (SC), Geranium thunbergii (GT) and their mixture (SC:GT=1:1, MIX) on inhibition of bone loss and chondral defect. To examine their activities, we measured the alkaline phosphatase (ALP) activity in human osteoblast-like MG-63 cells and performed tartrate-resistant acid phosphate (TRAP) staining in osteoclast differentiated from Raw264.7 cells. To investigate the influence on chondrocyte differentiation, we performed alcian-blue staining in chondrocyte differentiated from ATDC5 cells. All of SC, GT and MIX did not increase ALP activity in MG-63 cells. However, SC and mixture (SC:GT=1:1, MIX) significantly inhibited osteoclastic differentiation. And they also induced chondrocyte differentiation. These results suggest that SC and GT may have a potential for the treatment of bone loss and chondral defect by suppression of osteoclast differentiation and stimulation of chondrocyte differentiation. Therefore, clarification of their mechanisms and active components will be needed.

• Journal of the korean academy of Pediatric Dentistry
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• v.42 no.4
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• pp.291-301
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• 2015

This study compared the in vitro cell viability and differentiation potentials of human deciduous dental pulp cells (DPCs) on mineral trioxide aggregate (MTA)-like products (ProRoot MTA, RetroMTA and Endocem Zr). The experimental materials were prepared as circular discs, which were used to test the effects of the materials on the viability of human DPCs when placed in direct and indirect contact. Furthermore, the pH of the extracted materials was recorded, and their effect on cell differentiation potential was evaluated by evaluating the alkaline phosphatase (ALP) activity and Alizarin Red S staining of DPCs incubated with the test materials. In direct contact, the cell viability of human DPCs was higher with ProRoot MTA and RetroMTA than with Endocem Zr. However, when in indirect contact, the cell viability of human DPCs was generally higher in Endocem Zr than in ProRoot MTA and Retro MTA. With respect to pH, the alkalinity was lower for Endocem Zr than for the other test materials. The ALP activities of the cells were not enhanced by any of the experimental materials. Alizarin Red S staining of the tested human DPCs revealed that their differentiation potential was lower than for cells incubated with osteogenic induction medium. While there were differences in the responses of the human DPCs to the test materials, all displayed degrees of cytotoxicity and were unable to enhance either the viability or differentiation of human DPCs. However, Endocem Zr exhibited better cell viability and was less alkaline than the other test materials.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.2
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• pp.97-106
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• 2010

Aim of the study: An alternative source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Adipose tissue could be processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). This study was performed to confirm the availability of ATSCs in bone tissue engineering. Materials amp; Methods: In this study, adipose tissue-derived mesenchymal stem cell was extracted from the liposuctioned abdominal fat of 24-old human and cultivated, and the stem cell surface markers of CD 105 and SCF-R were confirmed by immunofluorescent staining. The proliferation of bone marrow mesenchymal stem cell and ATSCs were compared, and evaluated the osteogenic differentiation of ATSCs in a specific osteogenic induction medium. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific BMP-2, ALP, Cbfa-1, Osteopontin and osteocalcin were confirmed by RT-PCR. With differentiation of ATSCs, calcium concentration was assayed, and osteocalcin was evaluated by ELISA (Enzyme-linked immunosorbant assay). The bone formation by 5-week implantation of HA/TCP block loaded with bone marrow mesenchymal stem cells and ATSCs in the subcutaneous pocket of nude mouse was evaluated by histologic analysis. Results: ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. ATSCs could be easily identified through fluorescence microscopy, and bone formation in vivo was confirmed by using ATSC-loaded HA/TCP scaffold. Conclusions: The present results show that ATSCs have an ability to differentiate into osteoblasts and formed bone in vitro and in vivo. So ATSCs may be an ideal source for further experiments on stem cell biology and bone tissue engineering.

• Journal of Acupuncture Research
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• v.29 no.6
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• pp.11-21
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• 2012

Objectives : BHH10 is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify BHH10 extract induces osteogenic activity in human osteoblast-like MC3T3-E1 cells. Methods : MC3T3-E1, pre-osteoblast cell line, were treated with BHH10 of various concentrations($0.1{\mu}g/mL$, $1{\mu}g/mL$, $10{\mu}g/mL$). And then, the effect of BHH10 on osteoblast differentiation was examined by alkaline phosphatase(ALP) activity, von Kossa staining and RT-PCR for osteoblast differentiation markers such as osteocalcin(OCN), osteopontin(OPN). Results : BHH10 had dose-dependent effect on the viability of osteoblastic cells, and dose-dependently increased alkaline phosphatase(ALP) activity. BHH10 markedly increased mRNA expression for OCN, OPN in MC3T3-E1 cells. Also, BHH10 significantly induced mineralization in the culture of MC3T3-E1 cells. Conclusions : In conclusion, these results propose that BHH10 can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• Nutrition Research and Practice
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• v.10 no.2
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• pp.148-153
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• 2016

BACKGROUND/OBJECTIVES: Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS: A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS: The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS: The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

• Journal of Periodontal and Implant Science
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• v.43 no.4
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• pp.168-176
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• 2013

Purpose: The purpose of the current study was to examine the effect of dexamethasone (Dex) at various concentrations on the apoptosis and mineralization of human periodontal ligament (hPDL) cells. Methods: hPDL cells were obtained from the mid-third of premolars extracted for orthodontic reasons, and a primary culture of hPDL cells was prepared using an explant technique. Groups of cells were divided according to the concentration of Dex (0, 1, 10, 100, and 1,000 nM). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for evaluation of cellular viability, and alkaline phosphatase activity was examined for osteogenic differentiation of hPDL cells. Alizarin Red S staining was performed for observation of mineralization, and real-time polymerase chain reaction was performed for the evaluation of related genes. Results: Increasing the Dex concentration was found to reduce cellular viability, with an increase in alkaline phosphatase activity and mineralization. Within the range of Dex concentrations tested in this study, 100 nM of Dex was found to promote the most vigorous differentiation and mineralization of hPDL cells. Dex-induced osteogenic differentiation and mineralization was accompanied by an increase in the level of osteogenic and apoptosis-related genes and a reduction in the level of antiapoptotic genes. The decrease in hPDL cellular viability by glucocorticoid may be explained in part by the increased prevalence of cell apoptosis, as demonstrated by BAX expression and decreased expression of the antiapoptotic gene, Bcl-2. Conclusions: An increase in hPDL cell differentiation rather than cellular viability at an early stage is likely to be a key factor in glucocorticoid induced mineralization. In addition, apoptosis might play an important role in Dex-induced tissue regeneration; however, further study is needed for investigation of the precise mechanism.

• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.793-801
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• 2018

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.