• International Journal of Oral Biology
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• v.47 no.4
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• pp.55-62
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• 2022

Bioactive flavonoids have been shown to improve the biological activity of stem cells derived from different sources in tissue regeneration. The goal of this study was to see how naringin, a natural flavonoid discovered in citrus fruits, affected the biological properties of human dental pulp stem cells (HDPSCs). In this study, we found that naringin increases the migratory ability of HDPSCs. Naringin increased matrix metalloproteinase-2 (MMP-2) and C-X-C chemokine receptor type 4 (CXCR4) mRNA and protein expression in HDPSCs. ARP100, a selective MMP-2 inhibitor, and AMD3100, a CXCR4 antagonist, both inhibited the naringin-induced migration of HDPSCs. Furthermore, naringin increased osteogenic differentiation of HDPSCs and the expression of the osteogenic-related marker, alkaline phosphatase in HDPSCs. Taken together, our findings suggest that naringin may be beneficial on dental tissue or bone regeneration by increasing the biological activities of HDPSCs.

• BMB Reports
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• v.46 no.6
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• pp.328-333
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• 2013

Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material.

• 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.

• The Journal of Internal Korean Medicine
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• v.36 no.4
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• pp.518-526
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• 2015

Objectives This study investigated the effect of purified safflower (Carthamus tinctorius Linne) and safflower seed (Carthamus tinctorius L. seed; CS) extract, using hot water and ethanol extract methods , on the osteogenic differentiation of MC3T3E1 cells.Methods The safflower and safflower seed were extracted with hot water and ethanol. The samples were concentrated by a rotary evaporator and then freeze-dried using a freeze-dryer. The MC3T3E1 cells were propagated and maintained in DMEM (Gibco) containing 10% FBS and a 1% antibiotic antimycotic solution. To induce osteogenic differentiation, the cells were treated for 14 days with DMEM with 10 mM β-glycerophosphate and 50 μM ascorbic acid. Extract doses were confirmed by the results of an MTT assay, and treatment of the extracts was performed in a differentiation medium every two days. The ALP staining and activity were tested after osteogenic differentiation for five days, and after 14 days, osteogenic differentiation was determined by alizarin red S staining. The mRNA expressions of osteogenic-related genes were quantified using quantitative real-time PCR.Results In the results of the MTT assay, all concentrations of safflower extracts had no toxicity in the MC3T3El cells. But in the groups of 100 ng/ml and 200 ng/ml concentrations of safflower seed extracts, the cell viability was significantly reduced by up to 40-50%. So we fixed the treatment concentration of the extract at 50 ng/ml. In the ALP and alizarin red S staining, all extract groups increased osteogenic differentiation compared with the control group. The water-safflower extract group showed the highest mRNA level of Alp, Runx2, and Dlx5 genes. The mRNA level of Ocn, an osteogenic gene related to late-stage differentiation, in the ethanol-safflower extract group increased the mineralization more significantly than in other groups.Conclusions These data suggest that the extract of safflower increases the osteoblastic differentiation activates of MC3T3E1 cells like the extract of safflower seed. The water-extract and ethanol-extract of safflower have effects on different stages of osteogenesis in MC3T3El. Not only safflower seed but also safflower will be useful therapeutic reagents for age-associated chronic diseases such as osteoporosis.

• Journal of Periodontal and Implant Science
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• v.42 no.6
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• pp.224-230
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• 2012

Purpose: Various bone graft materials have been used for periodontal tissue regeneration. Demineralized freeze-dried bone allograft (DFDBA) is a widely used bone substitute. The current widespread use of DFDBA is based on its potential osteoinductive ability. Due to the lack of verifiable data, the purpose of this study was to assess the osteoinductive activity of different DFDBAs in vitro. Methods: Sarcoma osteogenic (SaOS-2) cells (human osteoblast-like cells) were exposed to 8 mg/mL and 16 mg/mL concentrations of three commercial types of DFDBA: Osseo+, AlloOss, and Cenobone. The effect of these materials on cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The osteoinductive ability was evaluated using alizarin red staining, and the results were confirmed by evaluating osteogenic gene expression using reverse transcription polymerase chain reaction (RT-PCR). Results: In the SaOS-2 cells, an 8 mg/mL concentration of Osseo+ and Cenobone significantly increased cell proliferation in 48 hours after exposure (P<0.001); however, in these two bone materials, the proliferation of cells was significantly decreased after 48 hours of exposure with a 16 mg/mL concentration (P<0.001). The alizarin red staining results demonstrated that the 16 mg/mL concentration of all three tested DFDBA induced complete morphologic differentiation and mineralized nodule production of the SaOS-2 cells. The RT-PCR results revealed osteopontin gene expression at a 16 mg/mL concentration of all three test groups, but not at an 8 mg/mL concentration. Conclusions: These commercial types of DFDBA are capable of decreasing proliferation and increasing osteogenic differentiation of the SaOS-2 cell line and have osteoinductive activity in vitro.

• Journal of Life Science
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• v.27 no.7
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• pp.767-782
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• 2017

MicroRNAs control the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs). However, the role of miR-200a and miR210 on the osteogenic differentiaton of hADSCs has not been determined. hADSCs were isolated from human adipose tissues. Direct binding of mircoRNA to target mRNAs was determined by luciferase assay of the constructs containing putative microRNA binding sites within 3' untranslated region of target mRNAs. Overexpression of miR-200a increased the proliferation and osteogenic differentiation of hADSCs, while causing downregulation of the levels of ZEB2. Inhibition of miR-200a with antisense RNAs inhibited the proliferation and osteogenic differentiation of hADSCs. Overexpression of miR-210 was found to inhibit the proliferation of hADSCs but increase the osteogenic differentiation, while causing downregulation of the levels of IGFBP3. Inhibition of miR-210 with antisense RNAs increased the proliferation but inhibited the osteogenic differentiation of hADSCs. Analysis of the luciferase activity of the constructs containing the miR-200a target site within the ZEB2 3' region and the miR-210 target site within the IGFBP3 3' region revealed lower activity in the miR-200a- or miR-210-transfected hADSCs than in control miRNA-transfected hADSCs. Downregulation of ZEB2 or IGFBP3 in the hADSCs showed similar effects on both their proliferation and osteogenic differentiation with that of miR-200a and miR-210 overexpression, respectively. The results of the current study indicate that miR-200a and miR-210 regulate the osteogenic differentiation and proliferation of hADSCs through the direct targeting of IGFBP3 and ZEB2, respectively.

• International Journal of Oral Biology
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• v.42 no.2
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• pp.71-78
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• 2017

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at $ED_{50}$ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

• Journal of Periodontal and Implant Science
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• v.49 no.2
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• pp.114-126
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• 2019

Purpose: The aim of this study was to evaluate the enhancement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG). Methods: The cell viability, differentiation, and mineralization of osteoblasts was tested with ErhBMP-2-/EGCG solution. Coated BCP surfaces were also investigated. Standardized, 6-mm diameter defects were created bilaterally on the maxillary sinus of 10 male New Zealand white rabbits. After removal of the bony windows and elevation of sinus membranes, ErhBMP-2-/EGCG-coated BCP was applied on one defect in the test group. BCP was applied on the other defect to form the control group. The animals were sacrificed at 4 or 8 weeks after surgery. Histologic and histometric analyses of the augmented graft and surrounding tissue were performed. Results: The 4-week and 8-week test groups showed more new bone (%) than the corresponding control groups (P<0.05). The 8-week test group showed more new bone (%) than the 4-week test group (P<0.05). Conclusions: ErhBMP-2-/EGCG-coated BCP was effective as a bone graft material, showing enhanced osteogenic potential and minimal side effects in a rabbit sinus augmentation model.

• Nutrition Research and Practice
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• v.4 no.5
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• pp.356-361
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• 2010

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 ${\mu}M$) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

• The Journal of Advanced Prosthodontics
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• v.6 no.4
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• pp.285-294
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• 2014

PURPOSE. The purpose of this study was to evaluate the properties of a porous zirconia scaffold coated with bioactive materials and compare the in vitro cellular behavior of MC3T3-E1 preosteoblastic cells to titanium and zirconia disks and porous zirconia scaffolds. MATERIALS AND METHODS. Titanium and zirconia disks were prepared. A porous zirconia scaffold was fabricated with an open cell polyurethane disk foam template. The porous zirconia scaffolds were coated with ${\beta}$-TCP, HA and a compound of ${\beta}$-TCP and HA (BCP). The characteristics of the specimens were evaluated using scanning electron microscopy (SEM), energy dispersive x-ray spectrometer (EDX), and x-ray diffractometry (XRD). The dissolution tests were analyzed by an inductively coupled plasma spectrometer (ICP). The osteogenic effect of MC3T3-E1 cells was assessed via cell counting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS. The EDX profiles showed the substrate of zirconia, which was surrounded by the Ca-P layer. In the dissolution test, dissolved $Ca^{2+}$ ions were observed in the following decreasing order; ${\beta}$-TCP > BCP > HA (P<.05). In the cellular experiments, the cell proliferation on titanium disks appeared significantly lower in comparison to the other groups after 5 days (P<.05). The zirconia scaffolds had greater values than the zirconia disks (P<.05). The mRNA level of osteocalcin was highest on the non-coated zirconia scaffolds after 7 days. CONCLUSION. Zirconia had greater osteoblast cell activity than titanium. The interconnecting pores of the zirconia scaffolds showed enhanced proliferation and cell differentiation. The activity of osteoblast was more affected by microstructure than by coating materials.