• Journal of Dental Anesthesia and Pain Medicine
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• v.16 no.4
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• pp.295-302
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• 2016

Background: Reactive oxygen species play critical roles in homeostasis and cell signaling. Dexmedetomidine, a specific agonist of the ${\alpha}2$-adrenoceptor, has been commonly used for sedation, and it has been reported to have a protective effect against oxidative stress. In this study, we investigated whether dexmedetomidine has a protective effect against $H_2O_2$-induced oxidative stress and the mechanism of $H_2O_2$-induced cell death in normal human fetal osteoblast (hFOB) cells. Methods: Cells were divided into three groups: control group-cells were incubated in normoxia without dexmedetomidine, hydrogen peroxide ($H_2O_2$) group-cells were exposed to $H_2O_2$ ($200{\mu}M$) for 2 h, and Dex/$H_2O_2$ group-cells were pretreated with dexmedetomidine ($5{\mu}M$) for 2 h then exposed to $H_2O_2$ ($200{\mu}M$) for 2 h. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone-related proteins were determined by western blot. Results: Cell viability was significantly decreased in the $H_2O_2$ group compared with the control group, and this effect was improved by dexmedetomidine. The Hoechst 33342 and Annexin-V FITC/PI staining revealed that dexmedetomidine effectively decreased $H_2O_2$-induced hFOB cell apoptosis. Dexmedetomidine enhanced the mineralization of hFOB cells when compared to the $H_2O_2$ group. In western blot analysis, bone-related protein was increased in the Dex/$H_2O_2$ group. Conclusions: We demonstrated the potential therapeutic value of dexmedetomidine in $H_2O_2$-induced oxidative stress by inhibiting apoptosis and enhancing osteoblast activity. Additionally, the current investigation could be evidence to support the antioxidant potential of dexmedetomidine in vitro.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.21-41
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• 2001

The goal of Periodontal treatment is predictable periodontal regeneration. But until now, many products including GTR materials and growth factors are beyond of complete regeneration. BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. BMP can promote periodontal regeneration by their ability to stimulate new bone and new cementum formation. But little is known about optimal conditions required for the application. Root conditioning is used for bioacive root change so altered root surface provides a substrate that promotes chemotaxis, migration and attachment of peridontal cells encouraging connective attachment to the denuded root surface. The aim of this study is to investigate whether the acid conditioning change effect of rhBMP-2 on human periodontal ligament cell and osteoblast cell line. 288 periodontally involved root dentin slices are divided into 6 groups, each 48, 1)control, 2)treated with BMP, 3)treated with citric acid 4)treated with citric acid+BMP 5)treated with tetracycline 6)treated with TC+BMP. Each group was devided half, so 12 root dentin slices were seeded with periodontal ligament cells and 12 were seeded with osteoblasts. At day 2 and 7, cell number, protein assay, ALP activitiy was measured. To investigate morphology of cultured cells, SEM was employed. Statistical analysis was performed with SPSS 8.0 either t-test or ANOVA test. The results are ; Protein assay and cell number was slightly decreased in CA+BMP group compared to Ca group but it was not statistically significant and ALP activity was much more increased in CA+BMP group compared to CA group so there was no statistically significance between BMP and CA+BMP group and statistically significant compared to control group. Cell number and protein assay was slightly increased in TC group and ALP activity was much less the BMP group and CA group. Cell number and protein and ALP activity was not much increased in TC+BMP group. TC group and TC+BMP group showed cell morphology change in SEM. This results suggested that application of root surface with citric acid before BMP treatment might give better result in periodontal regeneration.

• Journal of Periodontal and Implant Science
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• v.32 no.1
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• The korean journal of orthodontics
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• v.30 no.2 s.79
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• pp.197-204
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• 2000

[$K^+$]-selective ion channels were studied in excised inside-out membrane patches from human osteoblast-like cells (G292). Three classes of $K^+$channels were present and could be distinguished on the basis of conductance. Conductances were $270\pm27\;pS,\;113\pm12\;pS,\;48\pm8\;pS$ according to their approximate conductances in symmetrical 140 mM KCl saline at holding potential of -80 mV It was found that the small conductance (48 pS) $K^+$channel activation was dependent on membrane voltage. In current-voltage relationship, small conductance $K^+$channel showed outward rectification, and it was activated by the positive potential inside the membrane. In recordings, single channel currents were activayed by a negative pressure outside the membrane. The membrane pressure increased $P_{open}$ of the $K^+$ channel in a pressure-dependent manner. In the excised-patch clamp recordings, G292 osteoblast-like cells have been shown to contain three types of $K^+$ channels. Only the small conductance (48 pS) $K^+$channel is sensitive to the membrane stretch. These findings suggest that a hyperpolarizing current, mediated in part by this channel, may be associated with early events during the mechanical loading of the osteoblast. In G292 osteoblast-like cells, $K^+$channel is sensitive to membrane tension, and may represent a unique adaptation of the bone cell membrane to mechanical stress.

• Journal of Periodontal and Implant Science
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• v.36 no.2
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• pp.435-448
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• 2006

The purpose of this study was to investigate the effects of ICB(Irradiated frozen allogenic bone, Rocky Mountain Tissue Bank, USA) and MTF(Decalcified freeze-dried bone allograft, Musculoskeletal Transplant Foundation, USA) on the cell proliferation and differentiation of human fetal osteoblasts. Human fetal osteoblasts (hFOB1) were cultured with $10\;ng/m{\ell}$of ICB and MTF. The negatvie control group was cultured with DMSO and positive control group was cultured with BMF ($2\;ng/m{\ell}$). MIT was performed to examine the viability of the cell, and alkaline phosphatase activity was analyzed to examine the mineralization. Calcium accumulation was also evaluated. ICB and MTF did not increase the rate of the cellular proliferation of hFOB1s while they enhanced ALP and calcium accumulation. The expression of osteocalcin (OC) and bone silaloprotein (BSP) increased in hFOB1 treated with ICB and MTF ($10\;ng/m{\ell}$). These results suggest that ICB and MTF stimulate osteoblastic activity of the hFOBl.

• Molecules and Cells
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• v.38 no.3
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• pp.267-272
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• 2015

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC's), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC's led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I-IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC's.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.1
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• pp.10-18
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• 2011

Purpose: The purpose of this study is to find out the effects of bisphosphonates (BPs) on the proliferation and the alkaline phosphatase (ALP) activity of human bone marrow derived mesenchymal stem cells (hMSCs), and thus state its correlation with bisphosphonate related osteonecrosis of the jaw (BRONJ). Methods: hMSCs was obtained by collecting and culturing cancellous bone fragments from a patient undergoing iliac bone graft. Alendronate (Aln) and Pamidronate (Pam), Ibandronate (Ibn) were added to the culture media in the concentration from $10^{-3}$ M to $10^{-11}$ M and cell toxicity, viability were measured. For ALP activity evaluation, Aln and Pam were added to the culture media in the concentration from $5{\times}10^{-7}$ M to $1{\times}10^{-8}$ M and were cultured for 1 week, 2 weeks and 3 weeks. ALP activity data were standardized using protein assay. Control groups were prepared for each examination. Results: Aln, Pam and Ibn all failed to increase the proliferation of hMSCs. With 1 week, 2 weeks of $5{\times}10^{-8}$M of Aln treatment, the ALP activity increased. Pam treatment increased the ALP activity with 2 weeks of $5{\times}10^{-8}$ M and$1{\times}10^{-8}$M. Also Ibn treatment increased the ALP activity with 2 weeks of $5{\times}10^{-8}$ M and $1{\times}10^{-8}$ M. Conclusion: It is considered that BPs are not capable of improving the proliferation of hMSCs. Also, after a transient increase in the ALP activity with the lower concentration of BPs, the activity decreased again. Therefore, in patients on long-term medication of BPs, the proliferation and osteoblast differentiation of hMSCs are restrained, and thus delayed wound healing and increase in BRONJ complications may occur.

• Journal of dental hygiene science
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• v.19 no.4
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• pp.254-260
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• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.

• Journal of Periodontal and Implant Science
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• v.34 no.2
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• pp.317-332
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• 2004

Osteoblasts from alveolar bone may have an important role in the bone regeneration for periodontium, but their culture and characterization are not determined yet. The purpose of this study was to investigate the biological characteristics of primary explant cultured osteoblasts(PECO) from alveolar bone. Osteoblasts were isolated and cultured from alveolar socket of extracted tooth in children. To compare the characteristics, osteoblasts and gingival fibroblasts were cultured with DMEM at $37^{\circ}C$, 5% $CO_2$, l00% humidity incubator, and human fetal osteoblasts cell line(hFOB1) were cultured with DMEM at $34^{\circ}C$, 5%, $CO_2$ 100% humidity incubator. To characterize the isolated bone cells, morphologic change, cell proliferation and differentiation were measured. Morphology of PECO was small round body or cuboidal shape on inverted microscope and was similar with hFOB1. PECO became polygonal shape with stellate and had an amorphous shape at 9th passage in culture. PECO had significantly higher activity than that of gingival fibroblasts and hFOB1 in alkaline phosphatase activity. The expression of osteocalcin and bone sialoprotein in PECO was notably increased when compared with hFOB1 and gingival fibroblasts. These result indicated that PECO from alveolar bone in children has an obvious characteristics of osteoblast, maybe applied for the regeneration of bone.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.93-100
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• 2004

많은 연구들에서 hMSC를 얻기 위해 centrifugation, fluoroscence activated cell sorter(FACS), magnetic activated cell sorter(MACS)가 이용되어져 왔다. 그러나 centrifugation만을 이용한 경우 순도가 떨어지며 FACS나 MACS의 경우에는 비용, 시간이 많이 드는 단점이 있다. 따라서 이 연구에서는 antibody cocktail을 이용하여 hMSC를 좀더 쉽게 얻어내는 방법에 대해 알아보았다. 사람의 골반에서 12G의 바늘을 이용하여 골수를 흡입한 후 heparin이 들어있는 시험관에 넣고 처리과정을 시행하기 전에 냉장고에 보관하며 가능한 한 빨리 처리 과정을 실시한다. 얻은 골수에 적당량의 RosetteSep( Stemcell Technologies)을 첨가한 후 실온에서 20분간 반응시킨다. 그 후 적당량의 Ficoll-paque위에 골수와 RosetteSep의 혼합물을 섞이지 않게 올리고 원심분리를 이용하여 원하는 세포층을 얻어낸다. 이 세포층을 따로 분리한 뒤 배양한다. 배양 시 세포가 80%이상 차기 전에 계속 passage를 시행하며 배양한다. 이는 세포가 밀도가 높아져 원치 않는 세포로 분화되는 것을 막기 위함이다. 배양된 세포가 다양한 분화능력을 가지고 있는지 알아보기 위해 세 가지로 분화를 유도하였다. 적절한 배지와 적절한 환경에서 배양함으로써 얻어진 세포를 osteoblast, chondroblast, adipocyte로 분화를 유도하였다. 분화된 세포가 원하는 형질의 세포로 분화되었는지를 확인하기 위하여 osteoblast의 경우 alizarin red staining, alkaline phosphatase activity, chondroblast의 경우 toluidine blue staining, adipocyte의 경우 Oil-Red-O staining으로 염색하여 분화를 확인하였다. 분리해낸 세포는 각각 세 가지 세포로 분화가 되었으며 이는 RosetteSep이 hMSC를 성공적으로 분리해냈다는 것을 보여준다. 그러나 모든 세포가 분화를 보이지는 않았으며 따라서 hMSC의 순도를 높이기 위한 연구가 더 필요하다. RosetteSep을 이용하면 다른 방법들 보다 쉽게 hMSC를 얻을 수 있으나 기존의 방법과 순도의 측면에서 더 비교할 필요가 있다.