• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권5호
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• pp.453-456
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• 2001

Background: Autogenous alveolar bone cell transplantation may be suitable for tissue engineering for alveolar bone reconstruction. This study aimed to isolate human alveolar bone-derived cells (HABDCs) and to evaluate the ability of collagen gels to support HABDC proliferation and differentiation for human alveolar bone tissue engineering applications. Method: Cultures of primary HABDCs were established from alveolar bone chips obtained from 10 persons undergoing tooth extraction. These cells were expanded in vitro until passage 3 and used for the in vitro characterization of HABDCs and the in vitro analysis of collagen gels for alveolar bone tissue engineering. Results: Of the 10 attempts made to obtain HABDC cultures, eight were successful. HABDCs expressed the osteoblastic phenotype characterized by alkaline phosphatase activity, osteocalcin expression and the mineralization of the extracellular matrix in vitro. When seeded on collagen gels, HABDCs penetrated into the collagen gel matrices and proliferated inside the gels. Significantly, when HABDCs were embedded into the gels, collagen fibers and mineralization were produced within the gels. Conclusion: This study demonstrates the feasibility of using cultured HABDCs and collagen gels for human alveolar bone tissue engineering applications.

• Journal of Periodontal and Implant Science
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• 제25권2호
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• pp.210-221
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• 1995

The purpose of this study was performed to investigate the mineralization and differentiation of osteobalsts for bone regeneration in vitro and the effect of rate of the composition in periodontal cells on mineralization. For this study, healthy gingival tissues were surgically obtained from the patients during 1st premolar extraction for the purposes of orthodontic treament. Gingival tissue was washed several time with Phosphate buffered saline contained high concentration of antibiotics and antifungal agent, and cultured in Dulbecco's Modified Eagle's Medium(DMEM, Gibco, U.S.A.). Every cell were cultured in state at $37^{\circ}C$, 100% of humidity, 5% of $CO_2$ incubator. Bone marrow stromal cells were isolated from 5-clay-old rat femur with using medium irrigation mathod by syringe. Cell suspension medium were centrifuged at 1500 rpm for 5 min and then cultured in the petri dish. Two kinds of cell were freezed and stocked in the liquid nitrogen tank until experiment. Cell were incubated into the 24 multi-well plate with $5{\times}10^4$cell/well of medium at $37^{\circ}C$, 100% of humidity 5% $CO_2$ incubator for 24 hours. After discarded of the supernatent of medium, O.5ml of medium were reapplied and incubated. And counted the number of cell using the hemocytometer and inverted light microscope. We have measured the number of mineralized nodule with using Alizarin red S. staining in microscope. Furthermore every cell were observed the morphological change between every rate of co-culture of the two kinds of cell. The results were as follows; The rate of proliferation of co-culture cell revealed high rate tendency compared the bone marrow stromal cell only and low growth rate to compared with gingival fibroblast only. The tendency of formation of the mineralized nodule were observed dose-depend pattern of bone marrow stromal cell. It is concluded that the gingival fibroblast may inhibit the formation of mineralized nodule in the culture of the bone marrow stromal cell.

• 생명과학회지
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• 제26권3호
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• pp.331-337
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• 2016

섬유모세포성장인자-23(fibroblast growth factor 23, FGF23)은 뼈를 형성하는 세포에서 주로 생성되지만 그 작용은 신장에서 이루어진다. FGF23은 신장의 나트륨-인산염 공동수송체(Na-phosphate cotransporter)를 억제하여 인산염 재흡수를 감소시킨다. 이렇게 함으로써 인산염 항상성을 조절하는 작용과는 별개로 이것은 in vivo에서 뼈 형성을 억제하는 것으로 알려져 있다. 두개골 조골세포를 이용한 연구에서도 FGF23은 조골세포의 발달, 즉 분화 및 기질의 광화(mineralization)에 악영향을 미쳤다. 본 연구는 FGF23이 골수 유래 간엽줄기세포에서 조골세포로의 발달에 있어서도 유사한 영향을 줄 것인지를 조사한 것이다. 간엽줄기세포주인 D1 세포를 β-glycerophosphate, ascorbic acid, dexamethazone이 포함된 조골배(osteogenic medium)에 배양하여 alkaline phosphatase (Alp) 염색으로 분화를, Alizarin red 염색과 기질의 칼슘 함량의 분석을 통해 광화를 평가하였다. 분화 촉진 유전자인 Runx2, osteocalcin, Alp와 광화 억제 유전자인 Enpp1, Ank의 발현은 RT-PCR로 분석하였다. D1 세포의 증식과 조골세포로의 분화는 생리학적 농도를 훨씬 초과하는 FGF23의 농도에 의해서도 달라지지 않았다. FGF23 처치 1주, 2주, 3주 후 Alizarin red 염색에 의한 광화 정도의 평가에서도 대조군과 실험군의 차이는 발견되지 않았다. 그러나 두 군 모두 시간이 경과함에 따라 광화는 증가되었다. 기질에 침착된 칼슘의 양 또한 차이가 없었다. 분화 촉진 유전자와 광화 억제 유전자의 발현도 양 군 간에 다르지 않았다. 이러한 부정적인(negative) 결과는 FGF23에 의한 세포 내 신호전달의 장애가 아님이 Erk 인산화로 확인되었다. 이상의 결과로 미루어 두개골의 조골세포와 달리 FGF23은 간엽줄기세포에서 조골세포로의 분화와 광화에는 영향을 미치지 않을 것으로 사료된다.

• International Journal of Oral Biology
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• 제31권2호
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• pp.53-65
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• 2006

Calcium concentration in the bone resorption lacunae is high and is in the mM concentration range. Both osteoblast and osteoclast have calcium sensing receptor in the cell surface, suggesting the regulatory role of high extracellular calcium in bone metabolism. In vitro, high extracellular calcium stimulated osteoclastogenesis in coculture of mouse osteoblasts and bone marrow cells. Therefore we examined the genes that were commonly regulated by both high extracellular calcium and $1,25(OH)_2vitaminD_3(VD3)$ by using mouse oligo 11 K gene chip. In the presence of 10 mM $[Ca^{2+}]e$ or 10 nM VD3, mouse calvarial osteoblasts and bone marrow cells were co-cultured for 4 days when tartrate resistant acid phosphatase-positive multinucleated cells start to appear. Of 11,000 genes examined, the genes commonly regulated both by high extracellular calcium and by VD3 were as follows; 1) the expression of genes which were osteoclast differentiation markers or were associated with osteoclastogenesis were up-regulated both by high extracellular calcium and by VD3; trap, mmp9, car2, ctsk, ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and chemokine receptor genes such as sdf1, scya2, scyb5, scya6, scya8, scya9, and ccr1 were up-regulated both by high extracellular calcium and by VD3, 3) the genes such as mmp1b, mmp3 and c3 which possibly stimulate bone resorption by osteoclast, were commonly up-regulated, 4) the gene such as c1q and msr2 which were related with macrophage function, were commonly down-regulated, 5) the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were commonly down-regulated; slc8a1, admr, plod2, lox, fosb, 6) the genes which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were commonly up-regulated; s100a4, npr3, mme, 7) the genes such as calponin 1 and tgfbi which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were up-regulated by high extracellular calcium but were down-regulated by VD3. These results suggest that in coculture condition, both high extracellular calcium and VD3 commonly induce osteoclastogenesis but suppress osteoblast differentiation/mineralization by regulating the expression of related genes.

• Nutritional Sciences
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• 제9권4호
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• pp.233-239
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• 2006

Porous matrices of bioactive polymers such as polyglycolic acid (PGA) or polylactic acid (PLA) can be used as scaffolds in bone tissue growth during bone repair process. These polymers are highly porous and serve as a template for the growth and organization of new bone tissues. We evaluated the effect of PGA and PLA polymers on osteoblastic MC3T3-E1 cell extracellular mineralization. MC3T3-E1 cells were cultured in a time-dependent manner -1, 15, 25d as appropriate - for the period of bone formation stages in one of the five culture circumstances, such as normal osteogenic differentiation medium, PGA-plated, fetal bovine serum (FBS)-plated, PGA/FBS-coplated, and PLA-plated For the evaluation of bone formation, minerals (Ca, Mg, Mn) and alkaline phosphatase activity, a marker for osteoblast differentiation, were measured Alizarin Red staining was used for the measurement of extracellular matrix Ca deposit During the culture period, PGA-plated one was reabsorbed into the medium more easily and faster than the PLA-plated one. At day 15, at the middle stage of bone formation, cellular Ca and Mg levels showed higher tendency in PGA- or PLA-plated treatments compared to non-plated control and at day 25, at the early late stage of bone formation, all three cellular Ca, Mg or Mn levels showed higher tendency as in order of PGA-related treatments and PLA-plated treatments, compared to control even without significance. Medium Ca, Mg or Mn levels didn't show any consistent tendency. Cellular ALP activity was higher in the PGA- or PLA-plated treatments compare to normal osteogenic medium treatment PGA-plated and PGA/FBS-plated treatments showed better Ca deposits than other treatments by measurement of Alizarin Red staining, although PLA-plated treatment also showed reasonable Ca deposit. The results of the present study suggest that biodegradable material, PGA and also with less extent for PLA, can be used as a biomaterial for better extracellular matrix mineralization in osteoblastic MC3T3-E1 cells.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.801-809
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• 2002

The ultimate goal of periodontal therapy is to promote the regeneration of lost periodontal tissue, there have been many attempts to develop a method to achieve this goal, hut none of them was completely successful. The purpose of this study is to evaluate the effects of Bio-Oss(R) on alkaline Phosphatase (ALP) activity in human fetal osteoblasts (hFOB1). The results of this study were as follows, in ALP Activity, 100 ${\mu}g/ml$ Bio-Oss(R) treated group showed significantly increased value than negative control group, but positive group($10^{-7}$ M dexamethasone treated group) showed the highest ALP activity at 3 day. In mineralization assay, numerous mineralized nodules were identified as darkly stained spots in 100${\mu}g/ml$ Bio-Oss(R) treated group than two control groups, whereas a small number of mineralized nodules were showed in the positive control. ALP may relate to the initial phase of bone nodule formation. On the basis of these results, this study showed Bio-Oss(R) is capable of accelerating new bone formation through hFOBl differentiation in vitro.

• International Journal of Stem Cells
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• 제16권4호
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• pp.406-414
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• 2023

Dedifferentiated fat cells (DFATs) isolated from mature adipocytes have a multilineage differentiation capacity similar to mesenchymal stem cells and are considered as promising source of cells for tissue engineering. Bone morphogenetic protein 9 (BMP9) and low-intensity pulsed ultrasound (LIPUS) have been reported to stimulate bone formation both in vitro and in vivo. However, the combined effect of BMP9 and LIPUS on osteoblastic differentiation of DFATs has not been studied. After preparing DFATs from mature adipose tissue from rats, DFATs were treated with different doses of BMP9 and/or LIPUS. The effects on osteoblastic differentiation were assessed by changes in alkaline phosphatase (ALP) activity, mineralization/calcium deposition, and expression of bone related genes; Runx2, osterix, osteopontin. No significant differences for ALP activity, mineralization deposition, as well as expression for bone related genes were observed by LIPUS treatment alone while treatment with BMP9 induced osteoblastic differentiation of DFATs in a dose dependent manner. Further, co-treatment with BMP9 and LIPUS significantly increased osteoblastic differentiation of DFATs compared to those treated with BMP9 alone. In addition, upregulation for BMP9-receptor genes was observed by LIPUS treatment. Indomethacin, an inhibitor of prostaglandin synthesis, significantly inhibited the synergistic effect of BMP9 and LIPUS co-stimulation on osteoblastic differentiation of DFATs. LIPUS promotes BMP9 induced osteoblastic differentiation of DFATs in vitro and prostaglandins may be involved in this mechanism.

• Nutritional Sciences
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• 제8권4호
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• pp.242-249
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• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.

• Restorative Dentistry and Endodontics
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• 제39권3호
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• pp.187-194
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• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• Journal of Korean Dental Science
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• 제7권1호
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• pp.1-5
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• 2014

Purpose: Capseal I and Capseal II are calcium silicate and calcium phosphate based experimental root canal sealers. This study sought to evaluate the biocompatibility and mineralization potential of Capseal I and Capseal II. Materials and Methods: The biocompatibility and mineralization related gene expression (alkaline phosphatase [ALP], bone sialoprotein [BSP], and osteocalcin) of Capseal I and Capseal II were compared using methylthiazol tetrazolium assay and reverse transcription-polymerization chain reaction analysis, respectively. The results were analyzed by Kruskal-Wallis test. A P-value of <0.05 was considered significant. Result: Both Capseal I and Capseal II were favorable in terms of biocompatibility, influencing the messenger RNA expression of ALP and BSP. Conclusion: Within the limitation of this study, Capseal is biocompatible, with mineralization promoting potential; thus, it could be a promising root canal sealer.