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.
Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.
A recent report showed that nuclear factor of activated T cell (NFATc) 1 is a member of the NFAT family and is strictly implicated osteoblast differentiation and bone formation. Furthermore, the precise expression and function of NFATc1 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the function of NFATc1 in osteoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs). NFATc1 messenger RNA (mRNA) and protein levels were accessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. Cell proliferation determined using MTT assay. Differentiation was evaluated by alkaline phosphatase activity and formation of calcium nodule with alizarin red S staining. The mRNA expression of osteoblastic differentiation related genes were examined by RT-PCR. Marked upregulation of NFATc1 mRNA and protein was observed in cells grown in osteogenic medium (OS). NFATc1 transactivation was detected in hPDLCs that had been incubated in OS for 14 days. Treatment with $10{\mu}M$ cyclosporine A (CsA), a known calcineurin inhibitor, reduced the proliferation of hPDLCs, while $5{\mu}M$ CsA had no effect. Inhibition of the calcineurin/NFATc1 pathway by CsA, attenuated OS-induced osteoblastic differentiation in hPDLCs. In summary, this study demonstrates for the first time that NFATc1 plays a key role in osteoblastic differentiation of hPDLCs and activation of NFATc1 could provide a novel mechanism for periodontal bone regeneration.
The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at $37^{\circ}C$ for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
/
v.32
no.6
/
pp.524-529
/
2006
For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
/
v.32
no.6
/
pp.495-505
/
2006
In this study we evaluate the effects of bFGF-BB and PDGF on in vitro proliferation, differentiation and mineralization of mesenchymal stem cells (MSCs) from rat. MSCs were prepared from the bone marrow of 6 or 7-week-old male rats with a technique previously described by Maniatopoulos et al. in 1988. Lineage differentiation to osteogenesis, chondrogenesis and adipogenesis were performed. At first, we characterized the cultured cell on passage 1, 3, 5, 7 with immunocytochemical staining using CD29, 44, 34, 45, ${\alpha}$-SMA and type I collagen. And to study the effects of bFGF and PDGF-BB on proliferation, differentiation and mineralization, we seeded the expanded cell at a density of 6 $6{\times}10^3\;cells/cm^2$ to 100-mm dish for evaluation of cell proliferation and MTT assay was carried out on day 2, 4, 7, 9. We also resuspended the cells with same density $(6{\times}10^3\;cells/cm^2)$ to 24 well plates for subculture. On the following day, the attached cells were exposed to 2.5ng/ml bFGF and/or 25ng/ml PDGF-BB daily during 5 days. The osteocalcin (OC) level was assessed and mineral contents were evaluated with alizarin red S staining on subculture day 2, 7, 14, 21. We identified the mesenchymal stem cell from the bone marrow derived cells of rat through their successful multi-differentiation and stable display of its phenotype. And bFGF and PDGF-BB showed the effect that inhibited osteoblastic differentiation and mineralization mildly in above concentration at in vitro culture. This study was supported by grant 04-2004-0120 from the Seoul National University Hospital Research Fund.
Several experimental studies showed that the application of small amounts of electric current to bone stimulated osteogenesis at the site of the cathode and suggests that the application of electrical currents to periodontal defects could promote bone and cementum formation. The purpose of this study was to determine the effect of direct microcurrent to the periodontal regeneration of class III furcation defects in dogs. Class III furcation defects were surgically created on the third and the fourth premolars bilaterally in the mandibles of nine mongrel dogs. Experimental periodontitis were induced by placing small cotton pellets into the created defects for 3 weeks. The experimental sites were divided into three groups according to the treatment modalities: Group I-surgical debridement only; Group II-allogenic demineralized freeze dried bone grafting; Group III-allogenic demineralized freeze dried bone grafting and electrical stimulation. For fluorescence microscopic evaluation, calcein, oxytetracycline HCI and alizarin red were injected 2, 4 and 8 weeksfS days prior to sacrifice) after surgery. The animals were sacrificed in the 1st, 2nd, 4th and 8th week after periodontal surgery and the decalcified and undecalcified specimens were prepared for histological and histometrical examination. After the first and the second weeks, gingival recession was more severe in group I than groups II and III. After the fourth and the eighth weeks, there was no difference in the width of junctional epithelium and connective tissue attachment among the three groups, but the width of connective tissue attachment increased in group II at the eighth week, compared to the fourth week. The amount of bone repair in new attachment was significantly greater in group III, compared to groups I and II. New attachment formation was significantly greater in group III, compared to groups I and group II. These results suggest that electrical stimulation using microcurrent generator could be a useful tool for periodontal regenerative therapy in class III furcation defect.
Kim, Doo-Hyun;Kim, Heung-Joong;Jeong, Moon-Jin;Son, Ho-Hyun;Park, Joo-Cheol
Restorative Dentistry and Endodontics
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v.29
no.4
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pp.399-408
/
2004
Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORP) of OD314 by transient transfection analysis using green fluorescent protein (GPP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
/
v.28
no.3
/
pp.216-225
/
2002
Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in ${\alpha}-MEM$ medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.
Journal of Korean Academy of Oral and Maxillofacial Radiology
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v.29
no.1
/
pp.119-147
/
1999
The purpose of this study was to investigate the effects of the Co-60 γ irradiation on the osseointegration. 2.0 mm titanium alloy screw implants(Sankin Industry Co. Ltd., Japan) were placed in the tibial metaphysis of the rabbits, bilaterally. The mean length of the implants was 6.0 mm. The right tibia was irradiated with a single dose of 15Gy from 60Co teletherapic machine at 5th postoperative day. The experimental group was irradiated tibia. The control group was nonirradiated tibia. To observe the phase of bone formation, the bone labeling by intramuscular injection of 20mg/Kg of Tetracycline, Calcein, Alizarin red S, was performed. The rabbits were sacrificed on the 1st, 2nd, 4th, 6th, 8th week and the tibia including implants were taken, and then the specimens were examined by the microradiography, light microscopy, and fluorescent microscopy. The obtained results were as follows: 1. There were connective tissue between bone and titanium at 1st week, in both group. Especially, the many empty lacunae without nucleus and obscure cytoplasm in experimental group, were observed. 2. The osteons were observed at 4th week in control group, and at 6th week in experimental group. The bone formation in experimental group was retarded as compared to the control group. 3. In fluorescent microscopy, bone labelling band was observed as linear, arc or concentric shape. Occasionary interrupted labelling band was observed, which is demonstrated bone remodeling. 4. In microradiographic examination, the radiolucent image was found between bone and implant with widening of bone marrow spaces as compared to the control group.
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