• Journal of Periodontal and Implant Science
• /
• v.31 no.1
• /
• pp.163-180
• /
• 2001

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of periodontal ligament cells. Primary human periodontal ligament cells were cultured in dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells of 4th to 7th passage were inoculated in the multiwell plates coated with chitosan in concentration of 0.22, 0.2, and $2mg/m{\ell}$. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized modules was evaluated after 21 days of culture. The results were as follows : 1. The morphology of periodontal ligament cells on the chitosan coating was round or spheric. Round cells were aggregated after 6 hours of culture. Aggregated cells on the chitosan coated surface showed nodule-like appearance after 24 hours of culture and not achieved confluency at 7 days. 2. During early period of culture, the attachment of periodontal ligament cells were inhibited by chitosan coating. Inhibition of cell attachment tended to increase with the concentration of chitosan. 3. At the chitosan concentration of 0.02 and $0.2mg/m{\ell}$, periodontal ligament cells were more rapidly proliferated at 7 days, compared to the control group. At the concentration of $2mg/m{\ell}$, the proliferation of periodontal ligament cells was inhibitied(p<0.01). 4. Alkaline phosphatase activity of periodontal ligament cells was increased in chitosan coated group, especially at the concentration of $0.02mg/m{\ell}$after 4 days of culture.5. Periodontal ligament cells produced mineralized nodules on chitosan coated wells without the addition of mineralized nodule forming materials (ascorbic acid, ${\beta}-glycerophosphat$, dexamethasone). With the addition of mineralized nodule forming materials, periodontal ligament cells produced more mineralized nodules at the concentration of $0.02mg/m{\ell}$, compared to the control. In summary, the attachment, proliferation, cell activity, and alkaline phosphatase activity of periodontal ligament cells depended on the concentration of coated chitosan. Chitosan stimulated mineralized nodule formation by periodontal ligament cells. At the appropriate concentration($0.02mg/m{\ell}$), chitosan could increase alkaline phosphatase activity and stimulate the formation of mineralized nodule by periodontal ligament cells. These results suggest that chitosan can be used as an adjunct for bone graft material, and the matrix of tissue engineering for periodontal regeneration, especially bone regeneration.

• Journal of Periodontal and Implant Science
• /
• v.25 no.2
• /
• pp.210-221
• /
• 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.

• Journal of Periodontal and Implant Science
• /
• v.26 no.1
• /
• pp.89-102
• /
• 1996

The goal of periodontal therapy is to regenerate the loss of periodontal attachment appratus. Current theories suggest the cells of the periodontium have the capacity, when appropriately triggered, to actively participate in restoring connective tissues, including mineralized tissues. This study was performed to define the hard tissue regeneration effect of periodontal ligament(PDL) cells in vitro and the effect of rate of the composition in gingival fibroblasts(GF) on the hard tissue regeneration capacity of PDL cells. For this study, Cell growth rate, alkaline phosphatase(Al.Pase) levels and the ability to produce mineralized nodules in co-culture of PDL cells and GF were examined. The results were as follows : 1. At 7 and 15 days, Cell growth of co-culture of PDL and GF(50 : 50) was greater than that of PDL cells or GF alone(P>0.05). 2. Measurements of ALPase levels indicated that PDL cells had significantly higher activity when compared with that of co-culture groups and GF only(p<0.05). And, ALPase activity in 10 days was higher than that of 7 days(P>0.05) 3. The tendency of formation of the mineralized nodule were observed dose-depend pattern of PDL cells. There was statistically significant difference among group 1(PDL 100%), 2(PDL 70% : GF 30%), and 3(PDL 50% : GF 50%)(P<0.01). But, there was no difference among group 3, 4(PDL 30% GF 70%), and 5(GF 100%). 4. Also, the number of nodule was greater in co-culture of PDL 70% and GF 30% than in culture of PDL 70%(P<0.05) From the above results, it is assumed that the co-culture of PDL cells and GF stimulates the cell growth, which is not that of PDL cells but GF. And, the activity of ALPase depends on the ratio of PDL cells, and ALPase may relate to the initial phase of nodule formation. Also, it is thought that the calcified nodule formation principally depends on PDL cells, is inhibited by GF, and affected by cell density.

• Journal of Nutrition and Health
• /
• v.51 no.5
• /
• pp.379-385
• /
• 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 Periodontal and Implant Science
• /
• v.35 no.2
• /
• pp.289-298
• /
• 2005

Bone morphogenetic protein-7(BMP-7), a member of the transforming growth factor superfamily, stimulates osteoblast differentiation and bone formation. There are lots of evidences supporting a direct participation of periodontal ligament(PDL) cells on periodontal tissue regeneration. The purpose of this study was to evaluate the effect of recombinant human(rh) BMP-7 on primary rat PDL cells in vitro, with special focus on the ability of bone formation. The PDL cells were cultured with rhBMP-7 at the concentration of 0, 10, 25, 50, 100 and 200ng/ml for MTT assay. We evaluated the alkaline phosphatase activity at 3 and 5 days of incubation and the ability to produce mineralized nodules of rat PDL cells at 14 days of cell culture in concentration of 0, 10, 25, 50 and 100ng/ml. The cell activity was not reduced in cells treated with BMP-7 at $10{\sim}100ng/ml$, whereas the cell activity was reduced in the concentration of 200ng/ml than the control at day 1 and 3(p<0.01). At 3 and 5 day, alkaline phosphatase activity was significantly increased in cells treated with BMP-7 at 50ng/ml and 100ng/ml(p<0.05). The area of mineralized bone nodule was greater in cells treated with BMP-7 at 50 and 100 ng/ml than the control(p<0.01). These results suggest that rhBMP-7 stimulate rat PDL cells to differentiate toward osteoblast phenotype and secretion of the extracellular matrix of rat PDL cells.

• Journal of Periodontal and Implant Science
• /
• v.32 no.1
• /
• pp.235-247
• /
• 2002

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of human gingival fibroblasts. Primary culture of human gingival fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells were inoculated in the multiwell plates coated with chitosan in concentration of 0.02, 0.2, and 2 mg/ml. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized nodules was evaluated after 21 days of culture. The results were as follows : The morphology of cells on the chitosan-coated well was round or spheric. Round cells were aggregated since 6 hours of culture and showed nodule-like appearance after 24 hours of culture and did not achieved confluency at 7 days. The attachment of gingival fibroblasts was inhibited by chitosan coating with a tendency of dose dependent pattern. But, cellular activity of unit cell was higher than control. The proliferation of gingival fibroblasts was inhibited by chitosan coating at 2 mg/ml(P<0.01), while the cell proliferation at 0.02, 0.2 $mg/m{\ell}$ was comparable to the control well. Total alkaline phosphatase activity was inhibited by chitosan coating and decreased in the course of time. While ALP activity of unit cell was the highest at 2mg/ml after 4 days of culture. Finally, gingival fibroblasts produced the mineralized nodule at 2 mg/ml. In summary, the attachment, proliferation, and alkaline phosphatase activity of gingival fibroblasts were influenced differently by the concentration of coated chitosan. From this study, it could be used as the matrix of tissue engineering for gingiva without inhibition on proliferation of gingival fibroblasts using chitosan at the optimal concentration (0.02mg/ml).

• Journal of dental hygiene science
• /
• v.21 no.4
• /
• pp.243-250
• /
• 2021

Background: Endodontic sealers or their toxic components may become inflamed and lead to delayed wound healing when in direct contact with periapical tissues over an extended period. Moreover, an overfilled sealer can directly interact with adjacent tissues and may cause immediate necrosis or further resorption. Therefore, the treatment outcome conceivably depends on the endodontic sealer's biocompatibility and osteogenic potential. This study aimed to evaluate the cell viability and osteogenic effects of four different sealers in osteoblastic cells. Methods: AH Plus (resin-based sealer), Pulp Canal Sealer EWT (zinc oxide-eugenol sealer), BioRoot RCS (calcium silicate-based sealer), and Well-Root ST (MTA-based calcium silicate sealer) were mixed strictly according to the manufacturer's instructions, and dilutions of sealer extracts (1/2, 1/5 and 1/10) were determined. Cell viability was measured using the water-soluble tetrazolium-8 (WST-8) assay. Differentiation was assessed by alkaline phosphatase (ALP) activity and mineralized nodule formation by Alizarin Red S staining. Results: The cell viability of the extracts derived from the sealers excluding Well-Root ST was concentration dependent, with sealer extracts having the least viability at a 1/2 dilution. At sealer extract dilution of 1/10, the test groups showed the same survival rate as that control group, with the exception of BioRoot RCS. Among all experimental groups, BioRoot RCS showed the highest cell viability after 48 hours. The ALP activity was significantly higher in a concentration-dependent manner. Furthemore, all four materials promoted ALP activity and mineralized nodule formation compared to the control at 1/10 dilutions. Conclusion: This is the first study to highlight the differences in biological activity of these four materials. These results suggest that the composition of root canal sealers appears to alter the form of biocompatibility and osteoblastic differentiation.

• Journal of Periodontal and Implant Science
• /
• v.32 no.4
• /
• pp.801-809
• /
• 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.

• Journal of Periodontal and Implant Science
• /
• v.32 no.3
• /
• pp.445-456
• /
• 2002

The purpose of this study were to determine that dexamethasone(Dex) induces differentiation of periodontal ligament(PDL) cells to osteoblastic cells and to investigate expression of matrix Gla protein(MGP), which is one of bone matrix protein. The isolated human PDL cells and gingival fibroblasts were prepared and cultured. The fourth or sixth sub-passage cells were used in this experiments. control group, ascorbic acid and ${\beta}$-glycerophosphate treated group, ascorbic acid, ${\beta}$-glycerophosphate and l00nM Dex treated group, ascorbic acid, ${\beta}$-glycerophosphate, and 5 ${\mu}M$ Dex treated group were made for study. The results were as follows: Cellular morphological change of PDL cells according to time was investigated. At first, the cells exhibited confluent monolayer of spindle or polygonal appearance. The multilayer of cells were seen after 7 days of treatment. After 14 days, the cells lost polarity and were densely packed. The mineralized nodule formation was seen at 21 days in the only Dex treated PDL cell groups. In the gingival fibroblast groups and no Dex treated PDL cell groups, the mineralized nodule was not seen. The mineralized nodule formation of 5 ${\mu}M$ Dex treated group was higher than 100 nM Dex treated group. Alkaline phosphatase(ALP) activity was higher in the Dex treated PDL cell groups of 14 and 21 days than 0 and 7 days. MGP was expressed in the control and all experimental groups and the expression was constant at 0,7,14,21 day. The above results confirm that Dex is affected to differentiation of the PDL cells to osteoblastic or cementoblastic cells and has dose-dependent effect for mineralization. And, MGP is expressed in the PDL cells and is not affected to mineralization of PDL cells.

• International Journal of Oral Biology
• /
• v.33 no.1
• /
• pp.33-43
• /
• 2008

In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium ($Ca{^{2+}}_e$) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and ${\beta}$-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High $Ca{^{2+}}_e$(10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high $Ca{^{2+}}_{e^-}$induced expression of Ank, PC-1 and osteopontin. When high $Ca{^{2+}}_e$(5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high $Ca{^{2+}}_e$. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high $Ca{^{2+}}_{e^-}$treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high $Ca{^{2+}}_e$ stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high $Ca{^{2+}}_{e^-}$induced expression of Ank, PC-1 and osteopontin.