Biocompatibilidade do gel de quitosana associado ao glicerol fosfato na reparação de defeitos ósseos induzidos experimentalmente no rádio de coelhos (Oryctolagus cuniculus) / [Biocompatibility of chitosan gel associated with glycerol phosphate for the healing of experimentally induced radial defects in rabbits (Oryctolagus cuniculus)]

A engenharia de tecidos caracteriza-se como ciência interdisciplinar, a qual vem desenvolvendo biomateriais para a regeneração do tecido ósseo no âmbito das medicinas humana e veterinária. O objetivo desta pesquisa foi avaliar a regeneração óssea obtida da aplicação do hidrogel de quitosana associado ao glicerol fosfato em falha óssea experimentalmente induzida no rádio de coelhos. Foram utilizados 15 coelhos adultos, distribuídos aleatoriamente em dois grupos, representados por cada um dos rádios de cada animal, sendo um grupo tratado com hidrogel de quitosana associado ao glicerol fosfato (grupo biomaterial - GB) e um grupo que não recebeu tratamento com o biomaterial (grupo controle - GC). Os animais foram avaliados radiograficamente, por densitometria óptica e análise histológica, nos períodos 30, 60 e 90 dias pós-operatórios. Houve superioridade estatística na média geral das avaliações radiográficas do GB (2,33±0,48) sobre o GC (1,77±0,06). As médias gerais de avaliação densitométrica do GB foram superiores às do GC, sendo 6,207±1,374 e 5,71±1,512, respectivamente. A avaliação histopatológica do GB foi superior à do GC nos períodos de 30, 60 e 90 dias. Assim, é possível afirmar que o hidrogel de quitosana constitui biomaterial de características desejáveis, promovendo consolidação óssea mais rápida e eficiente, sem causar reações adversas.(AU)

Tissue engineering is an interdisciplinary science that has been developing biomaterials for bone regeneration in medicine and veterinary medicine, following an imminent need. The aim of this study was to evaluate bone regeneration after use of chitosan hydrogel associated with glycerol phosphate in experimentally induced bone gap in the radius of rabbits. Fifteen adult rabbits were randomly distributed in two experimental groups, represented by each radius of every single animal. The animals in the Biomaterial Group (GB) were treated with a glycerol phosphate-associated chitosan hydrogel and in the Control Group (GC) they received no treatment with the biomaterial. The animals were evaluated clinically, radiographically, histologically and by optic densitometry at 30, 60 and 90 days postoperatively. There was statistical superiority in the general average of the radiographic estimates of GB (2.33 ± 0.48) over the CG (1.77 ± 0.06). The general averages of GB densitometric evaluation were higher than the CG, being 6.207 ± 1.374 and 5.71 ± 1.512, respectively. Histopathological evaluation of GB was superior to CG in periods of 30, 60 and 90 days. Chitosan hydrogel constitutes a biomaterial of desired characteristics, promoting faster and more efficient bone repair when compared to GC.(AU)

Resin-modified glass ionomer containing calcium glycerophosphate: physico-mechanical properties and enamel demineralization

Abstract Sources of calcium and phosphate have been added to dental restorative materials to improve their anticaries effect. Objective This study evaluated the effect of adding calcium glycerophosphate (CaGP) to resin-modified glass ionomer cement (RMGIC) on the physico-mechanical properties, ion release, and enamel demineralization. Material and Methods: Specimens were fabricated for each experimental group: RMGIC without CaGP (Control), RMGIC with 1, 3 and 9% CaGP. To determine the release of fluoride (F), calcium (Ca) and phosphorus (P), six specimens were immersed in demineralization and remineralization solutions for 15 days. In another experimental trial, the following physico-mechanical properties were evaluated at time intervals of 1 and 7 days after fabrication: compressive strength (n=12), diametral tensile strength (n=12), surface hardness of material (n=6) and the degree of conversion of monomers (n=8). To study enamel demineralization, specimens (n=12) were attached to enamel blocks and submitted to pH-cycling. Subsequently, surface and cross-sectional hardness and the concentration of F, Ca and P in enamel were determined. Results The addition of CaGP to RMGIC led to higher mean release of F, Ca and P when compared with control (p<0.001). Mechanical properties were within the range of those of the ionomer cements after addition of 1% and 3% CaGP. The degree of conversion did not differ between groups at the 1st and the 7th day (p>0.439). The addition of 3% and 9% CaGP reduced mineral loss and increased F, Ca and P in the enamel when compared with control (p<0.05). Conclusion The addition of 3% CaGP in RMGIC increased the release of F, P and Ca, reduced enamel demineralization, and maintained the physico-mechanical properties within the parameters for this material.

Effect of bone morphogenetic protein-2 on human heart valve interstitial cells calcification in vitro / 中华心血管病杂志

<p><b>OBJECTIVE</b>To establish human heart valve interstitial cells calcification culture model in vitro, and observe the effect of bone morphogenetic protein-2 (BMP-2) on calcification of human heart valve interstitial cells.</p><p><b>METHODS</b>Human heart valve interstitial cells were cultured in vitro, and divided into control group: cells were cultured in conventional media plus recombinant human BMP-2 treatment and experimental group: besides above treaments, calcification inducers ( recombinant human BMP-2, β-glycerophosphate, L-ascorbic acid, dexamethasone) were added to the culture media. The two group of cells were cultured for 14 days and were stained by Von Kossa, then the cell calcification was observed in this valvular interstitial cells calcification culture model in vitro. Protein expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 8, BMP-2 and BMP-4 was determined by Western blot and BMP-2 secretion was measured by ELISA.</p><p><b>RESULTS</b>In the control group, the structure of human heart valve interstitial cells was clear, and the spindle and radial growth shaped cellular morphology was visible, and Von Kossa staining was negative. In the experimental group, the nuclei become darker in color, and granular sediment distribution was seen surrounding cells, and Von Kossa staining was positive, the cells were forming nodules of calcification. The protein expression of ICAM-1, interleukin 8, BMP-2 and BMP-4 in the experimental was significantly higher than that of the control group (all P < 0.05). The expression of BMP-2 in the experimental group was also significantly higher than that in control group ((92.5 ± 4.9) pg/ml vs. (22.2 ± 1.9) pg/ml, P < 0.05).</p><p><b>CONCLUSION</b>Human BMP-2, β-glycerophosphate, L-ascorbic acid, and dexamethasone can induce human heart valve interstitial cells calcification and enhance inflammation in vitro by stimulating the secretion of BMP-2.</p>

Effects and mechanism of fibroblast growth factor 21 on rat vascular smooth muscle cells calcification / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effect and mechanism of fibroblast growth factor 21 (FGF21) on rat vascular smooth muscle cells (VSMCs) calcification in vitro.</p><p><b>METHODS</b>VSMCs was treated with calcification medium containing calcium chloride and β-glycerophosphate to induce rat VSMCs calcification in vitro. VSMCs were divided into 5 groups: the control group (cultured in normal medium), the calcification group (incubated in calcified medium), the FGF21 group (cultured in calcified medium and FGF21), the PD166866 group (cultured in calcified medium and FGF21 and PD166866, inhibitor of fibroblast growth factor receptor-1 (FGFR1)), the GW9662 group (cultured in calcified medium and FGF21 and GW9662, inhibitor of peroxisome proliferators activated receptor-γ (PPAR-γ)). The calcification of VSMCs was detected by calcium content, alkaline phosphatase activity and alizarin red staining. The protein and mRNA expression of FGFR1, β-Klotho, osteocalcin and smooth muscle 22α (SM22α) were determined by western blot analysis and realtime-PCR, respectively.</p><p><b>RESULTS</b>(1) The mRNA (P < 0.01) and protein expressions of β-Klotho and FGFR1 were significantly downregulated in calcification group compared with control group (P < 0.05 or 0.01). (2) The protein levels and mRNA expression of calcium content, alkaline phosphatase activity and osteocalcin were significantly downregulated, while the protein levels and mRNA of SM22α were significantly increased in FGF21 group compared with calcification group (all P < 0.05). Moreover, alizarin red staining verified positive red nodules on calcified VSMCs was significantly reduced in FGF21 group than in calcification group. (3) Calcium content, alkaline phosphatase activity and alizarin red staining were similar between PD166866 group and calcification group (all P > 0.05). (4) Calcium content, alkaline phosphatase activity and alizarin red staining were similar between GW9662 group and calcification group (all P > 0.05).</p><p><b>CONCLUSION</b>The inhibition of VSMCs calcification by FGF21 is mediated by further downregulating FGFR1 and β-Klotho while activating PPAR-γ pathways.</p>

Fluoride varnishes with calcium glycerophosphate: fluoride release and effect on in vitro enamel demineralization

The aims of this study were (1) to assess the amount of fluoride (F) released from varnishes containing calcium glycerophosphate (CaGP) and (2) to assess the effect of the experimental varnishes on in vitrodemineralization. Six test groups using 5 varnishes: base varnish (no active ingredients); Duraphat® (2.26% NaF); Duofluorid® (5.63% NaF/CaF2); experimental varnish 1 (1% CaGP/5.63% NaF/CaF2); experimental varnish 2 (5% CaGP/5.63% NaF/CaF2); and no varnish were set up. In stage 1, 60 acrylic blocks were randomly distributed into 6 groups (n = 10). Then 300 µg of each varnish was applied to each block. The blocks were immersed in deionized water, which was changed after 1, 8, 12, 24, 48 and 72 hours. Fluoride concentration in the water was analyzed using a fluoride electrode. In stage 2, 60 bovine enamel samples were distributed into 6 groups (n = 10), and treated with 300 µg of the respective varnish. After 6 h the varnish was removed and the samples were subjected to a 7-day in vitro pH cycle (6 h demineralization/18 h remineralization per day). The demineralization was measured using surface hardness. The results showed that both experimental varnishes released more fluoride than Duofluorid® and Duraphat® (p < 0.05), but Duraphat® showed the best preventive effect by decreasing enamel hardness loss (p < 0.05). Therefore, we conclude that even though (1) the experimental varnishes containing CaGP released greater amounts of F, (2) they did not increase in the preventive effect against enamel demineralization.

Reimplante dentário após o preenchimento do canal com pasta de B-glicerofosfato de cálcio ou pasta de hidróxido de cálcio + MTA: estudo histomorfométrico em ratos / Tooth replantation after root canal filling with paste B-glycerophosphate calcium or calcium hydroxide paste: histomorphometric study in rats

A avulsão dentária acomete principalmente pacientes jovens e que possuem dentes com rizogênese incompleta, sendo frequente a necrose pulpar e a apicificação que é prejudicada. O amplo forame dificulta a inserção de curativos de demora. Assim é importante o uso de um curativo de demora a favorecer o controle da reabsorção radicular e a formação de uma barreira de tecido mineralizado permitindo o selamento apical. O hidróxido de cálcio tem sido o material mais utilizado para esse fim. O objetivo deste trabalho foi estudar o processo de reparo de incisivos de ratos com elementos dentais reimplantados tardiamente após a obturação do canal radicular com pasta de β-glicerofosfato de cálcio ou pasta de hidróxido de cálcio. Foram empregados 30 ratos machos divididos em 3 grupos de 10 animais. Os elementos dentais foram extraídos e mantidos em meio seco por 60 minutos. Após esse período os elementos dentais sofreram um preparo especifico descrito a seguir. No Grupo I os dentes foram preenchidos com soro fisiológico. No Grupo II, utilizou-se a pasta de hidróxido de cálcio no interior do conduto e plug apical de cimento MTA. E, no grupo III utilizou-se a pasta de β-glicerofosfato de cálcio. Os dentes foram reimplantados e a eutanásia dos animais foi realizada 60 dias após para a avaliação histomorfométrica em H.E (Hematoxicilina-Eosina). Os resultados demonstraram que os grupos I e III foram mais comprometidos pela reabsorção radicular inflamatória do que o grupo II. O grupo II apresentou menor comprometimento pela reabsorção total do que o grupo II (p<0,05). Nos grupos I e III a região periapical apresentou a maior extensão de tecido conjuntivo fibroso com a presença de infiltrado inflamatório. Conclui-se que o β-glicerofosfato de cálcio não foi mais eficiente do que o hidróxido de cálcio no controle da reabsorção radicular e no reparo da região periapical por tecido mineralizado

Thooth avulsion mainly affects young patients who have incomplete root formation, with frequent necrotic pulp and apexification impaired. The large foramen hinders the insertion of dressings delay. The use of a long time fill to facilitate control of formation and resorption of mineralized tissue barrier permitting the apical seal. Calcium hydroxide and MTA has been the most widely used material for this purpose. The objective of this work is to study the repair process of rat incisors with dental elements reimplanted late after root canal filling with paste β-glycerophosphate calcium or calcium hydroxide paste. Were used 30 male rats were divided into 3 groups of 10 animals. All the teeth were extracted and kept in dry for 60 minutes. In Group I, the root canals were filled with saline. Group II used the paste of calcium hydroxide and finished with a MTA plug and Group III were used β-glycerophosphate calcium paste. The teeth were reimplanted and euthanasia was performed 60 days later for histomorfometric analyse with HE(Hematoxiciline-Eosine). Results showed that the groups I and III were more impaired by inflammatory root resorption than group II. Group II had less involvement by total reabsorption of the group II (p <0,05). In groups I and III, the periapical region showed the greatest extent of fibrous connective tissue with inflammatory infiltrate. It is concluded that β-glycerophosphate calcium were less effective than calcium hydroxide in the control of root resorption and repair of the periapical area of mineralized tissue

Reimplante dentário após o preenchimento do canal com pasta de B-glicerofosfato de cálcio ou pasta de hidróxido de cálcio + MTA: estudo histomorfométrico em ratos / Tooth replantation after root canal filling with paste B-glycerophosphate calcium or calcium hydroxide paste: histomorphometric study in rats

A avulsão dentária acomete principalmente pacientes jovens e que possuem dentes com rizogênese incompleta, sendo frequente a necrose pulpar e a apicificação que é prejudicada. O amplo forame dificulta a inserção de curativos de demora. Assim é importante o uso de um curativo de demora a favorecer o controle da reabsorção radicular e a formação de uma barreira de tecido mineralizado permitindo o selamento apical. O hidróxido de cálcio tem sido o material mais utilizado para esse fim. O objetivo deste trabalho foi estudar o processo de reparo de incisivos de ratos com elementos dentais reimplantados tardiamente após a obturação do canal radicular com pasta de β-glicerofosfato de cálcio ou pasta de hidróxido de cálcio. Foram empregados 30 ratos machos divididos em 3 grupos de 10 animais. Os elementos dentais foram extraídos e mantidos em meio seco por 60 minutos. Após esse período os elementos dentais sofreram um preparo especifico descrito a seguir. No Grupo I os dentes foram preenchidos com soro fisiológico. No Grupo II, utilizou-se a pasta de hidróxido de cálcio no interior do conduto e plug apical de cimento MTA. E, no grupo III utilizou-se a pasta de β-glicerofosfato de cálcio. Os dentes foram reimplantados e a eutanásia dos animais foi realizada 60 dias após para a avaliação histomorfométrica em H.E (Hematoxicilina-Eosina). Os resultados demonstraram que os grupos I e III foram mais comprometidos pela reabsorção radicular inflamatória do que o grupo II. O grupo II apresentou menor comprometimento pela reabsorção total do que o grupo II (p<0,05). Nos grupos I e III a região periapical apresentou a maior extensão de tecido conjuntivo fibroso com a presença de infiltrado inflamatório. Conclui-se que o β-glicerofosfato de cálcio não foi mais eficiente do que o hidróxido de cálcio no controle da reabsorção radicular e no reparo da região periapical por tecido mineralizado...

Thooth avulsion mainly affects young patients who have incomplete root formation, with frequent necrotic pulp and apexification impaired. The large foramen hinders the insertion of dressings delay. The use of a long time fill to facilitate control of formation and resorption of mineralized tissue barrier permitting the apical seal. Calcium hydroxide and MTA has been the most widely used material for this purpose. The objective of this work is to study the repair process of rat incisors with dental elements reimplanted late after root canal filling with paste β-glycerophosphate calcium or calcium hydroxide paste. Were used 30 male rats were divided into 3 groups of 10 animals. All the teeth were extracted and kept in dry for 60 minutes. In Group I, the root canals were filled with saline. Group II used the paste of calcium hydroxide and finished with a MTA plug and Group III were used β-glycerophosphate calcium paste. The teeth were reimplanted and euthanasia was performed 60 days later for histomorfometric analyse with HE(Hematoxiciline-Eosine). Results showed that the groups I and III were more impaired by inflammatory root resorption than group II. Group II had less involvement by total reabsorption of the group II (p <0,05). In groups I and III, the periapical region showed the greatest extent of fibrous connective tissue with inflammatory infiltrate. It is concluded that β-glycerophosphate calcium were less effective than calcium hydroxide in the control of root resorption and repair of the periapical area of mineralized tissue...

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Calcium glycerophosphate supplemented to soft drinks reduces bovine enamel erosion

OBJECTIVE: This in vitro study evaluated the effect of calcium glycerophosphate (CaGP) supplemented to soft drinks on bovine enamel erosion. MATERIAL AND METHODS: Four pH-cycles were performed, alternating demineralization by the beverage and remineralization in artificial saliva. RESULTS: Mean wear (±SD, µm) was 7.91±1.13, 7.39±1.01, 7.50±0.91 and 5.21±1.08 for Coca-Cola® without CaGP or containing CaGP at 0.1, 1.0 or 2.0 mM, respectively, while no wear was detected for CaGP at 5.0 and 10.0 mM. Corresponding figures for Sprite Zero® without CaGP or containing CaGP at 0.1, 1.0, 2.0, 5.0 or 10.0 mM were 8.04±1.30, 7.84±0.71, 7.47±0.80, 4.96±0.81, 3.99±0.10 and 1.87±0.12, respectively. CONCLUSION: Supplementation of both beverages with CaGP seems to be an alternative to reduce their erosive potential.

A Trial of Screening of Genes Involved in Odontoblasts Differentiation from Human Dental Pulp Stem Cells

This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as beta-glycerophosphate (beta-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.

Controlled release of transforming growth factor-beta receptor kinase inhibitor from thermosensitive Chitosan-based hydrogel: application for prevention of capsular contracture / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Capsular contracture has become the most common complication associated with breast implant. Transforming growth factor-beta (TGF-β) is well known for a prominent role in fibrotic diseases. Due to the critical role of TGF-β in pathogenesis of capsular formation, we utilized thermosensitive C/GP hydrogel to controlled release of TGF-β receptor kinase inhibitor (SD208) and investigated their effects on capsular contracture.</p><p><b>METHODS</b>In vitro degradation and drug release of C/GP hydrogel were performed. Twenty-four rabbits underwent subpanniculus implantation with 30 ml smooth silicone implants and were randomly divided into four groups as follows: Group 1 received saline solution; Group 2 received SD208; Group 3 received SD208-C/GP; Group 4 received C/GP. At 8 weeks, the samples of capsular tissues were analyzed by hematoxylin and eosin and immunohistological staining. The mRNA expression of collagen III and TGF-β1 was detected by RT-PCR assay.</p><p><b>RESULTS</b>C/GP hydrogel could be applied as an ideal drug delivery vehicle which supported the controlled release of SD208. SD208-C/GP treatment showed a significant reduction in capsule thickness with fewer vessels. The histological findings confirmed that the lower amounts of inflammatory cells and fibroblasts infiltrate in SD208-C/GP group. In contrast, typical capsules with more vessel predominance were developed in control group. We did not observe the same inhibitory effect of SD208 or C/GP treatment on capsular contracture. Moreover, SD208-C/GP therapy yielded an evident down-regulation of collagen III and TGF-β1 mRNA expression.</p><p><b>CONCLUSIONS</b>This study demonstrated that controlled release of TGF-β receptor kinase inhibitor from thermosensitive C/GP hydrogel could significantly prevent capsule formation after mammary implants.</p>

Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes

The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-gamma, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-alpha were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes.

Gene expression pattern during osteogenic differentiation of human periodontal ligament cells in vitro

PURPOSE: Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro. METHODS: PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 microg/mL ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21. RESULTS: On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar. CONCLUSIONS: We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

Evaluation of osteogenic activity of periosteal-derived cells treated with inflammatory cytokines

INTRODUCTION: Skeletal homeostasis is normally maintained by the stability between bone formation by osteoblasts and bone resorption by osteoclasts. However, the correlation between the inflammatory reaction and osteoblastic differentiation of cultured osteoprogenitor cells has not been fully investigated. This study examined the effects of inflammatory cytokines on the osteoblastic differentiation of cultured human periosteal-derived cells. MATERIALS AND METHODS: Periosteal-derived cells were obtained from the mandibular periosteum and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured in an osteogenic induction Dulbecco's modified Eagle's medium (DMEM) medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. In this culture medium, tumor necrosis factor (TNF)-alpha with different concentrations (0.1, 1, and 10 ng/mL) or interleukin (IL)-1beta with different concentrations (0.01, 0.1, and 1 ng/mL) were added. RESULTS: Both TNF-alpha and IL-1beta stimulated alkaline phosphatase (ALP) expression in the periosteal-derived cells. TNF-alpha and IL-1beta increased the level of ALP expression in a dose-dependent manner. Both TNF-alpha and IL-1beta also increased the level of alizarin red S staining in a dose-dependent manner during osteoblastic differentiation of cultured human periosteal-derived cells. CONCLUSION: These results suggest that inflammatory cytokines TNF-alpha and IL-1beta can stimulate the osteoblastic activity of cultured human periosteal-derived cells.

A bioactivity study of Portland cement mixed with beta-glycerophosphosphate on human pulp cell / 대한치과보존학회지

The purpose of this study is to investigate the response of human pulp cell on Portland cement mixed with beta-glycerophosphate. To investigate the effect of beta-glycerophosphate and/or dexamethasone on human pulp cell, ALP activity on various concentration of beta-glycerophosphate and dexamethasone was measured and mineral nodule of human pulp cell was stained with Alizarin red S. MTS assay and ALP activity of human pulp cell on Portland cement mixed with various concentration of beta-glycerophosphate (10 mM, 100mM, 1M) was measured and the specimens were examined under SEM. Addition of beta-glycerophosphate or dexamethasone alone had no effect however, the addition of 5 mM beta-glycerophosphate and 100 nM dexamethasone had the largest increasement in ALP activity. There was no toxicity in all samples and the data showed that Portland cement mixed with 10 mM beta-glycerophosphate had more increase in ALP activity compared with control. In conclusion, Portland cement mixed with beta-glycerophosphate has no toxicity and promotes differentiation and mineralization of pulp cell compared with additive-free Portland cement. This implicated that application of Portland cement mixed with beta-glycerophosphate might form more reparative dentin and in turn it would bring direct pulp capping to success.

Effects of irradiation on TGF-beta1 mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line / 대한구강악안면방사선학회지

PURPOSE: To investigate the effects of irradiation on transforming growth factor beta1 (TGF-beta1) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. MATERIALS AND METHODS: Cells were cultured in alpha-minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with alpha-MEM supplemented with 10% FBS, 5 mM beta-glycerol phosphate, and 50 microgram/mL ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-beta1 mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. RESULTS: The amount of TGF-beta1 mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy, and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P.0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. CONCLUSION: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-beta1 mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

The effects of beta-TCP/rhBMP-2 on bone formation in osteoblast-like cells induced from bone marrow-derived mesenchymal stem cells

The present study aimed to investigate the osteogenic potentials of differentiated osteoblast-like cells (DOCs) induced from bone marrow-derived mesenchymal stem cells (MSCs) on beta-tricalcium phosphate (beta-TCP) with recombinant human bone morphogenetic protein (rhBMP-2) in vitro. Osteoblast differentiation was induced in confluent cultures by adding 100 nM dexamethasone, 10 mM beta-glycerophosphate, 50 mM L-ascorbic acid. The Alizarin red S staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were perfomed to examine the mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), receptor activator for nuclear factor kappaB ligand (RANKL), runt-related transcription factor 2 (RUNX2), collagen-I (COL-I). There were no significant differences in the osteogenic potentials of DOCs induced from MSCs on beta-TCP(+/-). According to the incubation period, there were significant increasing of Alizadin red S staining in the induction 3 weeks. The mRNA expression of ALP, RUNX2, and RANKL were higher in DOCs/beta-TCP(-) than DOCs/beta-TCP(+). According to rhBMP-2 concentrations, the mRNA expression of BSP was significantly increased in DOCs/beta-TCP(+) compared to that of DOCs/beta-TCP(-) on rhBMP 10 ng/ml. Our study presented the beta-TCP will have the possibility that calcium phosphate directly affect the osteoblastic differentiation of the bone marrowderived MSCs.

Effect of Various Inodizing Characteristics on Bone Integration of Titanium Implant Surface Design

Evaluation of angiogenic phenotypes in cultured human periosteal-derived cells under high-dose dexamethasone

Simultaneous determination of fluoride, chloride, sulfate, phosphate, monofluorophosphate, glycerophosphate, sorbate, and saccharin in gargles by ion chromatography / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Simple, reliable and sensitive analytical methods to determine anticariogenic agents, preservatives, and artificial sweeteners contained in commercial gargles are necessary for evaluating their effectiveness, safety, and quality. An ion chromatography (IC) method has been described to analyze simultaneously eight anions including fluoride, chloride, sulfate, phosphate, monofluorophosphate, glycerophosphate (anticariogenic agents), sorbate (a preservative), and saccharin (an artificial sweetener) in gargles. In this IC system, we applied a mobile phased gradient elution with KOH, separation by IonPac AS18 columns, and suppressed conductivity detection. Optimized analytical conditions were further evaluated for accuracy. The relative standard deviations (RSDs) of the inter-day's retention time and peak area of all species were less than 0.938% and 8.731%, respectively, while RSDs of 5-day retention time and peak area were less than 1.265% and 8.934%, respectively. The correlation coefficients for targeted analytes ranged from 0.999 7 to 1.000 0. The spiked recoveries for the anions were 90% approximately 102.5%. We concluded that the method can be applied for comprehensive evaluation of commercial gargles.