ALPase Activity Research Articles

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10 − 4 M, and 10 − 3 M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and beta-glycerophosphate, under 0.8%-15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1 h and 6 h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48 h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.

Enhancement Action of Sulfated Hyaluronan on the ALPase Activity of Rat Calvarial Osteoblasts

The purpose of this study was to clarify the effect of hyaluronan (Hya) and sulfated hyaluronan (SHya) on rat calvarial osteoblast (rOB) cells proliferation and differentiation in vitro. rOB cells were cultured in the presence of Hya with different molecular weights (0.2, 2, 30, 90, 120 x 104) for 10days. Hya did not affect the proliferation of rOB cells. However, SHya suppressed the proliferation of rOB cells. The alkaline phosphatase (ALPase) activity of rOB cells cultured with SHya for 10 days was significantly enhanced in comparison with control (in the absence of polysaccharides) and with Hya. Hya suppressed the ALPase activity of rOB cells. As a result, SHya can control rOB cells proliferation and differentiation. SHya suppressed the rOB cells proliferation in a few culture days and promoted the differentiation. It was suggested that these effects were based on the sulfate groups of SHya. Therefore, it is considered that SHya is useful for the biomedical material, which promotes the differentiation of rOB cells.

Fine Structural and Cytochemical Observations on the Dental Epithelial Cells During Cap Enameloid Formation Stages in Polypterus senegalus, a Bony Fish (Actinopterygii)

Tooth germs during cap enameloid formation stages in Polypterus senegalus were investigated by transmission electron microscopy and enzyme histo- and cytochemistry. Enameloid formation was divided into three stages: matrix formation, mineralization, and maturation. The enamel organ consisted of the inner dental epithelial cells, stellate reticulum, and outer dental epithelial cells during cap enameloid formation stages, but no stratum intermedium was found. During the matrix formation stage, the tall inner dental epithelial cells contained well-developed Golgi apparatus, abundant cisternae of rough endoplasmic reticulum and mitochondria. Spindle-shaped vesicles containing a filamentous structure were seen in the distal cytoplasm. During mineralization and maturation stages, many ACPase positive lysosomes were present in the cytoplasm, whereas the organelles were decreased in number. The infoldings of the distal plasma membrane of the inner dental epithelial cells were visible in the mineralization stage but were not marked in the maturation stage. The activity of nonspecific ALPase, Ca-ATPase, and K-NPPase was detected at the plasma membrane of the inner dental epithelial cells during the stages of mineralization and maturation. The results of fine structure and enzyme cytochemistry suggested that the dental epithelial cells were mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the enameloid matrix formation. The results also showed that the structure of the dental epithelial cells in Polypterus was different from that in teleosts and gars, but that the function of the dental epithelial cells was similar to that in teleosts possessing well-mineralized cap enameloid.

Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells

The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900±50 μm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers ( p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher ( p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased ( p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations.

Effect of β-alanyl-L-histidinato zinc on the differentiation of C2C12 cells

Although β-alanyl-L-histidinato zinc (AHZ) can promote osteoblast differentiation, the molecular mechanism responsible is not fully understood. The purpose of this study was to determine the effect of AHZ on undifferentiating mesenchymal cells. C2C12, a typical pluripotential mesenchymal cell line, was used. The cells were cultured in 5% serum-containing medium to induce differentiation, either with or without the addition of AHZ. Cell lineage was determined by immunostaining of type II myosin heavy chains, alkaline phosphatase (ALPase) activity, mRNA expression of cellular phenotype-specific markers using semi-quantitative reverse transcriptase-polymerase chain reaction, and core binding factor α1/runt-related transcription factor-2 (Cbfa1/Runx2) protein synthesis using Western blot analysis. C2C12 cells cultured in the presence of AHZ were strongly inhibited from developing into myoblasts, and showed high ALPase activity that was approximately double that in the vehicle. The expression of mRNA for Cbfa1/Runx2, ALPase, Sox9 and type X collagen was increased markedly by the AHZ-stimulated medium, whereas that of desmin and MyoD mRNA was drastically decreased. AHZ increased Cbfa1/Runx2 protein expression substantially. These results provide clear evidence that AHZ converts the differentiation pathway of C2C12 cells to the osteoblast and/or chondroblast lineage.

Effect of IL-1α on the expression of cartilage matrix proteins in human chondrosarcoma cell line OUMS-27

We examined the effect of the inflammatory mediator interleukin-1α (IL-1α) on cell proliferation, alkaline phosphatase (ALPase) activity, and the expressions of cartilage matrix proteins, bone morphogenetic protein-2 (BMP-2), and BMP-2 receptors in human chondrosarcoma cell line OUMS-27 (chondrocytes). The cells were cultured with Dulbecco's modified Eagle's medium containing 15% fetal bovine serum with 0, 1, 10, or 100 units/ml of IL-1α for up to 14 days. The expressions of cartilage matrix proteins, BMP-2, and BMP-2 receptors were estimated by determining mRNA levels using semiquantitative or real-time PCR and/or by determining protein levels using Enzyme-linked immunosorbent assay. Cell proliferation was decreased after 5 days in culture with IL-1α. The ALPase activity was decreased significantly in the presence of IL-1α until day 10 of culture. The expression of type II collagen was significantly decreased after 7 days in culture with IL-1α. The expressions of aggrecan and link protein were significantly decreased through day 14 of culture with IL-1α. The expression of BMP-2 was increased at days 3, 7, and 14 of culture with IL-1α, while the expression of type II receptor for BMP-2 was significantly decreased in the samples. These results suggest that IL-1α suppresses the expression of cartilage matrix proteins through a suppression of the autocrine action of BMP-2, brought about by the decrease in BMP-2 receptor expression in chondrocytes.

Transforming Growth Factor (TGF)- 1 Releasing Tricalcium Phosphate/Chitosan Microgranules as Bone Substitutes

Tricalcium phosphate (TCP)/chitosan composite microgranules were developed as bone substitutes and tissue engineering scaffolds with the aim of obtaining a high bone forming efficacy. The microgranules have the ability to fill various types of defect sites with closer packing. In addition, the transforming growth factor-beta 1 (TGF-beta1) was added to the microgranules in order to improve bone-healing efficacy. TCP/chitosan microgranules were fabricated by dropping a TCP suspended chitosan solution into a NaOH/ethanol solution. TGF-beta1 was incorporated into the TCP/chitosan microgranules by soaking the microgranules into the TGF-beta1 solution. Scanning electron microscopy (SEM) observations as well as experiments examining the release of TGF-beta1 from chitosan and TCP/chitosan microgranules were performed. SEM was used to examine the cell morphologies on the microgranules, and the extent of cell proliferation was evaluated using a dimethyl-thiazol tetrazolium bromide (MTT) assay. The differentiated cell function was assessed by measuring the alkaline phosphatase activity as well as performing an osteocalcin assay. The size of the prepared microgranules was 350-500 microm and TCP powders were observed on the surface of the microgranules. TGF-beta1 was released from the TCP/chitosan microgranules at a therapeutic concentration for 4 weeks. The proliferation of osteoblasts on the TGF-beta1 loaded microgranules was the highest among the microgranules. SEM indicated that the seeded osteoblastic cells were firmly attached to the microgranules and proliferated in a multilayer fashion. The ALPase activity and osteocalcin content of all the samples increased during the culture period. These results suggest that the TCP/chitosan microgranules are potential bone substitutes with a drug releasing capacity and a osteoblastic cells culture scaffold.

IL-1α affects mineralized nodule formation by rat osteoblasts

The present study was conducted to determine the effect of the inflammatory mediator interleukin 1α (IL-1α) on osteogenesis using rat osteoblasts. We examined the effect of IL-1α on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in rat osteosarcoma cell lines. The cells were cultured with α-minimum essential medium containing 10% fetal bovine serum with and without 0, 1, 10, and 100 units/ml of IL-1α for up to 14 days. The mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a Calcium C-Test kit. The expression of extracellular matrix proteins was estimated by determining levels of mRNAs using the semiquantitative reverse transcription-polymerase chain reaction. The mineralized nodule formation and the calcium content in mineralized nodules were remarkably suppressed by IL-1α after 5 days of culture. The ALPase activity decreased in a dose-dependent manner in the presence of IL-1α after 7 days of culture. The expression of type I collagen was decreased after 3 days of culture with IL-1α. The expression of bone sialoprotein was slightly decreased at days 3 and 5, and the expression of osteopontin was increased at days 3, 5, and 7 of culture with IL-1α. These results suggest that IL-1α suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

Inhibition of ossification in vivo and differentiation of osteoblasts in vitro by tributyltin

Tributyltin is ubiquitous in the environment and an endocrine disruptor for many wildlife species. However, minimal information is available regarding the effect of this chemical on bone formation. When tributyltin chloride (TBT) (1 mg/kg body weight) was administered subcutaneously to pregnant mice at 10, 12, and 14 days post coitus (dpc), fetuses at 17.5 days post coitus revealed the inhibition of calcification of supraoccipital bone. In contrast, 1 mg/kg body weight monobutyltin trichloride (MBT) did not affect the fetal skeleton. Therefore, we examined the effects of TBT and its metabolites (dibutyltin dichloride, DBT, and MBT) on bone metabolism using rat calvarial osteoblast-like cells (ROB cells). The viability of ROB cells was not affected by the exposure of the cells to 10 −10 to 10 −7 M TBT. However, TBT reduced the activity of alkaline phosphatase (ALPase) and the rate of deposition of calcium of ROB cells. In addition, the expression levels of mRNA for ALPase and osteocalcin, which are markers of osteoblastic differentiation, were depressed by the treatment with TBT. TBT inhibited ALPase activity and the deposition of calcium to a greater extent than did DBT. MBT had no effect on the osteoblast differentiation of ROB cells. Tributyltin is known to inhibit the activity of aromatase. However, the aromatase inhibitor aminoglutethimide did not reproduce the inhibitory effects of TBT on osteoblast differentiation. Our findings indicate that TBT might have critical effects on the formation of bone both in vivo and in vitro although its action mechanism is not clarified.

Substance P enhances the inhibition of osteoblastic cell differentiation induced by lipopolysaccharide from Porphyromonas gingivalis.

Substance P (SP) is a multifunctional neuropeptide that transmits pain signals, regulates the immune system, and may modulate emotional stress. SP stimulates bone resorption activity of osteoclasts, and SP level in gingival crevicular fluid is correlated with the degree of periodontal inflammation. However, the exact roles of SP in bone metabolism and periodontal diseases are poorly understood. To elucidate the effect of stress on bone metabolism, we investigated the effect of SP on osteoblastic cell differentiation in the presence of lipopolysaccharide from Porphyromonas gingivalis (P-LPS). The primary osteoblastic cells were isolated from fetal rat calvaria (RC) and cultured with SP, P-LPS, and an SP antagonist (SPa). The effects of SP on bone nodule (BN), alkaline phosphatase (ALPase) activity, mRNA expressions of SP receptor, bone matrix proteins, and Cbfa 1 were investigated. SP stimulated the expression of SP receptor mRNA in RC cells and enhanced its expression in the presence of P-LPS (50 ng/ml). SP inhibited BN formation and ALPase activity in a dose-dependent manner (10(-7) to 10(-5) M) and further suppressed mRNA expression of bone sialoprotein, osteopontin, and osteocalcin but not of type I collagen mRNA. The inhibitory effects were enhanced in the presence of P-LPS and blocked by Spantide III. Furthermore, the expression of Cbfa 1 mRNA was also markedly suppressed in the presence of SP and P-LPS. These findings suggest that SP inhibits osteoblastic cell differentiation and may be related to bone metabolism in periodontal diseases under conditions of stress.

Acceleration Effect of Human Recombinant Bone Morphogenetic Protein-2 on Differentiation of Human Pulp Cells Into Odontoblasts

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, bone morphogenetic protein-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and 100 ng/ml of human recombinant bone morphogenetic protein-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophosphoprotein (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by 1000 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by 10 and 100 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells.

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress.

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.

A novel function of N-cadherin and Connexin43: marked enhancement of alkaline phosphatase activity in rat calvarial osteoblast exposed to sulfated hyaluronan

In this study, we examined the interaction of the osteoblast which forms bone and sulfated hyaluronan (SHya). For the purpose of the creation of a new functional polysaccharide, we introduced a sulfate group in hyaluronan (Hya) of high molecular weight, and SHya of high molecular weight could be obtained for the first time. When rat calvarial osteoblast (rOB) cells were cultured with a high concentration of SHya, they formed aggregated spheroids after 4 h and the spheroids grew to about 200 μm after 24 h. We examined the expression of cell adhesion molecules in order to clarify the mechanism of aggregate formation. The N-cadherin (N-cad) and Connexin43 (Cx43) expression level of rOB cells cultured with SHya remarkably increased after 2 h. A difference in the expression of Integrin β1 (Intβ1) could not be observed between the SHya addition and control group. The alkaline phosphatase (ALPase) activity of rOB cells cultured with SHya after 8 h was significantly enhanced in comparison with control. Therefore, the sulfate group of SHya seems to enhance expression of cell adhesion protein such as N-cad and Cx43, resulting in aggregate formation and further remarkable induction of the ALPase activity of rOB cells.

Histopathological and Cell Enzyme Studies of Calcium Phosphate Cements

New types of self-setting calcium phosphate cement (N-CPC), which do not contain tetracalcium phosphate, were recently developed. N-CPCs harden in 10 minutes with phosphate solution as the cement liquid, and form hydroxyapatite as the set product. The objectives of the present study were to evaluate the biocompatibility (Study I) and cell enzyme activity of N-CPCs and a conventional CPC (Study II). Four experimental cements were tested: (1) dicalcium phosphate anhydrous (DCPA) and calcium oxide; (2) DCPA and calcium hydroxide; (3) tricalcium phosphate and calcium carbonate; and (4) DCPA and tetracalcium phosphate. Phosphate solution was used as the cement liquid for cements (1)-(3), and water for cement (4). Sintered hydroxyapatite particles (5) were used as a control. The test materials were implanted subcutaneously in rats. Four weeks after operation, the animals were sacrificed and histopathological observations were performed. Cements (2) and (3) showed no inflammatory reaction, and were surrounded only by very thin fibrous connective tissues. The histopathological reactions of N-CPCs were nearly identical and were similar to (4) and (5). In addition, effects of alkaline phosphatase (ALP-ase) activity--invoked by the presence of cements (3) and (4)--on osteoblast-like cells derived from dog alveolar bone were also examined because ALP-ase activity is closely related to new bone formation. These results indicated that (3) and (4) were highly compatible with subcutaneous tissues and suggested that these cements may enhance new bone formation.

N‐Acetyl Cysteine Regulates TNF‐α‐Inhibited Differentiation in ROS 17/2.8 Osteoblasts

Osteoblasts play a pivotal role in bone remodeling. The alkaline phosphatase(ALPase) activity was decreased in ROS 17/2.8 osteoblast treated with TNF‐α (2, 5 or 10 ng/ml). The treatment of TNF‐α inhibited osteoblast differentiation such as ALPase activity in ROS 17/2.8 osteoblast. TNF‐α (10 ng/ml) increased NF‐κB DNA binding activity in nuclear extracts of osteoblasts. The addition of NAC (N‐acetyl cysteine), free radical scavenger, completely prevented TNF‐α‐induced activation of NF‐κB. In addition, IκBα and IκBβ were rapidly degraded, allowing the activated NF‐κB to enter the nucleus and promote gene transcription. To determine whether IκBα signal transduction pathway is important in the differentiation, we generated IκB (KD)‐stably transfected ROS 17/2.8 cells. These IκB (KD) transfectants did not show any regulation of ALPase in osteoblasts. Here, we suggest that the degradations of IκBα and IκBβ and the following activation of NF‐κB are the targets of NAC and that NF‐κB transcription factor is a pivotal clue to regulation of differentiation in TNFα‐exposed osteoblasts.

泡セラミックス法と水熱処理法を組み合わせた多孔質リン酸カルシウムの合成とその細胞応答性

Four porous calcium phosphates (HAp, HAp/β-TCP, β-TCP, and α-TCP) were prepared using a ceramic foaming technique combined with a hydrothermal treatment. These porous calcium phosphates had enough pore size, high specific surface area, and high porosity (>80%) for the cell growth of osteoblast-like cells. The compositions of the porous calcium phosphates depended on the solution in hydrothermal treatment and the sintering temperature. The cell activities of MC3T3-E1 cells on porous HAp, HAp/β-TCP, and β-TCP were similar to that of monolayer cultivation as a control in MTT assay, ALPase activities, and the cell density.

Reduction of alkaline phosphatase activity in aged human osteogenic periodontal ligament fibroblasts exhibiting short telomeres.

The osteogenic cell type of human periodontal ligament fibroblasts (PDLF) undergoes senescence at finite population doubling numbers unrelated to donor ages. This study investigated telomere lengths of osteogenic PDLF from differently aged donors and alterations of the osteoblast-like properties in the aged PDLF with short telomeres. Telomere lengths of osteogenic PDLF were biased towards long or short among all 15- to 51-year-old individuals, and did not show a normal distribution by Pearson's test or a correlation to donor age by simple regression analysis. In osteogenic PDLF, senescence-associated beta-galactosidase was expressed in 78.5% of cells in the clones with short telomeres (mean 3.02 kbp), and in 9.4% of cells in the clones with long telomeres (mean 13.06 kbp). These results suggest that human periodontium comprises aged osteogenic PDLF without correlation to age. Osteogenic PDLF with long telomeres strongly expressed alkaline phosphatase (ALPase) activity whereas cells with short telomeres expressed ALPase activity to a weaker extent. Total activity of ALPase in the clones of osteogenic PDLF with long telomeres was significantly higher than that in the clones with short telomeres. The produced amounts of both osteopontin and osteocalcin in the clones of osteogenic PDLF with long telomeres were slightly but statistically significantly smaller than those in the clones with short telomeres. These findings suggest that aged osteogenic PDLF reduce the expression of ALPase activity but that there is not a critical alteration in bone-associated protein production. Aged osteogenic PDLF may impair the ability to induce ALPase-dependent calcification.

Human periapical granulation tissue contains osteogenic cells.

Bone defects caused by periapical inflammation can be treated and improved by endodontic therapy. However, the mechanism for osseous healing of periapical lesions after root canal treatment is unclear. In this study we examined whether fibroblastic cells from human periapical granulation tissue could produce calcified matrix in vitro. Periapical lesions from three patients were dissected in endodontic surgery, and fibroblastic cells (HFC) migrating from these lesions in vitro were used in this study. The HFC were cultured with or without beta-glycerophosphate (beta-GP) and ascorbic acid (AA), and the expression of human runt-related transcription factor-2 (Runx2), osterix (Osx), osteopontin (Opn), and osteocalcin (Ocn) mRNA, and alkaline phosphatase (ALPase) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) or by an enzyme-cytochemical technique. Furthermore, calcification in the cells was investigated by von Kossa staining. At the beginning of the culture, HFC expressed Runx2 mRNA faintly, but neither Opn mRNA nor ALPase activity. Immunocytochemical study also showed HFC expressed Runx2 more weakly, compared to SaOS2. However, the expression levels of ALPase, and Runx2, Osx, and Opn mRNA, were stimulated by 2 mM beta-GP and 50 microg/ml AA. After 4 weeks of culture with 2 mM beta-GP and 50 microg/ml AA, HFC formed von Kossa staining-positive calcified deposits on culture dishes, and also expressed Ocn mRNA. These results suggest that inflamed periapical granulation tissue contains osteogenic cells that have the potential to differentiate into mature osteoblastic or cementoblastic cells, and that such cells might contribute to osseous healing after root canal treatment.