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The purpose of this study was to evaluate the cytotoxicity of acrylic and methacrylic acid (AA and MA), propanoic acid and isobutylic acid (PRA and IBA) which were the corresponding saturated carboxylic acids, 2-hydroxylethyl methacrylate and acrylate (HEMA and HEA), and hydroquinone (HQ). The growth of the fibroblast when exposed to AA decreased with increase of the acid, and was less than when exposed to MA. The growth when exposed to their corresponding saturated acids, PRA and IBA, showed a similar tendency to AA and MA. The growth of fibroblasts exposed to 5 mmol/L HEMA after 4 days and 3 mmol/L HEMA after 6 days was significantly different compared with that without HEMA. The growth in 1, 3, and 5 mmol/L of HEA after 2 days was markedly decreased compared with that without HEA, and the cells were probably lifeless. The growth when exposed to HQ was extremely lower than their acids.

Osteocyte-like cells were prepared by sequentially treating calvaria from newborn rats with collagenase and chelating agents. On a reconstituted gel of basement membrane components, cells from the third collagenase digest displayed a round shape and expressed the highest level of alkaline phosphatase with minimal osteocalcin deposition into the matrix. On the other hand, cells derived from the interior after EDTA treatment exhibited well-developed dendritic cell processes and expressed essentially no alkaline phosphatase. The latter population also showed quite distinct characteristics such as higher extracellular activities of casein kinase II and ecto-5'-nucleotidase and the extracellular accumulation of a large amount of osteocalcin associated with mineral. These diverse phenotypic and protein expressions as well as the sites from which each population of cells were recovered strongly suggest that we have isolated osteoblastic and osteocytic cells. Bone sialoprotein II was extracellularly phosphorylated by casein kinase II in osteocytic cells but not in osteoblastic cells. We discuss the possibility that differentiation of young osteocytes from osteoblasts may facilitate the biochemical sequence of mineral deposition in the bone matrix.

HGKとHPLFにおけるanion exchanger(AE)の発現状態、ファミリーのタイプ、分布、などを観察し、更に、H_2O_2、ニコチンを用いてAEへの酸化刺激の影響を観察した。その結果、(1)ヒト抗AET、AE2抗体を用いて蛍光抗体法、RT-PCR法により観察した結果、両方法共に、両細胞上にAE2の存在が観察された。(2)AE2の分布を蛍光抗作法で観察した結果、蛍光は、HGKでは、細胞核周辺に多く、HPLFでは、細胞全体に観察された。(3)H_2O_2、ニコチン刺激によるHGK、HPLFの形態変化を観察した結果、H_2O_2刺激でHGKはH_2O_2濃度と作用時間に依存して、細胞間に隙間が出来、核を残す形で細飽が溶解した。HPLFは低濃度刺激では、細胞表面に網目構造を形成して次第に細胞が細くなり、細胞間に隙間を生じ、次第に核を残す形で溶解した。ニコチン刺激でも同様の結果が観察された。(3)RT-PCRを用いてH_2O_2、ニコチン刺激によるAE2への影響を観察した結果、両細胞共に、細胞が破壊寸前までAE2の存在が観察された。(4)H_2O_2、ニコチン刺激による細胞死を観察した結果、両細胞共に、細胞破壊の直前で、apoptosisが観察された。これらの結果より、HGK、HPLFのAEはAE2であり、AE2は酸化刺激より細胞を守り、細胞が破壊される直前まで機能していることが推測できる。, Since periodontal ligament fibroblasts and gingival keratinocytes are closely related to periodontal diseases among human cells constituting periodontal connective tissue, we clarified the presence, distribution and types of AE in human gingival keratinocytes and periodontal ligament fibroblasts. In addition, oxidative stress-induced dynamics of anion exchanger were investigated. The presence of AE on human gingival keratinocytes and periodontal ligament fibroblasts was investigated by the fluorescence antibody method and the RT-PCR method using anti-anion exchanger 1 (AE1) antibody and anti-anion exchanger 2 (AE2) antibody. In both human gingival keratinocytes and periodontal ligament fibroblasts, no expression of AE1 was detected by either method, while AE2 was expressed. The distribution of AE2 was investigated by the flurescence antibody method, and fluorescence was detected on the circumference of nuclear of human gingival keratinocytes, and entire cell body of human periodontal ligament fibroblasts. H_2O_2- and nicotine-induced dynamics of AE2 on human gingival keratinocytes and periodontal ligament fibroblasts were investigated by the RT-PCR method. No changes were observed after stimulation with H_2O_2 or nicotine, but AE was present until the cells were destroyed and lysed by stimulation with H_2O_2 and nicotine. These findings suggested that AE present on human gingival keratinocytes and human periodontal ligament fibroblasts is AE2, and AE2 protect the cell from oxidative stress.

Purpose: Bone marrow stromal cells (BMSCs) are a promising cell source in applications for tissue engineering and regenerative medicine. Optimization and control of the growth and differentiation of cultivated cells can be achieved by the administration of growth factors and hormones in vitro. This study provided experimental information on the enhancement of the osteogenic potential of rat BMSCs in vitro and in vivo.Methods: Mineralized nodule formation of rat BMSCs in culture for 3 weeks with dexamethasone (Dex)-treated media supplemented with both basic fibroblast growth factor (bFGF) and 17β-estradiol (E2) was examined by histology. In porous β-tricalcium phosphate (β-TCP), proliferation, migration, and differentiation of BMSCs were examined by histology and transmission electron microscopy. After culturing, the composites were subcutaneously implanted into syngeneic rats. The tissues with implants were harvested after 4 weeks and evaluated microscopically by using histological stain.Results: Dex-treated media supplemented with both bFGF and E2 was the most effective in mineralized nodule formation of BMSCs in vitro. Light and electron microscopy revealed the presence of many cells with developed rough endoplasmic reticulum. Bone formation in the BMSC/β-TCP composites in cultures in vitro for 3 weeks was observed histologically at 4 weeks after implantation. When BMSC/β-TCP composites were cultured in Dex-treated media supplemented with both bFGF and E2, the amount of bone formation at implants was substantially greater than that of composites cultured in Dex-treated media supplemented with bFGF.Conclusion: The combined use of bFGF and E2 could effectively improve the bone-forming ability of BMSCs.

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.

In order to evaluate the involvement of dysfunctional teeth in age-related deficits in hippocampal function, we examined the effect of removal of molar teeth (molarless condition) on neuronal degeneration and glial fibrous acidic protein (GFAP) expression in the hippocampus and on learning ability in a water maze test in young, middle-aged, and aged accelerated senescence-prone mice (SAMP8). The molarless condition enhanced an age-dependent decrease in both learning ability and the number of neurons in the hippocampal CA1 subfield and the age-dependent increase in the number and hypertrophy of GFAP-labeled astrocytes in the same subfield. These observations suggest that the molarless condition may be involved in the senile process in the hippocampus in SAMP8 mice.

Our objective in this study was to elucidate the mechanism underlying the decrease in dopamine (DA) levels in the brain with ageing We administered 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I to senescence-accelerated mouse-prones (SAMP8), to inhibit DA and serotonin syntheses, and following immunohistochemical staining, analyzed the immunoreactive intensities (IR-Is) for DA in the nigrostriatal dopaminergic neurons by microphotometry. The DA-IR-Is in the substantia nigra pars compacta and neostriatum of young mice (2 months old) reached a minimal value 3 h after DAHP administration and returned to the control value 12 h after the administration. However, in aged mice (10 months old), the minimal value was reached 6 h after the administration and the value remained at approximately 70 and 80% of the control value at 24 and 72 h, respectively, after DAHP administration. The results suggest that DA turnover is lower in aged mice than in young mice.

The localization of tyrosine hydroxylase (TH) -immunoreactive (IR) (TH-IR) neurons, which are transiently observed in the anterior olfactory nucleus (AON) of the mouse, was studied. A double-labelling method was used with antibodies against TH, L-3, 4-dihydroxyphenylalanine (DOPA), phenyletha-nolamine-N-methyltransferase (PNMT), γ-aminobutyric acid (GABA), calretinin (CR) and calbindin-D28k (CB). When the total number of TH-IR neurons observed in all regions of the AON obtained from 10- to 14- day-old mice was set at 100%, 30% of TH-IR neurons were observed in the lateral (AOL), 25% in the dorsal (AOD), 20% in the ventral (AOV), 15% in the medial (AOM) and 5% in both the posterior (AOP) and external (AOE) regions. Approximately 40% of the TH-IR neurons in the AOL to the AOV regions also exhibited GABA immunoreactivity. This corresponds to 20% of the total number of TH-IR neurons observed in all regions of the AON, and also corresponds to 12% of the total number of GABA-IR neurons observed in all regions of the AON. However, TH-IR neurons did not exhibit concomitant PNMT, CR, and CB immunoreactivities. These findings suggest that the TH-IR neurons in the AON of the mouse, a proportion of which is also GABA-IR, may function as a source of neuromodulators for the postnatal growth of GABA-IR neurons.

SPARC is known to be important in development and tissue remodelling. Here, we examined the effects of SPARC (secreted protein, acidic and rich in cysteine; osteonectin) derived from a rat osteosarcoma cell line on migration of renal cell carcinoma (RCC) by a Boyden chamber assay. YCR RCC cells migrated through type IV collagen-coated filters without stimuli (basal level). SPARC in the lower compartment stimulated chemotactic activity to 120% of the basal level, whereas premixing of YCR with purified SPARC before inoculation reduced their migration to 72% of the basal level. Furthermore, SPARC mixed with type IV collagen more efficiently stimulated their migration in a concentration-dependent manner (up to 170% of the basal level). This suggests that SPARC bound to type IV collagen plays a role in tumor invasion.

To explore lineage-dependent responses to mechanical stress in bone cells, newborn rat calvarial cells, exhibiting differential characteristics of osteoblastic and osteocytic cells, were compared in their immediate and late responses to stretching. Seven fractions of sequentially prepared cells were cultured on Matrigel to promote their differentiation. By cyclically stretching the flexible bottom of culture plates, cells were exposed to a physiological stress of approximately 4000 microstrain on Matrigel. Cells in fractions IV, V and VI exhibited striking responses; the levels of cAMP and insulin-like growth factor I, bone Gla protein, and mineral accumulation were significantly elevated in the stretched cells. Also, proliferation was significantly inhibited regardless of the presence of 10(-6)M indomethacin. In contrast, osteoblasts in fraction III and osteocyte-like cells in fraction VII exhibited no significant response. Thus, these intermediate cells, very mature osteoblasts to young osteocytes, are likely to serve as a mechanosensor in bone, controlling the metabolic aspects of physical stress. We conclude that the responses of these young osteocytes to low level, physiological strain are transmitted in a manner different from the responses of osteoblasts to higher magnitude of strain in which PGE2 induces cell proliferation, as reported by others.

enzymogram上におけるみかけの分子量は, ゲル基質の濃度によって異なることが判明した。われわれは, この変化は電気泳動中の酵素と基質の親和性に基づく泳動速度の遅延が原因であり, ミカエリス定数 (Km) 値に依存するのではないかと推察し, それに基づいて電気泳動上での真の分子量を推定する関係式を検討した。みかけの分子量から真の分子量を推定する方法としてゼラチン濃度を変化させ, その関係を調べたところ, 基質濃度およびKm値により導き出される関係式〓が成り立つことが示唆された。したがって本法は, 基質存在下で分子量が本来の値より大きく算出されてしまうenzymogramで, その基質濃度に依存しない, 基質が存在しないところの分子量を導き出す方法である。さらに, 二次元enzymographyを施行したところ, より正確な分子量を導き出せることが判明した。またこうした解析によって分子量の推定のみならず複数の酵素が共存している時でもそれぞれの酵素の基質に対するKm値を求めることが理論的に可能となった。

The effect of ipriflavone (IP) on the proliferation and differentiation of rat osteoblast-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) was studied in the presence and absence of estrogen. ROB cells were isolated from newborn rat calvaria by sequential collagenase digestion and HPLF from the outgrowth of human periodontal ligament in culture. The alkaline phosphatase (ALP) activity, employed as a marker of bone cell differentiation, was significantly enhanced by IP in both cell types; however, the concentration at which IP had a maximal effect was lower in ROB cells than in HPLF (10(-10) versus 10(-7) M, respectively). Cell proliferation judged by DNA content was either constant (ROB cells) or slightly increased (HPLF) by IP up to 10(-10) M, and decreased significantly above that concentration. In addition, the dose-dependent effect of estrogen on the growth and differentiation of each cell type in the presence and absence of IP was also tested. At the concentrations of IP which showed maximum effects in the induction of ALP, 10(-10) M for ROB cells and 10(-7) M for HPLF, IP inhibited DNA increase in an estrogen-independent manner. Estradiol (10(-11)-10(-9) M) itself increased the growth rate of both cell types significantly in a dose-dependent manner. Regardless of the concentrations of estradiol tested, ALP activities of both ROB cells and HPLF were elevated by the addition of IP. The ratio of ALP in the presence and absence of IP was similar over the range of estradiol concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to evaluate the durability of tubules occluded with FTLA treatment by toothbrush abrasion test on the applied surface and by measuring fluoride release from the FTLA components. Dentin specimens with simulated hypersensitive surfaces were treated with APF containing tannic acid. After which, the specimens received lanthanum-chloride-with-powdered-fluoroapatite-glass-ceramics treatment. The specimens were subjected to toothbrush abrasion test up to 6,000 strokes. SEM observation revealed that dentinal tubules of the FTLA treated specimens were completely occluded with fine deposits even after toothbrush abrasion of 6,000 strokes. EPMA analysis revealed that fluoride, lanthanum, and aluminum were the main FTLA components on the dentin surface after 6,000-stroke abrasion. To measure fluoride release from the FTLA components, a slurry was enclosed in a cellulose tube and suspended in deionized water at 37 degrees C. After fluoride was dialyzed against deionized water, a high concentration of fluoride was found to be released from FTLA the components, indicating FTLA treatment's prominent durability. These results suggested that FTLA treatment has a superior resistance against toothbrush abrasion and a high fluoride-releasing performance. These characteristics lend much weight to showing that the FTLA method is an effective and durable medicament for dentin hypersensitivity.

In order to evaluate the mechanism(s) responsible for senile impairment of cognitive function as a result of reduced mastication, the effects of the loss of the molar teeth (molarless condition) on the hippocampal expression of glial fibrous acidic protein (GFAP) and on spatial memory in young adult and aged SAMP8 mice were studied using immunohistochemical and behavioral techniques. Aged molarless mice showed a significantly reduced learning ability in a water maze test compared with age-matched control mice, while there was no difference between control and molarless young adult mice. Immunohistochemical analysis showed that the molarless condition enhanced the age-dependent increase in the density and hypertrophy of GFAP-labeled astrocytes in the CA1 region of the hippocampus. These effects increased the longer the molarless condition persisted. When the extracellular K+ concentration ([K+]o) was increased from 4 to 40 mM for hippocampal slices in vitro, the mean increase in the membrane potential was about 57 mV for fine, delicate astrocytes, the most frequently observed type of GFAP-positive cell in the young adult mice, and about 44 mV for the hypertrophic astrocytes of aged mice. However, there was no significant difference in resting membrane potential between these cell types. The data suggest that an impairment of spatial memory and changes in astroglial responsiveness occur following the loss of molar teeth in aged SAMP8 mice.

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF-Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation, 3-cycles/min for 24 h on d 7 in culture using a Flexercell strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF-beta 1) and macrophage colony stimulating factor (M-CSF) contents in the conditioned medium. The TGF-beta 1 level in the conditioned medium of HPLF was significantly higher than that of HPLF-Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF-Y and HGF. M-CSF secretion was inhibited by the stretching on all of HPLF, HPLF-Y and HGF. 1 alpha, 25 dihydroxy vitamin D3 (D3) stimulated M-CSF secretion into the culture medium of both HPLF and HPLF-Y, but the stretching inhibited M-CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.

In regenerating periodontal ligament (PDL) around the root of an artificial tooth, an important role is played by some physiologically active substance that promotes adhesion of the cells to the surface of the tooth root and induces cell proliferation and differentiation. In this study, the supernatant of the conditioned medium (CM) of dog periodontal ligament fibroblast (DPLF) was fractionated using an ion exchange chromatography-diethylaminoethyl (IEC-DEAE) column. DPLFs were cultured on hydrophobic dishes coated with each fraction. Cell proliferative activity and alkaline phosphatase (ALPase) activity, including electron microscopic features of the contact surface between the cells and the dish, were investigated. The DPLF-CM was separated by IEC-DEAE column into six fractions. Each fraction promoted an increase in DNA content and ALPase activity of the cultured DPLF, and especially remarkable were fractions 2 and 3. Fraction 2 at a molecular weight (Mw) of 210, 160, 85, 50 and 22 kD, and fraction 3 at Mw = 21 and 23 kD contained the type of proteins not found in other fractions. Electron microscopic analysis revealed that the cells in the coating group were in close contact with the surface of the dishes and that fine fibers protruding from the cell membrane clinged to the dishes. In the control group, a wide gap between the cells and the dishes was observed. These findings suggest that the DPLF-CM fractions contain specific physiological activating factors that induce proliferation and differentiation as well as cell adhesion of the DPLF cells.

There are two steps in the process of matrix-mediated bone and dentin mineralization. First, as in other soft tissues, osteoblasts/odontoblasts synthesize collagenous matrices and second, mineral deposits in these matrices at a location distant from the cells that synthesized the matrices. We suggest a sequence of events that lead the matrix to mineralization: the phosphoproteins of bone and dentin are posttranslationally processed by limited proteolysis, then they are extracellularly processed into a more phosphorylated species that, we believe, facilitates mineralization. Our in situ phosphorylation experiments done with [gamma-32P] GTP suggest the existence of extracellular phosphorylation by a casein kinase II (CKII)-like enzyme, the enzyme known to phosphorylate most of the phosphate residues in dentin phosphophoryn and bone sialoproteins (osteopontin and BSP II).

A series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.

骨芽細胞 (OB) のアルカリホスファターゼ (ALP) 発現に対する活性型ビタミンD3 (1, 25 (OH) 2D3) およびグルココルチコイドの調節機構について検討した。ラット新生児の頭蓋冠のコラゲナーゼ処理により得たOB-rich細胞の培養条件下にデキサメサゾンを添加し, 更に, 一部細胞を継代し1, 25 (OH) 2D3で処理した。その後, フローサイトメーター (FCS-10; 日本分光工業社製) にて細胞中のALP活性陽性細胞集団を分取し, 比活性の測定を行った。その結果, デキサメサゾン前処理による実験群では, コントロールと比較し1, 25 (OH) 2D3によりALP比活性は亢進し, 増殖が抑制される傾向を示した。また, アクリジンオレンジを用いた細胞周期解析によって, グルココルチコイドおよび1, 25 (OH) 2D3によりG1A細胞集団の割合の増加が認められた。即ち, グルココルチコイド処理によりOB中の1, 25 (OH) 2D3リセプター数が増加したため, 1, 25 (OH) 2D3により, 特定の細胞周期において, OBの分化した形質発現であるALPを誘導する可能性が示された。

Though fluorescent X-ray spectrometers have been applied for analysis of trace elements in many fields, few studies were reported on the odontological use of this method.In order to obtain a simple and quick analytical method for trace elements in deciduous teeth, pH of sample solution, APDC amount to be added, andaging time for precipitation were mainly examined.A deciduous tooth was dissolved by H NO3 and a metal complex was formed in which APDC bonded with trace elements in the tooth solution. Then, trace element analysis was carried out by a fluorescent X-ray spectrometer after the precipitation was collected on the Millipore filter.As a result, trace elements such as Cu, Zn, Pb, and Fe in primary teeth were determined quantitatively and it was elucidated that data were obtained without interference when excesses in the amounts of Ca and PO4 existing in teeth were removed, because the APDC did not form any precipitation with Ca2+and P8+.

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and P1 during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP. In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide. It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.

Phenytoin (PHT), administered as an anticonvulsant, has a side effect gingiva overgrowth in approximately 50% of patients. The present study was attempted to explore the biochemical mechanism on non-collagenous protein biosynthesis as affected by PHT. Responder cells (RES A3, RES C2) of a patient with gingival overgrowth were obtained by the method of Kawase et al. Normal human gingival fibroblasts (Gin-1), purchased from ATCC, were also used. All cells were inoculated at 1 x 10(4) cells/cm2 (12 multi-well plate or 60 mm tissue culture dish), and then cultured for 4, 8 and 12 days with or without PHT (5 micrograms/ml). Prior to harvesting at the indicated times, cells were incubated with 14C-amino acids (1.25 microCi/ml) for 24 hours. The 14C-labeled proteins were isolated from the cell layers including extracellular matrix, following Kurkinen et al. with a minor change. Each 14C-labeled fraction was dissolved in 3 ml of Aquasol-2 and the radioactivity by a liquid scintillation counter. The DNA content of cell layers affected by PHT was increased on Gin-1, RES A3 and RES C2 at the post-confluence, resulting also in an increase in cell number. Two morphologically different phenotypes of responder cells were observed, differing in nuclear and cell sizes. At 12 days culture, RES A3, were stimulated by PHT, showed increased synthesis of both total extractable proteins (EP) and binding proteins (BP) labeled with 14C-amino acids. Therefore, at least two distinct phenotypic responder cells are present in the PHT-induced overgrowth gingiva, alter the synthesis of non-collagenous proteins.