Initial dentin formation in human deciduous teeth

Dentin matrix formation is initiated as far as 11 deep in the prospective dental pulp. From this area, growth of the preodontoblast processes occurs toward the ameloblast. Process elongation is followed successively by 3 stages of dentin matrix formation: 1) fibril formation, 2) matrix maturation and 3) matrix calcification. These stages occur concurrently with differentiation of the odontoblasts. The bases of the odontoblast processes and distal surfaces of their cell bodies extrude cytoplasmic buds into the surrounding matrix. These function as initial sites of calcification.This study was supported by PHS Research Grant HD-01842-04, National Institute of Child Health and Human Development.     

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm²—16 s; 1.0 J/cm²—33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal–Wallis and Mann–Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm² promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm² groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm²) promote the proliferation of SHEDs and the maintenance of cell viability.     

Bone resorption by isolated human osteoclasts in vitro: effects of calcitonin

Human osteoclasts were isolated from 12- to 17-week-old fetal tissue and from transiliac crest bone biopsies for an in vitro study of their biology. A hypodermic needle was used to flush either the fetal long bones or the trabeculae of the iliac crest bone biopsy with tissue culture medium and the resulting cell suspension sedimented briefly either onto the surface of plastic tissue culture dishes, for time-lapse microcinematography, or onto slices of devitalized bovine cortical bone for quantitative assay of bone resorption. The osteoclasts were motile, tartrate-resistant acid phosphatase positive and capable of excavating pits in slices of devitalized bovine cortical bone. Human calcitonin, at doses of 1 ng/ml and 1 microgram/ml, caused a 70% inhibition of bone resorption by human fetal osteoclasts over a 24 h period but had no apparent effect on the morphology or motility of either fetal or adult osteoclasts.     

Production and characterization of new monoclonal antibodies to human osteoclasts

Summary Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.     

罗哌卡因对豚鼠心室肌细胞膜离子通道的影响

目的 研究罗哌卡因对豚鼠心室肌细胞膜离子通道的影响，探讨其心脏毒性作用的发生机理。方法 用酶解法获得单个的左心室肌细胞，用标准的全细胞膜片钳技术记录用药前后的钠流（ＩＮａ）、Ｌ－型钙流（ＩＣａ－ｌ）、延迟整流性钾流（ＩＫ）和内向整流性钾流（ＩＫ１）的变化。结果 罗哌卡因对ＩＮａ及ＩＣａ－Ｌ都有抑制作用，且呈浓度依赖性。５０ｕｍｏｌ．Ｌ＾－１罗哌卡因可使ＩＮａ、ＩＣａ－Ｌ分别下降３６．８％、７．６８     

Carbonic anhydrase activity in isolated osteoclasts

Osteoclasts were isolated from the long bones of chicks by a nylon mesh filtering system. The cell purity, in terms of area of the slide occupied, was on the average 77.5% osteoclasts, 22% aggregated osteoblasts and matrix debris, and 1.5% individual blood and marrow cells. Viability, as measured by cytochalasin-blockable phagocytosis, of up to 99% was obtained. Electron microscopic examination revealed good retention of ultrastructural features. The presence of carbonic anhydrase and acid phosphatase in osteoclasts was verified by selective staining methods; the aggregates were positive for alkaline phosphatase. Carbonic anhydrase activity was 0.89 ± 0.13 × 10⁻⁴ micro Wilbur-Anderson units per osteoclast, and red blood cells had 0.12 ± 0.03 × 10⁻⁴ units/cell. Neither calcitonin nor parathyroid hormone influenced the activity of carbonic anhydrase. A review of other hormonal effects on carbonic anhydrase is provided.     

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14− population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.     

lead levels in deciduous teeth of children from selected urban areas in the Cape Peninsula

The lead levels in shed deciduous teeth of children from two selected urban regions in the Cape Peninsula were compared. The average levels in the teeth of children living in the vicinity of two large industrial plants were: whole teeth 20,419 ppm, enamel 10,952 ppm, and dentine 22,733 ppm. The lead levels in teeth from children living in the vicinity of light industries were: whole teeth 16,556 ppm, enamel 2,919 ppm, and dentine 19,926 ppm. These differences were significant at the 1% level (teeth and enamel) and 5% level (dentine).     

Pharmacological characterization of isolated human prostate

PURPOSE: Human prostate contains alpha-1 adrenergic, cholinergic and nonadrenergic noncholinergic neuroreceptors. Using agonistic and antagonistic agents at these neuroreceptors we studied the resultant contractile responses in isolated human prostate. MATERIALS AND METHODS: Human prostate tissue was obtained at prostatectomy for benign prostatic hyperplasia in 37 adult male patients. Tissues were suspended in tissue bath chambers connected to force displacement transducers. Specimens were subjected to agonist induced contractions, the first always being norepinephrine (NE). Specimens were pretreated with antagonist (adrenergic, cholinergic, nonadrenergic noncholinergic or none if control), followed by contraction with a second agonist (NE or other). Contractile tensions were recorded on a polygraph and then statistically analyzed. RESULTS: The order of highest to lowest agonist induced tensile forces was NE, dopamine, acetylcholine, bethanechol, histamine and serotonin. Excitatory concentration EC(50) values were determined for each agonist tested. Significant differences were found between specific alpha-1 adrenergic receptor blockers (terazosin, prazosin and the experimental drug LY253352). In addition, many other agents antagonized the alpha-1 adrenergic receptor. Inhibitory concentration IC(50) values were obtained and the order of alpha-1 adrenergic antagonistic strengths from strongest to weakest was LY253352, prazosin, terazosin, ketanserin, SCH23390, diphenhydramine, DO710, dopamine, serotonin and histamine. CONCLUSIONS: Human prostate neuroreceptors were determined to be alpha-1 adrenergic, dopaminergic, muscarinic cholinergic, 2A serotonergic and H1 histaminergic. Dopamine, serotonin, histamine and their antagonists blocked the adrenergic response, indicating possible receptor-receptor interaction. Further study of the pharmacology of human prostate would likely identify new drugs for treating patients with bladder outlet obstruction due to benign prostatic hyperplasia.     

Differential expression of potassium ion channels in human renal cell carcinoma

Purpose  Ether-a-go-go (EAG) or EAG-related (ERG) voltage-gated potassium ion channels are involved in tumor generation and progression. Their over- and/or misexpression has been demonstrated in various tumors, and inhibition of these channels has suppressed proliferation of various cancer cells. We investigate and compare the pattern of expression of EAG and human ERG (HERG) channels in renal cell carcinoma and “normal” renal tissue. Method  Tissue samples, obtained at the time of radical nephrectomy from the tumor-bearing areas, and uninvolved renal tissue were preserved in 4% paraformaldehyde and cryosectioned at 20 μm. Immunohistochemical and Western blot analysis was performed on the tumor and uninvolved kidney parenchyma by incubating with polyclonal anti-HERG 1b (Alomone Lab, Israel), anti-EAG1, and anti-EAG2. Pattern of expression of EAG/HERG channels in normal renal tissue and carcinoma were noted and compared. Results  The study was performed on 16 radical and four partial nephrectomy specimens (n = 20). All tumors in the cohort were clear cell renal carcinoma. Normal renal tissue was found to exhibit heterogeneous cytoplasmic positivity for EAG1 and focal HERG immunoreactivity (IR) in the proximal (PCT) and distal convoluted tubules (DCT). EAG2 IR was absent in the normal renal tissue. Clear cell RCC demonstrated a loss of HERG expression while diffuse overexpression of EAG1 and EAG2 was noted. Western blot analysis corroborated the immunohistochemical observations. Conclusions  In our study both EAG1 and EAG2 potassium channels were overexpressed in clear cell renal cancer. In contrast to other adenocarcinomas, there is loss of HERG expression in clear cell RCC, which may possibly explain its chemoresistance. These ion channels may provide a potential for targeted therapy.     

High lead content of deciduous teeth in chronic renal failure

Lead is suspected to contribute to the progression of kidney disease. Lead content of blood and deciduous teeth was determined in 22 children aged 5–14 years at different stages of chronic renal failure (CRF). In addition, individual lead exposure was estimated from histories. The results were compared with a control group of 20 siblings or neighbours of patients living in the same environment (C1), and to a group of children known to be free of excessive lead exposure (C2). The mean blood lead concentration of patients was normal (mean 2.9 g/dl, range 1.1.–10.1). Mean dental lead content was 2.8, 1.7 and 1.4 g/g in CRF, C1 and C2, respectively. It always exceeded that of healthy peers. Increased dental lead content was associated with a high risk of exposure. It is suggested that both an increased lead uptake and renal dysfunction may contribute to the increased lead burden in children with CRF.     

乳牙滞留对牙颌面发育影响的临床分析

目的：探讨乳牙滞留对恒牙萌出及牙颌面发育的影响。方法：选择我科诊治的400例患者,共2011颗滞留乳牙进行分析,年龄5～18岁。结果：滞留乳牙以下颌乳中切牙最多,占24.99%;健康状况正常的有210例,仅占10.42%;其中在有继承恒牙的1994颗中,萌出正常的仅202颗,占10.13%。结论：乳牙滞留可影响患者的牙颌面发育,导致错畸形。     

Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.     

Enzymatic and electron microscopic analysis of isolated osteoclasts

A new method is described by which osteoclasts can be isolated for biochemical and electron microscopic analyses. As a source of cells for isolation by microdissection, imprints of metaphyseal bone were used. The use of imprints provides important advantages over bone sections, including a higher degree of cytologic and enzymatic preservation, and the delivery of whole cells which are more readily manipulated and which yield data that are more readily reproduced. By planimetric analysis of the histochemically-stained isolated cell samples, it was shown that osteoclasts represent over 90% of the sample mass. The levels of several of the pyridine nucleotide-linked enzymes involved in citric acid metabolism, as well as acid phosphatase, were determined in osteoclast samples weighing 0.2 to 2.0 g isolated from normal and parathyroid-treated rats. Aconitase activity measured in the direction of citrate to isocitrate was 0.5–0.8 M/K_d/H, the lowest of the activities studied. The activities of GDH and NADP-ICDH were 5 to 10 times higher than that of aconitase but only a tenth to a third that of acid phosphatase, lactic or malic dehydrogenase.Supported by USPHS Grant No. AM 12491.