Calcium-cobalt Method Research Articles

Streptozotocin Aggravated Osteopathology and Insulin Induced Osteogenesis Through Co-treatment with Fluoride.

The role of insulin in the mechanism underlying the excessive fluoride that causes skeletal lesion was studied. The in vitro bone marrow stem cells (BMSC) collected from Kunming mice were exposed to varying concentrations of fluoride with or without insulin. The cell viability and early differentiation of BMSC co-treated with fluoride and insulin were measured by using cell counting kit-8 and Gomori modified calcium-cobalt method, respectively. We further investigated the in vivo effects of varying dose of fluoride on rats co-treated with streptozotocin (STZ). Wistar rats were divided into six groups which included normal control, 10 mg fluoride/kg day group, 20 mg fluoride/kg day group, STZ control, STZ+10 mg fluoride/kg day group, and STZ+20 mg fluoride/kg day group. The rats were administered with sodium fluoride (NaF) by gavage with water at doses 10 and 20 mg fluoride/kg day for 2 months. In a period of one month, half of rats in every group were treated with streptozotocin (STZ) once through intraperitoneal injection at 52 mg/kg body weight. The serum glucose, HbA1c, and insulin were determined. Bone mineral content and insulin release were assessed. The results showed insulin combined with fluoride stimulated BMSC cell viability in vitro. The bone mineral content reduced in rats treated with higher dose of fluoride and decreased immensely in rat co-treated with fluoride and STZ. Similarly, a combination treatment of a high dose of fluoride and STZ decreased insulin sensitivity and activity. To sum up, these data indicated fluoride influenced insulin release, activity, and sensitivity. Furthermore, the insulin state in vivo interfered in the osteogenesis in turn and implied there was a close relation between insulin and bone pathogenesis in the mechanism of fluoride toxicity.

Preliminary Study on the Histology in the Head Sarcoma of Goldfish (Carassius auratus)

The sarcoma structure of goldfish was first analyzed by frozen section technique. The present study revealed the alkaline and acid phosphatase distribution in the head sarcoma of goldfish. And the histochemical staining in situ by calcium-cobalt method and lead nitrate method displayed a large amount of alkaline and acid phosphatases in the head sarcoma, with higher enzyme activity. The alkaline and acid phosphatases belong to hydrolases, which are widely present in various tissues. The results showed that the activity of external sarcoma is similar to the middle one, while there was more alkaline phosphatase near to the tissue sarcoma under the goldfish scales. And they have the strong activity. The study will lay the theoretical basis on enhancing the ornamental value of goldfish.

Salvianolic acid B promotes bone formation by increasing activity of alkaline phosphatase in a rat tibia fracture model: a pilot study.

BackgroundRadix Salviae miltiorrhizae is a herb frequently used within traditional Chinese medicine for the treatment of cardiovascular- and trauma-related diseases. Danshen is the dried root of Salviae miltiorrhizae, from which the polyphenolic compound Salvianolic acid B (Sal B) can be obtained. Sal B is a key component of Danshen. The aim of this study was to determine the effect of Sal B on the healing of long bones following trauma in a rat tibia fracture model.MethodsTibia fractures were created in 20 male Sprague Dawley rats. The animals were divided into two groups: (1) experimental group (n = 10); and (2) control group (n = 10). Rats in the experimental group were intraperitoneally administered with Sal B (40 mg/kg/d) for 3 weeks, while rats in the control group received an identical volume of physiological saline solution, administered in the same way. X-ray photographs were taken of all animals at the time points. Rats were euthanized at weeks 1, 3, 8 and 12 post-fracture. Fracture calluses were measured and callus sections were obtained and stained using hematoxylin and eosin (HE) and the calcium cobalt method. HE stained sections were observed and evaluated according to different grades of bone remodeling. Sections stained using the calcium cobalt method were analyzed with an imagine analysis system.ResultsData showed that callus growth was significantly greater in the experimental group compared with the control group (P < 0.05). Furthermore, histological scores in the Sal B-treated group were statistically higher than in the saline treated group at weeks 1, 3 and 8 post-fracture (P < 0.05). Alkaline phosphatase (ALP) activity was enhanced in the experimental group at weeks 1 and 3 post-fracture (P < 0.05).ConclusionsOur results suggest that Sal B may accelerate early-stage fracture healing. Increased activity of ALP may be one factor which promotes the healing process. This pilot study provides brief insight into the effect of Sal B in fracture healing. These findings will contribute to the development of more and enhanced treatment options for trauma fracture patients.

HTERT- and hCTLA4Ig-expressing human bone marrow-derived mesenchymal stem cells: in vitro and in vivo characterization and osteogenic differentiation.

Multipotent mesenchymal stem cells (MSCs) are commonly used as seed cells in studies of tissue engineering and regenerative medicine but their clinical application is limited, due to insufficient numbers of autogeneic MSCs, immune rejection of allogeneic MSCs and replicative senescence. We constructed two gene expression vectors for transfection of the human telomerase reverse transcriptase (hTERT) and cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4Ig) genes into human bone marrow-derived stem cells (hBMSCs). Successful transfection of both genes generated hTERT-CTLA4Ig hBMSCs that expressed both telomerase (shown by immunohistochemistry and a TRAPeze assay) and CTLA4Ig (demonstrated by immunocytochemistry and western blotting) without apparent mutual interference. Both hTERT BMSCs (92 population doublings) and hTERT-CTLA4Ig hBMSCs (60 population doublings) had an extended lifespan compared with hBMSCs (18 population doublings). Cell cycle analysis revealed that, compared with hBMSCs, a lower proportion of hTERT hBMSCs were in G0 /G1 phase but a higher proportion were in S phase; compared with hTERT hBMSCs, a higher proportion of hTERT-CTLA4Ig hBMSCs were in G0 /G1 phase, while a lower proportion were in S and G2 /M phases. hTERT-CTLA4Ig hBMSCs retained their capacity for osteogenic differentiation in vitro, shown by the detection of hydroxyapatite mineral deposition (labelled tetracycline fluorescence staining), calcareous nodules (alizarin red S staining), alkaline phosphatase (calcium-cobalt method) and osteocalcin (immunocytochemistry). Furthermore, subcutaneous transplantation of hTERT-CTLA4Ig hBMSCs in a rat xenotransplantation model resulted in the successful generation of bone-like tissue, confirmed using radiography and histological assessment. We propose that allogeneic hTERT-CTLA4Ig hBMSCs may be ideal seed cells for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Influence of glucocorticoids on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

BackgroundGlucocorticoid has been used extensively in clinical applications, because of its several pharmacologic actions, which include immunosuppression, anti-inflammation, anti-shock, and relief of asthma. However, the long-term or high-dose application of glucocorticoid can induce adverse effects such as osteoporosis, which is known in this case as glucocorticoid-induced osteoporosis (GIOP). It is a secondary osteoporosis that results in easy fracturing, and even disability. Therefore it became a thorny issue.MethodsThe rat model of glucocorticoid-induced osteoporosis (GIOP) was replicated to isolate BMSCs. Rats were assigned into four groups: normal, normal induction, GIOP, and GIOP induction. The growth cycle was monitored by using flow cytometry. Osteogenic differentiation was compared by using alkaline phosphatase (ALP) staining with a modified calcium cobalt method. The quantitative detection of osteoprotegerin and the receptor activator of nuclear factor kappa-B ligand (RANKL) was conducted by using enzyme-linked immunoassay. Finally, renal Klotho mRNA expression was assessed by using RT-PCR.ResultsBMSC proliferation was reduced in GIOP rats. The ALP-positive expression of normal BMSCs to the osteogenic induction solution was stronger than that of BMSCs from GIOP rats (P < 0.01). Osteoprotegerin expression was significantly higher in the normal induction group than in the normal, GIOP (P < 0.01), and GIOP induction groups (P < 0.05). RANKL expression was significantly higher in the normal induction group than in the other groups (P < 0.01) and significantly higher in the normal group than in the GIOP and GIOP induction groups (P < 0.01). RT-PCR analysis showed that renal Klotho mRNA expression was significantly reduced in the GIOP group compared with the normal group (P < 0.01).ConclusionBMSC proliferation, osteogenic differentiation, and reactive activity to an osteogenic inductor were reduced in GIOP rats. Klotho mRNA expression decreased during GIOP induction.

Effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells at different glucose concentrations: experiment with rats

To study the effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at different glucose concentrations. BMSCs of SD rats were isolated, cultivated in vitro, and divided into 6 groups to be induced to differentiate into osteoblasts under different conditions: (1) low glucose control group, (2) high glucose control group, (3) low glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (4) high glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (5) low glucose+drynaria total flavonoid group, and (6) high glucose with drynaria total flavonoid group. Alkaline phosphate (ALP) test kit was used to examine the level of ALP. The ALP staining positive rate was examined with modified calcium cobalt method. Alizarin red staining was adopted to observe the number of calcium nodes. Immunohistochemistry was used to detect type I collagen level. Advanced glycosylation end products (AGEs) were tested by ELISA. The A value indicating the ALP activity, ALP staining positive rate, calcium node number, and type I collagen expression score of the low glucose+drynaria total flavonoid group were (0.439±0.024), 48.7%, (9.75±1.71) nodes/HP, and (2.21±0.07) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.385±0.029), 35.0%, (6.25±0.96) nodes/HP, and (1.93±0.13) respectively, all P<0.05]. The A value, ALP staining positive rate, calcium node number, and type I collagen expression score of the high glucose with drynaria total flavonoid group were (0.352±0.022), 25.3%, (4.50±1.29)/HP, and (1.70±0.03) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.139±0.013), 22.7%, (3.25±1.50)/HP, and (1.28±0.27) respectively, all P<0.05]. The AGE expression levels of the high glucose classical induction group and high glucose+drynaria total flavonoid group were both significantly higher than those of the low glucose classical induction group and low glucose+drynaria total flavonoid group (both P<0.05). There were no significant differences in the AGE level among the low glucose control, low glucose classical induction, and low glucose+drynaria total flavonoid groups (all P<0.05); and among the high glucose control, high glucose classical induction, and high glucose+drynaria total flavonoid groups (all P<0.05). However, the AGE levels of the high glucose groups were all significantly higher than those of the corresponding low glucose groups (all P<0.05). Glucose increased the AGE levels dose- and time-dependently. The concentrations of AGEs were significantly negatively correlated with the expression of type I collagen (r=-0.410, P<0.05). Drynaria total flavonoid promotes the osteogenic differentiation of BMSCs and relieves the inhibitory effect of osteogenic differentiation by glucose at high concentration. Thus drynaria total flavonoid may provide a potential therapy for diabetic osteoporosis.

The Effect of the Major Components of Fructus Cnidii on Osteoblasts In Vitro

In traditional Chinese medicine, the cause of weak bones or bone loss is generally regarded as a result of kidney deficiency. Fructus Cnidii (FC), which is also known as She-Chuang-Zi, is a traditional herb that has been claimed to have kidney warming effects that invigorate Yang. In this study, we tried to determine the bone production-inducing effect of FC on osteoblastic cells in vitro using osthole, the main component of FC. Osteoblasts were isolated from neonatal Sprague-Dawley rat calvaria using the tissue piece culture method and treated with various concentrations of osthole ranging from 2.5 to 640 μg/mL, together with a blank control. Cell proliferation, alkaline phosphatase (ALP) activity, and bone nodules were measured. The cells were examined by hematoxylin-eosin staining, the Gomori Calcium-Cobalt method and immunofluorescent staining. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (or MTT) assay, ALP assay, and bone nodule results indicated significantly enhanced osteoblastic proliferation and differentiation at concentrations of osthole ranging from 40 to 320 μg/mL. Concentrations lower than 40 μg/ mL seemed less effective, and cytotoxicity to osteoblasts was observed at concentrations higher than 320 μg/mL. These results indicate that osthole is effective at inducing osteoblastic bone formation through the up-regulation of ALP activity. FC is a Chinese herb used to treat lumbar pain in clinical practice. Further studies concerning the effects and mechanism of osthole on osteoporotic patients and animals should be performed, as these studies may lead to the development of a drug treatment for osteoporosis in the future.

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique.

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.

Phosphatases in the central nervous system of spiders

The central nervous systems of web-building spiders (Araneidae, Agelenidae) and hunting spiders (Lycosidae, Salticidae) were tested for non-specific and specific phosphatases. Acid phosphatase exhibited weakly to moderately positive reactions in the neuronal cell bodies and in the neuropile fibre mass of all species investigated. Alkaline phosphatase could only be demonstrated in the external and internal neural lamellae of the brain and ventral cord of several specimens of the araneid species investigated. Tests for thiamine pyrophosphatase were negative with both the lead and calcium-cobalt methods. Distinctive positive reactions for adenosine triphosphatase were visible in the nervous system of all the species used, being especially strong in the optic ganglia of the hunting spiders. The demonstration of adenosine triphosphatase was only possible when applying the calcium-cobalt method after Padykula and Herman, while the lead method after Wachstein and Meisel did not produce any staining reaction at all. Controls of the histochemical reaction showed that the enzyme was activated by Ca2+ and inhibited by sulphydryl destroying reagents (e.g. PCMB), but was insensitive to ouabain. It could be probably classified as a mitochondrial proton-translocating adenosine triphosphatase.

Nonspecific alkaline phosphatase activityin normal and diseased human breast

Nonspecific alkaline phosphatase activity was identified in human normal and diseased breasts with the use of the calcium-cobalt, the lead-nitrate, and the azo-dye methods. The results varied not only with the staining method, but also with the functional status of the breast structures. In normal, dysplastic, and fibroadenomatous tissues there was a strong parallelism between myoepithelial and capillary enzyme activities. The calcium-cobalt method was the only technique which allowed staining of carcinoma cells; cancer stromal enzyme activity was evidenced only with the use of the same method. Our findings suggest that nonspecific alkaline phosphatase activity probably reflects a functional status of the labelled structures; the enzyme activity of myoepithelial cells is variable and not really specific.

ATPase activity in the breast: A comparison between three methods

Adenosine triphosphatase enzymatic activity was investigated in human approximatively normal, dysplastic and neoplastic mammary tissue, by three different methods. Staining intensity varied within wide limits; myoepithelial cells and blood vessels showed similar enzymatic activity. Epithelial cells reacted only faintly, or not at all; carcinoma cells were never labelled. Stromal response was highly variable. The calcium-cobalt method of Padykula and Herman gave more intense reactions than the lead-nitrate procedure of Wachstein and Meisel, either in the original form or according to the modifications recommended by Russo and Wells. With the latter method the sharpness of stain deposits on the different structures was markedly enhanced. The functional significance of ATPase activity is discussed.

Alkaline phosphatase reaction in hair follicles of male beagle dogs during hair cycle stages.

AbstractThe literature reviewed has revealed differences of opinion concerning the location of alkaline phosphates and its relationship to the hair cycle stages. This project was undertaken to determine alkaline phosphatase activity in the hair follicle stages: anagen, catagen, and telogen.Skin biopsy samples were collected over a period of 1 year from 9 purebred male Beagle dogs. At the beginning of the experiment 3 dogs were 2 weeks of age, 3 dogs were 12 months of age and 3 dogs were 21 months of age. The skin biopsies were taken monthly from alternate sides of each animal near the thoracolumbar region. Samples were fixed in 80% chilled alcohol and the alkaline phosphatase activity was determined by Gomori's calcium cobalt method. A total of 250 slides with seven sections per slide was examined. Each slide contained between 14–60 hair follicles.During anagen stage, the reaction of the dermal papilla for the presence of alkaline phosphatase was variable. A weak or negative reaction was observed in this study which was an unexpected finding. The reaction of the dermal papilla to alkaline phosphatase was positive in catagen stage. The cells of the dermal papillae in telogen stage were strongly alkaline phosphatase positive. The reaction was not confined to the blood vessels but occurred in all parts of the dermal papilla in both early and late stages.

Distribution and properties of an adenosine triphosphatase in the tanycyte ependyma of the IIIrd ventricle of the rat.

The distribution, histochemical properties and ultrastructural localization of adenosine triphosphate (ATP) hydrolyzing enzymes in the tanycyte ependyma of the third ventricle have been studied in female Wistar rats. Using a calcium-cobalt procedure and a lead capture technique, splitting of ATP could be demonstrated in perikarya and processes of tanycytes in the region of the ventromedial nucleus. The reaction showed no dependence on magnesium or sodium ions, did not occur with other monodi-, and tri-phosphates as substrates, and was inhibited by p-chlormercuribenzoate (PCMB) and sodium fluoride, but not by ouabain. With the calcium-cobalt method the highest intensity of reaction was found at pH 9.4, whereas the lead method gave optimal results at pH 6--8. At the ultrastructural level, the reaction product was found at the outer surface of the plasma membranes of tanycytes and reached its highest concentrations in the region of the region of the apical microvilli; From the findings it is concluded that splitting of ATP in tanycytes is due to a true ATPase. The enzyme might be involved in an active transport of substances by tanycytes.

A fine structural study on the extracellular activity of alkaline phosphatase and its role in calcification.

Nine day old experimental calluses in radii of young rats were treated with Gomori's (1939) calcium-cobalt method to demonstrate ultrastructurally the presence of alkaline phosphatase in a search for its possible role in the desposition of calcium. Enzyme activity first appeared as globule-like precipitates outside the cell membrane of early hypertrophic cartilage cells. This precipitate layer then seemed to give rise to spherical bodies of alkaline phosphatase which occur at a slight distance from the cell. The spherical bodies were also observed further away from the cell in an intermediate zone between neighboring cells. Needle-like crystal formation, apparently calcification, occurred inside single or aggregated bodies, leaving their peripheral rim clearly visible, even when calcification had increased to such an extent that individual crystals could no longer be recognised. In controls, treated in the same way but without substrate, or with EDTA, no enzyme precipitate or spherical bodies were seen. The appearance of crystalline deposits in bodies which contain alkaline phosphatase seems difficult to explain on any other basis than that there is a close functional association between the enzyme and calcification.

L-homoarginine, a specific inhibitor of liver-type alkaline phosphatase, applied to the recognition of liver-type enzyme activity in rat intestine.

l-Homoarginine, an inhibitor of alkaline phosphatase, has been applied for the study of the localization of alkaline phosphatase activity in rat liver and intestine. Five substrates were employed: Na α-naphthyl acid phosphate, naphthol AS-BI phosphate, Na β-glycerophosphate, o-carboxyphenylphosphate and O-ethanolamine phosphate. Diazonium coupling was employed with the naphthol substrates; the Gomori calcium-cobalt method was used with the others. Each substrate was also employed in the presence of 12.5 mMl-phenylalanine, an intestinal (brush border-specific) alkaline phosphatase inhibitor. The liver-type enzyme, in both the liver parenchyma and the lamina propria of the small intestine, was sensitive to 12.5 mMl-homoarginine in varying degrees, depending on the substrate used. With Na α-naphthyl acid phosphate, a total inhibition was observed histochemically; l-homoarginine inhibited partially purified liver alkaline phosphatase by 97%, as measured biochemically. The alkaline phosphatase of leukocytes and macrophages of the intestinal lamina propria was inhibited in the presence of l-homoarginine but not of l-phenylalanine. Reactive elements that resembled the macrophages of the lamina propria were present in the liver. The activity in these cells was also inhibited by l-homoarginine.

Histochemical studies of alkaline phosphatase in carcinoma of the liver.

Thirty-eight patients who had proven hepatic carcinomas (19 hepatomas, 4 of an unclassified type, 5 cholangiomas, and 10 metastatic carcinomas) were studied. Statistically significant, positive correlations were found between the serum alkaline phosphatase level and the compression effect of carcinoma nodules on the adjoining liver cells, and between the serum enzyme and the sinusoidal phosphatase activity in tissue section. There was no correlation between serum alkaline phosphatase values and serum bilirubin concentration. The seroflocculation tests and iodine test were not significantly abnormal. Neither hepatoma nor metastatic cancer cells took a stain by the calciumcobalt method of Gomori. The elevation of the serum alkaline phosphatase level in carcinoma of the liver most likely is caused by intrahepatic obstruction of biliary channels and a compression effect on the adjoining hepatocytes by carcinoma nodules.

A MODIFIED CALCIUM-COBALT METHOD FOR THE DEMONSTRATION OF ALKALINE PHOSPHATASE: THE USE OF PARA-NITROPHENYL PHOSPHATE AS A SUBSTRATE.

A MODIFIED CALCIUM-COBALT METHOD FOR THE DEMONSTRATION OF ALKALINE PHOSPHATASE: THE USE OF PARA-NITROPHENYL PHOSPHATE AS A SUBSTRATE.

APPLICATION OF THE STEREOSPECIFIC INHIBITOR L-PHENYLALANINE TO THE ENZYMORPHOLOGY OF INTESTINAL ALKALINE PHOSPHATASE.

The intestine-specific inhibitor of alkaline phosphatase, l-phenylalanine, has been employed in the study of alkaline phosphatase in rat intestine. Five substrates were used, β-glycerophosphate, o-carboxyphenylphosphate, α-naphthyl acid phosphate, naphthol AS-BI phosphate and naphthol AS-TR phosphate. The hydrolytic rate and specific l-phenylalanine inhibition was most marked in the case of the first three substrates. When formol-Ca fixed, gum-sucrose treated sections of rat intestine were incubated in these substrates in the presence separately of 0.05 Md- and l-phenylalanine and Gomori's calcium-cobalt method was employed, a striking inhibition by the l-isomer was regularly found. Azo dye methods were applied under different conditions of time, temperature and cation concentration in some instances. The contrast was marked but not dramatic in the case of α-naphthyl acid phosphate, and it was evident but not marked in the case of naphthol AS phosphate substrates. These findings have been discussed. The alkaline phosphatase of intestinal epithelial cells (human) grown in tissue culture exhibited great sensitivity to l-phenylalanine. Rat kidney and leukocyte alkaline phosphatase were relatively insensitive under the same conditions. Morphologically, the l-phenylalanine-sensitive alkaline phosphatase in rat intestine is largely confined to the striated border of the epithelial cells. In animals maintained on a high fat diet, azo dye methods demonstrated the presence of fine granules in the region of the Golgi apparatus and terminal web in the presence of d-phenylalanine. These reactions were absent in companion sections incubated in l-phenylalanine-containing substrate solutions. The possibility of comparing the morphology of a tissue in which both test and control solutions are identical except for the configuration of one component may contribute to a more precise interpretation of the results obtained in the test section.

A histochemical study of the odontoblast process

Serial ground sections of sound, recently erupted premolars were stained with 0.0005 M methylene blue and 0.01–0.1% toluidine blue at pH 2.6–7.0, with Schiff reagent, the periodic acid-Schiff method, the combined alcian blue-PAS method of Mowry (1956) and the alloxan-Schiff method. Sections were also examined for alkaline phosphatase activity using Gomori's calcium-cobalt method and the azo dye technique. In the predentine and in the dentine up to a distance of about 120–200 μ from the predentine-dentine junction the contents of the dentinal tubule stained solidly and intensely with methylene blue and toluidine blue at pH 7.0, the PAS method used alone or in combination with alcian blue, and the alloxan-Schiff method. The limit of this staining corresponds with the level by which a well-formed translucent peritubular zone has appeared. The contents of the dentinal tubule also showed alkaline phosphatase activity up to this distance from the predentine-dentine junction. Beyond this level the contents of the dentinal tubule stained much less strongly with the methods used. At the junction between the peritubular matrix and the contents of the dentinal tubule, however, a deeply stained circle was found. It is impossible at present to decide whether this structure forms a part of the peritubular matrix or is the outer limit of the odontoblast process as it showed certain staining differences compared with either the peritubular matrix or the contents of dentinal tubule.