We isolated cells from both calvaria and the outer cortices of long bones from 3-to 5-mo bovine fetuses . The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type I Collagen . In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by imunoprecipitation and fluorography of SIDS polyacrylamide gels . Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (-94%) type I collagen, with small amounts of types III and V collagens . In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and 3-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen . These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro .Several criteria have been used to characterize cells isolated and cultured from bone as osteoblasts, although none have proved specific . These are that (a) freshly isolated bone cells possess high alkaline phosphatase activity (1), (b) bone cells show a strong cyclic AMP response to parathyroid hormone (2), (c) bone cells secrete predominantly type I collagen when grown in the presence of ascorbic acid (3), and (d) bone cells produce and partially mineralize a matrix, given extended time in culture and defined culture conditions (4-9).Recently, the isolation and purification of several noncollagenous matrix proteins from fetal calf bone (10) have provided new tools to identify bone cells with increased certainty . Antibodies against these proteins have been used to establish their tissue specificity in fetal calf bone (11)(12)(13). In this study we confirm the criteria listed above using bone cells obtained from the calvaria and long bones of fetal calves, the first nonrodent system to be explored . We show by indirect im-THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 607-614 © The Rockefeller University Press -0021-9525/84/08/0607/08 $1 .00 munofluorescence and in biosynthetic experiments, that these cells produced three bone-specific, noncollagenous proteins ; osteonectin (11), the bone proteoglycan (12), and the bone sialoprotein (13), thus proving conclusively that this methodology can clearly identify bone cells in vitro. MATERIALS AND METHODSCulture Conditions : Fetal calves (3-5 mo in utero [8] and still in the fetal sac) were obtained from Schneider Packing Co ., S...

SUMMARY:The caudatoputamen (CP) and giobus paiiidus (GP) are supplied by vessels often involved with stroke in both rat and human. The pattern of vascular supply to the CP and GP in rat has, in contrast to humans, been only partially described. The vascular pattern to the rat CP and GP is described utilizing vascular endocasts and scanning electronmicroscopy in aging, nonnotensive rats. Endocasts were produced by intra-cardiac infusion of Batson's Corrosion Compound. The vascular pattern is complex, involving 1) recurrent vessels from the anterior cerebral artery, 2) branches from the arterial circle rostral or caudal to the origin of the middle cerebral artery (MCA), 3) up to 6 branches from the MCA, and 4) 2 major branches from the caudal part of the arterial circle. The vessels in groups 1-3 were serpentine, their luminal diameters abruptly reduced at branch points, and the angle of departure from the parent vessels approximated 90°. These vessels supplied much of the CP and GP, while group 4 supplied the caudal CP with vessels arranged in a lattice-like fashion from the 2 penetrating parental arteries.Stroke, Vol 12, No 6, 1981THE VASCULAR SUPPLY to the basal ganglia (caudate, putamen and giobus paiiidus) is involved in a large percentage of the cerebrovascular accidents that occur in man. 1 Patients with infarction or hemorrhage into the basal ganglia may show movement disorders and postural abnormalities.1 '' Although hypertension is a significant factor in cerebral vascular diseases, 1 '' the effects of hypertension on the structure and histochemistry of extracerebral vessels is better understood than the pathophysiology of stroke prone vessels.*""The rat is one of the principal models in the study of vascular pathophysiology associated with hypertension, and, most important, stroke mechanisms in rats and humans may be similar. '-4 The vascular supply to the rat basal ganglia has been only partially described. 4 The endocast data reported here are in accord with the microangiographical observations of Yamori et al.; 4 however, endocasts provide a more definitive picture of the vascular patterns in the basal ganglia. A detailed description of the vascular patterns in the basal ganglia is a prerequisite to an evaluation of ultrastructural and histochemical changes in the walls of these stroke prone vessels and their relation to the pathophysiology of hypertension. The vascular patterns in the caudatoputamen (CP) and giobus paiiidus (GP) are described utilizing vascular endocasts and scanning electron microscopy in aging, normotensive male rats. MethodsTwo techniques were used to gather the data for the present report. These techniques are described under I. Production of Endocast for Scanning MicroscopyThirteen male rats (13-20 months old) with normotensive blood pressures (systolic pressure range of 92-128 mm Hg, Narco Bio-systems, indirect blood pressure measurement system) were anesthetized with sodium pentobarbital (40 mg/kg i.p.). The thorax was opened and each animal was infused through the heart ...

This study of the in vitro synthesis and mineralization of bovine bone demonstrates that sheets of mineralized matrix can be produced consistently within 18-24 days of cell isolation. Mineralization surpasses that achieved by other systems with other species: The deposition of mineral extends beyond nodules to form branching trabeculae and then solid wafers of bone. Comparison of the fetal age of the bone source, enzyme digestion methods, seeding density, culture surface, nutritive media, and concentration of fetal calf serum and other additives, including insulin and ascorbic acid, has yielded a set of optimal culture conditions. In the presence of ascorbic acid and beta-glycerol phosphate, insulin has a dose-dependent effect on the morphology of the mineralized bone matrix produced. Quantitative analysis shows that in these cultures calcium accumulates most rapidly between days 6 and 10 after the introduction of mineralization medium but that mineral accretion continues throughout 14-16 days of culture. Alkaline phosphatase levels rise up to 200-fold, concomitant with a rapid increase in the number of cells per culture during the early mineralization phases; both fall as mineralization proceeds. This system has been used to study the induction of mRNA of type I collagen, alkaline phosphatase, and several noncollagenous bone proteins during the course of mineralization. Because of the degree of mineralization achieved with this system, it has many potential applications.

Tooth whiteners are considered as cosmetic agents to be used for bleaching teeth. Since tooth whitener may be swallowed during the whitening procedure, studies were conducted to determine whether ingestion of tooth whitener containing carbamide peroxide resulted in toxic effects. Adult female rats were used, and vaginal smears were examined daily to determine whether the animals were ovulating. Following an overnight fast, a single bolus of a commercial tooth whitener (5 g of tooth whitener/kg fasting body weight) was administered by gavage. Control rats received de-ionized water. After 2 h, mean respirations per min of animals receiving the tooth whitener Quik Start (contains 35% carbamide peroxide) decreased from 169 to 55, and body temperature decreased from 38.4 to 34 degrees C. Other distress signs included: labored breathing, loss of righting reflex, partial eye closure, bloody urine, and incontinence. Three of 22 animals (3/22) died within 48 h, of gastric hemorrhaging. Eight/10 rats stopped ovulating. At necropsy 2 weeks post-dosing, 10/19 animals had grossly bloated stomachs, and mucosal necrosis was observed histologically in 3. Animals receiving White & Brite or Nu-Smile (containing 10 or 15% carbamide peroxide, respectively) exhibited similar but milder symptoms. The data indicate that ingestion of large doses of commercial preparations of tooth whiteners may be acutely toxic, sometimes fatal, to female laboratory rats.

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