Leonard F. Bélanger

Staining Processed Radioautographs

Integrated radioautographs obtained by coating with melted nuclear emulsion (Eastmans NTB3) are serially processed and stained at 4° C. Kingsleys mixture of methylene blue-azure A at pH 6.9 is followed by 0.05% NaHSO3, which destains the emulsion. Basic fuchsin, 0.05% aqueous, is used as counterstain, followed by dehydration in absolute ethanol, clearing in cedarwood oil and mounting with Canada balsam. Basic fuchsin alone produces an oftentimes satisfactory monochromatic background.

Parathormone-induced Osteolysis in Dogs: A Microradiographic And Alparadiographic Survey

Fragments of the parietal bone and tibia of fourteen young dogs were studied by x-ray microradiography of undemineralized sections, alpharadiography, and acid mucopolysaccharide staining of demineralized sections. Eight of these dogs received parathormone in different doses and were killed after intervals ranging from three hours to four days. In the parathormone-treated animals, sites of resorption (osteolysis) were recognized in the middle of the trabeculae of cancellous bone and in the interstitial lamellae of compact bone. These sites were characterized by more numerous enlarged and confluent lacunae inhabited by hypertrophic osteocytes containing metachromatic material and surrounded by basophilic or metachromatic matrix of lower density. A dose of 300 units of parathyroid extract and a dose of 1000 units over three hours did not produce osteolysis. A dose of 600 units over three days and to a greater extent a dose of 1000 units over twenty-four hours produced the effects described. Osteocyte hypertrophy and hyperactivity caused by parathormone were often followed by degeneration and death of the cells.

Autoradiographic Visualization of in Vitro Exchange in Teeth, Bones, and other Tissues, Under Various Conditions:

A UTORADIOGRAPHIC studies in which the tracer has been administered in ViVol-5 have revealed an initial rapid, diffuse penetration of the radioelement to all parts of young mineralizing tissues. This reaction, interpreted to be the result of exchange, disappears when the specific activity of the blood decreases and it is replaced by definite, persistent reactions, in the form of bands, some of which are displaced through continuous growth. These areas were interpreted as being representative of formative centers (areas of incorporation of minerals). On the other hand, Newman and Mulryan6 and Underwood and Hodge7 have recently demonstrated the phenomenon of in vitro exchange between powdered bone, dentin, and enamel on the one hand and solutions containing P32 or Ca43 on the other. Furthermore, these authors have established differences between the exchange behavior of ashed tone and dentin as compared with powdered fresh bone and dentin. In order to verify the value of the physical exchange concept, sections of bones and teeth have been placed in radioactive solutions and autoradiograms have been obtained. Young and old tissues were examined, as well as pathologic material variouss teeth, calcified arteries and cartilage, bone tumors, and bone implants). Teeth treated with hyaluronidase were also studied.

Comparative effects of vitamin D, calcium, cortisone, hydrocortisone, and norethandrolone on the epiphyseal cartilage and bone of rachitic chicks.

Abstract Rachitic chicks were treated with vitamin D, calcium phosphate, cortisone, hydrocortisone, norethandrolone, or a combination of these factors. Vitamin D, norethandrolone, and calcium promote the maturation of cartilage matrix. Cortisone and hydrocortisone inhibit this process. Vitamin D and calcium promote the maturation of subepiphyseal bone cells and matrix. Norethandrolone promotes the growth of immature bone cells which secrete a small amount of matrix. Maturation of subepiphyseal bone is produced by the association of norethandrolone with calcium phosphate. The effects of vitamin D, the biochemistry of cartilage matrix maturation and its role in mineralization are discussed.

Autoradiographic studies of sulfated mucopolysaccharide metabolism in cartilage of osteolathyric rats and chicks.

Summary In lathyric rats and chicks, the in vivo sulfate fixation per individual cell was equal or slightly higher than that of normal animals. Since there were more active cells in the hypertrophic plate of the lathyric rat, the overall sulfur upake by cartilage was apparently greater. In 7 day lathyric chicks, the plate was not hypertrophied but the sulfate uptake seemed slightly greater. In vitro Ca45 uptake by demineralized cartilage of the upper extremity of the tibia was considerably less than that of the lower extremity and also less than that of controls, indicating a more rapid utilization or turnover.†

Autoradiographic Visualization of Ga45 Intake by Normal and Pathological Cartilage in vitro.

Summary Demineralized sections of normal and pathological cartilage from various sources, were soakes in weak solution of Ca45Cl2 and coated with fluid photographic emulsion. The autoradiographic record has shown an apparently specific pattern of Ca45 intake. Comparisons of these images and those obtained with S3504 in vivo and also the negatigating effect of hyluronidase seems to indicate a relationships between Ca45 intake and the relative local distribution of sulfated mucop9olysaccharides in the tissue.

Calcium regulation by the chick embryo. An experimental approach.

An artificial air-chamber was produced in White Leghorn chick embryos on the eighth day of incubation. On day twenty, the concentration of calcium in blood and in yolk was found to be lower in the treated embryos than in controls but concentration in bone was similar in both groups. The histological and microradiographic aspect of bone was also similar in both experimental and control embryos. The decrease in the concentration of calcium in blood and in yolk indicates that the experimental procedure succeeded in producing a decrease in calcium intake by the embryo. The fact that bones remained unaffected tends to support the idea that in case of deficient supply of shell calcium, bones can withdraw the calcium they need from the yolk deposit.RésuméUne poche à air artificelle est produite dans des embryons de poulet «White Leghorn», au 8ème jour de l’incubation. Au vingtième jour, la concentration en calcium du sang et du jaune d’oeuf est plus faible chez les embryons opérés que chez les témoins, mais son contenu dans l’os est identique dans les deux groupes. L’aspect histologique et microradiographique de l’os est identique chez les opérés et les témoins. La diminution de calcium dans le sang et le jaune d’oeuf indique que le procédé expérimental utilisé est efficace pour produire une diminution de l’incorporation de calcium par l’embryon. Le fait que les os ne sont pas touchés semble confirmer l’idée qu’en cas d’apport insuffisant de calcium de la coquille, les os peuvent prélever le calcium nécessaire au niveau du jaune d’oeuf.ZusammenfassungEine künstliche Luftkammer wurde am 8. Tag der Inkubation in Embryos von weißen Leghorn-Küken erzeugt. Am 20. Tag war die Calcium-Konzentration im Blut und im Dotter der behandelten Embryos niedriger als in den Kontrollen, aber im Knochen beider Gruppen war sie gleich. Das histologische und mikroradiographische Aussehen des Knochens war ebenfalls in beiden Gruppen ähnlich. Die Abnahme der Calcium-Konzentration in Blut und Dotter zeigt, daß durch das experimentelle Verfahren die Calciumaufnahme durch den Embryo herabgesetzt werden konnte. Die Tatsache, daß die Knochen nicht beeinflußt wurden, unterstützt die Ansicht, daß Knochen das benötigte Calcium dem Dotter entziehen können, wenn sie aus der Schale nicht genügend erhalten.

Detection of Phosphatase Activity in Polarized Light Following Glycerophosphate Incubation.

Summary A method of detection of phosphatase activity is described whereby some artefacts of diffusion, adsorption and nonspecific sulfide blackening are dealt with by examination of the original calcium or lead phosphate precipitates in polarized light. This technic demonstrates also the presence of natural mineral deposits in undecalcified material. Comparable estimates of the amount of salt deposited is made with the help of a densitometer.

Extratrabecular crystallization in rickets; effects of vitamin D, calcium and norethandrolone.

Summary Large extratrabecular crystals are present in rachitic bones of chicks. They disappear after treatment with Vit. D.

Impregnation of canaliculi and other bone constituents with iodinated starch

The venerable iodine react ion for s tarch (ConI~ and DE CLAUBRY, 1814; STRO~IEYE~, 1815)7 more recent ly explained as a physical intrusion of the amylose molecule (MEYE~ and ]~ERhTFELD~ 1941), has now been applied to bone with hope of producing a specific coloured impregnat ion of microspaces, especially canaliculi. EDTA-demiuera l ized 7 ~ sections of AFA-fixed specimens were thus soaked in a 5 % aqueous solution of Soluble Starch (Fisher Scientific Company) for 30 minutes. They were then dried and subsequent ly exposed for 1 hr. to iodine vapour in a closed jar containing s tandard Lugol solution. The sections were then cleared in toluene and eoverslipped with P e r m o u n t (Fisher). The results were more complex than expected. The canaliculi especially in the rat , chick, dog and other exper imenta l animals were admirab ly impregnated. H u m a n specimens were more or less successfully t reated. Unclemineralized mater ia l did not respond. The cytoplasmic por t ion of the osteocytes, the lacunar border, the inter lamellar mater ia l of compact bone, osteoid borders, carti lage ma t r ix and the mucoid product of mucous glands were all well impregna ted with iodinated starch. Sections exposed direct ly to iodine vapour showed a pa t t e rn of apparen t iodine up take bu t no blue colouring, indicating tha t in all instances, the starch was the only react ing agent and was somehow adsorbed a t different rates b y the various components of the tissues. A marked increase in the canalieul~r mass occurred in bones of magnesium deficient rats and in humans suffering f rom osteogenesis imperfecta . The adminis t ra t ion of pa ra thyro id ex t rac t in rats, the prolonged feeding of a calcium deficient diet in chicks and other conditions leading to osteocytic osteolysis (B~LA~GER~ ;[969) did not produce an apparen t increase in the canalicular mass. However , in the areas of osteocytic resorption, the residual ma t r ix became more heavily impregnated with the iodinated starch.