Milton B. Engel

Vital Staining of Growing Bones and Teeth with Alizarine Red S

The increasing use of alizarine Red S as a vital dye prompted this report. In contrast to the method of prolonged madder feeding which gives diffuse effects (2, 5, 13), each injection of alizarine Red S produces a sharp red line in the incremental zones of dentin, cementum and bone (14, 15) that are growing and calcifying at the time. The distance between 2 successive injections thus represents the amount of apposition that has occurred during the intervening period. Sharp effects were not found in the enamel. History. Robiquet and Colin (8) in 1826 reported alizarine to be the principal staining ingredient in madder, which was used in the dyeing of cloth by the early Egyptians. It was synthesized in 1869 (8) and Perkin and Story (18) synthesized 3-iodo-alizarin in 1931. By vital staining with alizarine Red S, studies have been made on bones and teeth of various species ranging from fish to monkey (1, 7, 10, 14, 17, 20). Alizarine has also been used as a non-vital stain in the Spalteholtz method of clearing (25), differential staining of bones (7, 17), and in ground sections of enamel (11). Chemistry. It is important to distinguish between alizarine Red S and other members of the alizarine group. The general formula of the latter is: O OH

Histochemical Studies of Phosphatase Distribution in Developing Teeth of Albino Rat.

Summary 1. Phosphatase activity and localization in developing tooth germs of rats (1 and 4 days old) is correlated with the degree of cellular differentiation and the status of the calcification process. 2. The enzyme is shown to be localized (a) in the stratum intermedium, (b) in the outer enamel epithelium of the incisors, (c) to a lesser extent in the stellate reticulum about the first molar in the more advanced stages (4 days old), and (d) in the pulp contiguous to those odontoblasts in the regions of dentin calcification. 3. No phosphatase activity was demonstrated in the ameloblasts or odontoblasts. 4. The connective tissue surrounding the crypt bone showed extensive phosphatase activity.

Interaction of ions and connective tissue.

The interaction of ions with tissues was studied by measuring liquid junction potentials between tissues and 0.15 M KCl, and LiCl, or 0.075 M CaCl2 and MgCl2, as well as with one tenth isotonic NaCl. For baseline values, 0.15 M NaCl was used. In this way a series of substitution and dilution potentials was established. As a connective tissue which varies in physicochemical state, use was made of the symphysis pubis of normal and castrate guinea pigs, and of guinea pigs in various stages of pregnancy. The former have tight, unrelaxed symphyses, while the latter show varying grades of relaxation. Experimentally, relaxation was also induced by the use of estrogens and relaxin in castrate guinea pigs. Unrelaxed symphyses showed high positive dilution potentials (+20 to + 30 mv), while highly relaxed symphyses showed zero or negative dilution potentials, approaching in some cases values calculated for aqueous salt junctions (—12.3 mv). These results are interpreted on the basis of a high concentration of negatively charged immobile colloid in tight connective tissue; following induction of the physiological change of relaxation, the potentials indicate low densities of immobile charged colloid. An estimate of 1250 for the average base binding equivalent weight of tissue colloids indicates a magnitude characteristic of mucopolysaccharides or mucoproteins. Evidence from other work indicates that relaxation in the symphosis is accompanied by a disaggregation (depolymerization) of components of ground substance, and by an increase in water soluble fractions. In relaxed symphyses, where colloid density is low, mobility data indicate that the cations approach their behaviour in water. In tight symphyses, selective effects appear, related to the high concentrations of immobile charge. Potassium in particular showed a marked decrease of mobility to approximately one-half its value in water. Effects with other ions were much smaller. Thus connective tissue in a state of high aggregation appears to act selectively as an ion-exchange resin for potassium; with breakdown of aggregates and uptake of water, selectivity is lost. From the NaCl dilution potentials, concentrations of immobile charges were estimated. Values ranged from 0.20 equivalents per liter in the tight symphysis to 0.05 equivalents per liter or less in the relaxed state. Values of this order of magnitude have been calculated from independent data.

THE REACTIVITY OF PROTEINS OF SOME CONNECTIVE TISSUES AND EPITHELIAL STRUCTURES WITH 2,4-DINITROFLUOROBENZENE:

2,4-Dinitrofluorobenzene was used without any coupling procedure as a histochemical reagent to localize some reactive groups of proteins in connective tissues and epithelial structures. Tissues were fixed by freezing-drying to avoid uncontrolled alterations of proteins and the reactions were modified in some sections by postfixation or pre-treatment with a variety of reagents, including alcohol, formalin, formaldehyde vapors, acetic anhydride, nitrous acid and N-ethylmaleimide. The staining and resolution were greatly enhanced by viewing the sections with monochromatic light at 410 mµ. When used in this way, 2,4-dinitrofluorobenzene is specific for: (1) α-amino groups of terminal amino acids; (2)ε-amino groups of lysine and hydroxylysine; (3) sulfhydryl groups of cysteine. However, the reaction seems to be dominated by the ε-amino groups of lysine in most of the tissues. In the native state not all functional groups of tissue colloids react presumably because the groups are masked by the interractions involved in forming tertiary structures or covalent bonds. This is specially notable in calcified tissues. Pyknotic nuclei, pre-dentin, carious dentin and newly formed bone are more reactive suggesting a less highly organized state of their structural proteins or some other modification in the lysine and hydroxylysine containing proteins. 2,4-Dinitrofluorobenzene is valuable as a specific cytochemical reagent and, in some instances, as an indicator of the state of organization of cellular and extracellular colloids.

Excretion of urinary mucoprotein following parathyroid extract in rats.

Summary Control rats excrete, in a 72-hour period. 0.35-1.92 mg (mean: 1.08 mg) urinary mucoprotein determined as mannosegalactose standard. Following injection of 1000 units of parathyroid hormone, mucoprotein excretion increased significantly to 0.52-4.52 mg (mean: 2.84 mg). This increase is correlated in time with a dissolution of bone matrix, with an increase in plasma mucoprotein, and with the appearance of mucoprotein granules in kidney tubule cells and mucoprotein-containing casts in tubules.

FIXATION OF CELLS AND TISSUES BY CHLORO-S-TRIAZINES

Cyanuric chloride and related water-soluble dichloro-s-triazines (Procion dyes and Lissatan PR) or monochloro-s-triazines (Procion H and Cibacron dyes, were utilized as fixatives for fresh and freeze-dried animal tissues. With these reagents, endogenous and exogenous substances were irreversibly insolubilized and rendered resistant to chemical and enzymatic degradation. Certain components have been preserved heretofore lost by other methods. Fixation may depend mainly on the formation of covalent and chelate bonds with the tissue colloids. These chloro-s-triazines can be applied in various ways to meet specific fixation needs. In order to avoid the effects of solvents, cells and thin segments of tissues can be cyanurated in the vapors of cyanuric chloride. The water-soluble Procion dyes and Lissatan PR are effective fixatives for some constituents in fresh tissues; enamel matrix is especially well preserved. The most effective of these cyanurating agents for general use appears to be cyanuric chloride in nonpolar solvents when applied to freeze-dried tissues. Following some cyanurations, unreacted striazine halogen appears available for further condensation with active hydrogen in reactants containing certain functional groups (i.e.,—NH2, —SH, —OH, etc.) By condensations with suitable reactants it appears possible to establish crosslinked polymers in tissues. There is evidence that reaction of fresh hydrated tissues with dichloro-s-triazines imparted some water-repellency to the tissues. Through unreacted halogen in the previously condensed s-triazinyl rings, it appears possible at ambient temperature to effect additional condensations with water-soluble agents utilized in hydrophobing treatments.

A microprobe analysis of inorganic elements in Halobacterium salinarum

Halobacterium salinarum were grown on peptone agar containing 4.28 M NaCl, 0.036 M K and other salts. Stationary phase organisms were lifted onto carbon planchets, freeze‐dried, carbon coated and examined in a scanning electron microscope equipped with an X‐ray spectrometer. Intracellular element concentrations (mol/kg H2O) were determined using a bulk analysis program with appropriate standards. The cell K concentration was 110 times that of the medium. For Na this value was 0.3 and for Cl, 1.1. When Rb was present in the medium, its intracellular concentration was 77 times higher than the external value. The cation minus anion value suggests a high fixed negative charge, 0.72 equivalents. Intracellular apparent dielectric constants were calculated using cellular EMFs derived from the literature, and sodium concentration. The determined values ranged from 22–28 (vs 80 for normal water) suggesting phases of structured cell water. Ionic distributions in these extremophiles are treated according to the classical principles elucidated by Willard Gibbs and represents a heterogeneous system in thermodynamic equilibrium with the hypersaline environment. Factors to be considered are: (1) composition of Halobacterium and its immobile negative charge; (2) the physicochemical properties of the individual ions (charge, ionic radius, hydration energy, standard chemical potential); (3) the dielectric constant of the dispersion medium (water); and (4) the binding of ions, particularly potassium.

THE HISTOCHEMICAL VISUALIZATION OF PROTEIN-BOUND SULFHYDRYL GROUPS WITH AN AZOMERCURIAL REAGENT

An azomercurial reagent, 4-(p-dimethylamino benzene azo) phenyl mercuric acetate has been adapted for the histochemical demonstration of protein bound sulfhydryl groups. Sections were made from freeze-dried tissues including various keratinized structures, muscle and some parenchyma. They were stained directly in an alcoholic glycine buffered solution of the azomercurial. High resolution and good contrast were insured by viewing the sections with monochromatic light at 430 mµ. The method, which is based on the work of Horowitz and Klotz, appears to be highly specific. Tissues which are very low in cysteine or virtually cysteine-free, do not react, and pretreatment of thiol containing sections with N-ethylmaleimide, p-chloromercuribenzoate or H2O2 prevents staining. Visualization of disulfide linkages following reduction was only partially successful due mainly to loss of soluble materials. Preliminary microphotometric studies suggest that the method could be used for semi-quantitative determinations. In many keratinizing tissues, cells and intercellular cement underlying the keratinized structures contain a high concentration of protein-bound thiol dispersed homogeneously as granules, or in fibrous form.

Histochemical Studies Of Cartilage Implants

The behavior of cartilage transplants and the cellular reaction in the surrounding connective tissue have been studied in rabbits. Costal cartilage was used as a control. Histochemical methods, together with freezing-drying fixation, were used to visualize some tissue components of interest. These included the carbohydrate-protein complexes of the ground substance, inorganic phosphate-carbonate, and intracellular glycogen. Dissolution of the mucoprotein ground substance was observed on the periphery of autografts sixty days after implantation and of costal cartilage of comparable age. In boiled autocartilage implants, a more general and extensive dissolution occurred and this began much earlier. In these instances the changes in the matrix were accompanied by deposition of phosphate-carbonate, presumably as the calcium salt. In vitro experiments in which a bacterial collagenase was used resulted in striking disintegration of boiled cartilage and somewhat lesser changes in non-boiled autocartilage grafts and costal cartilage. In a like manner enzymatic activity by connective-tissue cells could lead to changes in the matrix of implants. The fate of cartilage implants appears to be determined by their inherent physicochemical characteristics and by the connective-tissue reaction which is provoked in the host. Cells of the boiled cartilage implants lost their glycogen early and resynthesis was impossible. In the autografts, evidence for sustained metabolic activity comparable with that in costal cartilage was deduced from the continued presence of intracellular glycogen.

Relations between the metabolism and structure of bone.

Despite its rigid physical character, bone, like most other connective tissues, is a highly labile substance capable of rapid structural transformations. Although these morphologic changes have been investigated extensively, the means by which they are accomplished still is understood poorly. There are numerous places in nature, other than in bone, where problems of solubilization also In many of these instances, it has been suggested that the dissolution of the insoluble matter occurs through chelation or complex formation with certain of the intermediate products of carbohydrate metabolism?, 4-6 We have considered the possibility that a similar situation exists in bone resorption. Thus, it has been postulated that accumulated diand tricarboxylic acids could compete for cations with the mucoproteins and other negatively charged colloids of the bone matrix, thereby bringing the calcium salts into To develop this hypothesis, certain aspects of the carbohydrate metabolism of bone were studied during the metaphyseal dissolution caused by parathyroid extract and in the sequence of apposition and resorption in pigeon bone during egg-laying.