Reprecipitation Phenomena Arising During the Preparation of Demineralised Sections. III. Scanning Electron Microscopic Examination of Secondary Calcium Phosphate Deposits

Sections of teeth partly demineralised in 10% formic acid were examined by X-ray diffraction, microradiography and scanning electron microscopy. In the undemineralised circumpulpal dentin, the tubules were empty, lying in a matrix containing hydroxyapatite. In the 'plume' areas of remineralisation, the tubules were filled with mineral deposits. X-ray diffraction revealed the presence of brushite and monetite in these areas. In the outer layers of dentin the tubules were empty, lying in a matrix containing some residual hydroxyapatite. These findings confirmed that the remineralisation process occurred within the dentinal tubules.     

Comparison of decalcifying agents and techniques for human dental tissues

Teeth are among the hardest animal tissues, because they are composed of large amounts of inorganic compounds. Consequently, teeth are difficult to prepare for microscopic examination. Acids and chelating agents traditionally have been used to remove calcium ions. We compared decalcifying agents including strong acids, weak acids, chelating agents, techniques using electric current, agitation and heat. Freshly extracted teeth were fixed and decalcified using formic acid-formalin, formal-nitric acid, formalin-EDTA, Von Ebner’s fluid and Perenyi’s fluid. Three additional techniques including formic acid with agitation, formic acid with heat and formic acid with electric current also were evaluated. Decalcified teeth were evaluated histologically for tissue preservation and staining characteristics. Formic acid with gentle agitation produced the best decalcification overall based on time required for decalcification, ease of sectioning, hard and soft tissue staining and tissue preservation. Our findings support the use of agitation with formic acid decalcification, because it reduces significantly both the time required and the deleterious effects of prolonged immersion.     

Rapid demineralization in acidic buffers.

The demineralization of routine histological specimens in buffers of weakly ionized organic acids, unbuffered formic acid, and EDTA was investigated. The rate of demineralization was measured by a chemical method and from radiographs. Lactate-containing buffers and buffers of formic acid with its potassium salt were more rapid in effect than any other agent. Acidic buffers and unbuffered formic acid produced rapid diffuse demineralization with secondary precipitation of calcium salts. Preservation of dental enamel in such buffers resulted from the significantly slower rate of enamel demineralization than that for bone and dentine. In rapid demineralizing agents the secondary salts were quickly redissolved while in slow buffers these salts persisted. Multivalent ions such as citrate and maleate slowed the rate of demineralization, and a citrate-containing buffer was the slowest of all the agents tested. Demineralization in EDTA exhibited a different pattern with the establishment of a well-defined front of demineralization without apparent reprecipitation. EDTA attacked enamel, bone and dentine at the same rate. An attempt was made to relate the observed rates of demineralization to current theories of the demineralization process.     

A Method for Decalcification with Citric Acid

Psammoma bodies and small calcifications are frequently seen in a wide variety of tissues. These deposits of calcium salts render tissues difficult to section. Conventional decalcification alters the tissues; consequently it is not advisable to decalcify tissues containing only small calcium deposits. A simple and rapid method to remove small amounts of calcium salts using citric acid alone is described. This method does not alter the antigenic properties of the substances studied.     

Accurate Localization in Ultrathin Sections by Direct Observation of the Block Face for Trimming

Psammoma bodies and small calcifications are frequently seen in a wide variety of tissues. These deposits of calcium salts render tissues difficult to section. Conventional decalcification alters the tissues; consequently it is not advisable to decalcify tissues containing only small calcium deposits. A simple and rapid method to remove small amounts of calcium salts using citric acid alone is described. This method does not alter the antigenic properties of the substances studied.     

Covalent modification of Alzheimer's amyloid beta-peptide in formic acid solutions.

beta-peptide is a normal component of amyloid deposits in Alzheimer's disease. In aqueous solution, beta-peptide is extremely insoluble and rapidly aggregates forming oligomeric beta-sheet structures that eventually precipitate from solution. Presumably, this process is related to the production of amyloid deposits in Alzheimer's disease. Formic acid is commonly used to dissolve the beta-peptides and prevent aggregation in biological and biophysical studies. However, a side-reaction which covalently modifies beta-peptide is encountered with formic acid. In this report, fast atom bombardment mass spectrometry and tandem mass spectrometry demonstrate that both Ser8 and Ser26 become O-formylated in 70% aqueous formic acid solutions. The implications of this O-formylation upon the aggregational properties of beta-peptide are discussed.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

Rapid demineralization in acidic buffers

Summary The demineralization of routine histological specimens in buffers of weakly ionized organic acids, unbuffered formic acid, and EDTA was investigated. The rate of demineralization was measured by a chemical method and from radiographs. Lactate-containing buffers and buffers of formic acid with its potassium salt were more rapid in effect than any other agent. Acidic buffers and unbuffered formic acid produced rapid diffuse demineralization with secondary precipitation of calcium salts. Preservation of dental enamel in such buffers resulted from the significantly slower rate of enamel demineralization than that for bone and dentine. In rapid demineralizing agents the secondary salts were quickly redissolved while in slow buffers these salts persisted. Multivalent ions such as citrate and maleate slowed the rate of demineralization, and a citrate-containing buffer was the slowest of all the agents tested. Demineralization in EDTA exhibited a different pattern with the establishment of a well-defined front of demineralization without apparent reprecipitation. EDTA attacked enamel, bone and dentine at the same rate. An attempt was made to relate the observed rates of demineralization to current theories of the demineralization process.Supported by the British Medical Research Council     

An electron microscopic histochemical and X-ray microprobe study of spherites in a mussel

Transmission electron microscopic, histochemical and X-ray analytical microprobe techniques were used to study the inorganic-organic relationship in the spherites (calcospherules) from the mantle, i.e. subadjacent to the outer mantle epithelium, of the fresh-water mussel Amblema. These structures were shown to contain calcium which could be chelated by the flotation of sections on solutions of either formic acid or ethylene glycol bis-(beta-amino ethyl ether)-N, N1-tetra-acetic acid (EGTA), Analysis of both non-chelated and sections revealed a significant sulfur peak. Chelated spherites were also intensely stained with acid phosphotungstic acid (PTA), Such data is indicative of the presence of an organic glycoprotein (proteoglycan) matrix which could serve to bind mineral ions, thus forming organic-inorganic aggregates for calcium transport and homeostasis. In this regard, the spherites are analogous to both calcium phosphate containing mitochondrial granules and the initial calcification sites in vertebrate mineralizing tissues.     

The effects of spaceflight on the mineralization of rat incisor dentin

The lower incisors of Male Wistar rats flown for 18.5 days on the Soviet Cosmos-1129 Biosatellite were sectioned and chemically analyzed with an electron microprobe in order to determine whether there were specific effects of spaceflight on dentin formation/mineralization. Control tissues were obtained from rats housed under identical conditions in a land-based mock-up of the Biosatellite. The profiles of calcium (Ca), phosphorus (P), and sulfur (S) concentrations in dentin were measured in continuous traverses (1.0 micron intervals) from the pulp to the dentinoenamel junction. The incisor dentin formed during spaceflight had higher than normal (at 1G) concentrations of Ca (+ 10-15%) and P (+ 20-30%), particularly in the temporally youngest tissues within 80 micron of the pulp which had been least affected by secondary mineralization. The S-concentration profiles tended to decrease with tissue age. Fourier analysis (to determine the growth rhythms) revealed abnormal distributional patterns of S in the recently formed dentin from the Flight rats. The sulfur fluctuations in Flight animals alone periodically peaked above the irregular background fluctuations. These observations indicate that spaceflight has measureable effects on dentinogenesis, and they may also bear on the problem of the regulatory role of proteoglycans in mineralization and in the maturation of mineral and matrix moieties in skeletal tissue.     

Structure and mechanical properties of the soft zone separating bulk dentin and enamel in crowns of human teeth: insight into tooth function

The 200-300 microm soft zone of dentin, found beneath enamel in crowns of human teeth, is thought to fulfill important roles in tooth function, but little is known about its structure-mechanical relations. Scanning electron microscopy images of fracture surfaces showed that near the dentino-enamel junction (DEJ), a porous reticulate matrix of intertubular-dentin contains tubules with no peritubular lining. Peritubular-dentin however is found at some distance from the DEJ, and it gradually thickens with increasing depth into the bulk dentin. Concurrently, tighter packing of the collagen fibers is observed with a gradual increase in mineral deposits on and between the fibers. This structurally graded zone is known to be softer when tested for micro-hardness. It undergoes greater strain compared to bulk dentin, when measured using Moiré interferometry. We investigated the deformation and stiffness of this zone by means of non-contact laser-speckle interferometry (ESPI), and nanometer-scale deformations were tracked during compression-testing performed in water. We report a significantly reduced stiffness of this zone compared to bulk dentin, with mid-buccal regions of teeth averaging 3.5 GPa compared with 9.7 GPa in mid-lingual regions. Our results support and expand upon the hypothesis that the durability of the whole tooth relies upon a bucco-lingual asymmetric matching of stiffness by means of an interphase: a cushioning soft layer between enamel and bulk dentin.     

Comparison of the calcium content of different tissues present in the human mandible

Qualitative and semi-quantitative comparisons of calcium content in the developing human mandible were performed by means of microradiographic and histophotometric analysis. Differences in calcium content between enamel, calcified cartilage, chondroid tissue and dentin are significant at the 1% level. Chondroid tissue and woven bone are almost similarly mineralized tissues.     

Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling

To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the tissues first. However, the effects of decalcifying reagents on the integrity of DNA are largely unknown. In the present study, we evaluated the usefulness of various decalcifying reagents including 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), 5% formic acid, 5% HCl, 10% nitric acid, Plank–Rychlo's solution, Morse's solution and K-CX solution in TUNEL staining. Mouse maxilla was selected as the experimental system. Apoptotic cells naturally occurring in the epithelium were analysed. Tissues were assessed by soft X-ray imaging to confirm complete decalcification. The time required for decalcification of the tissue was 7 days with 10% EDTA and 2 days with other decalcifiers. Decalcified tissues were stained with Methyl/Green–Pyronine Y or 4, 6-diamidino-2-phenylindole for assessment of DNA integrity. Nuclei of epithelial cells were strongly positive for both dyes after decalcification with 10% EDTA, 5% TCA, Morse's solution and 5% formic acid. The other reagents failed to retain DNA. Our results demonstrated good TUNEL staining of the maxilla treated with 10% EDTA or 5% TCA . Based on the required time for processing and the signal-noise ratio, we recommend 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)     

Production of a monoclonal antibody to an attachment protein derived from human cementum.

Cementum is the mineralized structure that covers the surface of the roots of teeth; it serves as the attachment site for collagen fibers of adjacent soft connective tissues. Very little is known about how cementum formation is regulated or how it affects other periodontal structures. We have raised a monoclonal antibody that may aid in studies to determine the biology and function of cementum. Mice were immunized with a 55-kDa attachment protein partially purified from human cementum and a monoclonal antibody, H166, was produced. Incubation of tissue sections with this antibody and fluorescein isothiocyanate-conjugated secondary antibody revealed that it immunostains cementum but not dentin, gingiva, or periodontal ligament. Alveolar bone did not bind the antibody, although a few paravascular cells were positive. Long bones, kidney, liver, skin, and several other tissues were negative. Protein fractions separated from cementum extracts by binding to immobilized H166 column contained 55-, 49-, 39-, 29- to 31-, and 23- to 26-kDa components that cross-reacted with the antibody in Western blots; these components were previously shown to be derived from a common precursor. We conclude that the antibody recognizes a group of proteins related to 55-kDa attachment protein in cementum. Our data show that the antibody could serve as a marker for cementum.     

Mitochondrial matrix granules in soft tissues : I. Elemental composition by X-ray microanalysis

The elemental composition of individual matrix granules in mitochondria of rat brown fat, mouse gall bladder and guinea pig kidney has been examined by X-ray microanalysis. The matrix granules showed a similar elemental composition that was strongly dependent upon the method of sample preparation. Low-temperature oxygen plasma microincineration or wavelength-dispersive X-ray spectrometers were used to demonstrate the presence of phosphorus in matrix granules of osmium tetroxide-fixed specimens. Matrix granule osmiophilia was retained in glutaraldehyde-fixed brown fat only if exposure to polar organic solvents was avoided during subsequent steps, e.g. by cryosectioning. As normally prepared, matrix granules lack detectable calcium but had bound this at detectable levels after fixation in osmium tetroxide, but not glutaraldehyde, supplemented with 5 mM Ca²⁺. The results demonstrate that mitochondrial matrix granules of normal soft tissues are not calcium phosphate deposits and contain phospholipids, apparently as a major constituent. Thus they provide evidence against the hypothesis that matrix granules are primarily involved in mitochondrial calcium sequestration and, indirectly, for the hypothesis that the granules may be related to inner membrane assembly.     

Histochemical studies of uric acid in some insects. I. Storage in the fat body of Periplaneta americana and the action of the symbiotic bacteria

An improved histochemical method for uric acid consists in precipitation as silver magnesium urate combined with fixation of the tissues in formol/glutaraldehyde followed by argentaffin reaction with silver nitrate buffered with "tris" to pH 9.5. This reveals urates both in solid deposits and in solution in the tissues. Polyphenols concerned in sclerotin formation also react. In Periplaneta, uric acid synthesized in the trophocytes is carried by intracellular and intercellular channels to form the intercellular deposits of solid spheres. The symbiotic bacteria in the mycetocytes in contact with the deposits appear to metabolize the uric acid and they disperse and eliminate the deposits.     

Autometallographic localization of protein-bound copper and zinc in the common winkle,Littorina littorea: A light microscopical study

Summary Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrare for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and dithizone procedures exhibited poor sensitivity: there was no positive reaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in sperm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.     

Decalcification of Snail Shells to Facilitate Sectioning of the Whole Organism

This note describes the application of several decalcification procedures used on snail shells to permit histologic sectioning of the intact soft tissues. The configuration of the body parts, the integrity of the cells and their capacity to react to standard staining procedures is not impaired (figures 1, 2). The following bone decalcifers were used in a comparison test: Pereny's method (Luna 1968), nitric acid method I & II (Luna 1968), formic acid-sodium citrate method (Luna 1968), and two commercial products: RDO—DuPage Kinetic Laboratories, Downers Grove, IL 605 15; and Decal—Omega Chemical, Cold Spring On Hudson, NY 10516     

Spatially and temporally controlled biomineralization is facilitated by interaction between self-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution

Bone and dentin biomineralization are well-regulated processes mediated by extracellular matrix proteins. It is widely believed that specific matrix proteins in these tissues modulate nucleation of apatite nanoparticles and their growth into micrometer-sized crystals via molecular recognition at the protein-mineral interface. However, this assumption has been supported only circumstantially, and the exact mechanism remains unknown. Dentin matrix protein 1 (DMP1) is an acidic matrix protein, present in the mineralized matrix of bone and dentin. In this study, we have demonstrated using synchrotron small-angle X-ray scattering that DMP1 in solution can undergo oligomerization and temporarily stabilize the newly formed calcium phosphate nanoparticle precursors by sequestering them and preventing their further aggregation and precipitation. The solution structure represents the first low-resolution structural information for DMP1. Atomic force microscopy and transmission electron microscopy studies further confirmed that the nascent calcium phosphate nuclei formed in solution were assembled into ordered protein-mineral complexes with the aid of oligomerized DMP1, recombinant and native. This study reveals a novel mechanism by which DMP1 might facilitate initiation of mineral nucleation at specific sites during bone and dentin mineralization and prevent spontaneous calcium phosphate precipitation in areas in which mineralization is not desirable.