Silver methenamine staining for scanning electron microscopy of bone sections containing biomaterials.

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.

[Coloration of cytologic thick sections containing biomaterials with silver methenamine. Usefulness for scanning electron microscopy]. / Coloration de coupes histologiques épaisses contenant des biomatériaux par la silver methenamine. Utilité pour l'examen en microscopie électronique à balayage.

UNLABELLED: Prostheses implanted in hard tissues cannot be processed for electron microscopic examination or microanalysis in the same way as those in other tissues. For these reasons, we have developed a method allowing light and electron microscopic studies as well as microanalysis of the interface between bone and biomaterials after the sections glycoproteins have been stained with silver methenamine. Silver can be evidenced by SEM in back scattered mode. MATERIALS AND METHODS: Tranverse sections of a human femur containing an HA-coated prostheses were obtained with a diamond saw and ground to a thickness of 50-100 microns. The sections were stained in a microwave oven using a 1% silver methenamine solution. They have been examined by back-scatter SEM operated at 25 KV. EDS has been performed on cellular inclusions and extracellular bone matrix. RESULTS: Type I and III collagen fibers, and reticulin fibers were stained. The mineralized matrix was heavily colored. At the cell level, the nuclear and cytoplasm membranes, the chromatin and ribosomes were shown. The characteristic peaks of the Ag spectrum are distinct from those of the elements used in orthopaedic biomaterials and did not impair their identification.

Biomaterials and biomedical devices.

This review discusses the factors important in the incorporation or integration of biomaterials and devices by tissue. Methods for surface modification and surface-sensitive techniques for analysis are cited. In vitro methods to evaluate the biocompatibility or efficacy of certain biomaterials and devices are presented. Present and future directions in neural prostheses, cardiovascular materials, blood or bone substitutes, controlled drug delivery, orthopedic prostheses, dental materials, artificial organs, plasma- and cytapheresis, and dialysis are discussed.

Evaluation in cats of a new material for cranioplasty: a composite of plaster of Paris and hydroxylapatite.

The materials ordinarily used to reconstruct bone defects in the calvaria and facial bones either are difficult to shape, are partially resorbed by the body, or are likely to become infected if used near a contaminated area such as the frontal sinus. Calcium sulfate hemihydrate (plaster of Paris) has been known for years to have excellent reparative qualities in bone defects, but ordinarily it is quickly resorbed. Consequently, a new material, a composite of a dense form of plaster of Paris and hydroxylapatite, was devised to provide nonabsorbable hydroxylapatite particles for bone to form around and within during the phase of plaster absorption. Two types of this material were evaluated in cranial defects in cats. Each of the plaster of Paris/hydroxylapatite mixtures was placed into a surgically unroofed frontal sinus and into a contralateral parietal trephine hole in a group of 32 cats. Two cats in each group succumbed to anesthesia, leaving two sets of 30 cats. During the entire follow-up period there was only one other death, with no evidence of wound infection, wound dehiscence, implant rejection, or cerebral dysfunction among the survivors. The cats in each group were sacrificed at 1, 2, 3, 5, 7, 8, 9, 10, or 12 months after operation. Following sacrifice, both the frontal and parietal defects were exposed and examined visually, histologically, and with histomorphometric analysis for new bone formation. New bone formation was present as early as 1 month after operation and continued to increase during the 12 months of the study. Based upon these osteogenic qualities, the ease of shaping the composite, and the lack of infection in the frontal sinus region, it is concluded that this substance could be a valuable new material for human cranioplasty.

Peroxidases induced in rat uterus by estrogen administration. II. Cytochemical and biochemical heterogeneity.

In estrogen and diethylstilbestrol-treated rats, uterine peroxidases originate from two sources, the infiltrating eosinophils (exogenous) and the uterine tissue itself (endogenous). The study reported here distinguished the exogenous peroxidases by biochemical means and by cytochemistry. Eosinophil peroxidases are confined to the stromal and myometrial regions and appear simultaneously with endogenous peroxidases. At 48 h after estrogen-administration, the clear uterine luminal washings contain five peroxidase isoforms; this increases to 7-15 isozymes by 72 h. Uterine fluid peroxidase isozymes are acidic proteins with pI values ranging from pH 4.0 - 7.2, while the principal eosinophil peroxidase is a basic protein with a pI value ranging from pH 8.0-8.9. Eosinophil peroxidase is electrophoretically demonstrable only in the presence of the cationic detergent cetyltrimethyl ammonium bromide (CTAB) and has a spectrophotometric optimum of pH 4.4. In contrast, uterine fluid peroxidases have a pH optimum of 7.2. and no requirement for CTAB. Uterine tissue peroxidase extracted in the presence of Ca2+, showed a minor electrophoretic peroxidase band in the acidic pH range; however, a CTAB-activated peroxidase similar to the principal eosinophil peroxidase appeared as a basic protein. The data strongly suggest that uterine fluid peroxidases are estrogen-induced peroxidases (EIP) distinct from the eosinophil peroxidases that are largely restricted to the stromal compartment. This conclusion is supported by cytochemical studies that show two eosinophilperoxidases. The one shown by DAB was resistant to cyanide whereas the one shown by PPD/PC was inhibited by cyanide. A uterine tissue peroxidase, which was demonstrated only in stromal cells by the DAB medium, was more sensitive to cyanide than the eosinophil peroxidase shown by DAB.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidases induced in rat uterus by estrogen administration. I. Synthesis, secretion and immunofluorescent demonstration of fluid enzymes.

Uterine secretions are of fundamental importance in reproductive processes. Uterine activity is related to the hormonal status of the animal, and peroxidase is a major estrogen-regulated glycoprotein in the uterine fluid. Estrogen-induced peroxidase (EIP) can be separated into several isoelectric variants. SDS-PAGE electrophoresis suggested that EIP has a molecular weight of approximately 70kd. The observed pattern of EIP isoforms appears to result from differential glycosylation of a single protein species because neuraminidase treatment resulted in a gradual shift of EIP to the more basic isoelectric variants prior to abolishing enzymatic activity. Antibody raised against one of the major isoelectric variants of EIP demonstrated uterine epithelial cells by immunofluorescence indicating an epithelial cell localization for the enzyme. Radioactive bands comigrating with major EIP isozymes were detected in uterine fluid by in vivo labeling after estrogen treatment. The results of these studies support the hypothesis that EIP is a major estrogen-regulated secretory protein of the uterine epithelium.

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy.

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.

Histochemical demonstration of monoaminergic and cholinesterase-positive nerve fibres in regenerating rat submandibular gland autografts.

A cholinesterase localization method and a monoamine histofluorescence technique were used to locate nerve fibres in regenerating rat submandibular gland autografts. Experimental rats had a portion of one submandibular gland excised and cut into small fragments which were autografted immediately into the middle one-third of the tongue. Control rats had a portion of one submandibular gland removed and discarded, and their tongues were sham-operated. Seven to ten weeks later, the rats were killed and the tongues were removed, frozen and sectioned in a cryostat. A light microscopical study of the tongue sections subjected to the cholinesterase technique showed that the submandibular gland autografts contained many nerve fibres that exhibited cholinesterase activity. These cholinesterase-positive nerve fibres were distributed throughout the autografts. The fibres were associated with the numerous duct-like structures and the less numerous acini. In addition, ultraviolet illumination of tongue sections after treatment with a glyoxylic acid mixture revealed histofluorescent monoaminergic nerves within the autografts. These fibres were less prominent than the cholinesterase-positive fibres and appeared to run primarily along blood vessels within the autografts. The results suggest that autonomic nerves are present within regenerating submandibular gland autografts.

Evaluation of peripheral nerve regeneration following crushing or transection injuries.

Infraorbital nerves were subjected to experimental transection or crushing injury. Repair was evaluated using horseradish peroxidase as a tracer. Labeled neurons in the trigeminal ganglion were compared in both types of injury. Results showed that regeneration of the infraorbital nerve after crushing injury was better than when there was nerve transection with passive reapproximation.

Autoradiographic and cytochemical studies of phagocytic cells in selected fibre tracts of the mouse periodontium.

Proliferative and protein synthetic activities of phagocytic cells of specific fibre tracts of the periodontium of C57Bl mice were employing autoradiographic techniques; these were combined with a histochemical technique for horseradish peroxidase (HRP) as a marker for phagocytic activity. Animals were injected either with [3H]thymidine as a marker for proliferative activity, or with [3H]proline as a marker for protein synthetic activity prior to HRP injection. Blocks from the maxillae of experimental and control animals were fixed, decalcified, and sectioned at 50 micrometers. These were incubated with HRP localization media, dehydrated and flat embedded in Epon 812 wafers. The entire length of the periodontium, including adjacent tooth and bone, were selectively cut from the wafers, mounted on epoxy blocks and serially sectioned at 2 micrometers. Slides containing these sections were then dipped in NTB-3 nuclear track emulsion, and after appropriate exposure times, were developed and post-stained. Sections were examined microscopically, employing an ocular grid, and phagocytic cells within each area examined were delineated as either 'fibroblast-like' (FL cells) or 'endothelial/macrophage-like' (EML cells) according to criteria such as morphology, location, orientation and proximity to a vascular channel. They were then subclassified as labelled or unlabelled with respect to the autoradiographic markers. The thymidine labelling index obtained for non-phagocytic FL cells was 3.09%; this was more than twice that for phagocytic FL cells (1.35%). Similarly phagocytic FL cells in all regions studied incorporated less than half as much [3H]proline as did their non-phagocytic counterparts. This was determined by silver grain counts over HRP-stained and unstained cells using a matched pair system. In addition, the variation of the relative number of phagocytic FL cells in specific fibre tracts suggested a relationship to functional demand. The distribution of these cells was closely related to experimentally determined rates of protein turnover. Phagocytic FL cells have a markedly reduced proliferative rate and synthesize proline-containing proteins at a reduced rate. This may reflect protein production primarily for the purpose of cell maintenance. These findings are consistent with the presence of subpopulations of fibroblasts (or fibrocytes) developmentally or functionally modified for phagocytosis; alternatively, this could signify modulation of fibroblasts from primarily biosynthetic activities to degradative functions in response to varying microenvironmental conditions.

Neutrophil pseudoplatelets: their discrimination by myeloperoxidase demonstration.

Neutrophils, especially in acute infection or the myeloid leukemias, may shed platelet-sized particles that can readily be distinguished from true platelets because they contain neutrophil myeloperoxidase. This enzyme, unlike platelet peroxidase, is not inhibited by glutaraldehyde. The myeloperoxidase and acid hydrolase levels and continuous plasma membranes of these cell-like particles suggest that they are functional cellular entities. They further differ from platelets in that they contain nuclear remnants, occur in bacteria-laden pus and inflammatory exudates, are ingested by macrophages, and do not adhere to each other or aggregate. They could be involved in the immune response to pathogens or contribute to trauma and healing by facilitating deployment of neutrophil acid hydrolase, neutral protease, and myeloperoxidase.

Studies of eosinophil medusa cells by electron imaging modes.

Medusa cells, amoeboid variants of the eosinophil with pseudopod-like processes, were examined by light microscopy (LM), transmission electron microscopy (TEM), the secondary electron imaging (SEI) and the backscattered electron imaging (BEI) modes of the scanning electron microscope. TEM was performed on rare medusa cells found in leukocyte concentrate preparations where the ion contents of the collection and fixation media were balanced so that divalent cations such as calcium and magnesium were not sequestered. LM, SEI and BEI studies were performed principally on cytochemically-stained films of leukocyte concentrate preparations on microscope slides or coverslips. These films of patients with eosinophilia contained many medusa cells and much higher ratios of medusa cells to eosinophils than critical point-dried specimens, if they were prepared as for routine hematologic examination, and precautions were taken to insure that calcium and magnesium ions in collection and fixation media were not sequestered. After brief glutaraldehyde fixation, the smears were stained with either osmium tetramethylethylenediamine (Os-TMEDA) for acid mucopolysaccharides, or 3,3'-diaminobenzidine (DAB)/hydrogen peroxide medium for peroxidases. The Os-TMEDA was sufficiently conductive for SEM. Chelation of the oxidatively-polymerized DAB dye with copper nitrate rendered it conductive. These conductive and electron-opaque stains permitted the correlation of SEI with BEI on individual cells, their unambiguous identification as eosinophils or medusa cells and their differentiation from other leukocytes by virtue of content and/or size of their granules and their degree of nuclear segmentation.

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice.

Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining using p-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice. The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate. The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.

The adrenal medulla of the diabetic mouse (C57BL/KsJ, db/db): biochemical and morphological changes.

1. Anatomic and biochemical indices of adrenal medullary function were studied in mice (Mus musculus) with hereditary mellitus (C57BL/KsJ, db/db). 2. In the diabetic mice increases in medullary catecholamine content and in the activities of tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase were accompanied by increases in adrenal weight and size. 3. Morphometric study of adrenals from diabetic mice showed that the medulla was increased in size but had a lower cell density indicating that medullary hypertrophy as well as hyperplasia were probably responsible for this size increase. 4. These observations are consistent with the occurrence of chronic adrenal medullary stimulation in the diabetic syndrome of these mice.

Medusa cells: cytostructure and cytochemistry of amoeboid eosinophils with pseudopod-like processes.

Eosinophils having one or more pseudopod-like processes of various lengths are observed in bone marrow, peripheral blood, sputum, nasal smears, and in other exfoliative cytology and tissue specimens after fixation and staining for histochemical study; they are called medusa cells. Although the conformations and lengths of the processes vary, they resemble protozoal pseudopodia. The form which first called our attention to these cells is a conical determinate projection that tapers to a fine tip, much like a protozoal axopod. A basal specialization in the form of a vesicle or thickening may be frequently observed where the process protrudes from, or appears attached to, the cell body. The processes of these eosinophil variants appear morphologically specialized to interact with other cellular elements of the blood and are occasionally seen in contact with, or engulfing, erythrocytes, platelets or other leukocytes. Two hydroperoxidases have been elucidated in eosinophils and medusa cells by virtue of different substrate specificities, subcellular localizations and inhibitor sensitivities. One of these hydroperoxidases is shown by 3,3'-diaminobenzidine, is cyanide resistant, and is never observed in granules or rods in the medusa cell processes; it is frequently polarized to the sites of contact of medusa cells with other cellular elements of the blood. The other hydroperoxidase is revealed by p-phenylenediamine-pyrocatechol, is sensitive to cyanide and is frequently observed in granules and rods in medusa cell processes as well as in a population of larger granules in the cell bodies; the granules in the processes appear to be precursors to the rods, which may be related to Charcot-Leyden crystals. The extrusion of medusa cell processes is facilitated by the divalent cations calcium and magnesium and is inhibited by anions which sequester them such as phosphate, EDTA, citrate and oxalate. Medusa cells have been observed in samples from both rodents and humans and can be very prominent when eosinophilia accompanies allergy, parasitosis and malignancy.

Peripheral neuropathy in mouse hereditary diabetes mellitus. II. Ultrastructural correlates of degenerative and regenerative changes.

The fine structural changes in some peripheral nerves and sensory ganglia from mice (C57BL/KsJ, db/db) with an hereditary diabetic syndrome, similar to human maturity-onset diabetes mellitus, were studied during development of the mild peripheral neuropathy. The abnormalities observed included axonal degeneration, disruption of myelin, accumulation of electro-dense material in axons, satellite cells and Schwann cells, increased frequency of pi granules of Reich in Schwann cells, enlarged mitochondria, and proliferated and thickened Schwann cell basal laminae. Distal hind limb nerves were most affected. Sensory ganglion neurons were normal except for occasional chromatolytic cells, so that nerve cell loss was not present in this peripheral neuropathy. Morphological indications of Schwann cell hyperplasia, hypertrophy, and axonal sprouting supported the contention that a continuous cycle of axonal degeneration and regeneration was occurring. The ultrastructural changes and accumulation of electron-opaque, lipid material suggested that a defect in lipid metabolism, secondary to the diabetic condition, could be an important factor in the peripheral neuropathy in the diabetic mouse.