Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain

Reticular and collagenous fibers stain differently when subjected to ammoniacal silver reduction. A variety of tissues were subjected to such a “reticulin” technique and the association of reaction product with intercellular connective tissue elements was studied with the electron microscope. The reaction with reticular fibers was primarily associated with the interfibrillar matrix, and was globular in form having a wide variety of particle sizes. Conversely, in dermal collagen the unit fibrils were stained rather than the interfibrillar matrix. The precipitate was punctate in form and was associated with the cross striations of unit collagen fibrils. Large microfibrils also reacted positively with the stain, imparting a faint periodicity. Basement membranes were stained uniquely. The underlying plasmalemma and the lamina densa were heavily stained with silver while the lamina lucida was relatively unstained. The unit fibrils of the lamina reticularis stained in the same manner as dermal unit collagen while the ground substance remained unstained. This represents a clear distinction between the argentophilic characteristics of collagenous fibers, reticular fibers, and basement membranes.     

Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain.

Reticular and collagenous fibers stain differently when subjected to ammoniacal silver reduction. A variety of tissues were subjected to such a "reticulin" technique and the association of reaction product with intercellular connective tissue elements was studied with the electron microscope. The reaction with reticular fibers was primarily associated with the interfibrillar matrix, and was globular in form having a wide variety of particle sizes. Conversely, in dermal collagen the unit fibrils were stained rather than the interfibrillar matrix. The precipitate was punctate in form and was associated with the cross striations of unit collagen fibrils. Large microfibrils also reacted positively with the stain, imparting a faint periodicity. Basement membranes were stained uniquely. The underlying plasmalemma and the lamina densa were heavily stained with silver while the lamina lucida was relatively unstained. The unit fibrils of the lamina reticularis stained in the same manner as dermal unit collagen while the ground substance remained unstained. This represents a clear distinction between the argentophilic characteristics of collagenous fibers, reticular fibers, and basement membranes.     

The length of parallel fibers in the cat cerebellar cortex. An experimental light and electron microscopic study

Summary Experimental light and electron microscopic studies were carried out to determine the length of parallel fibers in the cat cerebellar cortex. Using a fine surgical knife, vermal and hemispheral folia were cut perpendicular to their long axis. The animals were sacrificed 1–10 days after the operation. Sections of the transected folia were then stained with a Fink-Heimer procedure. The resulting degeneration appeared as fine dots that extended lateral to the lesion, as predictable from the course of the parallel fibers. Densitometer readings indicate that the density of degeneration declines gradually lateral to the lesion. The specificity of the silver impregnation was checked by processing silver stained sections for electron microscopy. This confirmed the location of the silver precipitate on degenerating parallel fibers. The pattern of parallel fiber degeneration in the molecular layer has a trapezoidal configuration centered on the lesion. The shorter parallel fibers are located at the base of the molecular layer and extend for 5 mm. The parallel fibers become progressively longer as they approach the pial surface where they attain a maximum length of 7 mm. Our studies suggest that in folia longer than 7 mm parallel fibers are 6 mm long on the average.In addition, it was determined on Golgi sections that the average center-to-center distance between en passant boutons of individual parallel fibers is 5.2 m. The data indicate that an average parallel fiber, 6 mm long, forming approximately 1100 boutons, may synapse with each Purkinje dendritic tree it traverses.     

Improved periodic acid-silver methenamine staining procedure for electron microscopy.

Mucosubstances can be stained for electron micrsocopy with silver methenamine after periodic acid oxidation in ultrathin Epon sections mounted on nickel grids. The grids are transferred from a solution to another during the staining procedure conveniently in a container made of polyvinyl chloride plastic. This staining procedure is easy to perform and gives reproducible results. It is especially suitable for simultaneous handling of a large number of grids.     

Bone induction by porous glass ceramic made from Bioglass (45S5)

Porous glass ceramic, which was prepared from Bioglass powder (45S5, U.S. Biomaterials) by foaming with diluted H(2)O(2) solution and sintering at 1000 degrees C for 2 h, was implanted as cylinders (5 mm in diameter and 6 mm in length) in thigh muscles of dogs for 3 months. Histological observation was made on thin un-decalcified sections. Bone formation was histologically found in pores of all implants (X16) retrieved from 16 dogs. The bone tissue was also identified with backscattered scanning electron microscopy observation (BSE) and energy dispersive X-ray microanalysis (EDX). This is the first report of bone induction in soft tissues of animals by glass ceramic that has long been recognized as a bioactive (osteoconductive) biomaterial. The present results justify the impetus to investigate the osteoinductivity of calcium phosphate-based biomaterials, to study the mechanism of bone induction (osteoinduction) by calcium phosphate-based biomaterials, to develop osteoinductive calcium phosphate-based biomaterials, and to examine the relation between osteoinduction and osteoconduction.     

Relationships between tooth eruption, occlusion and alveolar bone resorption: cytological and cytochemical studies of bone resorption on rat incisor alveolar bone facing the enamel

The rat labial incisor alveolar bone facing the enamel and bearing the occlusal force was examined by electron microscopy after being compared with the lingual alveolar bone by histological and scanning electron microscopic (SEM) observations. On the labial side, shallow resorptive lacunae were recognized all over the bone surface; these were mainly covered by osteoclasts and some mononuclear cells. The cement line was absent from the bone matrix. On the lingual side, residues of Sharpey's fibers, the bone formation surface and deep resorptive lacunae were observed by SEM. Histologically, bone remodeling areas showing both osteoclasts and active bone-forming osteoblasts on the bone surface, as well as many cement lines in bone matrix, were recognized. Furthermore, electron microscopic and cytochemical studies demonstrated that mononuclear cells located close to osteoclasts displayed osteoblastic characteristics such as alkaline phosphatase activity, a developed Golgi apparatus, and a rough endoplasmic reticulum. These findings indicate that continuous bone resorption occurs on the labial bone surface, while active bone remodeling occurs on the lingual surface. Even on the labial surface, osteoblastic cells close to osteoclasts seem to play an important role in the differentiation and or activation of osteoclasts.     

Ten-minute silver stain for Pneumocystis carinii and fungi in tissue sections

A ten-minute methenamine silver staining procedure for the rapid demonstration of Pneumocystis carinii and fungi in tissue sections has been developed. The procedure is optimal for both urgent and routine histologic diagnosis of diffuse, progressive, and life-threatening pulmonary infiltrates, especially in the compromised host, and of fungal lesions in general. Moreover, it is simple and brief, is applicable to both paraffined and frozen sections, and can easily be used by the pathology resident or pathologist if the need arises. It consistently yields results that are far superior to those obtained by Grocott's original methenamine silver method, and as good as or superior to those of modified silver methods and such nonsilver procedures as Giemsa's, Gram-Weigert, and toluidine blue stains.     

Correlative evaluation of variable pressure-scanning electron microscopy to histology methods for vascular tissues

Abstract

For pathological investigation, vascular tissues implanted with biomaterials or medical devices require microscopic histology and scanning electron microscopy (SEM) assessment to evaluate biocompatibility with the vascular luminal surface. However, these valuable specimens are often susceptible to SEM and histology tissue preparation artifacts. Correlative variable pressure-scanning electron microscopy (VP-SEM) and histology methods were developed and evaluated, using current SEM technology, allowing direct imaging of hydrated samples without lengthy tissue processing, labor, equipment, and their sequela artifacts. Unstained and silver-stained, fixed swine carotid arteries were assessed using a cooling stage and an environmental secondary electron detector. After VP-SEM imaging, arteries were processed for histology evaluation. Assessment of luminal endothelial surface SEM images and tissue histology of the underlying mural wall was made to determine if low vacuum, cryo-freezing, electron beam exposure, and/or staining affected tissue morphology. Conventional SEM requires lengthy tissue processing with dedicated equipment for solvent dehydration, critical point drying, and metallic sputter coating in order to image under high vacuum for secondary electron imaging. This process causes notable artifacts when samples are later processed for microscopic histology. In contrast, silver staining of swine carotid arteries, followed by aldehyde fixation, facilitated VP-SEM imaging and histology preparation with results that were comparable to conventional SEM and had acceptable tissue morphology. VP-SEM with a cooling stage and an environmental secondary electron detector allows efficient SEM imaging and correlative histological assessment of vascular tissue samples. This correlative method may be further developed and implemented for research applications with biomaterial and medical device implanted tissues.     

The role of mesenchyme-like tissue in the pathogenesis of thanatophoric dysplasia

We have studied the light microscopic, transmission, and scanning electron microscopic (SEM) findings in 13 cases of thanathophoric dysplasia (TD) and 4 control infants. In the TD growth plate, areas with less abnormal cartilage and bone alternated with areas of severely abnormal cartilage and bone. These latter abnormal areas were always found around tongues of apparent mesenchymal tissue that appeared to penetrate from the investing perichondrium and periosteum. The ultrastructure of the less abnormal areas was similar to that of the control infants, including cell and matrix structure as well as mineralization. The abnormal cartilage and bone had many ultrastructural abnormalities that were also found in the adjacent mesenchymal tissue. The mesenchymal cells, adjacent chondrocytes, and osteoblasts contained dilated endoplasmic reticulum and moderately large intracytoplasmic vacuoles. In the area adjacent to the cartilage, the matrix of the apparent mesenchyme contained thin collagen fibers and proteoglycan granules, whereas the matrix adjacent to the bone contained thick bundles of short collagen fibers. The matrix of the surrounding cartilage and bone resembled the adjacent matrix in the mesenchyme. In addition, many vesicular structures or osmiophilic particles were found in the matrix of the mesenchyme and adjacent cartilage and bone. SEM examination showed normal and abnormal bone trabeculae adjacent to each other. In the abnormal trabeculae, there were large, densely packed osteoblastic and osteocytic lacunae. The calcified collagen fibers had a random orientation, in contrast to the longitudinal orientation in the relatively normal bone. Chemical studies of collagen in the metaphyses of bones from five infants with TD showed a small amount of collagen type III (less than 5%), which was not found in three control infants. Thus, a basic pathogenetic mechanism in the skeletal abnormalities of TD appears to be the focal replacement of the growth plate and periosteum by persisting abnormal mesenchymal-like tissue from which the abnormal bone originates.     

Synthesis of alveolar bone Sharpey's fibers during experimental tooth movement in the rat

There is little information concerning the effects of tooth movement on the relative synthesis of bone matrix and Sharpey's fiber collagenous proteins. The purpose of this study was to investigate this situation using radioautographic techniques. The maxillary first molar tooth in rats was tipped toward the midline using an appliance and the animals were injected with 3H-proline after 3 days and sacrificed 24 hr later. Maxillae were sectioned and silver grain proportional areas (grain density/5,000 microm2) evaluated over Sharpey's fibers and adjacent alveolar bone matrix using computerized densitometry and histomorphometric techniques. These data were compared to a group of untreated animals by Fisher's exact test. At depository surfaces of experimental tissues, the silver grain proportional area over bone matrix was significantly greater than over Sharpey's fibers (P<0.05) and control bone matrix (P<0.01). The silver grain proportional area over Sharpey's fibers was not different between the groups. At resorptive surfaces, the silver grain proportional area over both bone matrix and Sharpey's fibers was significantly greater in experimental tissues compared to controls (P<0.01). Thus, movements of adjacent teeth affect both the quantity and ratios of collagenous protein incorporation into Sharpey's fibers and adjacent alveolar bone, which is dependent on the intensity and characteristics of the force.     

Accumulation of aluminium in lamellar bone after implantation of titanium plates, Ti-6Al-4V screws, hydroxyapatite granules

Titanium plates, Ti6Al4V screws and surrounding tissues, and biopsies of hydroxyapatite (Osprovit) grafts of maxillary sinus lifting were investigated to evaluate the release and accumulation of ions. Optical microscopy, SEM and X-ray microanalysis were carried out to evaluate the plates and screws removed from patients presenting inflammation and biopsies. Ions release from metallic appliances or leaching from granules towards soft tissues was observed. An accumulation of aluminium but not titanium was found in soft tissues. A peculiar accumulation of aluminium in the dense lamella of newly formed bone was recorded. The results seem to indicate that biological perturbations may be related to aluminium release from the tested biomaterials. The aluminium content of these biomaterials, its diffusion and accumulation are discussed. Further studies on ion release from biomaterials and aluminium fate in skeletal tissues are suggested.     

Backscattered electron imaging: A new method for the study of cardiomyocyte architecture using scanning electron microscopy.

Scanning electron microscopy (SEM) with secondary electron emissions is useful for the study of cardiomyocyte architecture, however, the information is limited from the cell surface. Whereas backscattered electron (BSE) emission can give a high-resolution image of the specimen's intracellular structure after heavy metal staining. In this study, we applied BSE imaging analysis to the study of the arrangement of cardiomyocytes in the myocardium. The tissue specimens from a normal fresh monkey heart, normal human heart obtained at autopsy, and surgically resected tissue from a patient with old myocardial infarction in the left ventricular aneurysmectomy were used. The tissue specimens were fixed in neutral formalin, treated with NaOH and then stained with Gomori's silver methenamine reagent followed by tannic acid and osmium tetroxide. After dehydration and drying, the specimens were coated with carbon and examined by SEM with a BSE detector. In the tissue preparations, the A bands of sarcomeres were selectively stained with silver so that the arrangements of subsarcolemmal myofibrils and the intercalated discs were clearly seen in the BSE images. In the left ventricular aneurysmal walls of old myocardial infarction, atrophied cardiomyocytes with disarray of subsarcolemmal myofibrils were observed. The results strongly suggest that BSE images are further applicable to the study of the architecture of cardiac myocytes and their branches, and the arrangement of intracellular myofibrils in various diseased myocardium.     

Developing Elastic Tissue: An Electron Microscopic Study

Electron microscopic identification of elastic tissue in normal and disease states has been uncertain due to the lack of a specific electron-dense stain. Recently we introduced a silver porphyrin electron microscopic stain (silver tetraphenylporphine sulfonate) for the identification of adult elastic tissue. This stain has now been employed to study the development of elastic tissue with the aim that new and old elastica can be differentiated at the electron microscopic level. Present observations showed that developing elastica consisted of two distinct morphologic components. Each portion exhibited different staining properties with the silver porphyrin and lead citrate. One component was fibrous and the other amorphous. The fibrous component stained with lead citrate while the amorphous stained with the silver porphyrin. The fibrous component was the first to appear; the amorphous portion appeared later in development and was formed within the fibrous matrix. Mature elastic tissue was devoid of the fibrous component. Based upon the morphologic appearance and staining properties, one can now differentiate between newly formed elastic tissue and the existing one in various disease states.     

A Scanning Transmission Microscopy and Energy-Dispersive X-ray Microanalysis of Idiopathic Ocular Calcification and Oxalosis in AIDS Patients

In a series of 98 consecutive eyeballs enucleated at postmortem from 86 patients dying with AIDS, the incidence of calcium deposits was 14 and 18.6%, respectively, for oxalates and calcium hydroxyapatite. The calcific eyes were examined by scanning electron microscopy (SEM) coupled with energy-dispersive X-ray microanalysis to confirm the elemental nature of the precipitates. Transmission electron microscopy was used in 2 of the cases with oxalosis. Oxalates with a free end exhibited a plate-like shape at SEM and appeared acicular at TEM, due to the reduced thickness of ultrathin sections. Crystals that were embedded in tissues such as the sclera or degenerate detached retinal tissue formed either spherules or plates at SEM. No clear relationship with intracellular structures could be found at TEM, possibly due to postmortem autolysis phenomena. Calcium hydroxyapatite deposits appeared at SEM as fine granules distributed over the collagen fibers of the corneal and conjunctival stroma and the scleral lamellae, but were also present intracellularly, both in the nucleus and cytoplasm of epithelial cells.     

A scanning transmission microscopy and energy-dispersive X-ray microanalysis of idiopathic ocular calcification and oxalosis in AIDS patients.

In a series of 98 consecutive eyeballs enucleated at postmortem from 86 patients dying with AIDS, the incidence of calcium deposits was 14 and 18.6%, respectively, for oxalates and calcium hydroxyapatite. The calcific eyes were examined by scanning electron microscopy (SEM) coupled with energy-dispersive X-ray microanalysis to confirm the elemental nature of the precipitates. Transmission electron microscopy was used in 2 of the cases with oxalosis. Oxalates with a free end exhibited a plate-like shape at SEM and appeared acicular at TEM, due to the reduced thickness of ultrathin sections. Crystals that were embedded in tissues such as the sclera or degenerate detached retinal tissue formed either spherules or plates at SEM. No clear relationship with intracellular structures could be found at TEM, possibly due to postmortem autolysis phenomena. Calcium hydroxyapatite deposits appeared at SEM as fine granules distributed over the collagen fibers of the corneal and conjunctival stroma and the scleral lamellae, but were also present intracellularly, both in the nucleus and cytoplasm of epithelial cells.     

Effects of orthodontic forces on the morphology and diameter of Sharpey fibers of the alveolar bone of the rat

There is little information available concerning the effects of functional and therapeutic forces on Sharpey fibers and adjacent bone matrix. In the present study, springs were placed between the left first and second maxillary molar teeth of rats and retained for 1, 2, 3, 4, or 5 days. The right side served as a control. Tissues from sham-operated, untreated animals were also studied. Maxillae were removed, fractured, rendered anorganic with sodium hypochlorite, and then examined by scanning electron microscopy (SEM). Some tissues were demineralized and examined by high-voltage electron microscopy (HVEM). Sharpey fibers were studied at the alveolar wall and at the midline of the interdental septum (intra-septal Sharpey fibers). In 5-day experimental tissues, SEM showed intra-septal Sharpey fibers had either a reduced number of, or lacked, unmineralized cores. Unit collagen fibrils in 5-day tissues viewed by HVEM were densely packed into Sharpey fibers which had no afibrillar areas. Sharpey fibers at the alveolar wall demonstrated no observable changes in morphology or in pattern of mineralization. After 5 days of spring placement, the mean diameters of intra-septal fibers were significantly less than those at the alveolar wall (p less than 0.001). The disparity in Sharpey fiber diameters of treated and untreated control animals suggests that untreated controls are essential to the design of studies of rodent tooth movement. This study suggests that orthodontic tooth movement produces changes in the morphology and mineralization patterns of Sharpey fibers which might affect the mechanical strength of the periodontium.     

组织化学染色技术在超薄生物塑化标本中的应用

目的 探讨组织化学染色技术是否可以应用于塑化标本并验证染色塑化标本是否具有自发荧光。 方法 选取1个手掌标本经超薄生物塑化技术做成组织块,进行连续切片,切片数量56张,并按切片顺序进行染色处理：原始切片,苏木素-伊红染色,凡尔霍夫- 酸性品红染色,亚甲蓝-天青Ⅱ号染色,在扫描仪、光学显微镜和激光扫描共焦显微镜下观察染色效果和组织结构特点并进行比较。 结果 3种染色技术都可应用于塑化切片染色。苏木素-伊红染色显示肌肉、结缔组织呈红色或紫红色,骨质呈紫蓝色或蓝色。凡尔霍夫-酸性品红染色显示弹力纤维呈黑色,胶原呈红色,其他组织为黄色或棕黄色。亚甲蓝-天青Ⅱ号染色显示肌腱呈紫红色,骨质呈粉红色,软骨呈紫罗兰,其他组织呈紫色。但细胞内结构的染色效果并不理想。在激光扫描共焦显微镜下,胶原纤维、弹力纤维和肌肉纤维具有自发荧光,结构清晰可辨。结论 常用的组织化学染色技术适合于超薄塑化切片染色,染色切片的各种组织结构比未染色的观察效果好。染色后,塑化切片中具有自发荧光的结构在激光扫描共焦显微镜下亦可清晰显影。     

Histopathological and microanalytical study of zirconium dioxide and barium sulphate in bone cement.

AIMS: To report the appearances of zirconium dioxide and barium sulphate in interface membranes, synovium, and other tissues around joint prostheses. METHODS: Histological sections from 23 specimens were reviewed by light microscopy and polarisation. Scanning electron microscopy and x ray microanalysis were performed on routinely processed paraffin wax sections. RESULTS: Polyethylene, metals, and polymethylmethacrylate cement debris were easily recognisable. Almost all the cement remnants contained either zirconium dioxide or barium sulphate, confirmed by microanalysis. The contrast media had characteristic light microscopic appearances. Zirconium was identified in macrophages away from cement remnants. CONCLUSION: The presence of radiographic contrast media in tissues around prosthetic joints is common but not widely recognised.     

Deposition of bone-like apatite on silk fiber in a solution that mimics extracellular fluid

The fabrication of apatite-organic polymer hybrids is one of several attractive methods for the development of biomaterials as a substitute for bone. Such materials have both bone-bonding ability and mechanical properties analogous to natural bone. The biomimetic process has focused attention on fabricating such hybrids, where bone-like apatite is deposited on an organic polymer surface in solutions that mimic physiological conditions. In this process, a bone-like apatite layer can be coated onto organic substrates either by using a simulated body fluid (SBF) with ion concentrations nearly equal to those of human extracellular fluid, or by using fluids that are supersaturated with respect to apatite at ambient conditions. In this study, we investigated the ability of natural silk and its related materials to facilitate apatite deposition under biomimetic conditions. Cloths made of raw silk or normal silk fibers were soaked in 1.5SBF, which has 1.5 times the ion concentration of SBF. Sericin film, which is made from an extract of degummed raw silk, was soaked in 1.5SBF. The cloth and the film soaked in 1.5SBF then were characterized by scanning electron microscopic (SEM) observation, energy dispersive X-ray microanalysis (EDX), and thin-film X-ray diffraction (TF-XRD). Apatite deposition was observed on the surface of cloth made from raw silk fiber after it was soaked in 1.5SBF, but it was not observed on cloth made from normal silk fibers. The apatite deposition on the raw silk fiber cloth was accelerated when the fibers were subjected to treatment with CaCl(2) solution at a concentration of at least 1 kmol/m(3) before immersion in 1.5SBF. Apatite deposition also was observed on the sericin film after the film was soaked in 1.5SBF for 7 days. These results indicate that apatite deposition on raw silk cloth is attributable to the catalytic effect of sericin because the surface of raw silk consists of sericin whereas that of normal silk contains fibroin. The deposition of the apatite and its crystal growth are accelerated by the presence of calcium ions on the sericin after treatment with CaCl(2) solution. Thus, sericin on natural silk fiber has the potential to facilitate apatite deposition and can be useful as a polymer material in the fabrication of hybrid materials analogous to bone through biomimetic processes.     

Acid fast inclusions in tissues of dogs dosed with lead

Liver, kidney and bone from dogs with experimental lead toxicosis were examined to study the frequency of occurrence of acid fast inclusions and the results were correlated with the lead content of these tissues. Light and electron microscopic studies revealed that bone and kidney had characteristic acid fast lead inclusions in 95 and 68 per cent of cases, respectively. Liver, although it had a high lead content, did not often show inclusions. For histopathological diagnosis of lead poisoning in dogs, we suggest that Ziehl-Nielsen stained sections of the epiphyseal area of long bone be examined for the presence of acid fast inclusions in osteoclasts.