Scanning electron microscopy evaluation of the link between a new dentinal desensitizer and dentine.

AIM: The purpose of this study was to evaluate the interaction of a novel dentinal desensitizer Twin Pro with enamel-dentinal adhesive preparations or filling materials and dentine using scanning electron microscopy (SEM) technology. METHODS: Black's I class cavities were drilled extracted molar teeth free of caries or fissures, and the cavities disinfected. The specimens were divided into 4 groups of 2 teeth each treated as follows: Group A: Twin Pro, fluid (Tetric-Flow Vivadent) and micro-hybrid (Tetric-Ceram Vivadent) composites placed on the etched (Liner Bond 2V Clearfil) cavities. Group B: Twin Pro, PQ1-Ultradent loaded Primer-Bonding plus fluid and micro-hybrid composites applied to the acid-etched (37% orthophosphoric acid) cavities. Group C: Twin Pro plus Silver amalgam alloy (Phasealloy- Sybram Kerr). Group D (control group): self-etching primer plus fluid and micro-hybrid composite. Specimens were investigated by SEM. RESULTS: The results obtained in all groups show that the application of Twin Pro does not alter the adhesiveness of the restorative composite materials to the dental wall. In fact the desensitizer and the adhesive layers are indistinguishable at SEM observation, and well adherent to the dentinal surface of the cavities. CONCLUSION: The results of SEM investigations show that Twin Pro does not decrease the adhesion of restorative materials to the tooth surface, as it establishes an efficient interconnection with the different materials used, and it forms a uniform layer covering and occluding dentine tubules, and this might constitute an efficacious sealing of dentinal tubules which possibly contribute to decrease dentinal sensitivity to environmental nociceptive stimuli.

Evidence of rat tail tendon proteoglycans at emission field scanning electron microscopy.

Alcian blue (AB) in critical electrolyte concentration (CEC) was used for scanning electron microscopy (SEM) on rat tail tendon. Segments with diameters of 4 to 9 nm are evident in the perifibrillar area with their lengths ranging from 180 to 400 nm. Frequently the segments adhere closely to collagen gap zones. Segments are not evident either in specimens fixed in glutaraldehyde or in specimens stained with AB in CEC solutions previously treated with glycanolytic enzymes. The segments suggestively correspond to electrodense filaments evident in transmission electron microscopy (TEM) if the rat tail tendon is similarly treated (Scott, 1981). It may be concluded that AB in CEC solutions can be utilized for SEM investigations and represents a good method for the detection of the three dimensional disposition of proteoglycans (PG) and their interaction with collagen.

The paracoronal (marginal) cells of fungiform papilla of Rana esculenta.

Paracoronal secretory cells can be observed outside the sensorial area of the fungiform papilla of Rana esculenta. The morphology of these cells, the type of secretion and their function have, to date, only been incidentally described. By scanning electron microscopy (SEM), the paracoronal cells appear as swallow's-nest-shaped formations with openings 10-15 microns in diameter. The walls of paracoronal cells are characterized by laminar processes subdividing the interior hollow. The cavity of these formations is occupied by amorphous material as demonstrated by light microscopy (LM) pictures. The secretory material fills 7/8 of the upper part of the cytoplasm and appears rather transparent. The secretory material is PAS-negative, unlike the secretory granules contained in laminar processes. By transmission electron microscopy (TEM), they appear as clear ovoid structures, the nucleus of which is situated in the deeper part of the cell, enveloped by a thin cytoplasmic layer and characterized by secretory apparatus and the presence of secretory granules of middle electron-opacity. The apical part of these cells presents large mucous droplets. These cells adhere both to ciliated and parietal cells. Following cytochalasin-B treatment, cells do not show any considerable ultrastructural modification, while after terbutaline treatment the profiles of secreted paracoronal cells increase greatly. Histochemical properties of their secretory products are similar to those of parietal cells and their particular anatomical localization may exclude the direct implication of these cells in taste transduction.

The role of proteoglycans in maintaining collagen fibril morphology.

The aortic wall contains various heterogenous proteoglycan populations which interact in different ways with other components of extracellular matrix. Proteoglycans (PGs) are known to provide structural support to the vessel wall as well as to influence specific physiological functions of the tissues. The aim of the present study was to investigate the effects of Chondroitinase AC (Chase), Streptococcal Hyaluronidase (Hyase) and Heparanase on human aortic wall collagen which had been treated previously with 4M GuHCl, in order to verify the effects of selective glycanolytic treatment on type I collagen fibril ultrastructure. Following 4M GuHCl treatment, collagen fibrils are seen to have a clearly visible period. Subsequent to GuHCl and Streptococcal Hyase treatment all collagen fibrils appear to be completely swollen in thin aperiodic filaments; the typical 64 nm collagen period is completely undetectable. After GuHCl and Chase treatment a small number of collagen fibrils are seen to be swollen in thin fibrils which are mainly localized at some distance from elastic fibres. Following GuHCl and Heparanase/Heparitinase III treatment a considerable number of collagen fibrils appear to be swollen in thin fibrils; the majority of which are situated in the vicinity of elastic fibrils. The swelling of collagen fibrils underlines the fundamental role of proteoglycans in maintaining collagen fibril integrity and periodicity. It is as yet impossible to precisely map interactions between these proteoglycans and collagen fibres. The role of Hyaluronic acid requires further investigation, although the nature of this interaction is undoubtedly a matter of considerable interest.

Morphological features of different regions in frog crista ampullaris (Rana esculenta).

The cellular organization of different regions of the crista epithelium from the frog posterior semicircular canal was studied by light, transmission and scanning microscopy. The sensory epithelium consists of hair cells surrounded by supporting cells and basal cells located close to the basement membrane. Three types of hair cells, namely club-like, cylindrical and pear-like cells differentially distributed along the crista could be recognized on the basis of their shape. Club-like cells are located only in the peripheral regions, cylindrical cells both in the central and in the peripheral regions, and pear-like cells appear segregated into the intermediate regions. Sensory cells of the central region are characterized by a ciliary apparatus consisting of stereocilia usually shorter--and in some cases less numerous--than those of cells of the other regions. The presence of large evaginations of the apical membrane of hair cells and of several vesicles of microexocytosis demonstrates that receptor cells have a considerable secretory activity. This secretory activity is also proven by the presence in the supranuclear region of hair cells of numerous Golgi complexes. Moreover, the presence of two kinds of Golgi complexes, one constituted by dilated cisternae containing a moderately electron-dense material and the other made up of flattened electron-transparent cisternae, suggests a diversified secretion of material by the hair cells. This heterogeneous material may provide substances important for cupula formation and the composition of the endolymph.

Collagenase-extractable proteoglycans from lesion-free areas of human aorta.

Different proteoglycan (PG) populations were isolated from normal human aorta by extraction of minced tissue with 4M GuHCl and by further digestion of the residue with collagenase. Dissociative extraction induced a complete disappearance of Alcian Blue positive material, which was demonstrable by transmission electron microscopy before the treatment around collagen fibrils and in pericellular areas. However, 4M GuHCl extraction solubilized only an average of 60% of aorta total hexuronate content. Collagenase treatment of the residue resulted in a complete loss of collagen fibril organization, which was coupled with a further hexuronate recovery, accounting for about one third of total tissue content. The bulk of PGs obtained in collagenase digest was retained by Sepharose CL-4B column. Their sulphated glycosaminoglycan (GAG) composition differed from PGs extracted with 4M GuHCl, containing only chondroitin sulphate (CS) and heparan sulphate (HS), without detectable traces of dermatan sulphate (DS). Moreover, they contained hyaluronic acid. The results obtained by agarose polyacrylamide gel electrophoresis (APGE) and Octyl-Sepharose chromatography, followed by further APGE and Sepharose CL-4B gel-filtration, carried out before and after treatment with Chondroitinase ABC and AC and Heparinase I and III, suggested that collagenase digest contained different PG populations, carrying mainly either CS or HS chains. Moreover, HS containing PGs showed higher hydrodynamic size and stronger properties of hydrophobic interactions than CS containing PGs.

Primary osteoma cutis. Clinical, morphological, and ultrastructural study.

Primary osteoma cutis arises in the deeper dermis for no apparent reason and presents as mature, lamellar, and osteonic bone; secondary cutaneous osteomas are correlated with inflammatory processes, scars, or dysembryoplasia and are always composed of osteoid. Ultrastructural findings of primary cutaneous osteomas have not been reported to date. Light and electron microscopic findings of a case of primary osteoma cutis are described: mineralized areas may be divided into macrocalcification and microcalcification. Macrocalcification consists of lamellar bone. Osteocytes populate the lamellae, whereas collagen fibril distribution is bone-like. Hydroxyapatite deposition presents as globular or needle-like electron-dense material progressively masking the connective tissue matrix. Microcalcifications, which are found in macroscopically normal dermis around the calcified plaque, consist of osteoid tissue inhabited by osteoblast-like cells. Microcalcifications may be interpreted as metastatic calcifications related to the primary osteoma calcified plaque. Primary osteoma cutis may be considered as true bone amartothic formation rather than dermal mineralization.

Collagen fibril ultrastructure alters after glycanolytic digestion.

Fixed fragments of bovine nasal septum cartilage were digested for six hours either with testicular hyaluronidase or streptomyces hyaluronidase or flavobacter chondroitinase ABC, and observed with a transmission electron microscope. Collagen fibril diameters (D) were measured to evaluate the effect of enzymatic digestion on the fibril size. This resulted in an increased frequency (17% to 47%) of "thin" fibrils (80 to 32 nm), followed by a decrease (65% to 31%) of the frequency of "mid" fibrils (32 to 64 nm). The frequency of "thick" fibrils (over 64 nm) showed a moderate increase (18% to 22%). Considering the relationship between fibril diameter, fibril volume and collagen content, the apparently relevant increase in number of the "thin" fibrils corresponds to an alteration of only 4% of the total collagen. On the other hand the increase of the "thick" fibrils implies a conspicuous alteration of 20% of the total collagen. The observed fibril rearrangement after digestion may be explained in terms of the wrap of matrix proteoglycans around each fibril. The enzymatic removal of the proteoglycans could make "mid" collagen fibrils free to regress into "thin" as well as to merge together into "thick" fibrils.

Scanning electron microscopy of proteoglycans in metaphyseal cartilage.

Alcian blue (AB) was used for scanning electron microscope investigations on metaphyseal cartilage. In the pericellular area three-dimensional network connecting the cell membrane surface to the lacunar wall is evident. The network is formed by very long rod-like filaments about 50 nm thick. The segments may be interpreted as the proteoglycans (PGs) of the pericellular area. In the pericellular area in hypertrophic and degenerative zones, the rod-like segments are closely connected to "chain granules" which are the morphological expression of Ca-P non crystalline compounds. The rod-like segments are not at all evident either in glutaraldehyde-osmium fixed fragments or in predigested-(streptococcal hyaluronidase and chondroitinase) AB stained ones. It is concluded that AB is a good method to detect the three-dimensional spatial disposition of cartilage PGs.

The dependency of collagen fibrillogenesis in vitro on fibroblast culture conditions. Fibroblasts in mono- and multi-layers.

The extracellular matrix produced by monolayer and tridimensional cultures of fibroblasts was investigated using histochemical and ultrastructural methods. In monolayer cultures, collagen and proteoglycans produced by fibroblasts could not be organized into morphologically recognizable structures. Tridimensional fibroblast cultures produced a well organized matrix with periodic, parallel ordered collagen fibrils of 50 nm diameter, criss-crossed by alcianophylic segments 6-10 nm thick in diameter and 100-300 nm in length, parallel to each other, perpendicular to the collagen fibrils and spaced 67 nm from each other. Some alcianophylic segments lay perpendicular to the above described ones, with maximum lengths of 65-70 nm. Alcianophylic segments are the ultrastructural evidence of structural proteoglycans. These observations suggest that the culture conditions influence the collagen and proteoglycans secretion, so that the final organization of the matrix results quite different.

Morphology of epiphyseal apparatus of a ranid frog (Rana Esculenta)

Morphological, histochemical and ultrastructural investigations on epiphyseal apparatus of Rana Esculenta were made. The most important findings were the following: 1) metaphyseal cartilage is localized inside proximal diaphyseal compact bone as a plug; 2) metaphyseal cartilage do not reduce in thickness during ageing; 3) metaphyseal cartilage do not show vascular invasion and do not mineralize in degenerative zone; 4) trabecular bone was not at all evident in this animal; 5) external periosteum is well vascularized and proliferates in correspondence to marginal epiphyseal end of the diaphyseal. From these results the hypothesis that the ranid frog bone growth is not due to metaphyseal metabolism (as in avian and mammals) but to bone periosteal marginal mineralization is reached.

Ultrastructural findings of congenital dyserythropoietic sickle cell beta thal-associated anemia.

The ultrastructural findings of erythroblasts and reticulocytes in one case of congenital dyserythropoiethic anemia (CDA) associated with a haemoglobinopathy, sickle cell beta thalassemia minor (Type V CDA), is described. The observations can be summarized as follows: 1) A lot of large breaks are present in the erythroblast nuclear envelope. 2) Nuclear membrane evaginations are filled with dense loose chromatin. 3) Electron-transparent areas (moth eaten chromatin) are evident in dense chromatin. 4) Electron-dense granular material, related to altered haemoglobin chain storage, is evident in the nucleus and in the cytoplasm. 5) Iron deposits are present in mitochondrial matrix. 6) Myelinic figures are present in reticulocyte cytoplasm. For the first time the ultrastructural findings in this type of associated CDA are described and related to the double origin of clinical symptoms.

Ultrastructure of periprosthetic Dacron knee ligament tissue. Two cases of ruptured anterior cruciate ligament reconstruction.

Light- and electron-microscopic investigations were performed on two failed Dacron ligaments that had been removed from 2 patients shortly after failure of the implant 2-3 years after reconstruction of the anterior cruciate ligament. Two different cell populations and matrices were correlated with closeness to the Dacron threads. Fibroblasts surrounded by connective tissue with collagen fibrils were located far from the Dacron threads. Roundish cells, appearing to be myofibroblasts surrounded by a more lax connective tissue and elastic fibers, were found close to the Dacron threads. The presence of myofibroblasts and the matrix differentiation could be attributed to the different mechanical forces acting on the Dacron and on the connective tissue because of their different coefficients of elasticity. The sparse occurrence of inflammatory cells in the synovial membrane and in the connective tissue surrounding the Dacron supports the biologic inertness of this artificial material. However, the repair tissue was not structured to resist tension stresses.

Ultrastructural aspects of human nonunion.

A histological study on the tissue of nonunion of tibias of two young patients was performed to evaluate the ability of cells to start the mineralization of the matrix. The observations can be summarized as follows: 1) Tissue vessels often appear occluded by thrombotic material; 2) Fibroblasts and chondrocytes found in the nonunion tissue seemed normal, with a good secretion apparatus; 3) The cell membranes were able to produce matrix vesicles; 4) Matrix vesicles and cell membrane looked positive to ALPase reaction, 5) Hydroxyapatite crystals could be observed in the cell matrix or inside matrix vesicles. It may be concluded that cells populating nonunion tissue are well equipped to induct the mineralization of the matrix, but the absence of a blood supply, enough to bring them a normal calcium amount, is the real reason for the nonunion.

Morphological aspects of rat metaphyseal cartilage pericellular matrix.

In order to verify whether it is possible to observe morphological evidence of a Ca-P amorphous phase (the first step of Ca-P crystalline deposition), the pericellular area of metaphyseal cartilage was investigated. In the pericellular zone of proliferative, maturation, hypertrophic, degeneration and calcification cartilage, many electron-opaque granules, having a very regular diameter of about 12 nm, disposed in closely-packed chains (chain granules) and increasing in number from the proliferation to the calcification zone, are evident. These chain granules, which are closely connected with proteoglycans, disappear after decalcification and are spatially related to ALPase and ATPase activities. They may be the morphological reflection of the Ca-P amorphous phase.

Infantile cortical hyperostosis (Caffey disease): ultrastructural and immunohistochemical characterization of the peritrabecular cells.

The ultrastructure and the immunohistochemical pattern of the cells which are responsible for the bone resorption in the cortical infantile hyperostosis were investigated. The osteoclasts present a great positivity to MB1 antigen and a low positivity to OKM5. Mononuclear cells with primary lysosomes, looking like osteoclast ones are present in high concentration in peritrabecular spaces. These cells show a high positivity to OKM5 antigen and a low positivity to MB1 antigen. The mononuclear granulated cells are positive to tartrate-resistent acid phosphatase. The possible common origin and their co-operation in bone resorption is discussed.

Recognition of cell surface modulation by elliptic Fourier analysis.

A development of elliptic Fourier analysis, consisting in an alignment of harmonics according to their clockwise or counter-clockwise rotation, resolves the discrepancy between calculated harmonic frequencies and observed morphological periodicities. The new technique is supported by consistent data of empiric and fractal contours, comparatively analyzed and visualized with and without harmonic alignment. The method is particularly suitable for the recognition of periodic modulations of the cell surface. A preliminary analysis of two different cell populations (echinocytes and chondrocytes) shows distinct patterns of surface modulation that allow an effective discrimination of the cell type, while providing relevant information about the respective cytological configurations.

Type I procollagen in the severe non-lethal form of osteogenesis imperfecta. Defective pro-alpha 1(I) chains in a patient with abnormal proteoglycan metabolism and mineral deposits in the dermis.

We have screened type I procollagen synthesized in vitro by skin fibroblasts from several patients with the severe non-lethal form of osteogenesis imperfecta. Cells from one patient synthesized and secreted both normal and a larger amount of abnormal type I procollagen. The abnormal alpha chains are larger in size due to post-translational overmodifications involving the whole triple helical domain. Abnormal collagen heterotrimers had a melting temperature 2.5 degrees-3 degrees C lower than normal ones or from controls. Chemical analysis of collagen in the medium showed a greater degree of both lysyl hydroxylation and hydroxylysyl glycosylation, the major increase in molecular mass of overmodified alpha chains being due to the higher hydroxylysine-bound hexose content. The proband's cells modify proteoglycan metabolism and mineral crystals form in the dermis, possibly a response to abnormal collagen-proteoglycan interactions. These findings can be explained by a small defect in the product of one allele for pro-alpha 1(I) chains: three-quarters of the synthesized type I procollagen molecules are composed of trimers containing one or two chains defective near the C-terminus of the triple helix or in the C-propeptide. The data obtained for this patient confirmed that the severity of clinical manifestations in osteogenesis imperfecta strongly depends on the location and nature of the mutations, and that the phenotype could be a consequence of a collagen defect(s) and its influence on collagen-collagen interactions and collagen interactions with other connective tissue components.