Effect of enamel matrix derivative on human periodontal fibroblasts: proliferation, morphology and root surface colonization. An in vitro study.

BACKGROUND: Several clinical trials have shown the effectiveness of Emdogain(R) (EMD) in promoting tissue regeneration, even though the underlining biological mechanism is still poorly known. OBJECTIVES: The aim of the present study was to verify the effect of EMD on the proliferation of human periodontal ligament (PDL) fibroblasts and on their colonization and differentiation following contact with the root surface of extracted teeth in vitro. METHODS AND RESULTS: Fibroblasts from PDL were seeded on Petri dishes and cell growth was evaluated by cell counting in the presence and absence of EMD, after 1, 3 and 8 d of culture. A significant effect of EMD upon cellular proliferation at d 3 and 8 was detected. When PDL cells were grown for 12 d with EMD on etched human root surface, a change in cell morphology was observed. Scanning electron microscopy revealed that cells grown on root EMD-treated surface present a body with a flattened surface closely adherent to the substrate and an outer smooth surface rounded in shape. From the flattened surface some thin and elongated cellular processes connecting with the substrate were also observable. PDL cells grown on EMD-treated surface showed lack of alkaline phosphatase activity, as some authors noticed in cementoblasts in vitro. CONCLUSIONS: In conclusion, our data indicate that EMD enhances human PDL fibroblast proliferation. Furthermore, the cells in the presence of EMD show morphological changes that make them more similar to cementoblasts than to fibroblasts, suggesting a process of cellular differentiation that could play an important role in periodontal tissue repair.

Expression of vascular endothelial growth factor in human dysfunctional temporomandibular joint discs.

A high density of blood vessels is found in specimens of temporomandibular joint (TMJ) disc at any stage of internal derangement of the joint, but the factors responsible for angiogenesis in the disc have not been described. The purpose here was to investigate, in human TMJ discs, the expression of vascular endothelial growth factor (VEGF), a multifunctional cytokine that contributes to angiogenesis. Specimens, free of significant morphological alterations and with varying degrees of disc tissue degeneration/regeneration, were studied by immunohistochemistry for VEGF in order to correlate immunohistochemical with histopathological findings. In normal discs and discs with minor pathological changes, fibroblast-like cells, fibrochondrocytes and chondrocyte-like cells were either not or only weakly immunostained by VEGF antibody. In disc specimens from internal derangement of the TMJ with significant tissue degeneration/regeneration, VEGF was consistently expressed. In these specimens, immunoreaction products for VEGF were observed both in the disc and in the endothelial cells of newly formed vessels. This VEGF immunolocalization is consistent with the stimulation of angiogenesis and the morphogenesis and differentiation of chondrocytes. Therefore VEGF expression by disc chondrocyte-like cells might reflect the action of the cytokine as an inducer of angiogenesis and as an autocrine signal for cells of the chondrogenic lineage.

Immunolocalization of vimentin and alpha-smooth muscle actin in dysfunctional human temporomandibular joint disc samples.

The expression of vimentin and alpha-smooth muscle (alpha-SM) actin was examined in 10 human temporomandibular joint (TMJ) disc samples, with internal derangement and in two control specimens, in order to evaluate the phenotypic characteristics of TMJ disc cells in relationship to histological findings. This was accomplished by means of monoclonal antibodies specific for vimentin and alpha-SM actin and immunocytochemical technique. The study, revealed that every disc cell constantly expressed vimentin. Scattered alpha-SM actin positive cells could be appreciated in normal TMJ discs and tissues with minor pathological findings. In TMJ discs with severe alterations, i.e. tears and clefts, almost fibroblast-like cells, fibrochondrocytes and chondrocyte-like cells were strongly immunolabelled by anti-alpha-SM actin antibody. According to these findings it can be assumed that vimentin is expressed by all disc cell populations and it appears not to be influenced by any disease condition of the disc; on the other hand the up-regulation alpha-SM actin immunolabelling seems to be correlated to histopathological findings of tears and clefts. Cells, with a contractile phenotype, close to such defects, could be involved in disc tissue contraction and repair. The plasticity of disc cell populations which evolve towards a different phenotype when subjected to action of macro- and micro-environmental factors is also supported.

Deep lamellar keratoplasty.

PURPOSE: To describe a new technique of lamellar surgery and to compare the results of 150 cases treated with this technique with those observed in as many cases of penetrating keratoplasty (PK). PATIENTS AND METHODS: One hundred and fifty deep lamellar keratoplasty (DLKP) procedures were performed in patients affected by various diseases of the corneal stroma but having a preserved endothelium. Each case was matched with a patient of similar age, gender and pathology who had undergone PK. The following parameters were evaluated: number of rejection episodes, density of endothelial cells, degree of ophthalmometric astigmatism, visual acuity and ocular tension. Statistical analysis was performed with Student's t test to compare patients in a similar clinical setting. RESULTS: No rejection episodes were recorded in the 150 patients who underwent DLKP surgery, whereas rejections occurred in 4% of the patients after PK. A statistically significant higher density of endothelial cells was found in DLKP cases, who also experienced a lower degree of astigmatism than PK patients. A slight but significant improvement in visual acuity was achieved in DLKP patients. CONCLUSION: Our results confirm that DLKP is the preferential procedure when no endothelial damage is involved.

CD44 standard form (CD44H) expression and distribution in dysfunctional human temporomandibular joint discs.

The expression pattern of the cell adhesion molecule CD44 standard form (CD44H) in dysfunctional human temporomandibular joint (TMJ) discs was studied immunohistochemically and compared with normal disc pattern in order to evaluate the expression of this adhesion molecule and correlate it to histopathological changes. Immunohistochemistry with anti-CD44H antibodies was performed on paraffin sections of pathological and normal discs. In normal TMJ discs, a moderate immunolabelling with anti-CD44H antibodies was detectable in fibroblastlike cells, in the few fibrochondrocytes and in chondrocytelike cells. In dysfunctional discs, the staining pattern and intensity varied according to the histopathological findings of the specimens. The TMJ discs showing abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern of normal discs. In the discs showing areas of fibrocartilaginous metaplasia, CD44H expression was upregulated in fibrochondrocytes and fibroblastlike cells, especially around the chondroid tissue. Overall, these results suggest that CD44H mediates the binding of some ECM proteins in TMJ disc cells. The up-regulation of CD44H observed in some dysfunctional TMJ discs seems to indicate a prevention of apoptosis in fibroblastlike cells and an important role in phenotypical change of fibrochondrocytes into chondroblastlike cells, enabling the aggregation of chondroid tissue pericellular matrix components.

Cellular S-100 protein immunostaining in human dysfunctional temporomandibular joint discs.

S-100 protein was detected immunohistochemically in diseased human temporomandibular joint discs with different degrees of pathology, and the findings compared with those of normal discs. In normal discs, large nerve trunks in the posterior ligament were strongly stained by anti-S-100 antiserum; the very few chondrocyte-like cells sometimes showed faint staining, while no staining was observed in any fibrochondrocyte-like or fibroblast-like cell. In dysfunctional discs, S-100 protein immunostaining seemed to correlate with structural pathological findings. The discs showing an abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern as normal discs. In discs with fibrocartilaginous metaplasia and dystrophic cartilage formation, fibrochondrocyte cells showed a very strong immunoreaction for S-100 protein and fibroblast-like cells in some instances were also positive. These findings suggest that S-100 upregulation in disc cells can be considered an attempt at tissue repair by chondroid metaplasia following an injury in that it enables fibroblast-like cells and fibrochondrocytes to acquire a chondrogenic phenotype.

A scanning electron microscopy comparison of enamel polishing methods after air-rotor stripping.

In the last few years, orthodontic literature has shown particular interest in the interproximal enamel reduction technique described as stripping or slenderizing. Most researchers have shown, by scanning electron microscopy (SEM) studies, the difficulties encountered while attempting to remove coarse abrasions left after stripping with the first instrument. The objective of this SEM study was to compare the different polishing methods proposed in the literature and to assess the efficiency of our own procedure. For this purpose, 48 healthy human teeth (premolars and molars) were used after removal for orthodontic or periodontal reasons. The teeth were divided into eight groups of six teeth each (two molars and four premolars), and mounted on a typodont to simulate a clinical situation. Each group underwent stripping according to one of the following techniques: 16-blade tungsten carbide bur and fine and ultrafine diamond burs; coarse diamond bur and fine and ultrafine diamond burs; coarse diamond disk and Sof-Lex disks (Dental products/3M, St. Paul, Minn.); 16-blade tungsten carbide bur and phosphoric acid on finishing strip; and 8-straight blade tungsten carbide diamond bur and Sof-Lex disks. The SEM investigations demonstrated that it is not possible to eliminate, with normal polishing and cleaning methods, the furrows left on the enamel both by the diamond burs and the diamond disks and the 16-blade tungsten carbide burs. Mechanical and chemical stripping as well did not prove to be effective. By contrast, with the use of a 8-straight blade tungsten carbide bur followed by Sof-Lex disks for polishing the enamel, it is possible to obtain well-polished surfaces that many times appear smoother than the intact or untreated enamel.

Endoarticular loose bodies and calcifications of the disk of the temporomandibular joint: morphological features and chemical composition.

We studied articular disks and endoarticular loose bodies taken from patients suffering from different types of temporomandibular joint (TMJ) pathology. The scanning electron microscopy (SEM) analysis of the disks and the endoarticular loose bodies was followed by a chemical-compositional analysis using an energy dispersive spectrometer (EDS) and by characterization of the crystalline phases by X-ray powder diffraction (XRD). The articular disks were composed of a central radiopaque area lacking any evident structural features, surrounded by compact bundles of collagen fibers. EDS and XRD analyses showed that endodiscal radio-opaque areas were hydroxyapatite. By SEM, we observed a fibrous network only in circumscribed areas of the endoarticular loose bodies. The chemical-compositional analysis showed that the loose bodies were composed of calcite (CaCO3). The results of this investigation, along with the clinical history of the patients, allow us to formulate some hypotheses regarding the etiopathogenesis of these structural anomalies. The endodiscal calcifications could be the result of a chronic inflammatory process that produces displastic alterations of the articular disk. Moreover, an acute inflammatory process with modifications in the mechanisms of the synovial fluid turnover seems to be the event that leads to the formation of endoarticular loose bodies.

A microscopic and immunocytochemical study of structural changes in dysfunctional human temporomandibular joint discs.

The central part of 12 articular discs from patients with serious alterations in function of the temporomandibular joint were investigated. The control discs were removed at autopsy from individuals who did not have any such functional defects. The anomalous discs had an increased cellular component; fibrocytes, fibroblasts, numerous myofibroblasts and, less frequently, smooth muscle cells and mast cells were present. The myofibroblasts, which had numerous bundles of thin filaments in the cytoplasm, were positive to the immunocytochemical reaction with antibody to alpha-smooth muscle actin. In one disc only a large part had changed into a mostly fatty tissue. In the majority of the altered discs, the part examined, which usually is neither vascularized nor innervated, was characterized by the presence of numerous blood vessels. Besides the capillary network several larger vessels were present. In one disc, several myelinated and unmyelinated fibres, isolated or in nerve bundles, were also seen. These observations show that the disc fibrous tissue may undergo deep structural modifications that appear to indicate not only a capacity for repair but also an ability to adapt to new functional conditions.

Lymphatic capillaries of the pig lung: TEM and SEM observations.

Pulmonary lymphatic vessels extend within the connective tissue sheets surrounding airways and blood vessels. Frequently in this location they also border the lobular parenchyma, but not lymphatic vessels have been found within intralobular compartments between blood capillaries and alveoli. The presence and distribution of lymphatic vessels in pulmonary tissue are consistent with an important role for the lymphatic system in the clearance of interstitial fluids in the lung. Pulmonary lymphatic channels have structural characteristics of initial lymphatics; their walls are formed only by an endothelial layer, and no muscular cells are present. A network of anchoring filaments and collagen and elastic fibers surrounds the vessel walls. Because the lung is a mobile organ the tissue undergoes compression and distension during respiratory phases. These modifications could have a role in the mechanisms for lymph formation and flow.

Collagen fiber arrangement in temporo-mandibular joint (TMJ) disks from human subjects with functional diseases. Scanning electron microscopy investigations.

Twelve articular disks from patients with temporo-mandibular joint (TMJ) arthropathy were studied and compared with two normal disks. Scanning electron microscopy (SEM) examination of the surfaces and of longitudinal and cross-sections of the disks allowed the observation of the arrangement of the collagen fiber component in different parts of the disk. The superficial part of the articular disks appears to be formed by rather compact fibers. The internal portion is usually formed by bundles of collagen fibers in sheets, alternating with isolated fibers arranged in a parallel or irregular way. In some samples, blood vessels were observed. Our investigations suggested that the appearance of vascularization is the first remarkable histological change that can be observed in functionally abnormal articular disks.

Observations on the enamel of odontomas.

The morphological study of odontomas provides an alternative model for observing the formation of dental tissues, since different maturing stages are present simultaneously. Investigations were performed on decalcified samples (using light microscopy and transmission electron microscopy) and on undecalcified samples of complex odontoma enamel (using transmission electron microscopy). Simultaneous presence of prismatic enamel at various maturing stages with different structural characteristics was observed. Such enamel was sometimes associated with layers of ameloblastic cells with characteristics of cells in functional activity. In other sites, the enamel did not present a prismatic structure but it appeared as unstructured material clusters with abundant organic component. It was concluded that the theory according to which an ecto-mesenchymal inductive failure occurs in odontomas is not confirmed. The defect seen at the beginning of the differentiated and anomalous tissue maturation may be related to latest events in the development of the enamel organ. In this regard, it was concluded that such events involve the efficiency of the ameloblasts and the possible alterations in the organic matrix.

Morphometric computerized analysis on the dentinal tubules and the collagen fibers in the dentine of human permanent teeth.

A morphometric analysis has been performed on important components of human dentine using an image computerized analyzer. The dentinal tubule diameter and their area percentage were calculated. Moreover the area percentage of the collagen fibers in the dentinal matrix was measured. These parameters have been evaluated in different areas of the coronal and the radicular dentine in permanent teeth. Measurements have been performed on undecalcified and decalcified teeth and on teeth treated with enzymatic digestion to remove the organic non collagen matrix and to evidentiate the collagen fiber network. The values obtained in different areas of the tooth and in samples submitted to different treatments were evaluated by statistical analysis. Dentinal tubule diameter and area percentage significatively decrease from the inner to the peripheral dentine both in the undecalcified teeth as in the decalcified ones and in the samples undergone to enzymatic digestion. The collagen fiber percentage in the organic matrix is significatively lower in the mantle dentine.

[Light and electron microscopy observations on different tissues of a complex odontoma]. / Observations en microscopie photonique et électronique sur différents tissus d'un odontome complexe.

Several aggregates of dental tissues constitute the complex odontoma. They are almost completely covered by a layer of prismatic enamel. By the observations at transmission and scanning electron microscope enamel present the characteristics of a not fully developed tissue. The organic component is still abundant and the enamel surface is covered by a cellular layer having the morphological features of the mature ameloblasts. Therefore this enamel results to be yet actively engaged in the maturation phase although the tissues of the odontoma have generally a limited developing time.

[An ultrastructural study of the fibrillary component of dental tissues]. / Etude ultrastructurale de la composante fibrillaire de certains tissus dentaires.

The recent interest for highly sophisticated techniques of dental tissue preparation aiming to display very particular structures, moved the AA. to improve the literature suggestions. In particular they made TEM and SEM observations of transitional zones between healthy and normal pulp and dentin after decalcification and trypsin at different concentrations treatment. The images obtained draw in the attention the study facilities of a technique that really removes all the non collagenic material. The data obtained in the pericellular zones also allowed some interventions in the recent literature discussion about inter-odontoblastic fibres.

[Guided tissue regeneration: an ultrastructural observation by transmission electron microscopy and scanning electron microscopy]. / Régénération tissulaire guidée: observation ultrastructurale au microscope électronique à transmission et au microscope électronique à balayage.

PTFE membranes are used for guided tissue regeneration in order to treat angular bone defects or forcation involvements in surgical treatment. Ultrastructural investigations have been performed by means of electron transmission and scanning microscopy. In agreement with previous reports, fibroblast cells adhering to the reticular structure of PTFE membrane were observed; these were interposed among coagulated clusters of fibrinous material and blood cells round shaped. Elongated bacterial cells were always present in the microscope fields analysed. These observations were confirmed by means of transmission microscopy; moreover specific techniques enabled us to demonstrate that fibroblast cells were synthetizing collagen, which was present in the form of extracellular fibers mixed to fibrin clusters. Roundish and elongate bacterial cells were always observed both in the extracellular matrix and into macrophages.

[Dental tissue-composite bonding. SEM study]. / Giunzione tessuti dentari-composito. Osservazioni al SEM.

The Authors investigated the different operative methods effects in the transition zone between human fresh teeth calcified tissues and a composite material. Morphological observations, done by means of standard and back scattered scanning electron microscopy, demonstrated the Gluma adhesion system efficiency towards enamel and dentin if manufacturing's applying systems are carefully followed, by showing a structure with no solutions of continuity between tooth and restoration.

Scanning and transmission electron microscopy of rabbit heart lymphatic capillaries.

The ultrastructural characteristics of the lymphatic capillaries of the rabbit heart were examined by scanning and transmission electron microscopy (SEM and TEM) using similar technical preparation. SEM defined the interrelationships between endothelial cells including the pocket-like structures formed by overlapping between cells and the corresponding intercellular bridges. Except for rare spindle cells apposed on the luminal surface of the endothelium seen only on SEM, these features were confirmed by TEM. On the abluminal side of the lymphatic capillary wall, a rich network of filaments and fibrils was detectable using both ultrastructural techniques. These two modalities (SEM and TEM) complement one another in defining the microanatomy of the lymphatic capillary.

Lymphatic vessels in inflamed human dental pulp.

Investigation has been performed on both the light and electron microscopic characteristics of the lymphatic vessels present in the dental pulp of human teeth which have been affected by serious carious lesions. These conditions provoke a severe inflammatory response resulting in structural and functional modifications of the tissue; increase of the tissue pressure is followed by the need for a more intensive lymphatic drainage. In the inflamed pulps, dilated lymphatic vessels with distended walls and "open junctions" between endothelial cells are detectable. On the other hand they lack certain endothelial structures which characterize the morphology of these vessels under normal conditions. In the pulpal regions affected by fibrotic proliferation shrunken vessels with irregular profiles are present. From these observations it is possible to obtain other information on the mechanisms regulating the lymphatic drainage in different structural and functional conditions of the interstitium.