Successful regenerative response of a severe bone defect in a right lower central incisor affected by a cemental tear.

Cone-beam computed tomography and clinical examinations including pulp vital testing and pocket probing depth showed a cemental tear with a severe labial alveolar bony defect, but no endodontic lesions, in #25, which had a sinus tract at the labial site, in a 75-year-old woman.

Endodontic Approach and Periodontal Regenerative Therapy for a Mandibular Right Central Incisor Affected by a Perforation and Cemental Tear.

A cemental tear involves complete or incomplete separation of the cementum on the root surface along the cementodentinal junction. Because a cemental tear can lead to periodontal breakdown and mimic endodontic and periodontal lesions, diagnosing clinical cases can be difficult and requires special examinations. A 72-year-old woman presented with a localized periodontal defect on the labial and interproximal surfaces of the mandibular right central incisor. Performing CBCT scans and a biopsy during periodontal surgery allowed definitive diagnosis of a cemental tear and perforation of the site. First, the perforation was repaired with endodontic therapy. Periodontal regenerative therapy using recombinant human fibroblast growth factor-2 (rhFGF-2) was then performed after removing granulomatous tissue and cementum fragments. Examination of the biopsy specimen showed bacterial colonies. This case showed successful clinical and radiographic outcomes at the 18-month follow-up.

Elastic system fibers in rat incisor periodontal ligament--immunohistochemical study using sections of fresh-frozen un-demineralized tissues.

The aim of this study was to examine the localization and distribution of the components of elastic system fibers in the periodontal ligament of continuously erupting rat incisors in an effort to understand the mechanism of the eruption of the tooth. Sections of fresh-frozen, un-demineralized incisors of the rat mandible were prepared for immunohistochemical localization of elastin, fibrillin-2 and microfibril-associated glycoprotein-1 (MAGP-1). The structure of the periodontal ligament was well preserved in sections of fresh-frozen tissues. At the basal region of the ligament, intense immunolabelling for fibrillin-2 and MAGP-1 was observed as dot-like structures (transversely sectioned fibers) mainly on the tooth side of the ligament close to the cementum. These dot-like structures gradually increased in number towards the incisal area and were distributed throughout the tooth side of the ligament. This pattern of distribution was the same as that of reported oxytalan fibers. Elastin-immunopositive fibers were also detected in the ligament, although the labelling was limited and distribution was sparse. In conclusion, both fibrillin-2 and MAGP-1 immunopositive fibers may serve as a scaffold for deposition of tropoelastin during elastogenesis in the periodontal ligament. They may also provide guidance for the migration of fibroblasts to the occlusive side, which generates contractile forces for the movement of the tooth for continuous eruption of incisors.

Ultrastructural study of tissues surrounding replanted teeth and dental implants.

OBJECTIVES: The aim of this study was to describe the ultrastructure of the dentogingival border at replanted teeth and implants. MATERIAL AND METHODS: Wistar rats (8 weeks old) were divided into groups for replantation and implantation experiments. In the former, the upper right first molars were extracted and then immediately replanted. In the latter, pure titanium implants were used. All tissues were fixed, demineralized and embedded in epoxy resin for ultrastructural observations. RESULTS: One week after replantation, the junctional epithelium was lost, and the oral sulcular epithelium covered the enamel surface. The amount of the epithelium increased in 2 weeks, and resembled the junctional epithelium, and the internal basal lamina and hemidesmosomes were formed in 4 weeks. One week after implantation, peri-implant epithelium was formed, and in 2 and 4 weeks, this epithelium with aggregated connective tissue cells were observed. In 8 weeks, the peri-implant epithelium receded, and aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers appeared to seal the implant interface. CONCLUSION: In replantation of the tooth, the internal basal lamina remained at the surface of the enamel of the replanted tooth, which is likely to be related to regeneration of the junctional epithelium and the attachment apparatus at the epithelium-tooth interface. Following implantation, a layer of cells with characteristics of connective tissue cells, but no junctional epithelium and attachment apparatus, was formed to seal the site of the implant.

In situ Abeta pores in AD brain are cylindrical assembly of Abeta protofilaments.

According to the amyloid pore hypothesis, pores formed by small oligomers of misfolded amyloidogenic proteins cause membrane leakage with the unregulated rapid influx of ions leading to cell death. Ultrastructurally, pores reconstituted in vitro have mainly been characterised so far, and the presence of in situ pores in the amyloid tissues has not yet been demonstrated. In this study, the presence of in situbeta amyloid (Abeta) pores was shown with high resolution transmission electron microscopy, in the neuronal cell membrane as well as in the membrane of mitochondria-like organelles in the brain with Alzheimer's disease. They are 16 nm wide and 11 nm long flat columnar structures made up of a single cylindrical layer (wall) of laterally associated Abeta protofilaments which surrounds a 10 nm wide opening or lumen. Protofilaments are the basic unit of the fibrils of all amyloid-forming proteins and peptides. Individual extracellular Abeta protofilaments were 2-3 nm wide straight tubular structures with helical wall formed by the tight coiling of 1 nm wide Abeta filaments. These in situ Abeta pores are similar but not identical to in vitro reconstituted Abeta pores.

Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.

Formation of experimental murine AA amyloid fibrils in SAP-deficient mice: high resolution ultrastructural study.

Previously, the role of the serum amyloid P component (SAP) in the deposition of murine AA amyloid has been examined in SAP-deficient mice in which the deposition was significantly retarded. In this study, AA amyloid fibrillogenesis in SAP-deficient mice was examined ultrastructurally. The fibrils of wild type mice were made up of a microfibril-like main body composed of SAP, chondroitin sulfate proteoglycan (CSPG), and outermost heparan sulfate proteoglycan (HSPG), and associated on its surface were 3 nm wide AA protein 'helical rods', a possible suitable form for Congo red staining. In SAP-deficient mice, fibrils of a similar appearance were also noted among an overwhelming amount of amorphous material, but the AP-containing main body of the fibril was replaced by elongated irregular aggregates of CSPG. The mechanism of retardation of AA amyloid induction in SAP-deficient mice has not yet been clear. It may be caused by possible slower formation of a 'substitute' core. Also, slower formation of AA helical rods may be possible due to the difference in the core material to which AA protein is attached. If it is so, it may limit the extent of Congo red staining, resulting in underestimation of the actual amount of AA protein.

Ultrastructural characterization of an artificial basement membrane produced by cultured keratinocytes.

A recent study in our laboratories on the growth of keratinocytes at the culture medium/air interface has led to the identification of a novel thin sheet-like matrix that supports adherent cells. This novel matrix consists of components secreted by keratinocytes, including type IV collagen, and laminins 1 and 5, that self-assembled to a membrane structure. In the present study, a detailed ultrastructural characterization of this membrane was done with high-resolution electron microscopy after negative staining. The basic organization of the membrane was found to be a dense network of 8- to 10-nm-wide irregular rod-like elements. High-resolution examination and immunolabeling showed that type IV collagen filaments form the core of these elements, and other components including heparan sulfate proteoglycan in the form of 4.5- to 5-nm-wide ribbon-like "double tracks" are aggregated around it. These detailed features of the membrane strikingly resembled those of the basement membrane in vivo. These ultrastructural similarities indicate that the membrane may also have basement membrane-like functional properties, and suggest that it should be considered for testing in future medical applications.

A direct interaction between transforming growth factor (TGF)-betas and amyloid-beta protein affects fibrillogenesis in a TGF-beta receptor-independent manner.

Transforming growth factor-beta (TGF-beta) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-beta protein (Abeta) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-beta receptors. Surprisingly, TGF-beta potentiated the neurotoxic effects of the 40-residue Abeta peptide, Abeta-(1-40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-beta and Abeta-(1-40). Surface plasmon resonance confirmed that TGF-beta binds with high affinity directly to Abeta-(1-40) and electron microscopy revealed that TGF-beta enhances Abeta-(1-40) oligomerization. Immunohistochemical examination of mouse brain revealed that hippocampal CA1 and dentate gyrus, two regions classically associated with Abeta-mediated pathology, lack TGF-beta Type I receptor immunoreactivity, thus indicating that TGF-beta receptor-mediated signaling would not be favored in these regions. Our observations not only provide for a unique, receptor-independent mechanism of action for TGF-beta, but also help to reconcile the literature interpreting the role of TGF-beta in Abeta function. These data support a critical etiological role for this mechanism in neuropathological amyloidoses.

A basement membrane-like matrix formed by cell-released proteins at the medium/air interface supports growth of keratinocytes.

Epithelial cells require adherence to a matrix for regular growth. During standard keratinocyte cell culture in serum-free medium, we observed that cell colonies formed not only on the bottom of the culture vessels but also at the medium/air interface. Coomassie blue staining detected a protein membrane that extended up to several centimeters between the colonies of floating cells. Ultrastructural investigation of this membrane revealed structures closely resembling those of basement membranes, and immunochemical staining confirmed the presence of laminins-1 and -5 as well as collagen IV, representative components of basement membranes. Cells attached to the floating membrane proliferated and could be cultivated for up to six months. When keratinocyte-conditioned medium was filtered and transferred to a culture vessel without cells, the protein membrane at the liquid/air interface formed within one week suggesting self-assembly of cell-released proteins. Our findings provide a basis for the production of epidermal basement membranes for potential medical uses.

Neurologic defects and selective disruption of basement membranes in mice lacking entactin-1/nidogen-1.

Entactin-1 (nidogen-1) is an ubiquitous component of basement membranes. From in vitro experiments, entactin-1 was assigned a role in maintaining the structural integrity of the basement membrane because of its binding affinity to other components, such as type IV collagen and laminin. Entactin-1 also interacts with integrin receptors on the cell surface to mediate cell adhesion, spreading, and motility. Targeted disruption of the entactin-1 gene in the mouse presented in this study revealed a duplication of the entacin-1 locus. Homozygous mutants for the functional locus lacked entactin-1 mRNA and protein and often displayed seizure-like symptoms and loss of muscle control in the hind legs. The behavior patterns suggested the presence of neurologic deficits in the central nervous system, thus providing genetic evidence linking entactin-1 to proper functions of the neuromuscular system. In homozygous mutants, structural alterations in the basement membranes were found only in selected locations including brain capillaries and the lens capsule. The morphology of the basement membranes in other tissues examined superficially appeared to be normal. These observations suggest that the lost functions of entactin-1 result in pathologic changes that are highly tissue specific.

Development of oxytalan fibers in the rat molar periodontal ligament.

Although oxytalan fibers are known to be a ubiquitous component of the periodontal ligament, little information has been available concerning their organization in the developing periodontal ligament. In the present study, growth and distribution of oxytalan fibers were examined in the developing periodontal ligament of rat molars aged 11, 14, 19, 21 and 28 days. A quantitative analysis of the fibers was made and the spatial relationship between the fibers and blood vessels was studied by means of a three-dimensional reconstruction of serial sections. At the beginning of root formation, oxytalan fibers appeared at first as dot-like structures around the root sheath as well as in areas very close to blood vessels. These structures were resolved in the electron microscope to be made up of 12-nm-wide microfibrils in the vicinity of the surface of the cells of the root sheath. In the process of development, these dot-like structures elongated into entities with helical appearances. As the development further proceeded, longer oxytalan fibers were produced in the apico-occlusal direction along with blood vessels. Quantitative analysis showed that an increase in oxytalan fibers coincided with an increase in the density of the vascular network in the developing periodontal ligament. Based on the results of the present study, the role of oxytalan fibers in the developing periodontal ligament may be in the maintenance of the integrity of the vascular system as previously suggested.