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Research projects focusing on biomaterials related factors; the bulk implant material, the macro-design of the implant and the microsurface roughness are routinely being conducted at our laboratories. In this study, we have investigated the bone tissue reactions to turned commercially pure (c.p.) titanium implants with various thicknesses of the oxide films after 6 weeks of insertion in rabbit bone. The control c.p. titanium implants had an oxide thickness of 17-200 nm while the test implants revealed an oxide thickness between 600 and 1000 nm. Routine histological investigations of the tissue reactions around the implants and enzyme histochemical detections of alkaline and acid phosphatase activities demonstrated similar findings around both the control and test implants. In general, the histomorphometrical parameters (bone to implant contact and newly formed bone) revealed significant quantitative differences between the control and test implants. The test implants demonstrated a greater bone response histomorphometrically than control implants and the osteoconductivity was more pronounced around the test implant surfaces. The parameters that differed between the implant surfaces, i.e. the oxide thickness, the pore size distribution, the porosity and the crystallinity of the surface oxides may represent factors that have an influence on the histomorphometrical results indicated by a stronger bone tissue response to the test implant surfaces, with an oxide thickness of more than 600 nm.

Pathogenesis of in-stent restenosis remains poorly understood because information from human histopathologic studies is scarce. We used an improved saw-grinding and cutting method on methacrylate-embedded samples containing metal stents, which allows in situ hybridization and immunohistochemical analysis of in-stent restenosis. Twenty-one samples were collected 3 hours to 3 years after stenting from 6 patients aged 36 to 81 years. Except in very early samples collected within hours after the stent deployment, neovascularization was present in all segments studied. At advanced stages, extensive neovascularization was located mainly at the luminal side of the stent struts and was only rarely accompanied by inflammatory cells. The neovessels colocalized with vascular endothelial growth factor (VEGF)-A mRNA and protein expression as well as with iron deposits and oxidation-specific epitopes, which imply the presence of chronic oxidative stress. VEGF-A expression was detected in the same areas containing macrophages, endothelial cells, and, to a lesser extent, smooth muscle cells, which also showed platelet-derived growth factor-BB expression. We conclude that in-stent restenosis features neovascularization, VEGF-A and platelet-derived growth factor-BB expression, and iron deposition, which is most probably derived from microhemorrhages. These mechanisms may play an important role in the development of neointimal thickening and could provide useful targets for the prevention and treatment of in-stent restenosis.

Introduction: The salivary duct carcinoma (sdc) represents a rare variant of the group of adeno-carcinomas of the salivary glands. Histopathologically, it is marked by solid and cribriform cell nests with central necrosis, displaying distinct similarity with the ductal carcinoma of the breast, where prognosis can be correlated with Her-2 gene-amplification. Based on this histopathological similarity, the prognostic value of Her-2 gene amplification in SDC was examined in the presented pilot-study., Patients and Methods: Four own patients with different clinical courses were examined in regard to their histopathological features, Her-2 gene-amplification and proliferation (Ki67)., Results: Three of the four patients died tumor related 2.4, 5.5 and 8.2 years after initial diagnosis. The remaining patient died tumor-free 6 year after diagnosis (myocardial infarct). The two patients with an early recurrent disease and distant metastasis showed a high Her-2 expression and proliferation (Ki67), compared to the other two patients., Conclusion: In the presented pilot-study a distinct correlation between Her2-gene-amplification, proliferation (Ki67) and clinical course could be observed. Additional analysis to evaluate this aspect seems rectified, especially under recognition of therapy decisions.

Bone tissue repeatedly formed in titanium 6-aluminum 4-vanadium rabbit bone harvest implants was collected in vivo at various times between 12 days and 5 weeks. Qualitative and quantitative examinations on undecalcified thin sections were performed in the light microscope. The amount of bone tissue was calculated on routinely stained sections. Alkaline (ALP) and acid phosphatase (ACP) enzyme activities were investigated. We also performed immunohistological detection of bone matrix proteins. Increasing bone density as well as an increasing mineralization of the tissue was observed in the biopsies with increasing time. The ALP and ACP activities were similar at short times (12 days and 2 weeks). With increasing time the ALP activity was stronger than that of ACP. The results from the immunohistochemical detection of osteonectin, osteopontin, bone sialoprotein, and collagen I and II demonstrated changes in the tissue differentiation with time. The tissue formation in the canal became more mature with time of ingrowth, as observed with the various techniques used in this study. Owing to these methodical developments, undecalcified ground sections may be used for detailed analysis of various phases of tissue formation in close proximity to implants., (Copyright 2000 John Wiley & Sons, Inc.)

Extraoral implants are used increasingly frequently in the wake of ablative tumor surgery and adjuvant radiation or chemotherapy for craniofacial rehabilitation with facial prostheses and epitheses. However, high rates of nonintegration and implant loss have been reported for extraoral implants, especially for those in the periorbital region following irradiation. This case report and corresponding histologic evaluation describe the osseointegration pattern in irradiated periorbital bone, based on the example of 3 retrieved, clinically integrated, stable titanium screw implants.

Background: Examination of the tissue surrounding retrieved implants involve routine investigations on cut and ground sections. Undecalcified sections with implants in situ are histologically stained followed by qualitative and quantitative observations of the tissue response to the implants by light microscopy., Purpose: A novel technique that allows for the accurate definition and quantification of enzymes involved in bone formation (alkaline phosphatase) and resorption (acid phosphatase) in the tissue is presented., Materials and Methods: Commercially pure titanium and titanium alloy (Ti6Al4V) implants were retrieved after 6 and 12 weeks of healing in rabbit bone. In addition, 4-week specimens from commercially pure titanium bone harvest chambers placed in rabbit bone were used. Undecalcified cut and ground sections were produced and evaluated with enzyme and immunohistochemical staining techniques., Results: The titanium implants retrieved after 6 weeks of insertion in rabbit bone revealed a higher activity of both alkaline phosphatase and acid phosphatase activity compared to the implants followed for 12 months. The former samples revealed ongoing bone-tissue remodeling in the interface, whereas the latter ones showed steady-state bone conditions. Applying the new technique allowed for investigation of various bone proteins present in the tissue that had formed inside titanium canals of harvest chambers at various times of follow-up., Conclusions: The combination of routine histologic stainings with enzyme and immunohistochemical technique of cut and ground specimens is a valuable tool in the investigations of retrieved implants from humans and animals. This novel technique now may be used to describe the state of bone regeneration in the interface zone associated with implant research.

The composition of collagen was analyzed and the degree of lysyl hydroxylation of individual collagen chains was determined in four osteosarcomas and two osteofibrous dysplasias. In addition, the tumor proliferation (number of mitoses, proliferating-nuclear-antigen-positive cells, MIB) as well as the response to chemotherapy (morphological regression grade) were checked. All tumors contained a high proportion of collagen III and, in all but one osteosarcoma, pepsin-extracted collagens I and III were overmodified. Furthermore, the proportion of diglycosides in collagen I was about four times higher than in controls. The collagen composition and modification resembled those of bones at early stages of human development. One osteosarcoma and both osteofibrous dysplasias were in the normal range of lysyl hydroxylation. There was no correlation between the collagen properties and the histopathological marker of tumor proliferation.

Whole-mount electron microscopy was used in conjunction with immunogold labeling to characterize the cytoskeleton architecture of C6 rat glioma cell subclones. These subclones differ in intermediate filament (IF) protein composition and either contain vimentin (subclone C6D8) or do not express any of the known cytoplasmic IF proteins (subclone C6D10) (Röser et al., 1991). In C6D8 cells short thin (3 nm) connecting filaments frequently linked vimentin to actin filaments and, in addition, connected vimentin filaments to each other. Occasionally, direct contacts were noticed between actin and vimentin filaments. Thin connecting filaments were present at a significantly higher number in IF-deficient C6D10 cells, forming a dense cytoplasmic network in conjunction with actin filament bundles as the dominating structure. The data indicate that thin connecting filaments are present in C6 cells independent of the expression of cytoplasmic IF proteins. They suggest that structural linkages between vimentin and actin filaments mediated by thin connecting filaments could play a major role in determining the cytoskeleton architecture of these cells.

Kashin-Beck disease is an acquired, chronic and degenerative osteoarticular disorder. Selenium deficiency and fulvic acid in drinking water have been implicated in the cause of this disease. Pathologically, chondronecrosis of the growth plate and articular cartilage and subconsequent disturbance of ossification were observed in the joints. In this animal model study, mice were fed with a selenium deficient diet and fulvic acid supplemented drinking water for two generations. In undecalcified histological preparations of bone we carried out histological staining to detect mineralized and unmineralized bone and cartilage. The results revealed that selenium deficiency and fulvic acid supplementation induced degeneration of the articular cartilage in the knee joints of mice. Dynamic fluorescent labelling of ossification, enzyme histochemical detection of alkaline phosphatase activity in osteoblasts and a typical immunohistochemical localization of collagens type I and II indicated the development of fibrocartilage at the articular surface of knee joints, resembling the early stages of osteoarthrosis. This became obvious by disturbed development of the articular space and meniscus, markedly impaired formation of subchondral bone and early differentiation failure during enchondral ossification. This animal model provides an approach to study the molecular pathogenesis of Kashin-Beck disease.

A simplified method of low temperature methyl and butyl methacrylate embedding (up -20 degrees to -15 degrees C) is demonstrated using a proper redox system of benzoyl peroxide and aromatic amine. This method combines the morphological superiority of plastic-embedded bone tissue and bone marrow sections with the advantages of specific enzyme histochemical and immunochemical markers. The method permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activities. The specimens were fixed in 1.6% formaldehyde and 5% sucrose in 0.02 M phosphate buffer at pH 7.4, washed in 0.02 M phosphate buffer and 5% sucrose, dehydrated with acetone and impregnated with monomers of embedding medium. All these steps were carried out at +4 degrees C. The method presented is especially suitable for enzyme histological and immunohistological diagnosis of primary and secondary bone tumours, soft tissue tumours, as well as myelo- and lymphoproliferative disorders of bone marrow biopsies. Examples are demonstrated with mono- and polyclonal antibodies and reaction products of hydrolytic enzymes.

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.