Culture of primary human gingival fibroblasts on biodegradable membranes.

Repair and regeneration of periodontal tissues by tissue engineering is dependent on the use of biodegradable polymer scaffolds which serve as a carrier for cells or bioactive substances. There is a need to understand how a specific biomaterial may influence gene expression. The aim of this investigation was to develop and to optimize an in vitro technique for the adherance and proliferation of primary human gingivaL cells on implantable and biodegradable matrices. Square pieces of Bio-Gide matrix (BG) and slices of Ethisorb tamponade (ET) were coated with poly-L-lactide. The stability of coated and uncoated scaffolds was investigated by incubation in standard culture medium. Various concentrations of the cells were seeded onto coated and uncoated polymer matrices in tissue culture dishes without shaking ("static seeding") or continuous shaking ("agitated seeding"). Cultures were grown for 4 week and were then evaluated by light and scanning electron microscopy. After a culture period of 10 d, BG-carriers showed a delicate consistency which made histological processing difficult. Cells were grown only sparsely in coated and non-coated BG-scaffolds. Contrary. ET-specimens were stable during a 4 week culture period. After "static seeding" a significantly higher number of cells resulted in comparison to those in "agitated" cultures. The cells were evenly distributed throughout the ET-carriers and produced extracellular matrix compounds as well. Furthermore, the examination with RT-PCR (reverse transcription-polymerase chain reaction) revealed that the cells synthesized and secreted type I collagen, and expressed genes implicated in transducing bone morphogenetic protein (BMP) signals. Messenger RNAs for BMP-2, -4, -7, the BMP type I receptors Act R-1 (alk 2, activin-like kinase receptor), BMPR-IA (alk 3), -IB (alk 6), and the type II receptor BMPR-II were detected. These data reveal that static seeding favors the adherence and proliferation of primary gingival cells on polyglactin matrices. This system may serve as a valuable tool for periodontal tissue engineering.

Immunohistological and morphometric analysis of inflammatory cells in rapidly progressive periodontitis and adult periodontitis.

The purpose of this study was to localize, characterize, and quantify in situ the inflammatory cells in the gingival connective tissue prior and subsequent to the initial therapy of ten patients with rapidly progressive periodontitis (RPP) and five patients with adult periodontitis (AP). Using immunohistological techniques, the amount of T lymphocytes, alphabeta-T lymphocytes, gammadelta-T lymphocytes, B lymphocytes, and plasma cells was determined at the beginning of the periodontal therapy (baseline) and at the time of periodontal surgery. Furthermore, the distribution of collagen types I, III, V, and VI was investigated using transmission electron microscopy. At baseline, patients with RPP revealed much higher numbers of inflammatory cells than patients with AP. During initial therapy of patients with RPP, the amount of T cells, alphabeta-T cells, and gammadelta-T cells was reduced significantly (P<0.05). Biopsies of patients with AP revealed a statistically significant reduction of all cell types, except alphabeta-T cells and gammadelta-T cells in the deep connective tissue. The transmission electron microscopy of biopsies from patients with RPP and AP with severe inflammation taken at baseline revealed that collagen types I and III were destroyed nearly completely in areas with leukocyte infiltration, whereas collagen types V and VI revealed a more pronounced labeling reaction. The results revealed that, during initial therapy, the amount of inflammatory cells was reduced significantly more in biopsies of patients with AP than in patients with RPP. At baseline, the inflamed gingival tissue consists mainly of collagen types V and VI in areas with infiltrates of inflammatory cells.

Perspectives in the biological function, the technical and therapeutic application of bone morphogenetic proteins.

The bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of growth and differentiation factors and have been characterized by their ability to induce new bone formation in ectopic (non-skeletal) sites. BMPs are secreted molecules and are key regulators in early embryogenesis and organogenesis. One of the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in vertebrates. This osteo-inductive capacity of BMPs has long been considered very promising for applications in bone repair, in the treatment of skeletal diseases, and in oral applications such as dentiogenesis and cementogenesis during regeneration of periodontal wounds. We discuss here biological roles of the BMPs in the organism and their signaling cascades leading to bone and cartilage formation in particular. It is also the aim of this review to evaluate the potential and the problems of BMPs in skeletal tissue engineering for the regeneration of bone damaged by disease or trauma and to serve as therapeutic agents for periodontal defects.

Infection of primary human gingival fibroblasts by Porphyromonas gingivalis and Prevotella intermedia.

Adhesion and penetration of clinical isolates of Porphyromonas gingivalis and Prevotella intermedia in human gingival fibroblast monolayers were studied by transmission electron microscopy (TEM). Fibroblasts were cultured from biopsies of human healthy gingiva. Porphyromonas gingivalis and Prevotella intermedia were isolated from patients with periodontitis. Fibroblasts were incubated with microorganisms in an antibiotic-free medium for 24 h. Then cultures were washed to remove nonadherent bacteria. Consecutively, infected cultures were grown for another 24 h. Thereafter, the treated monolayers were prepared for TEM investigations. Internalized Porphyromonas gingivalis and Prevotella intermedia were visible after 24 h of incubation. Prevotella intermedia showed only division in cytoplasm of fibroblasts after 24 h and 48 h incubations. Infected fibroblasts revealed various morphological alterations such as extensive vacuolization and breakdown of mitochondria. These findings demonstrate that Porphyromonas gingivalis and Prevotella intermedia may invade human gingival fibroblasts and thus may damage these cells directly or due to the release of microbial cytotoxic components.

Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model.

The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques. The results were compared with monolayer cultures. Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy. Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions. The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation. Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system. Frequently, the fibers were oriented parallel to the long axis of the cells. Type VI collagen formed thin fibers and revealed a reticular pattern. In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers. Their shape and spatial distribution resembled that of cells in tissue biopsies more closely. The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information.

Histopathological investigation of gingival tissue from patients with rapidly progressive periodontitis.

In this study, fine structural features of the pocket walls in rapidly progressive periodontitis (RPP) and adult periodontitis (AP) in 20 cases were compared using light and transmission electron microscopy. Gingiva was also obtained from a control group of periodontally healthy teeth. Clinical parameters were assessed in both RPP and AP patients and in controls. Bone destruction and attachment loss were more marked in RPP than in AP. Light microscopical observations of inflamed RPP tissue as compared to AP showed gross histological distortions in the pocket walls. Micro-ridges within the epithelium and large intercellular spaces between the epithelial cells were observed in most RPP biopsies. Epithelial cells surrounding the microclefts and adjacent keratinocytes were found to produce interleukin-1beta (IL-1beta). Prevotella intermedia and Porphyromonas gingivalis were identified in the RPP biopsies using immunohistological methods. These microorganisms were localized outside the epithelium and inside intercellular spaces. Furthermore, the effect of inflammation on the distribution of collagen types I, III, IV, V, and VI in the human gingiva was studied after staining them with antibodies to these proteins. In RPP and AP tissues, the staining was sparse in areas of inflammation and leukocytic infiltration. Collagen type I and III were almost entirely lost at sites of inflammation. Type V and VI collagen antibodies were retained in inflamed areas. Type IV collagen was restricted to basement membrane structures. These observations demonstrated numerous structural features indicative of more pronounced degenerative changes in RPP than in AP.

Light-microscopical investigation of the distribution of extracellular matrix molecules and calcifications in human dental pulps of various ages.

The distribution of extracellular matrix molecules, especially collagen types I, III, V, and VI, in the extracellular matrix of the connective tissue of human dental pulp of various ages was studied by polarization and indirect immunofluorescence microscopy by using a conventional fluorescence microscope and a confocal laser scanning microscope. Polarization and immunofluorescence microscopy of paraffin sections showed thick fibers of collagen type I, which represented the main component of the connective tissue matrix of the dental pulp. By indirect immunofluorescence, thin fibers and small bundles of collagen type III were determined to be one of the main fibrillar elements present in the dental pulp matrix. Collagen type IV was detected by a clear intense staining of the basement membrane of blood vessels at all ages examined. Collagens type V and VI formed a dense meshwork of thin microfibrils throughout the stroma of the connective tissue of the dental pulp. These fibers were localized around blood vessels and appeared to be enriched in the subodontoblastic layer. Investigations by means of confocal laser scanning microscopy revealed fibers of collagen type VI spiralling between fully differentiated odontoblasts toward the predentin layer. With advancing age, the connective tissue matrix appeared to be condensed and aggregates of thick fiber bundles could be observed. Furthermore, the participation of various collagen types in the composition of pulp stones was shown. These calcifications and diffuse calcifications increased in frequency with advancing age in a statistically significant manner.

Pathohistology of undecalcified primary teeth in vitamin D-resistant rickets: review and report of two cases.

The basic dental defects in vitamin D-resistant rickets seem to be manifested in dentin. Enamel is usually reported to be normal. This histologic examination showed the penetration of microorganisms through the calcified structures of the enamel layer without visible caries. The microorganisms passed through the dentinoenamel junction and invaded dentin, which was characterized by calcospherites and large amounts of interglobular dentin. Furthermore, microorganisms could be detected in dentinal tubules, which were exposed to the oral cavity when enamel was removed. However, large areas of tertiary dentin extended between such tubules and the pulp. These light microscopic results suggest that clinical manifestations, such as, pulp recrosis and periapical lesions (without carious defects) may be caused by the penetration of microorganisms through microclefts of the enamel layer as well as pathologically altered enamel microstructures of affected teeth.

Clinical course of hypophosphatemic rickets in 23 adults.

Twenty-three adult patients (19 females, 4 males) with x-linked hypophosphatemic rickets (HPR) underwent a retrospective evaluation of the clinical course and a clinical examination by a nephrologist, orthopedic surgeon and dentist. Blood and urine analysis, bone density measurements with QCT and DEXA, ultrasonic examination of the kidneys were performed and the patients were asked to fill in a standardized questionnaire on pain and psychosocial rehabilitation. Mean final height was 152.4 cm +/- 8.5 SD in females and 157.3 cm +/- 8.9 SD in males. Decreased joint mobility was seen in all patients, deviations of the normal leg axis in 18/23 patients in spite of 69 correcting osteotomies in the past. Dental (n = 14) and psychosocial problems were associated with inability to work (n = 8). There was a trend that patients with a very low Tp/GFR had a more severe course of the disease. Early therapy with vitamin D metabolites and phosphate had a beneficial effect on growth, bone density and deformations. Eight patients had nephrocalcinosis due to vitamin D and phosphate therapy and had normal kidney function. Four patients had urinary tract abnormalities. We conclude that patients with HPR should receive continuous interdisciplinary care given by nephrologists, orthopedic surgeons, physiotherapists and dentists not only during childhood but also as adults.

Immunohistological determination of interleukin-1 beta in inflamed human gingival epithelium.

Interleukin-1 beta (Il-1 beta) is the predominant form of Il-1 produced by monocytes of the blood and by macrophages of various tissues. Human keratinocytes express both Il-1 alpha and Il-1 beta mRNA, but appear to produce mainly Il-1 alpha. The aim of this study was to determine the localization of interleukin-1 beta and interleukin-1 beta receptors in human gingival epithelium by different immunohistochemical methods. The alkaline phosphatase and the immunogold staining technique, as well as fluorescence microscopy, were used to investigate active participation of gingival epithelial cells in the development of periodontitis. Biopsies of human interdental papillae showed some activity of epithelial cells in the production of Il-1 beta. Single cells, clusters or larger areas of the sulcular and oral epithelium appeared to produce Il-1 beta at inflamed sites, and in these areas the normal epithelial structure was disturbed. Epithelial cells grown from the same biopsies appear able to express specific receptor molecules for Il-1 beta under normal culture conditions. It is concluded that gingival keratinocytes might be activated by inflammatory irritants and participate actively in the inflammatory processes.

Enzyme, lectin and immunohistochemistry of plastic embedded undecalcified bone and other hard tissues for light microscopic investigations.

Enzymes and tissue antigens were localized on plastic embedded undecalcified bones and teeth using Technovit 7200 VLC (Kulzer, Germany). This resin is hard enough for cutting and grinding procedures on rotating plates with diamond layers. The pores between the diamond grains are not obstructed with this resin. The procedure described here permits localization of antigens in the soft tissues adjacent to, or in the biological hard tissues themselves and in dental implants (ceramic or metallic) on the light microscopic level. The undecalcified bone is fixed and embedded in plastic and cut at 100-150 microns. The slices are ground automatically by a grinding machine to a thickness of 5-10 microns. After application of the substrates for alkaline and acid phosphatases and the required dyes, the distribution of these enzymes can be demonstrated. Tissue antigens also can be detected with slightly modified standard techniques of immunohistochemistry and lectin histochemistry using the peroxidase technique or fluorescence microscopy.

[Clinical course, histology and prognostic assessment of odontomas]. / Zur Klinik, Histologie und prognostischen Beurteilung der Odontome.

In our opinion ameloblastic fibro-odontoma seems to be a true neoplasia. Complex composite odontomas and compound composite odontomas represent hamartomatous malformations. The ameloblastic fibro-odontoma seems not to differentiate into a complex composite odontoma. Since ameloblastic fibro-odontomas bear great resemblance to common odontomas, it is suggested that all odontomas be sent to a qualified oral pathologist for microscopic examination.

[Development of plaque-induced gingival pockets in an animal experiment]. / Die Entstehung plaquebedingter Gingivataschen im Tiermodell.

Single teeth and resected jaw specimens including periodontal tissue were prepared as saw-cut thin-ground specimens without previous decalcification. The specimens were classified into 4 stages of pathogenesis to allow studies into the mechanisms of gingival pocket formation. Examination of the preparations under the light microscope indicated that the pathologic gingival pockets caused by microbial plaque might be formed by degenerative alterations in the second or third innermost cell layer of the junctional epithelium. The intercellular contacts were dissolved and, thus, cyst-like cavities were formed within the epithelium. The contact between tooth enamel/cementum--basal membrane--epithelial cells, however, was maintained. Artifacts could be ruled out by comparing preparations of single teeth with those of entire jaw segments.

Effect of mitotic inhibitors on the ultrastructure of root meristem cells.

After 5 h in 10(-3)M vinblastine the most obvious effects upon Vicia faba L. cells are seen in the spindle apparatus. These include the microtubules themselves as indicated by c-type metaphases and the pole regions of otherwise intact spindles, leading to multipolar anaphases and to telophases with more than two daughter nuclei. Vesicle transport may be undisturbed and new cell walls can be formed, although cases of disturbed cell plate and cell wall formation were noted occasionally, accompanied by myelinizations in phragmosomes. After 24 h in the same concentration of vinblastine, divisions are no longer observed and the plasma membranes are severely affected. They show extensive myelinizations, accumulations of lipids and dehiscence from the cell walls which are frequently thickened and irregularly formed. Of the other cellular membranes, the nuclear envelope and, more frequently, the tonoplast may be affected. Electron-dense deposits appear in the vacuoles. Comparable, though less severe, changes including multipolar anaphases and myelinizations result from treatment with lumicolchicine, but not with colchicine. Vinblastine leads to the appearance of filament bundles both in cytoplasm and karyoplasm, lumicolchicine to morphologically identical filaments in the cytoplasm alone. The nature of these filaments is unknown.

[Report on 4055 cataract operations in a department with treatment by non-resident physicians, with special consideration of anesthesia problems (author's transl)]. / Bericht über 4055 Kataraktoperationen auf einer Belegarztabteilung - mit besonderer Berücksichtigung der Anästhesie.

In addition to eye clinics and ophthalmic wards under the supervision of a senior hospital surgeon, wards where patients are treated by their own, non-resident physicians, play an important part in the treatment of eye conditions in Germany. This is a very old tradition which was introduced by von Graefe. Especially in the present situation, now that the number of hospital beds is no longer being increased without very careful thought, there is much talk about the efficiency of differently run wards. In such discussions ophthalmic wards with treatment by non-resident physicians are also being subjected to critical examination. While reports from eye clinics and ophthalmic wards under the management of hospital surgeons frequently appear on the various journals and at conferences, information on wards where beds are available to non-resident physicians is rather scanty. Yet a large proportion of in-patient ophthalmic treatment is administered in such wards. This report covers more than 20 years' experience in a ward of this kind in a large West German city, taking cataract operations as an example.