RESUMEN
In a series of experimental studies, the bone formation around systematically modified titanium implants is analyzed. In the present study, three different surface modifications were prepared and evaluated. Glow-discharge cleaning and oxidizing resulted in a highly stoichiometric TiO2 surface, while a glow-discharge treatment in nitrogen gas resulted in implants with essentially a surface of titanium nitride, covered with a very thin titanium oxide. Finally, hydrogen peroxide treatment of implants resulted in an almost stoichiometric TiO2, rich in hydroxyl groups on the surface. Machined commercially pure titanium implants served as controls. Scanning Auger Electron Spectroscopy, Scanning Electron Microscopy, and Atomic Force Microscopy revealed no significant differences in oxide thickness or surface roughness parameters, but differences in the surface chemical composition and apparent topography were observed. After surface preparation, the implants were inserted in cortical bone of rabbits and evaluated after 1, 3, and 6 weeks. Light microscopic evaluation of the tissue response showed that all implants were in contact with bone and had a large proportion of newly formed bone within the threads after 6 weeks. There were no morphological differences between the four groups. Our study shows that a high degree of bone contact and bone formation can be achieved with titanium implants of different surface composition and topography.
RESUMEN
OBJECTIVE: Proinflammatory cytokines are known to affect the follicular epithelium in autoimmune thyroid disease. Here we investigated the effect of interferon-gamma (IFN-gamma) on the barrier function of primary cultured human thyrocytes. DESIGN: Graves' thyroid follicle segments were cultured as a tight and polarised monolayer on the filter of a bicameral chamber, thereby allowing the in vivo epithelial characteristics to be maintained. METHODS: Transepithelial electrical resistance was measured with a Millicell ERS ohmmeter. The tight junction proteins claudin-1 and occludin were analysed by immunofluorescence and Western blotting. Cell morphology was studied by transmission electron microscopy. RESULTS: Thyrotrophin (TSH; 1 mU/ml) promoted the development of a tight epithelium monitored as a persistent increase in the transepithelial resistance to about 800 omega x cm2. IFN-gamma (100 U/ml), on the other hand, decreased the resistance to 60-150 omega x cm2 after 48 h. In IFN-gamma-treated cells the expression of claudin-1, but not that of occludin, was decreased along with a diminished intracellular and cell surface immunostaining. In addition, claudin-1 was disrupted at cell-cell contacts. IFN-gamma also caused profound cell shape changes and a multilayered cellular organisation, without ultrastructural or biochemical (caspase-3 activity) signs of cytotoxicity. TSH was unable to counteract the effects of IFN-gamma. CONCLUSIONS: IFN-gamma destroys the barrier function of filter-cultured human thyroid epithelial cells. The loss of barrier involves down-regulation and an altered distribution of claudin-1. This novel effect of IFN-gamma on target cells in thyroid autoimmunity might be of pathophysiological relevance to the exposure of hidden autoantigens.
Asunto(s)
Enfermedad de Graves/metabolismo , Interferón gamma/farmacología , Proteínas de la Membrana/biosíntesis , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/fisiología , Uniones Estrechas/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Claudina-1 , Regulación hacia Abajo/efectos de los fármacos , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Enfermedad de Graves/patología , Humanos , Inmunohistoquímica , Proteínas de la Membrana/inmunología , Microscopía Electrónica , Microscopía Fluorescente , Ocludina , Proteínas Recombinantes , Glándula Tiroides/metabolismo , Tirotropina/inmunología , Uniones Estrechas/metabolismo , Uniones Estrechas/fisiologíaRESUMEN
The determination of secreted levels of reactive oxygen species by implant-adherent cells in vivo is required for understanding of the role(s) of such reactive oxygen species for the tissue response around medical devices. A model with subcutaneous implants of c.p. titanium (Ti) or polystyrene (PS) (cell culture grade) inserted on the back of rats were used. Implants and associated cells were retrieved and assayed after 1, 3, 5, 7, 14, 21 and 28 days. Morphological analysis of exudate cells showed that polymorphonuclear leukocytes (PMN) predominated after one day whereas macrophages were predominant after three days. The number of implant-adherent cells, as reflected by measurement of DNA, decreased with time. Ultrastructural observations showed that macrophages were predominant cells in contact with the implant surface. Measurement of hydrogen peroxide (H(2)O(2)) secretion by implant-adherent cells during 40 min incubation ex vivo revealed a constitutive generation of 40-400 pmol H(2)O(2)/microg DNA, depending on implantation time. Stimulation with protein kinase C agonist phorbol myristate acetate (PMA) caused an increased H(2)O(2) generation by adherent cells at early (up to five days) but not later (7-28 days) time periods. No major differences between Ti and PS were observed. Taken together, these findings show that Ti and PS implant-adherent cells secrete H(2)O(2) under in vivo conditions. Further, a reduced capacity to mount an enhanced H(2)O(2) secretion upon stimulation was demonstrated at late time periods. The role of this mediator for biocompatibility remains to be established.
RESUMEN
The aim of the present experimental study was to evaluate the tissue response to hafnium (Hf) a reactive metal closely related to titanium (Ti) and zirconium (Zr). Hf has not been previously evaluated as implant material in a biologic environment. In a first experiment, 21 machined Hf non-threaded implants (test) and 21 similar Ti implants (control) were inserted in the abdominal wall of 21 rats. Animals were sacrificed after 8 days (6 rats), 6 (7 rats) and 12 weeks (8 rats). In a second experiment, 18 rabbits received 18 Hf and 18 Ti threaded implants in their tibiae, one implant in each tibia. The rabbits were sacrificed after 6, 12 and 24 weeks (6 animals/time interval). The bulk metal of the abdominal wall implants, embedded together with the surrounding tissue, was electrolytically dissolved and semithin (1 microm) sections of the intact tissue-implant interface were evaluated by light microscopy (morphometry). Bone-implant contact and bone area within threads were evaluated in ground sections. In soft tissues, a fluid space containing predominantly monocytes/macrophages surrounded the abdominal implants at 8 days. At 6 and 12 weeks, a fibrous capsule, consisting of layers of macrophages and fibroblasts, surrounded the implants. Macrophages, including multinuclear giant cells, always formed the innermost layer in contact with the implant surface. No quantitative or qualitative difference in the tissue organization was detected between Ti and Hf implants. In rabbits, 6 weeks after insertion, the proximal two threads located within the cortical bone were filled with bone in contact with Hf and Ti. The distal threads contained bone marrow. After 12 and 24 weeks, mature bone was present in the proximal 3-4 implant threads. No statistically significant difference was found between Hf and Ti implants at any time periods. It is concluded that Hf is an interesting metal for biomedical applications in bone and soft tissue.
RESUMEN
Thyrotropin [thyroid-stimulating hormone (TSH)] receptor on-off signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and potential difference (PD) were used as parameters to monitor the effect of altered TSH concentrations on vectorial electrolyte transport. TSH induced rapid but long-lasting changes in R (decrease) and PD (increase) that were cAMP-dependent and related to enhanced transcellular conductance of sodium and chloride. Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for 48 h resulted in restitution of R to control level within 30 min. Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the TSH concentration (0.01-0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface trypsinization. The observations indicate that, during continuous exposure to physiological concentrations, TSH exerts a close minute-to-minute surveillance of thyroid function and the rate-limiting step of deactivation is the dissociation of ligand from the TSH receptor at the cell surface. TSH-deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction.
Asunto(s)
Células Epiteliales/fisiología , Receptores de Tirotropina/fisiología , Transducción de Señal/fisiología , Glándula Tiroides/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Amilorida/farmacología , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/fisiología , Diuréticos/farmacología , Electrólitos/metabolismo , Filtración , Furosemida/farmacología , Cinética , Porcinos , Glándula Tiroides/citología , Tripsina/farmacologíaRESUMEN
BACKGROUND: Despite good success rates of osseointegrated oral implants, failures do occur. To minimize losses, failure mechanisms should be elucidated. PURPOSE: This study sought to describe the morphology of tissues surrounding late failed Brånemark implants in relation to their clinical and radiographic findings to acquire a better understanding of the etiologic factors. MATERIAL AND METHODS: Ten failed implants and their surrounding tissues were consecutively retrieved from nine patients after prosthesis placement (late losses). On radiographs, a radiolucent line was visible around nine clinically mobile implants. Tightening of the abutment screw evoked pain at seven mobile implants. Clinically, no other visual inflammatory sign or symptom was manifest. A fistula originated from one stable implant, surrounded on radiographs by a diffuse bone rarefaction. Retrieved implants were electrochemically dissolved. Intact tissue-implant thin (1 micron) and ultrathin (70-80 nm) sections were analyzed with light and transmission electron microscopy. RESULTS: Peri-implant marginal tissues displayed moderate inflammatory infiltrates located adjacent to and beneath the junctional epithelium. One patient affected by oral lichen planus displayed an intense lymphocyte/plasma cell-dominated immune reaction. Deep peri-implant tissues surrounding mobile implants consisted of a dense, fibrous tissue capsule with minimal inflammation. Epithelial downgrowth was observed around four implants. Small areas of nonmineralized bone in contact with the implant were noticed in the apical portion of two implants. One implant was almost entirely colonized by bacterial plaque with the exception of its apical portion, where bone-implant contact was observed. The stable implant was characterized by bone-implant contact. CONCLUSION: Altogether clinical, radiographic, and histologic findings indicated that two major etiologic factors might have been implicated in the failure process of the investigated implants: excessive occlusal load in relation to the bone-supporting capacity and, in two cases, infection.
Asunto(s)
Implantes Dentales , Fracaso de la Restauración Dental , Mandíbula/patología , Maxilar/patología , Periodoncio/patología , Anciano , Fuerza de la Mordida , Pilares Dentales , Fístula Dental/patología , Placa Dental/microbiología , Diseño de Prótesis Dental , Retención de Prótesis Dentales , Electroquímica , Epitelio/patología , Femenino , Fibrosis , Humanos , Liquen Plano Oral/patología , Masculino , Mandíbula/diagnóstico por imagen , Maxilar/diagnóstico por imagen , Microscopía Electrónica , Persona de Mediana Edad , Oseointegración , Periodontitis/patología , Periodoncio/diagnóstico por imagen , Radiografía , Factores de Tiempo , Insuficiencia del TratamientoRESUMEN
The purpose of this study was to morphologically describe the tissues surrounding 20 early failed (prior to prosthesis placement) Brånemark System oral implants. The implants and their surrounding tissues were consecutively retrieved and analyzed with light microscopy and transmission electron microscopy. Failures were chronologically divided into those occurring prior to, at, and after abutment connection. The clinical conditions varied from osteomyelitis to totally asymptomatic but mobile implants. Different histopathologic pictures were observed, ranging from a stratified, almost acellular, connective tissue layer, via a capsule with a great number of inflammatory cells, to a heterogeneous interface with areas of highly vascularized connective tissue and portions of poorly mineralized bone detached from the implant surface. The histopathologic variation may reflect different etiologies and/or time stages of the failure process. Epithelial downgrowth was occasionally observed for asymptomatic submerged implants. Epithelial cells were attached to the failed implant surface via hemidesmosomes. The histologic, clinical, and radiographic findings together indicated that 3 major etiologies might have been implicated in the failure processes: impaired healing ability of the host bone site, disruption of a weak bone-to-implant interface after abutment connection, and infection in situations with complicated surgery.
Asunto(s)
Implantación Dental Endoósea/efectos adversos , Implantes Dentales/efectos adversos , Fracaso de la Restauración Dental , Periodontitis/patología , Adulto , Anciano , Pérdida de Hueso Alveolar/etiología , Desmosomas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oseointegración , Periodontitis/etiología , Periodoncio/patología , Infecciones Relacionadas con Prótesis/patología , Dehiscencia de la Herida Operatoria , Infección de la Herida Quirúrgica/etiologíaRESUMEN
The early healing phase of hard tissue implants is important to their long-term success. Problems during this phase can result in a so-called primary biological failure. In 24 New Zealand white rabbits, the healing in cortical bone of noncoated TiAlV and cpTi cylinders and of TiAlV cylinders plasma-spray-coated with hydroxyapatite (HA) of fluorapatite (FA) was investigated histologically and histomorphometrically after 3, 7, 14, and 28 days. Histomorphometry consisted of bone contact measurements and the use of a new semi quantitative scoring system that discriminated various tissues in contact with the implant. The results demonstrated that the most important parameter in initial implant healing is the bone itself and not the characteristics of the implanted material. For all implants, healing was characterized by a sequence of hematoma formation, bone resorption, and new bone formation where the initial press-fit situation revealed more bone-implant contact than after 7 and 14 days. There were only minor differences between the implant types: the new bone formation directly on the implant surface was qualitatively histologically superior to the CaP-coated implants, but this could be confirmed with the scoring method only for the HA-coated implants. It is concluded that initial press-fit fixation in cortical bones is not an end situation; rather, what happens is that as a result of interface remodeling, early postoperatively implant integration in the bone will decrease temporarily prior to a subsequent phase of new bone formation.
Asunto(s)
Huesos/anatomía & histología , Prótesis e Implantes , Animales , Femenino , Cinética , Masculino , Oseointegración , ConejosRESUMEN
A commonly accepted hypothesis is that acute pharyngotonsillitis is caused by bacteria which first adhere to the epithelial surface and then invade the tonsillar parenchyma; however, evidence directly supporting this hypothesis is not available. In previous studies on acute pharyngotonsillitis, we found that the secretion in crypts and at the surface was infected in acute pharyngotonsillitis while no bacteria were detected in the parenchyma. Based on these results, we have proposed a new hypothesis stating that the infection is restricted to the crypt and surface secretions in acute pharyngotonsillitis. To evaluate this hypothesis further, in the present study we examined tonsillar tissue and secretion from patients with acute pharyngotonsillitis, recurrent pharyngotonsillitis and healthy tonsils. Surface secretion was studied after sampling by an imprint technique followed by routine histological preparation. Tonsillar tissue was examined by fluorescence microscopy after staining with acridine orange and by transmission electron microscopy. There were high numbers of bacteria and moderate or extensive ongoing phagocytosis in the crypt and surface secretion from patients with acute pharyngotonsillitis. Bacteria, leucocytes and phagocytosis were also present, but to less extent in the secretion from patients with recurrent pharyngotonsillitis and to even less extent in the healthy controls. In none of all the investigated tonsils were bacteria present in the parenchyma. Bacterial adherence to the epithelial surface was only very rarely observed. This study supports the hypothesis that acute pharyngotonsillitis is an infection restricted to the crypt and surface secretion and that bacterial adherence is not of significant importance in the pathogenesis of acute pharyngotonsillitis.
Asunto(s)
Tonsila Faríngea/microbiología , Exudados y Transudados/microbiología , Faringitis/microbiología , Tonsilitis/microbiología , Tonsila Faríngea/inmunología , Tonsila Faríngea/ultraestructura , Bacterias/aislamiento & purificación , Bacterias/ultraestructura , Exudados y Transudados/inmunología , Humanos , Recuento de Leucocitos , Microscopía Electrónica , Microscopía Fluorescente , Neutrófilos/citología , Neutrófilos/ultraestructura , Faringitis/inmunología , Tonsilitis/inmunologíaRESUMEN
Locally produced proinflammatory cytokines are likely to play a pathophysiological role in autoimmune thyroid disease. An important feature of the thyroid, not previously considered in cytokine actions, is the barrier created by the follicular epithelium, which secludes two lumenal autoantigens [thyroglobulin (Tg) and thyroperoxidase] from the extrafollicular space. We examined the influence of recombinant cytokines on the barrier function of human thyrocytes cultured as a tight and polarized monolayer in bicameral chambers. Whereas interleukin (IL)-6 (100 U/mL), interferon-gamma (100 U/mL), tumor necrosis factor-alpha (10 ng/mL), and transforming growth factor-beta1 (10 ng/mL) had no effects, exposure to IL-1alpha for 24-48 h reduced the transepithelial resistance from >1000 to <50 omega x cm2 and increased the paracellular flux of [3H]inulin and exogenous 125I-Tg. This response to IL-1alpha, which was dose dependent (1-1000 U/mL) and reversible, was accompanied by dramatic morphological changes of the epithelial junction complex, including aberrant localization of the tight junction protein zonula occludens-1. At the same time, IL-1alpha decreased the apical secretion of endogenous Tg and stimulated the basolateral release of a novel high-molecular-mass protein. We conclude that IL-1alpha reduces the thyroid epithelial barrier without signs of general cytotoxicity. The observation suggests a mechanism by which IL-1alpha may promote the exposure of hidden autoantigens to the immune system in thyroid autoimmunity.
Asunto(s)
Citocinas/fisiología , Uniones Intercelulares/efectos de los fármacos , Interleucina-1/farmacología , Glándula Tiroides/fisiología , Cadherinas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Técnicas Citológicas , Epitelio/efectos de los fármacos , Epitelio/fisiología , Humanos , Técnicas Inmunológicas , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Permeabilidad , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Distribución Tisular , Proteína de la Zonula Occludens-1RESUMEN
The bone formation around titanium implants with varied surface properties was investigated after 1 year in rabbits. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits. Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads. There were no significant differences between the differently prepared implant groups. Our study shows that a high degree of bone contact and bone formation is achieved after 1 year with titanium implants which are modified with respect to oxide thickness and surface topography. There is no indication that a reduction of surface roughness, which in the initial phase decreases the rate of bone formation, had any influence on the amount of bone after 1 year in rabbit cortical bone.
RESUMEN
The role of surface properties (chemical and structural) for the interaction between biomaterials and tissue is not yet understood. In the present study, implants made of titanium, zirconium (transition metals with surface oxides) and gold (metallic surface) were inserted into the rabbit tibia. Light microscopic (LM) morphometry showed that after 1 and 6 mo the gold implants had less amount of bone within the threads and a lower degree of bone-implant contact than the titanium and zirconium implants, which did not differ from each other. These quantitative differences were supported by LM and ultrastructural observations of the interface. The ultrastructural observations in addition demonstrated that the layer of non-collagenous amorphous material located between the implant and the calcified bone was appreciably thicker around zirconium than around titanium implants. The factors potentially responsible for the observed morphological differences in the bone around the different material surfaces are discussed.
RESUMEN
The objective of the present investigation was to characterize the cellular composition of the soft tissues surrounding consecutively retrieved late failures of Brånemark implants. Criteria for implant failure were signs of loss of osseointegration (radiographic peri-fixtural radiolucency and mobility). The clinical history of the implants did not include adverse symptoms. At the time of retrieval, percussion-induced pain was experienced at 4/8 implants, but no macroscopical signs of inflammation or infection or infection was observed. Immunohistochemistry was applied on 6 marginal peri-implant specimens and on specimens of deeper tissues associated with the previously load-bearing implant surface from 8 failed implants, whereas 6 clinically healthy mucosal specimens and 4 hyperplastic biopsies from stable implants served as controls. The immunohistochemical evaluation showed that the soft tissues surrounding failed implants contained a large number of macrophages (CD68), HLA-DR positive cells, lymphocytes and plasma cells preferentially accumulated towards the removed implant surface. PMNs were a rare finding. Downgrowth of epithelium, in some cases encapsulating the whole fixture, was observed in sections where an intact implant/soft tissue interface was preserved. Healthy control mucosal specimens always contained labelled cells, albeit in a low amount, whereas hyperplastic control samples displayed an intense inflammatory and immunological response with numerous positive cells and PMNs scattered throughout the biopsy. In conclusion, failed implants were characterized by a chronic inflammatory response of the surrounding tissues with macrophages as the predominant labelled cell type, while hyperplastic tissues around stable implants were distinguished by an acute inflammatory process. These findings suggest that an on-going infection is unlikely to be the etiological factor for the late failures of dental implants examined in this study.
Asunto(s)
Implantes Dentales/efectos adversos , Fracaso de la Restauración Dental , Oseointegración/inmunología , Periodontitis/etiología , Anciano , Implantación Dental Endoósea , Retención de Prótesis Dentales , Femenino , Antígenos HLA-DR/aislamiento & purificación , Humanos , Inmunohistoquímica , Macrófagos , Masculino , Persona de Mediana Edad , Neutrófilos , Periodontitis/inmunología , Periodoncio/inmunología , Linfocitos TRESUMEN
Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4-6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200-500 omega.cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 omega.cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM-or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma stimulation for 48 h. We conclude that human thyrocytes form a tight and polarized epithelium when cultured on permeable filters. The polarized structure and function of the cells are positively regulated by TSH. The culture system may be useful in studies addressing the role of the epithelial phenotype (cell polarity and tight barrier) in normal thyroid function as well as in pathological processes in the thyroid, such as autoimmunity, cell transformation and tumor progression.
Asunto(s)
Técnicas Citológicas/instrumentación , Glándula Tiroides/citología , Tirotropina/farmacología , Polaridad Celular , Células Cultivadas , Epitelio/metabolismo , Enfermedad de Graves/inmunología , Enfermedad de Graves/patología , Antígenos HLA-DR/análisis , Humanos , Proteínas de la Membrana/metabolismo , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismoRESUMEN
Time-dependent distribution of extracellular proteins (albumin, fibrinogen, fibronectin, collagen-I and IgG) in the interface zone between implant and soft tissue has been investigated utilizing a recently developed method. Commercially pure (c.p.) titanium and polytetrafluoroethylene (PTFE) implants were inserted in the abdominal wall of rats for 1, 6 and 12 weeks followed by a mild fixation, cryoprotection, rapid freezing in LN2-cooled propane, cryosubstitution and low-temperature infiltration with UV curing of the methacrylate LR-Gold. Before sectioning, the bulk part of the titanium was removed by an electrolytical dissolution technique (electropolishing), while the PTFE implants were removed by a fracture technique. Employing a cryosubstitution method combined with postembedding immunohistochemistry, a light microscopic analysis was allowed. The selected proteins had an apparently varying distribution in the implant-close tissue and their distribution changed during the follow-up period. There was also a difference in the distribution pattern for each protein around titanium and PTFE implants. Insertion of the c.p. titanium implants elicited an inflammatory reaction in many respects similar to a normal wound healing response, while the PTFE implants caused a more pronounced, persistent inflammation.
Asunto(s)
Colágeno/análisis , Fibrinógeno/análisis , Fibronectinas/análisis , Inmunoglobulina G/análisis , Politetrafluoroetileno , Prótesis e Implantes , Albúmina Sérica/análisis , Titanio , Músculos Abdominales , Animales , Materiales Biocompatibles , Inmunohistoquímica , Masculino , Fibras Musculares Esqueléticas/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
The effects of insulin-like growth factor I (IGF-I) on mitogenesis, epithelial barrier function and transepithelial iodide transport were studied in confluent, polarized monolayers of pig thyrocytes cultured on filter in Transwell bicameral chambers. The growth rate in controls cultured in 1% fetal calf serum was low. Insulin-like growth factor I stimulated dose-dependently the incorporation of [3H]thymidine, maximally at 100 ng/ml, which corresponded to an increase of DNA content by 60% after 6 days. Thyrotropin (1 mU/ml) alone did not stimulate cell multiplication but inhibited partially the stimulatory effect of IGF-I. Insulin-like growth factor I (100 ng/ml) increased within 10 min the transepithelial potential difference, which remained elevated for several days, but did not significantly change the transepithelial resistance. When added together, IGF-I reinforced the effects of TSH on potential difference (increase) and resistance (decrease). A preserved epithelial barrier in IGF-I-treated cultures was confirmed by observing a normal immunolocalization of the tight junction protein ZO-1 and an unchanged ultrastructure of the junctional complex. Insulin-like growth factor I increased the transepithelial flux of 125I- in the basal-to-apical, but not in the opposite, direction. Stimulation of iodide transport by IGF-I was modest after 2 days and pronounced after 6 days. In comparison, TSH-stimulated iodide transport was higher after 2 days but lower after 6 days. Both TSH and IGF-I were strongly synergistic, after 6 days amounting to a 90-fold increase over the control basoapical 125I- transfer. The simultaneous accumulation of 125I- in the cell layer was increased two- to fourfold by IGF-I and/or TSH. In conclusion, IGF-I is able to induce growth in preformed monolayers of pig thyrocytes cultured on permeable filter. During these conditions, the mitogenic effect of IGF-I is partially inhibited by TSH, which has no growth-promoting action on its own. The transepithelial transport of iodide and bulk electrolytes is altered by IGF-I without affecting the epithelial barrier function. Specifically, IGF-I up-regulates the activity of the basolateral iodide pump and increases the iodide permeability of the apical plasma membrane. The action of IGF-I on iodide transport is independent of, although synergistic with, that of TSH. The findings support the notion that IGF-I may be an important regulator of thyroid growth and differentiated functions.
Asunto(s)
Polaridad Celular , Factor I del Crecimiento Similar a la Insulina/farmacología , Yoduros/farmacocinética , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/fisiología , Animales , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Electrofisiología , Células Epiteliales , Epitelio/fisiología , Femenino , Permeabilidad/efectos de los fármacos , Porcinos , Glándula Tiroides/citología , Tirotropina/farmacologíaRESUMEN
The release of interleukin-1 alpha (IL-1 alpha) by human peripheral blood monocytes cultured for 24 and 48 h on polystyrene (PS) and titanium-sputtered polystyrene (Ti) was evaluated. Magnetron sputtering of the PS surfaces resulted in a formation of a 50-nm-thick coat, consisting of an outer layer of TiO2. Monocytes released IL-1 alpha without the addition of exogenous stimuli. A doubling of the culture time from 24 to 48 h did not have a major effect on the amount of IL-1 alpha released. The IL-1 alpha levels were increased by addition of lipopolysaccharide (LPS). High concentrations of PS particles (1 and 3 microns diameter) were equally effective stimuli for IL-1 alpha release as LPS. Preadsorption of fibronectin to culture plates augmented LPS-stimulated IL-1 alpha secretion, whereas preadsorbed fibrinogen had an inhibitory effect. Our observation indicate a direct activation of monocytes by PS and Ti, resulting in IL-1 alpha secretion, which is modified by protein adsorption and exogenous stimuli.
Asunto(s)
Materiales Biocompatibles , Interleucina-1/metabolismo , Monocitos/fisiología , Poliestirenos , Titanio , Adsorción , Células Cultivadas , Fibrinógeno , Fibronectinas , Humanos , Técnicas In Vitro , Lipopolisacáridos/farmacología , Ensayo de Materiales , Microscopía Electrónica , Monocitos/efectos de los fármacos , Monocitos/ultraestructura , Prótesis e Implantes , Propiedades de Superficie , Cicatrización de Heridas/fisiologíaRESUMEN
The integrity of epithelial cell junctions is controlled by E-cadherin-mediated (Ca2+-dependent) cell-cell adhesion. In thyroid follicular cells the dissociation of junctions induced by transfer to low Ca2+ medium (Ca2+ switch) is prevented by thyrotropin acting via cyclic AMP/protein kinase A (cAMP/PKA) (Nilsson et al., Eur. J. Cell Biol. 56, 308-318, 1991). In MDCK kidney epithelial cells protein kinase inhibitors elicit a similar response which, however, is cadherin-independent (Citi, J. Cell Biol. 117,169-178,1992; Citi et al., J. Cell Sci. 107, 683-692, 1994). As such inhibitors also may interfere with PKA, we examined in a single cell type, filter-cultured pig thyrocytes, the effects and possible interactions of the cAMP/PKA agonist forskolin (or thyrotropin) and the kinase inhibitor H-7 in Ca2+ switch experiments. We found that the epithelial barrier dysfunction, comprising loss of transepithelial resistance, increased transepithelial flux of [3H]inulin and redistribution of junction proteins (cadherin and ZO-1), which follows Ca2+ removal were inhibited by TSH, forskolin, and H-7. All agents were also able to induce recovery of resistance in low Ca2+. The maximal recovery effects of forskolin and H-7 were additive when given simultaneous with Ca2+ chelator. In contrast, forskolin-induced recovery initiated 10 min after Ca2+ removal was antagonized by H-7. The protection of junctions by forskolin in low Ca2+ was rapidly abolished by light trypsinization (0.001%), whereas the same concentration of trypsin had little or no effect on the corresponding action of H-7 or staurosporine, another potent kinase inhibitor. In H-7-treated cells kept in low Ca2+, trypsin caused redistribution of ZO-1 from the plasma membrane to the cytoplasm while the transepithelial resistance remained high. Taken together, the data indicate that TSH via cAMP/PKA and the protein kinase inhibitor H-7 reinforce the thyroid epithelial barrier under low Ca2+ conditions by distinct although interacting mechanisms. The high sensitivity to proteolysis in the absence of Ca2+ suggests that the cAMP-regulated mechanism is cadherin-dependent. H-7 promotes or inhibits the cAMP/PKA-mediated recovery of transepithelial resistance depending on the duration of the preceding low Ca2+ period. The trypsin-induced displacement of ZO-1 in H-7-treated cells in low Ca2+ suggests that the localization of ZO-1 to the tight junction is not necessary for the maintenance of junctional tightness.
Asunto(s)
Calcio/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glándula Tiroides/fisiología , Uniones Estrechas/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Animales , Células Cultivadas , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Sinergismo Farmacológico , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Epitelio , Isoquinolinas/farmacología , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Piperazinas/farmacología , Estaurosporina , Porcinos , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/enzimología , Tirotropina/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/enzimología , Tripsina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
The bone formation around titanium implants with varied surface properties is investigated. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits (1, 3 and 6 weeks). Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition, between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads at 6 weeks. The smooth, electropolished implants, irrespective of anodic oxidation, were surrounded by less bone than the machined implants after 1 week. After 6 weeks the bone volume as well as the bone-implant contact were lower for the merely electropolished implants than for the other three groups. Our study shows that a high degree of bone contact and bone formation are achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, the result with the smooth (electropolished) implants indicates that a reduction of surface roughness, in the initial phase, decreases the rate of bone formation in rabbit cortical bone.
Asunto(s)
Regeneración Ósea/fisiología , Prótesis e Implantes , Tibia/fisiología , Titanio/metabolismo , Animales , Electroquímica , Femenino , Masculino , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Oxidación-Reducción , Conejos , Propiedades de Superficie , Tibia/cirugía , Tibia/ultraestructura , Titanio/químicaRESUMEN
The effects of epidermal growth factor (EGF) and phorbol ester (tetra-O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance (RTE > 6.10(3) omega.cm2) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [3H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained (RTE 1-3.10(3) omega.cm2; impermeability to [3H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca2+ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 microM) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [3H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca(2+)-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.