Inflammatory response to titanium surfaces with fibrinogen and catalase coatings: an in vitro study.

The aim of the present study was to evaluate the possibility to modulate the early inflammatory response in vitro by coating titanium surfaces with candidate proinflammatory (fibrinogen coated turned titanium "Fib") and antiinflammatory proteins (catalase on top of fibrinogen coated turned titanium "Cat"). Additionally, turned titanium surfaces (Ti) were used as controls. The discs were incubated with human mononuclear cells. Adhered cells were investigated with respect to number, viability, differentiation (acute marker 27E10 vs. chronic marker RM3/1), and cytokine production (TNF-alpha and IL-10), after 24 and 72 h. The results indicated that it is possible to modulate the inflammatory response with protein coatings. However, the strongest inflammatory response, indicated by increased number of adhered cells and release of pro and antiinflammatory mediators, was induced by Cat. Furthermore, the cytokine production on this surface was not sensitive to LPS stimulation. Differentiation measured as the expression of the chronic cell surface marker, dominated after 72 h for all surface modifications and Cat displayed an increased number compared to the others. A decrease in the total number of adhered cells and amounts of TNF-alpha were observed on all surfaces over time. The cell viability was, in general, high for all tested surfaces. In conclusion, the study proved it possible to influence the early inflammatory response in vitro by immobilizing protein coatings to titanium surfaces. However, the catalase surface demonstrated the strongest inflammatory response, and the possibility to selectively use the potent antiinflammatory capacity of catalase needs to be further evaluated.

Elevation of cytokines in peritoneal fluid and blood in patients with liver cirrhosis.

BACKGROUND/AIMS: Liver cirrhosis, described as the endstage of a necroinflammatory process, is often accompanied by ascites formation. The rationale for this study was the hypothesis that patients with liver cirrhosis have a low-grade chronic inflammatory response, which leads to an increased amount of proinflammatory cytokines accumulated in ascites. Twenty-five patients with liver cirrhosis complicated by ascites and twelve healthy volunteers were prospectively included in the study. METHODOLOGY: Ascites and blood samples from the patients were obtained for analysis of inflammatory cytokines using enzyme-linked immunosorbent assay methodology. Blood samples were taken from the healthy volunteers to obtain reference values. RESULTS: Plasma and ascites concentrations of interleukin-1alpha, interleukin-6, and tumor necrosis factor-alpha were significantly elevated in the patients compared with plasma levels in the group of healthy controls. Significant elevation of interleukin-10 concentrations was found in ascites but not in plasma in the patients. There was no significant difference in interleukin-10 levels between patient and control plasma. CONCLUSIONS: The findings suggest that elevated cytokine concentrations in ascites and serum could perpetuate an inflammatory reaction that may be a source of preservation of an ongoing systemic inflammatory reaction. This may contribute to the maintenance, and even progress, of the liver dysfunction, leading to exaggerated ascites development.

Adhesion, apoptosis and cytokine release of human mononuclear cells cultured on degradable poly(urethane urea), polystyrene and titanium in vitro.

Early interactions between materials and mononuclear cells may influence the viability and secretory response of the cells. Such effects may in turn influence the subsequent inflammatory and repair phases around the materials. In the present study, it was examined if mononuclear cells cultured in vitro either unstimulated or stimulated with lipopolysaccharide (LPS) (10ng/ml) revealed differences regarding cell viability and apoptosis. A major interest was to study the influence of different material properties on the parameters of the inflammatory response upon cell adhesion to materials with widely different surface chemical properties but similar surface topography: degradable poly(urethane urea) (PUUR), cell culture treated polystyrene (PS) surfaces, and commercially pure (c.p.) titanium (Ti). Finally, the secretion of the proinflammatory tumor necrosis factor-a (TNF-alpha) and the downregulating interleukin-10 (IL-10) cytokines was examined in the supernatants from 24h mononuclear cell cultures. No differences in cell viability as measured by lactate dehydrogenas (LDH) were observed between the three materials. The number of material-surface adherent cells was higher on PUUR than the more hydrophilic PS and Ti as judged by quantification of material surface-associated DNA, light microscopic morphological examination of DAPI-stained cells and SEM. LPS increased the number of adherent cells, irrespective of the type of material. The lowest number of apoptotic (annexin-V) and necrotic (propidium iodide) mononuclear cells was detected on PUUR. LPS decreased the number of both apoptotic and necrotic cells, irrespective of material. Low TNF-alpha levels were detected in unstimulated conditions, irrespective of material types. A significantly lower amount of TNF-alpha was found with unstimulated cells on PUUR than on Ti. A significantly higher IL-10 level was detected in unstimulated Ti cultures compared with PUUR and PS. Secretion of IL-10 was predominantly stimulated by LPS on PUUR and Ti. The data indicate that material-related differences are expressed in differences in cell adherence, apoptosis and cytokine secretion. Further, degradable PUUR has equal or less cell-activating properties than Ti and PS under in vitro conditions.

IL-1alpha, IL-1beta and TNF-alpha secretion during in vivo/ex vivo cellular interactions with titanium and copper.

Titanium (Ti) and copper (Cu) were used to evaluate cytokine secretion around materials with different chemical properties. Ti disks were coated with Cu or left uncoated. The disks were inserted subcutaneously in rats for 1, 3, 12, 18, 24 and 48 h. Interleukin-1alpha (IL-1alpha), IL-1beta and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in vivo around the materials, in sham operated sites, and after ex vivo incubation of surface adherent cells. Ti and Cu revealed distinct cytokine expression patterns. Cu recruited cells showed higher and prolonged release of IL-1alpha than Ti at longer times (>24 h), whereas Ti exhibited a transient IL-1alpha response at earlier periods (<24 h). An early enhanced secretion of TNF-alpha characterized Ti. Low amounts of IL-1beta were found around both materials. Sham site recruited cells produced lower levels of cytokines. The results after ex vivo incubations were similar to those in vivo. This study shows that material chemical properties influence early cytokine production. The Ti-associated transient rise of IL-1alpha and TNF-alpha may be of importance for the early tissue response around biocompatible materials, while a delayed high IL-1alpha expression could be a marker of inflammation induced by toxic materials.

Apoptosis and cytokine release in human monocytes cultured on polystyrene and fibrinogen-coated polystyrene surfaces.

The effects of polystyrene (PS) material surface preadsorption with fibrinogen (3 mg/ml) and a low concentration of lipopolysaccharide (LPS; 10 ng/ml) and polystyrene particles (PS; 10(5)/ml) on human monocyte adhesion, viability and cytokine release were studied during 24h culture in vitro. LPS caused an upregulation of CD14 in adherent cells. In comparison with unstimulated cells on uncoated polystyrene surfaces, LPS did not alter the number of adherent cells but caused a markedly increased release of the proinflammatory cytokines (IL-1alpha and TNF-alpha) and the down-regulating IL-10. The expression of indicators of various stages of cell death, TdT, annexin-V, propidium iodide (PI) and lactate dehydrogenase (LDH), were unaltered, decreased, decreased and increased, respectively, after LPS stimulation. PS particles (3 microm psi) caused an increased DNA fragmentation but had a reduced proportion of annexin-V and PI positive cells in comparison with unstimulated cells on uncoated PS. In contrast, 1microm psi particles had a similar proportion of TdT, annexin-V and PI expressing cells as unstimulated controls. Cultures stimulated with particles (irrespective of size), had a similar concentration of proinflammatory cytokines as unstimulated controls, whereas a higher level of IL-10 was detected. Precoating of PS with fibrinogen revealed an enhanced cell adhesion and a concomitant reduction of CD14 expression. irrespective of stimulation with various agonists. The proportions of TdT, annexin-V and PI positive cells were unaltered or reduced on fibrinogen-coated PS in both unstimulated and agonist-challenged cultures. However, depending on the presence and type of agonist, fibrinogen mediated either a markedly increased (LPS) or equivalent (particles and unstimulated) IL-1alpha and TNFalpha release. Further, in comparison with uncoated substrates, fibrinogen was associated with a reduction of IL-10 release, irrespective of the type of stimuli. These observations, using low concentrations of bacterial and material products, indicate that fibrinogen modulates cell material interactions and up- and down-regulates specific events depending on the nature/ type of immediate stimuli.

H2O2 production by cells on titanium and polystyrene surfaces using an in vivo model of exudate and surface related cell function.

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.

In vivo/ex vivo cellular interactions with titanium and copper.

Machined, commercially pure titanium (Ti) disks were coated with approximately 400 nm copper (Cu) by physical vapor deposition or left uncoated. The kinetics of inflammatory cell recruitment, distribution and viability was evaluated around Ti, Cu, and in sham sites after 1, 3, 12, 18, 24, and 48 h in a rat subcutaneous (s.c.) model. Further analysis of the cells on implant surfaces was performed by ex vivo incubation of the disks. Ti and Cu stimulated an increased recruitment of inflammatory cells in comparison with sham sites. A markedly higher amount of cells, predominantly polymorpho-nuclear granulocytes (PMN), was detected around Cu after 18 h and onwards. More cells were found at the implant surfaces than in the surrounding exudates after 18 h. The total amount of lactate dehydrogenase (LDH), an indicator of plasma membrane injury, was higher in Cu exudates after 18 h in comparison with Ti and sham. In contrast, no differences in the proportion of dead cells (trypan blue dye uptake) were detected in the exudates. Further, LDH levels were higher around Ti than Cu during the initial 18 h of ex vivo incubation. The results of this study indicate that the early inflammatory process associated with a cytotoxic material in soft tissues is largely attributed to the induction of a markedly strong and prolonged chemotactic response. In contrast, this process is characterized by a higher amount of inflammatory cells around a biocompatible material than in sham sites, but with a transient course and total LDH similar to sham sites.

Secretion of IL-1 and H2O2 by human mononuclear cells in vitro.

The secretion of hydrogen peroxide (H2O2) and interleukin-1alpha (IL-1alpha) was evaluated during in vitro culturing of human monocytes. The oxidative metabolism and cytokine secretion were correlated to the cell distribution (number of surface-associated cells), the DNA content and their integrity, evaluated by lactate dehydrogenase (LDH) assay. The differentiation of cultured monocytes was determined by the expression of CD14, 27E10 and RM3/1. After 24 h cultivation, unstimulated cells had a low production of H2O2 and IL-1alpha. A four-fold increase in the production of H2O2 was detected with 5 and 10 microg/ml of lipopolysaccharide (LPS) and polystyrene (PS) particles. PS particles induced a concentration-dependent increase in IL-1alpha after 24 h. In contrast, cultivation for 48 h, did not result in any measurable production of H2O2, irrespective of the type of stimulus. A decreased viability of monocytes was shown after stimulation with PS particles in high concentrations. Our results indicate that the phenotype expression, adhesion, integrity and secretory pattern of human monocytes is dependent on the culture time and the type and concentration of stimulus.

Co-culture of human monocytes and thyrocytes in bicameral chamber: monocyte-derived IL-1alpha impairs the thyroid epithelial barrier.

Pro-inflammatory cytokines are important mediators in tissue responses to a wide range of endogenous (e.g. autoantigens) and exogenous (e.g. infections, wounds, biomaterials) stimuli. The complex interactions taking place between different cell types in such processes are difficult to examine in vivo. Here we studied the effect of human monocytes on thyroid epithelial cells co-cultured in bicameral chambers. Freshly isolated monocytes (1x10(6)/ml) added to the basal compartment reduced the transepithelial resistance (from 300-600 to <100 Omega.cm(2)) and caused a disruption of the tight junctions in apically grown thyrocyte monolayers after co-culture for 24 h. The barrier function was further attenuated by monocytes exposed to lipopolysaccharide (10 microg/ml) or polystyrene microspheres (size: 3 microm; 1x10(7)/ml). Loss of transepithelial resistance was accompanied by release of interleukin 1alpha (maximally 550 pg/ml) from the monocytes. Conversely, the resistance remained high when co-cultures were simultaneously incubated with neutralizing anti-human interleukin 1alpha antibodies. The results show that the integrity of cultured thyroid epithelium is impaired by monocytes without requirement of direct cell-to-cell contact. This action, mediated by interleukin-1alpha, suggests a mechanism by which hidden (lumenal) autoantigens might be exposed to interstitial antigen-presenting cells in autoimmune thyroid disease. In perspective, the model provides a tool in which humoral and cell-cell dependent processes generated by bioactive agents and particulate materials, for instance, during the healing and repair of tissue around biomaterials and hybrid implants, can be selectively examined.

Immunohistochemistry of soft tissues surrounding late failures of Brånemark implants.

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.

Monocyte activation on titanium-sputtered polystyrene surfaces in vitro: the effect of culture conditions on interleukin-1 release.

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.

Inhibitory effects of amide local anaesthetics on stimulus-induced human leukocyte metabolic activation, LTB4 release and IL-1 secretion in vitro.

The anti-inflammatory effects of the amide local anaesthetics lidocaine and bupivacaine were evaluated in vitro by examination of the metabolic activation and secretory responses of human polymorphonuclear granulocytes (PMNGs) and mononuclear cells. Pretreatment with lidocaine or bupivacaine had a dose-dependent inhibitory effect on PMNG luminol-amplified chemiluminescence stimulated by bovine serum albumin (BSA)/anti-BSA immune complexes (IC) or by serum-opsonized zymosan (SOZ) particles. Both lidocaine and bupivacaine inhibited the release of the inflammatory mediators leukotriene B4 (LTB4) and interleukin-1 (IL-1) evaluated by radioimmunoassay (RIA). Pretreatment of suspended PMNGs and monocytes with the anaesthetics caused a marked inhibition of LTB4 release when the cells were stimulated with SOZ. In short-term (24 h) cultures of mononuclear cells the addition of lidocaine or bupivacaine reduced, in a dose-dependent manner, the level of IL-1 detected after stimulation with lipopolysaccharide (LPS). In all three assays (chemiluminescence, LTB4 and IL-1 RIA) bupivacaine was found to be more potent than lidocaine. The present results show that amide local anaesthetics have marked suppressive effects on the metabolic activation and secretory functions of leukocytes stimulated by different agonists. Although the detailed mechanisms for these effects are not known, they may explain part of the potent anti-inflammatory actions of local anaesthetics previously described in vivo.

Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances.

A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.

Detection of single antibody-secreting cells generated after in vitro antigen-induced stimulation of human peripheral blood lymphocytes.

Culture and assay procedures are described for the generation and the subsequent detection of single antigen-specific antibody-secreting cells after in vitro stimulation of human peripheral blood lymphocytes with tetanus toxoid. Simple, specific and sensitive, this new assay-culture system is well suited for the analysis of specific antibody production in man.

An immunoenzyme procedure for enumerating fibronectin-secreting cells.

A recently described solid phase immunoenzyme procedure (ELISPOT) has been adapted for the detection of individual cells secreting fibronectin. Simple and sensitive, this technique should find useful application for studying fibronectin production at the cellular level.