Mia Källtorp

In vivo cell recruitment, cytokine release and chemiluminescence response at gold, and thiol functionalized surfaces

Hydroxylated and methylated surfaces were prepared by the self-assembled monolayer technique (SAM) of alkane thiols on gold. The surfaces were used to evaluate the influence of implant surface chemistry on protein deposition and inflammatory cell response. Implants were inserted subcutaneously in the rat for 3 and 24 h. The surface chemical properties influenced the in vitro rat plasma protein adsorption (ellipsometry/antibody) with few exceptions (albumin not found and fibrinogen always found). The number of recruited cells and their distribution (DNA from implant versus from exudate) was influenced by the different chemistries at 24 h, but not at 3 h. HIS48+, ED1+, ED2+ and small numbers of CD5+ cells were present in the exudate at both time periods (flow cytometry). The cellular oxidative metabolism was low, although cells on -OH surfaces responded with the highest phorbol ester-stimulated chemiluminescence (CL)/DNA. The levels of cytokines IL-1alpha, IL-1beta and TNFalpha (ELISA) were not influenced by material surface chemistry. Sham operated sites had a higher cytokine concentration/DNA compared with exudates from an implant milieu. The results of this study show that surface chemical functionalization modifies specific events in the inflammatory response around implants in soft tissues.

IL-1α, IL-1β and TNF-α 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 su ...

In vitro study of monocyte viability during the initial adhesion to albumin- and fibrinogen-coated surfaces

Surface adherent monocytes and macrophages play a central role in the inflammatory response to biomaterials. In the present study the adhesion, viability and apoptotic changes in material surface adherent monocytes during the first hours of cell-surface interactions in vitro were studied, using tissue culture polystyrene surfaces coated with human albumin and fibrinogen. Human peripheral blood monocytes were enriched by a two-step gradient centrifugation and resuspended (1 x 10(6)/ml) in RPMI with 10% fetal bovine serum. The cells were added to polystyrene surfaces coated with human fibrinogen or albumin and incubated in 37 degrees C (5% CO2, 100% humidity) for 30 min, 1, 2, 3 and 24 h. The adherent cells were stained for early apoptotic changes (exposed phosphatidylserine) and cell death using Annexin-V-fluorescein and propidium iodide staining, respectively. A bi-phasic adhesion was observed on the fibrinogen coated surface, having the highest number of adherent cells after 30 min and 24 h, while the cell number was markedly reduced after 1-3 h. The number of adherent cells on albumin was relatively low after all short time incubations but had reached a high level after 24 h. The number of adherent dead cells was highest after I h on both albumin (approximately 30%) and fibrinogen (approximately 15%). In the 24 h cultures, the viability of adherent cells was high on both surfaces (95-100%). Viable cells staining positive for early apoptotic changes could only be clearly observed on the albumin coated surface, after 30 min of cell-material surface interaction. Cell death, including apoptotic death, thus seems to play an important role during the initial interactions between monocytes and a foreign surface.

Inflammatory cell recruitment, distribution, and chemiluminescence response at IgG precoated- and thiol functionalized gold surfaces

The role of complement activation by artificial surfaces relative to inflammatory response is not well understood. This study was performed to evaluate the inflammatory cell recruitment, distribution, and ex vivo metabolic activation of surfaces with different plasma protein adsorption and complement activation properties in vitro. The implants were (1) pure gold (reference), (2) albumin-precoated (3) IgG-precoated gold, and (4) 3-mercapto-1, 2-propanediol [mercaptoglycerol (MG)] and (5) glutathione (GSH) immobilized to gold. The implant disks were inserted subcutaneously in rats for 24 h, and the number of inflammatory cells that were recruited to the implant adjacent to the surrounding fluid phase (exudate) and the surfaces were quantified by DNA measurements. The oxidative burst was analyzed ex vivo using spontaneous and phorbol myristate acetate (PMA)-stimulated, luminol-enhanced chemiluminescence (CL). The in vitro surface-induced anti-rat C3 binding was evaluated by ellipsometry and antibody techniques after plasma incubations for 1 and 30 min. The ellipsometric results showed that immobilized mercaptoglycerol and IgG-coated, but not the immobilized glutathione or the reference Au, bound anti-C3. The in vivo results revealed that the largest amount of cells was associated with the IgG-coated surfaces, followed by immobilized GSH and MG, albumin-coated, and gold surfaces, respectively. No spontaneous ex vivo luminol-enhanced CL was recorded from the cells irrespective of surface functionality or localization. A down-regulation of surface-associated and exudate leukocyte CL was observed ex vivo, irrespective of surface functionality. The results do not indicate a clear relationship between the degree of complement activation in vitro and leukocyte recruitment and adhesion in vivo for differently functionalized surfaces.

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.© 2001 Kluwer Academic Publishers

Analysis of rat plasma proteins desorbed from gold and methyl- and hydroxyl-terminated alkane thiols on gold surfaces.

It is believed that adsorbed blood or plasma components, such as water, peptides, carbohydrates and proteins, determine key events in the concomitant inflammatory tissue response close to implants. The aim of the present study was to develop a procedure for the collection and analysis of minor amounts of proteins bound to solid metal implant surfaces. The combination of a sodium dodecyl sulfate washing method coupled with a polyacylamide gel electrophoretic protein separation technique (SDS–PAGE), Western blot and image analysis enabled the desorption, identification and semiquantification of specific proteins. The analyzed proteins were albumin, immunoglobulin G, fibrinogen and fibronectin. Concentration procedures of proteins were not required with this method despite the small area of the test surfaces. The plasma proteins were adsorbed to pure gold and hydroxylated and methylated gold surfaces, which elicit different tissue responses in vivo and plasma protein adsorption patterns in vitro. The image analysis revealed that the pure gold surfaces adsorbed the largest amount of total and specific proteins. This is in accordance with previous ellipsometry/antibody experiments in vitro. Further, the principles described for the protein analysis can be applied on implant surfaces ex vivo. ©©2000 Kluwer Academic Publishers

Titanium in Soft Tissues

Titanium is a biomedical material, which has been investigated in different types of tissues and used in a large number of applications (Part V, Chapters 21–27). It is therefore a suitable reference material in studies focused on the mechanisms of integration of materials. As previously reviewed and discussed (Part III, Chapters 7–13), titanium is a material, which is amenable to a large number of modifications. These include surface modifications intended for systematic studies of the biological effects of specific surface properties, a course which has been pursued by us and others. Titanium is also an interesting material to use in combination with other materials with different properties, such as coatings of various types. The most important aspect to be discussed in the following biologically oriented chapters is that titanium has documented and beneficial properties in a biological environment during long-term clinical follow-ups. On the other hand, implanted medical devices may fail, and so do occasionally implants made of titanium. An understanding of the mechanisms, which determine a clinically successful medical device as well as the processes, which lead to such failures is of crucial importance for the future development of safe and functional implants.

On the formation of fibrous capsule and fluid space around machined and porous blood plasma clot coated titanium

Machined and machined submicron porous titanium, with and without a thin blood plasma coating (100 nm), were implanted for 7 or 28 days in subcutaneous pockets on the back of the rat. After explantation the specimens were analyzed by light microscopy with respect to thickness of the fibrous capsule, the fluid space width between implants and fibrous capsule, and formation of blood vessels. The results at 7 days indicate a thinnest fluid space for the plasma clot coated porous titanium surface, and the spaces vanished at the light microscopic level after 28 days outside all the analyzed surfaces. The thickness of the fibrous capsule increased outside the different surfaces at 7–28 days, and in this respect no significant differences were observed between the different surfaces at any time. Analysis of neovascularization showed that the number of vessels and proportion of vessels in the fibrous capsule increased with time at all surfaces, except machined Ti where the number instead decreased from 7 to 28 days. The average distance between the blood vessels and the fluid space increased with time for all types of surfaces. The results in the present study indicate that the healing process around titanium can be modulated by porosity and thin pre-prepared plasma coatings.© Kluwer Academic Publishers

Ellipsometric studies in vitro on kinetics of rat complement activation.

The role of complement activation may be important during the early interactions between implantable materials and blood and during the acute inflammatory phase, but it is not well understood. This applies especially to rats that are extensively used in in vivo animal models for materials and surface testing. Features of the kinetics of rat complement activation were studied and compared with human complement by the ellipsometry and antibody techniques. The results indicate that the rat classical pathway is rapidly activated, but it is not as fast as the human system. The activation of the alternative pathway was observed within 5 min in the rat system and within 15 min for the human. Thus, the observations indicate substantial differences in the kinetics between the two species. This may influence the choice of the rat experimental model and the tissue response to materials during in vivo conditions.

Ex vivo PMA-induced respiratory burst and TNF-α secretion elicited from inflammatory cells on machined and porous blood plasma clot-coated titanium

The release of inflammatory mediators around implants and normal wounds may differ due to the presence of the solid surface. In this study, machined and sub-micron porous titanium implants with and without a 100 nm thick blood plasma clot were inserted subcutaneously in rat for 3 or 24 h. The cell recruitment to the interfaces, in vivo secretion of TNF-alpha and the ex vivo PMA-induced production of reactive oxygen species were subsequently investigated. The thin plasma clot coating gave rise to an increased ex vivo PMA-stimulated oxygen radical production by implant-associated cells at both implantation times, and an increased cell recruitment at 24 h. The total TNF-alpha secretion was highest at sham sites and plasma clot-coated porous titanium at 24 h. After 24 h, the cell-type pattern in the exudate around the porous plasma-coated implant was more similar to that found at sham sites than that adjacent to the non-coated implants. No differences were observed between the machined Ti and the machined sub-micron porous Ti.