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

Biomoterids 17 (1996) 651-658 0 1996 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0142-9612/96/$15.00

ELSEVIER

Mono&e activation on titaniumsputterkd polystyrene surfaces in vitro: the effect bf culture conditions on interleukin-1 release C. Gretzer*, AS. Eriksson*t, and P. Thomsen*

B. Alldbnt, L.E. Ericson*

‘Institute of Anatomy and Cell Biology, Gothenburg University, S-413 90 Gothenburg, Sweden; iDepartment Medicine, &tra University Hospital, Sweden; iDepartment of Physics, Chalmers University of Technology, Gothenburg, Sweden The release

of interleukin-lcr

polystyrene

(PS) and titanium-sputtered

PS surfaces

resulted

Monocytes from

released

in a formation IL-la

without

increased

by addition were

the addition

culture

plates

inhibitory

Keywords: Received

effective

augmented

effect.

secretion,

Monocytes,

stimuli

is modified titanium,

12 May 1993; accepted

IL-la

polystyrene,

whereas

of the

A doubling

of the culture

released.

The IL-la

of PS particles

preadsorbed

of monocytes

and exogenous

LPS, particles,

for 24 and 48 h on sputtering

layer

as LPS. Preadsorption

activation

adsorption IL-l,

stimuli. of IL-la

cultured Magnetron

of an outer

High concentrations

secretion,

a direct

monocytes

consisting

of exogenous (LPS).

indicate

10 June

coat,

for IL-lee release

by protein

blood

(Ti) was evaluated.

on the amount

LPS-stimulated

Our observations which

effect

of lipopolysaccharide

equally

peripheral

polystyrene of a 50-nm-thick

24 to 48 h did not have a major

diameter)

IL-la

(IL-ICC) by human

of

of TiOl. levels

time were

(1 and 3pm

of fibronectin fibrinogen

to had an

by PS and Ti, resulting

in

stimuli.

proteins

1995

main fibroblast stimulatory factor. Two distinct forms in the IL-l family have been of polypeptides characterized: IL-la and IL-l/?. Both forms are produced by mononuclear phagocytes and they have, similar biological activities, with some exceptions, including pro-inflammatory and immunomodulatory effects’. In order to determine the role of the monocyte during the healing of implants it is of importance to analyse the function of these cells in more detail. However, since the in vivo situation is highly complex it is important to develop in vitro models which allow a selective manipulation of the different implant-tissue components found during in vivo conditions. In addition, several technical prerequisites have to be fulfilled: the model surfaces have to be manufactured by simple techniques and in great numbers, the surfaces should be well characterized and be able to be modified with regard to surface structure and chemical composition, and the surfaces should be clean and be used during sterile cell culture conditions. Moreover, from an analytical point of view, it is important to be able to study the morphology of the monocytes before and after fixation. Thus, thin metal coatings which allow studies using transmitted light are preferred.

The factors which govern the tissue response around implants are not fully understood. During the first week after implantation of titanium in soft tissues, a fluid space, which contains proteins’ and scattered inflammatory cell.s?, separates the implant surface from the tissue. The majority of cells in the fluid space are not in direct contact with the surface during the first week of healing3. However, during later time intervals, macrophages are adherent to the surface of and other biomaterials (reviewed by titanium’.* Anderson and Miller5). One factor which may influence the regenerating tissue around the implant is the activity of the macrophages and the release of mediators from macrophages adherent on the implant surface. It is possible that these cells act as ‘messenger cells’, transmitting information about the implant surface structure and composition to the surrounding tissue. Evidence for an effect of biomaterials on macrophage-derived stimulation of fibroblasts has supernatants from human been presented6, 7: monocytes cultured on polymers stimulate fibroblast proliferation and collagen synthesis. In these latter studies, interleukin-1 (IL-l) was implicated as the Correspondence to Dr P. Thomsen. 851

Biomaterials

1996, Vol. 17 No. 9

852

Monocvte

In the present study an in vitro model for studies on monocyte-implant interactions is presented.

MATERIALS AND METHODS Preparation of surfaces Polystyrene culture dishes (24 wells; Nunc@, Nunc, Denmark) were coated with titanium by a combination of reactive and non-reactive magnetron sputtering in a Leybold-Heraeus 2550 unit. The culture dishes were placed on a rotating substrate table. After evacuation to < 5 x 10-6mbar, sputter gases were introduced, pressure of lo-‘mbar. maintaining a constant Sputtering was conducted until the desired coating thickness was obtained. Argon and Oz (both with a 86.688% purity) were used as sputter gases. The target was a 15cm diameter pure titanium plate (69.5% purity) separated from the substrate by 4 cm. The coating was made by a two-step procedure. Firstly, the argon was mixed with 20% Oz in order to obtain a good adherence between substrate and coating. Secondly, in the deposition step, sputtering was performed in pure argon. The average deposition rate was 2.5 nm min-‘, as determined by surface profilometry across a coating edge created by masking during the deposition. The typical coating thickness was 50nm. The adherence of the coating was assessed by use of the conventional scotch tape test and it was verified that no failure occurred during 10 consecutive tests. The elemental composition of the coatings was studied by Auger electron spectroscopy (AES) using a Perkin-Elmer PHI 660 instrument with a 3.OkV electron beam.

Monocyte activation Buffy coats from blood donors were subjected to a twostep gradient centrifugation according to the method of Pertoft et ~1.~. In brief, 7ml of undiluted blood was carefully layered over 4ml of 60% v/v Percoll in conical centrifugation tubes and centrifuged at room temperature at 800g for 30min. The interface was harvested and washed once with cold phosphatebuffered saline (PBS; without Ca2+ and Mg’+). The monocytes were resuspended in cold PBS and 7ml of the cell suspension was layered over 3ml of 50% v/v Percoll and centrifuged at 800g for 60 min at +4”C. After washing and counting a 99% cell viability (trypan blue dye exclusion) was determined. Seventyfive percent of the monocytes were reactive with M3 monoclonal antibody (Becton and Dickinson, USA), assessed by flow cytometry. Glass smears were fixed with a citrate-acetone-methanol fixative and stained with a-naphtyl acetate esterase (Sigma, USA). Staining was observed in 75% of the cells. The monocytes were washed, counted and kept at +4”C in PBS until culturing. The isolated mononuclear cells were suspended at 105, lo6 and 5 x lo6 cells per ml in complete RPM1 1640 with 5% fetal calf serum and 1% each of streptomycin and fungizone (Gibco, UK), and added to the culture dishes (1 ml final volume). In separate experiments polystyrene and titanium-coated Biomaterials

1996, Vol. 17 No. 9

activation

on titanium

and Dolvstvrene

in vitro: C. Gretzer

et al.

wells were incubated at +4”C overnight with human fibronectin (Sigma) and fibrinogen (Sigma) in normal plasma concentrations (0.25 and 3 mg ml-‘, dissolved in sterile Hanks’ balanced salt solution, respectively). Before use, the wells were washed three times with Hanks’ balanced salt solution and kept wet until incubation with monocytes. The effect of exogenous agents on the activation of cells on titanium and evaluated adding was polystyrene by lipopolysaccharide (LPS; Sigma) in different concentrations (O-20 pg ml-‘) or polystyrene particles (1 and 3pm diameter; Sigma). The monocytes were cultured in 5% CO1 and 100% humidity at 37°C for 24 or 48 h. The supernatant was carefully harvested, centrifuged free of monocytes, immediately frozen and until used in IL-la kept at -70°C the radioimmunoassay (Amersham, UK). Mean values were determined from duplicate wells. The assay is based upon competition between unlabelled IL-la in the sample and the 1251-labelled analyte for sites on the specific antibody. This means that the binding of the labelled analyte to the binding of the antibody is inversely related to the amount of the labelled analyte in the sample. The standard curve was created by using a visual best fit method. The plot was performed by plotting ?&B/B,, as a function of the log IL-la concentrations on logarithmic graph paper. Monocytes adherent to the different substrata were fixed by 2.5% glutaraldehyde in 0.05~ sodium cacodylate, pH 7.2, and post-fixed in osmium tetroxide. After dehydration in a graded series of ethanol, the cells were embedded in epoxy resin (Agar 100, Agar Aids, Stansted, Essex, UK) for light microscopy (l-pm-thick sections) and transmission electron microscopy. Sections were cut either of the whole specimen including the polystyrene dish or after the dish was fractured away from the embedded culture.

Statistics All data, throughout the study, were subjected to the Wilcoxon range sum test for determination of significant differences at the 1 and 5% levels.

RESULTS Surface characterization AES showed the presence of carbon, titanium and oxygen, but no other elements in any of the coatings analysed (Figure 1). Quantitative elemental abundances were derived by analysis of the AES spectra using standard calibration factors. Elemental depth profiles were obtained by sequential sputter etching of 1.5nm between each consecutive AES recording. The depth profiles showed the presence of carbon, titanium and oxygen (Figure 2). The carbon signal was low in the outer part of the coating but was strongly increased at a depth of about 50nm. This increase is caused by the polystyrene substrate. For a depth < 50nm the sample was dominated by titanium and oxygen. In the outer part of the coating the oxygen content was relatively high. The central part of the

Monocyte

i 5 iii

activation

on titanium

and polystyrene

in vitro:

C. Gretzer

-

f

400 Kinetic

200

600 energy (eV)

600

Figure 1 A typical Auger electron survey spectrum oxidized titanium coating made by sputtering polystyrene, showing the differential versus kinetic for the Auger electrons.

0

200

400 Depth

800

1000

for an onto energy

853

et al.

plasma membrane from the substream (Figure ~a). Defects in the titanium coat were never observed, but the layer had an undulating appearance (Figure 5b). Most probably this was induced during sectioning as the polystyrene was compressed, resulting in wrinkles in the cell-containing plastic section. For this reason most observations were made on specimens in which the culture dish was removed before sectioning. Treatment with LPS resulted in a change in the morphology, characterized by monocyte appearance of slender cytoplasmic extensions in most cells (Figure 5~). Monocytes were found to release IL-let when cultured on both polystyrene and titanium surfaces. In initial experiments the effects of different cell culture time and different LPS concentrations, were concentrations examined on polystyrene

800

(AI

Figure 2 Auger electron spectroscopy depth profile for an oxidized titanium coating made by sputtering onto polystyrene. The relevance abundances are shown for three elements.

coating was dominated by titanium, whereas at the interface between the coating and the polystyrene substrate, the composition of the coating was again consistent with a TiOz stoichiometry. The transmission through the titanium-coated polystyrene culture plates was measured at normal Cary 5 incidence on a UV-vis/near-IR spectrophotometer operating in the 0.3 < i < 2.5 pm wavelength range. The transmission was found to have a constant value across the 0.4 < A < 0.7pm range pertinent to visible light (Figure 3). In the IR there were sharp dips in the transmittance.

0.5

1

2

1.5

Wavelength

2.5

(pm)

Figure 3 Spectral transmittance through oxidized titanium coating on polystyrene.

a

50-nm-thick

Monocyte activation Electron microscopy after culture for 24 h showed that the predominant cell type present in the cultures was monocytes (> 90%). In addition, lymphocytes and occasional pollymorphonuclear granulocytes were present (Figures 4 and 5). On both types of substrata the monocytes occurred either as single cells or in aggregates. No apparent difference in the ultrastructure of monocytes could be distinguished in monocytes grown on polystyrene (Figure 4) or on titanium-coated polystyrene (Figure 5d). Neither did preadsorption of fibrinogen or fibronectin to culture wells affect the general monocyte morphology in any apparent manner. The monocytes were closely adherent to the substrata ,with a narrow gap (30-50 nm) separating the

Figure 4 Electron micrograph of aggregate of cells kept in culture for 24h. Monocytes (Me) predominate but a lymphocyte (L) is also present. A fragment-of polystyrene (PS) remains after the dish bottom was removed from the embedded culture (original magnification x7500). Biomaterials

1996, Vol. 17 No. 9

Monocyte

854

activation

on titanium

and polystyrene

in vitro:

C. Grefzer

et al.

IL-la

(fino1/100 Ia

lo5-

0

0 0 12.5 5

10

20Lps WJmo

Figure

Figure 5

a, Detail of Figure 4 showing the close contact between a monocyte (Me) and the polystyrene (PS) dish (original magnification x18000). b, Electron micrograph showing the titanium layer (ti) sputtered on polystyrene. A monocyte (Me) is attached to the titanium layer. The undulating appearance is regularly seen when cells and culture dish are sectioned without being separated and is probably an artefact caused by chatter during cutting of the polystyrene (original magnification x40000). c, Monocyte on polystyrene dish and exposed to LPS, which induces the appearance of long, slender cytoplasmic extensions. A similar effect of LPS is also seen in monocytes attached to titanium (original magnification x7000). d, Aggregate of monocytes cultured on titanium-coated polystyrene. The culture was separated from the dish before sectioning and the major part, except for some fragments (ti), was therefore removed (original magnification x 10 000).

6 The effect of different concentrations of LPS (added to the wells together with the monocytes). Monocytes were cultured for 24 h on polystyrene and the amount of IL-la determined in the supernatants. The data points represent the values obtained in four separate experiments with different blood donors (‘P < 0.05; n.s., non-significant).

IL-la

(fmouloo pl)

20n.s.

n

lS*

surfaces. When monocytes were stimulated with LPS an augmented IL-lcr level was detected after 24 h. The with an increasing stimulation of monocytes concentration of LPS (O-20 pg ml-‘) revealed a plateau effect between 1 and 10 pgml-’ (Figure 6). Five and 1O~mmll’ of LPS elicited the highest level of IL-1~. When increasing the culture time from 24 to 28 h, no significant increase in the amount of IL-l% was detected (Figure 7), irrespective of LPS stimulation or not. The number of monocytes had a significant effect on the IL-la levels (Figure 7). When the number of non-LPS-treated cells was increased 10 times (from lo5 to lo6 monocytes per ml) only a two-fold increase in IL-lx was observed. In wells with LPS-stimulated monocytes, on the contrary, a marked increase (sevenfold) was detected when increasing the cell number from lo5 to lo6 monocytes per ml. When further increasing the cell concentration to 5 x 10’ monocytes Biomaterials

1996, Vol. 17 No. 9

lo-

l 24h 046h

5ns.

O-

Number of

.:'i 1w

, 1w 5xlV

L unstimulated’

1w I

,

,monocytes

(/ml)

1w Sxl(r LPS 4 (10 MM)

Figure 7 The effect of different monocyte concentrations and culture times on the amount of IL-la. Monocytes were cultured on polystyrene for 24 and 48 h in medium alone (unstimulated) or with LPS (1O~gml~‘) added to the medium together with the cells. The data are mean values from at least six experiments.

Monocyte

activation

on titanium

in vitro: C. Gretzer et al.

and polystyrene

per ml, the IL-la level was greatly increased, irrespective of LPS treatment or not (Figure 7). In subsequent experiments the IL-la levels were measured using 1 x lo6 monocytes per ml after a 24 h culture period on both polystyrene and titanium surfaces, with and without the addition of LPS (1Opg ml-‘). These experiments showed that on both surfaces cells were activated to such a degree that measurable levels of IL-la were obtained without the addition of LPS (Figure 8). Preadsorption of the substrata with fibronectin (0.25 mgmll’) markedly increased the IL-la level after stimulation with LPS (lO~grnl_~; Figure 8). No such further increase by LPS was detected when monocytes were cultured on polystyrene and titanium surfaces which were preadsorbed with fibrinogen (3 mg ml-‘) and stimulated with LPS. In order to examine the effect of particulate agents on monocyte function, IL-la levels were measured 24 h after the addition of polystyrene particles. Polystyrene particles (1 and 3 ,um diameter) were detected intracellularly after 24 h. The addition of lprn large particles in concentrations between 10” and 10s particles per ml did not cause a significant increase in the IL-la levels after 24 h in comparison with unstimulated monocytes (Figure 9). However, the addition of 10’ particles per ml resulted in approximately six-fold increase of IL-la in comparison with unstimulated monocytes. The IL-la levels reached with this particle size and concentration were higher than the IL-la levels obtained after stimulation with LPS (10 pg ml-‘). A concentration-dependent increase in IL-

IL-la

IL-la (W100

855

IJJI)

i

polfltyrene ti-sputtered

30 20

10 n unstim LPS (1Opg)

l(r -

10’ l@ lo’ 3pm

-

106 10’ 18 -

lum

lo’ -

Figure 9 IL-la in the supernatant after 24h cultivation of 1 x lo6 monocytes per ml on polystyrene and titaniumsputtered surfaces. The monocytes were unstimulated or stimulated with LPS (1Opg ml-‘) or polystyrene particles (1 and different diameter) in concentrations. 3pm Mean f s.e.m. of six separate experiments (*P < 0.05).

la levels was observed after the addition of 3 vrn large particles (lo”-10’ particles per ml). The IL-la levels detected after stimulation with 10’ particles per ml were about equal to those obtained after LPS stimulation. During the experiments with particle stimulation, no major differences in IL-la levels were observed between monocytes adherent to polystyrene and titanium.

memopII DISCUSSION

30

5 0 mwtim L

LPS (10I49

Maled

A

unstim

LPS unstim (10I&@

-fibrioogemd (3 mg/ml)

LPS (10M)

-fibrondaA CO.25Wmb

Figure 8 The IL-la level detected in the supernatants from 1 x 10” monocytes per ml cultured for 24 h on uncoated, fibrinogenand fibronectin-coated polystyrene and titanium-sputtered surfaces with and without addition of 1Opg ml-’ LPS. Mean fs.e.m. of six separate experiments using cells from six different blood donors (‘P < 0.05; n.s., non-significant).

The present study shows that human peripheral blood monocytes cultured on both a conventional tissue culture-grade polystyrene and a TiOz surface secrete IL-la without the addition of exogenous cellstimulating substances. In a similar experimental system to ours, using polymer surfaces7, release of IL-l was not detected unless the monocytes were stimulated by the addition of LPS. Although we do not have an explanation for this discrepancy, major differences in analytical methods (biological versus immunoassay) and material substrates may have an influence. Our finding of IL-la release without the addition of exogenous stimuli corroborate previous observations on the stimulatory effect of macrophage adherence to plastic surfaces: macrophage adherence has been shown to induce rapid (within 1 h) IL-l mRNA expression and protein synthesis’0711. Furthermore, our finding supports the assumption that a biomaterial surface may be a direct activator of macrophages, previously suggested by the in vitro observations of Murray et al.“. In the latter study the release of factors that stimulated bone resorption was markedly higher by murine peritoneal macrophages adherent on glass, stainless steel, polytetrafluoroethylene, epoxy resin, polyethylene and Riomaterials 1996, Vol. 17 No. 9

Monocyte

856

poly(methy1 methacrylate) than by cells in suspension. The mechanism for the material surface activation of cells is as yet unknown. The expression of membraneassociated IL-l is decreased by inhibition of a divalent cation-dependent spreading of murine peritoneal macrophages on plastic13. As indicated by the data presented by Murray et al.“, the degree of surface hydrophilicity and surface roughness may be important. IL-1 and tumour necrosis factor are produced by macrophages in response to LPS, although they are The precise mechanisms for regulated differently’*. release are not known. LPS-stimulated cytokine However, interactions between LPS and LPS-binding sites on the macrophage surfacer5,16 and LPShave been suggested. LPS binding proteins’7-‘g stimulation of IL-la (and IL-lp) production is downregulated by protein kinase C inhibitors in murine peritoneal macrophages” and human monocyteszl. Recent data indicate that protein kinase C activation sufficieint for an increased mRNA alone is expression of IL-l/?, whereas additional stimuli (provided by LPS) are required for mRNA of IL-la in human expression and production mononuclear phagocytes in vitrozl. In order to study cell-biomaterial surface interactions in vitro, the choice of cell type is crucial. In vivo data indicate that the macrophage plays a pivotal role during healing of implants in tissues. After insertion of solid, machined titanium implants in soft tissues in the rat, an inflammatory reaction is elicited. After 1 week the majority of inflammatory cells in the fluid space, predominantly monocytes and macrophages, are either located on the surface of the reorganized tissue or adherent to the implant surface. However, after 6 weeks the fluid space is largely absent and the tissue is in immediate contact with the implant surface2,*. During this phase of healing the macrophage is by far the most common cell type in contact with the surface, whereas fibroblasts (and macrophages) are detected further out from the surface in the surrounding fibrous capsule. Macrophages persisted at the titanium implant surfaces and exhibited different phenotypes, as judged by their ultrastructure*. However, the function of the macrophages, for instance possible factors secreted by the cells, during the healing of the implant in soft tissues is not understood. In contrast to the cellular reactions around titanium in soft tissues, morphological observations of the tissue response in bone (cortical bone in rabbit tibia) indicate that the presence of macrophages and multinuclear giant cells at the titanium surface is a transient event, succeeded by a mineralization of the which is directed towards the implant tissue surface22,23. In vitro studies have shown that titanium surfaces system C3’*, with a activate the complement subsequent generation of C3a and C5aZ5. Complement component C5a stimulates the release of IL-l by mononuclear cells and IL-l is implicated as a potent substance (osteoclast-activating bone-resorbing The direct contact between bone and factor)8. titanium implants in vivo and the long-term stability Biomaterials

1996. Vol. 17 No. 9

activation

on titanium

and polystyrene

in vitro: C. Gretzer et al.

of loaded implants in patients would speak against a continuous generation of high concentrations of IL-l vivo. in the titanium-tissue interface in Our observations that endotoxin augments the IL-la release by the macrophages on the titanium surface and findings that C5a, together with endotoxin, cause a markedly increased production of IL-l” suggest that in a situation with bacterial infection and exudation at the implant surface a bone resorption may be mediated by IL-l. In fact, several studies indicate that bacterial infection may be one important cause of early failures (mobility and loss of bone support) of titanium implantsz7. Moreover, recent immunohistochemical studies have also revealed the presence of macrophages and immunocompetent cells around failed (mobile) Brinemark implants (unpublished). Polystyrene (latex) particles are considered to be inert and non-inflammatory agentsz8-“‘. However, the present observations show that human monocytes released IL-lx after stimulation with 1 and 3pm diameter polystyrene particles. These findings are in agreement with those of Murray and Rushton3’. In their study latex particles (1.1 pm diameter), especially in high concentrations, were potent activators of mouse peritoneal macrophages, stimulating an increased release of prostaglandin E2 and even greater release of stimuli of bone resorption in vitro in comparison with control macrophages. To the authors’ knowledge, the presence of material particles has not been demonstrated around dental titanium implants. However, interactions between macrophages and particles have been implicated in the mechanisms of failure of cemented and non-cemented arthroplasties. In these instances, inflammatory cells, including macrophages, are localized in the tissue around the implant33-35. In such locations, bone is resorbed and a fibrous capsule is formed, resulting in a mobile implant, pain and loss of function. A crucial point in the present study was the preparation of the culture wells. However, a titanium coating thickness of 50nm was regularly attained. Moreover, the coating was firmly adherent to the underlying polystyrene substrate and was found to consist of three parts. The high oxygen content in the outer surface of the coating on the polystyrene surfaces and the AES data are consistent with a Ti02 stoichiometry. Most likely, this oxide was formed by reactions with the sputter-deposited titanium and ambient air when the coater was ventilated to atmospheric pressure after the coating process. The central part of the coating was titanium rich and hence metallic. In the inner part of the coating, towards the substrate, the coating was again consistent with a TiOz stoichiometry. Presumably, this oxide was formed during the reactive sputtering in the presence of O2 gas. Optical transmittance through metal-coated culture wells is advantageous since it allows microscopic examination of monocytes on the surface. The constant and high transmittance in the visible is interesting; it is conceivably associated with percolative effects among metallic components in the titanium-rich part of the coating3’, as well as with induced transmission caused

Monocyte

activation

on titanium

and polystyrene

in vitro: C. Gretzer et a/.

by the dielectric Ti02-like surface layers on both sides of the metallic coating37. An advantage with the metal-sputtered substrates is that the possible influence of different metals on monocytes morphology function may be analysed and compared. Furthermore, the surface of the substrate may be systematically modified with respect to, for example, surface composition, oxide thickness and microtopography, and the effects on cell adherence, morphology and secretory response evaluated. The role of material surface properties and biological components, for instance surface-adsorbed proteins, for cell activation in the implant-tissue interface in soft tissues is not understood. The present study showed that preadsorption of fibronectin markedly increased the level of LPS-stimulated IL-la release, whereas preadsorbed fibrinogen inhibited the LPSinduced augmentation of IL-la. The mechanisms of these effects are not known. One possibility might be that the stimulatory effect of fibronectin is related to its function as non-immune opsonin and stimulator of phagocytosis38-40 or via binding to the cell surface. Another possibility is that the monocyte adherence and/or cell morphology might have been altered by the preadsorption. A rapid increase of human monocyte adherence to matrix-bound fibronectin following treatment with LPS has been demonstrated*l, possibly mediated via an increased fibronectin receptor synthesis and expression*‘. Obviously, the present observations have to be followed by systematic studies on the effect of different concentrations of proteins, coupled with immunocytochemical studies on the relationships between the inflammatory cell, surface and protein. Recent immunocytochemical studies’ have shown that the macrophages in the fluid space are closely associated with strands of fibrin and fibronectin which are found on the surface of the tissue (extracellular matrix). These morphological observations suggest that interactions between the macrophage and proteins may be of importance for the localization of cells adjacent to implant surfaces and, as indicated by the present observations, also for their secretory response. However, it may also be argued that direct material surface-macrophage interactions may be of less relevance for the (early) events which occur after implantation3. Thus, in order to simulate the in viva conditions the introduction of other extracellular matrix components and/or other cells may be considered. In summary, sputtering of titanium onto polystyrene surfaces resulted in an outer Ti02 surface. Human monocytes cultured on these surfaces released IL-la in vitro without the addition of exogenous stimuli, but the release could be further augmented by exogenous agents, like LPS and polystyrene particles. Polystyrene particles in high concentrations were equally effective stimuli as the optimal concentration of LPS. Moreover, the amount of IL-la detected was dependent on the cell concentration and the type of protein adsorbed on the surface. A doubling of the culture time 24 to 28 h did not have any major effect on the IL-la response. The experimental system may provide one means whereby the mechanisms for monocyte activation on biomaterials can be analysed.

857

ACKNOWLEDGEMENTS The financial support from the IngaBritt and Arne Lundberg Science Foundation, the King Gustaf V 80year Fund, the Medical Faculty, University of Gothenburg, the Gothenburg Medical Society, the Swedish National Association against Rheumatism, the Swedish Society of Medical Sciences, the Swedish National Board for Technical Development (NUTEK) and the Swedish Medical Research Council (9289 and 9495) is gratefully acknowledged. The authors wish to thank the Blood Central, Sahlgren’s Hospital for supplying buffy coats.

REFERENCES 1

2

3

4

5 6

7

8 9

10

11

12

13

14

Rosengren A, Johansson BR, Thomsen P, Ericson LE. A method for immunolocalization of extracellular proteins in association with the implant-soft tissue interface. Biomaterials 1994; 15: 17-24. RBstlund T, Thomsen P, Bjursten LM, Ericson LE. Difference in tissue response to nitrogen-ion implanted titanium and c.p. titanium in the abdominal wall of the rat. J Bjomed Mater Res 1990; 24: 847-860. Eriksson AS, Lindblad R, Ericson LE, Thomsen P. Distribution of cells around implants in soft tissue in the rat. J Mater Sci: Mater Med 1994; 5: 269-278. Johansson CB, Albrektsson T, Ericson LE, Thomsen P. A quantitative comparison of the cell response to commercially pure titanium and Ti6A14V implants in the abdominal wall of the rat. J Mater Sci Med 1992; 3: 126-136. Anderson JM, Miller KM. Biomaterial biocompatibility and the macrophage. Biomaterials 1984; 5: 5-10. Miller KM, Anderson JM. In vitro stimulation of fibroblast activity by factors generated from human monocytes activated by biomedical polymers. J Biomed Mater Res 1989; 23: 911-930. Bonfield TL, Colton E, Anderson JM. Plasma protein adsorbed biomedical polymers: activation of human monocytes and induction of interleukin 1. J Biomed Mater Res 1989; 23: 535-548. Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunoll989; 44: 153-195. Pertoft H, Johnsson A, WtimegCd B, Seljelid R. Separation of human monocytes on density gradients of Percoll. J Immunol Meth 1980; 33: 221-229. Fuhlbrigge RC, Chaplin DD, Kiely JM, Unanue ER. Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide. J Immunol 1987; 138: 3799-3802. Haskill S, Johnson C, Eierman D, Becker S, Warren K. Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes. J Immunol 1988; 140: 1690-1694. Murray DW, Rae T, Rushton N. The influence of the surface energy and roughness of implants on bone resorption. J Bone Joint Surg fBr) 1989; 71B: 632-637. Labadia M, Faanes RB, Rothlein R. Role of adherence vs spreading in the induction of membrane-associated interleukin-1 on mouse peritoneal macrophages. J Leukocyte Bioll990; 48: 420-425. Takasuka N, Tokunaga T, Akagawa KS. Preexposure of macrophages to low doses of lipopolysacharide inhibits the expression of tumor necrosis factor c( mRNA but not of IL-lb mRNA. J Immunol 1991; 146: 3824-3830. Biomaterials

1996, Vol. 17 No. 9

Monocyte activation on titanium and polystyrene

858 15

16

17

18

19

20

21

Akagawa KS, Kamoshita K, Tokunaga T. Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-l on the proliferation and differentiation of murine alveolar macrophages. ] ImmunoI 1988; 41: 3383-3390. Akagawa KS, Kamoshita K, Tomita T, Yasuda T, Tokunaga T. Regulatory mechanism of expression of LPS binding site(s) and signalling events by LPS in macrophages. Adv Exp Med RioI 1990; 256: 467-480. Lei MG, Morrison DC. Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. II. Membrane localization and binding characteristics. J ImmunoI 1988; 141: 1006-1011. Hampton RY, Golenbock DT, Raetz CRH. Lipid A binding sites in membranes of macrophage tumor cells. J Riol Chem 1988; 263: 14802-14807. Hara-Kuge S, Amano F, Nishijima M, Akamatsu Y. Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells. J Biol Chem 1990; 265: 66066610. Kovacs EJ, Radzioch D, Young HA, Varesio L. Differential inhibition of IL-l and TNF-r mRNA expression by agents which block second messenger pathways in murine macrophages. J Immunol 1988; 141: 3101-3105. Hurme M, Serkkola E. Different activation signals are required for the expression of interleukin-1 r and /I genes in human monocytes. Stand J Immunol 1991;

28

29

30

Sennerby L, Thomsen P, Ericson LE. Early tissue response to titanium implants inserted in rabbit cortical bone. I. Light microscopic observations. J Mater Sci: Mater Med 1993;

23

32

Sci: Mater Med 1993;

24

34

35

36

Nishihara T, Ishihara Y, Noguchi T, Koga T. Membrane IL-1 induces bone resorption in organ culture. J 1989;

Preprosthetic

Biomaterials 1996, Vol. 17 No. 9

RA, Gray AB, Wright S, Railton GT, Freeman MAR. Sensitivity to titanium. A cause of implant failure. J Bone Joint Surg 1991; 73B: 25-28. Yagil Y, Yosefin M, Bergman DJ, Deutscher G, Gadanne P. Scaling theory for the optical properties of semicontinuous metal films. Physiol Rev 1991; B43:

38

1984; 39

40 41

43: 1881-1886.

Oral

and

Maxillofacial

42

Principles AppI Opt 1983;

of

design

of

architectural Rev Med

35: 561-575.

Proctor RA. Fibronectin: a brief overview of its structure, function and physiology. Rev Infect Dis 1987; 9 (Suppl. 4): S317-321. Hynes R. Molecular biology of fibronectin. Annu Rev CeII RioI 1985; 1: 67-90. Roth P, Polin RA. Lipopolysaccharide enhances monocyte adherence to matrix-bound fibronectin. Clin Immun

Brlnemark P-I, Lausmaa J, Thomsen P, Ericson LE, B&remark R, Skalak R. Anatomy of osseointegration and the transfer of load. In: Fonseca RJ, Davis WH, eds. Reconstructive

71A: 1337-1342. Lalor PG, Revel1

coatings.

Perala D, Chapman R, Gelfand J. Complement activation by dental implants. Int J Oral Maxillofac Implants 1991;

ImmunoI

27

1988; 70A: 347-356. Lombardi AV, Mallory TH, Vaughan BK, Drouillard P. Aseptic loosening in total hip arthroplasty secondary to osteolysis induced by wear debris from titaniumalloy modular femoral heads. J Bone Joint Surg 1989;

24: 4127-4141. Mosher DF. Physiology of fibronectin. Annu

4: 494-502.

McAlarney ME, Machlin ES, Skalak R, Kim S. Adsorption of C3 versus metallurgical structure of titanium oxides (abstract). J Dent Res 1989; 68:

72B: 988-992.

Agins HJ, Alcock NW, Bansal M et al. Metallic wear in failed titanium-alloy total hip replacements. A histological and quantitative analysis. J Bone Joint Surg

11342-11352. Berning PH.

6: 136-141.

26

Rae T. The biological response to titanium and titanium-aluminium-vanadium alloy particles. II. Long-term animal studies. Riomaterials 1986; 7: 37-40. Murray DW, Rushton N. Macrophages stimulate bone resorption when they phagocytose particles. J Bone

37

306.

25

Surgery, Ch. 8. Philadelphia: WB Saunders, 1995: 165224. Humes JL, Bonney RJ, Pelus L et al. Macrophages synthesis and release of prostaglandins in response to inflammatory stimuli. Nature 1977; 269: 149-151. Baggiolini M, Schnyder J, Dewald B. Role of phagocytosis in macrophage activation. In: Karnovsky LM, Bolis L, eds. Phagocytosis-Past and Future. London: Academic Press, 1982: 339-355. Schorlemmer HU, Davies P, Hylton W, Gugig W, Allison AC. The selective release of lysosomal acid hydrolases from mouse peritoneal macrophages by stimuli’ of chronic inflammation. Br J Exp Pathol 1977;

Joint Surg I&J 1990; 33

4: 240-250.

Sennerby L, Thomsen P, Ericson LE. Early tissue response to titanium implants inserted in rabbit cortical bone. II. Ultrastructural observations. J Mater

et al.

58: 315-26. 31

33: 713-718.

22

in vitro: C. Gretzer

Immunopatholl990;

57: 363-373.

Holers VM, Ruff TG, Parks DL, McDonald JA, Ballard LL, Brown EJ. Molecular cloning of a murine fibronectin receptor and its expression during inflammation. JExp Med 1989; 169: 1589-1605.