titanium-hydrogen peroxide interaction: model studies of the influence of the inflammatory response...

Titanium-hydrogen peroxide interaction: model studies of the influence of the inflammatory response on titanium implants

Pentti TengvaJl, Ingemar LundstrCim, Lws Sjaqvist and Hans Eilwing Department of Physics and Measurement Technology, Linkc’iping Institute of Technology, S-58 1 83 Linkaping, Sweden

Laxs Magnus Bjursten Institute for Applied Biotechnology, PO 80x 33053, S-400 33 Giiteborg, Sweden (Received 2 November 1987; accepted 21 January 1988)

In vitro studies of titanium and TiO, as well as other metals were carried out to investigate the role of these metals in the inflammatory response through the Fenton reaction. The TiOOH matrix formed traps the superoxide radical, so that no or very small amounts of free hydroxyl radicals are produced. Ellipsometry and spin trapping with spectrophotometry and electron spin resonance (EM) were used to study the interaction between Ti and t1202. Spectrophotometry results indicated that Ti, Zr, Au and Al are low free OH-radical producers. We propose a new model for the titanium-tissue interface where the oxidized titanium surface is covered with a hydrated TiOOtl matrix after the inflammatory reaction. This matrix is suggested to possess good ion exchange properties, and extracellular components may interact with the Ti(lV)-H202 compound before matrix formation. The TiOOH matrix is formed when the H202 coordinated to the Ti(IV)-H20, complex is decomposed to water and oxygen. Superoxide (O,-) may be bound therein. The oxide layer initially present may be partly reformed to a TiOOH matrix due to the interaction with hydrogen peroxide.

Keywords: Titanium, hydrogen peroxide, oxygen radicals. inflammato~ response

When implants are inserted into living tissue, the tissue reaction is influenced by the presence of the foreign material as well as the insertion procedure itself. The implantperse is also modified by the tissue. Examples of such reactions are the oxidation of metals’.’ and the degradation seen in polymers’. Furthermore, the influence on the metal implant from the surrounding is larger in tissue in vivo than in saline aqueous solution in vitro3. Since the insertion of an implant is always associated with an inflammatory response caused by the surgical trauma, it is of general interest to investigate how metals behave under such conditions. Clinical experience shows that the response is different at different locations in the body and for different materials. One of the features of an inflammatory response is the release of superoxide and hydrogen peroxide from inflammatory cells into the extracellular space4-” and an interaction between these species and the foreign body is expected. Hydrogen peroxide is dismuted from superoxide (Oz-), occurring during the

Correspondence to Dr P. Tengvall.

respiratory burst through a reaction catalysed by the enzyme superoxidase dismutase (SOD) as follows:

SOD

Oz- + Oz- + 2H+ + H202 + Oz (1)

We suggest that the possible occurrence of so-called Fenton reactions is of special importance after reparative surgery where alloyed or pure metals are implanted into the human body, e.g. as in hip prostheses and dental bridges. In a Fenton reaction, a metal ion (M”+) takes part in the formation of a hydroxyl radical (OH’) during the hydrogen peroxide (HzOz) decomposition through the following reaction:

M”+ + H202 + M(“+ “+ + OH- + OH* (2)

Transition metals in aqueous solutions may take part in the OH’ generation in at least two ways. The metal surfaces (or the surface oxide layers) may decompose HP02 thereby creating OH’, or metal ions leaking out to the surroundings of the metal implant may catalyse the decomposition of H202

Q 1989 Butterworth Et Co (Publishers) Ltd. 0142-9612/89/030166-l 0$03.00

166 Biomaterials 1989, Vol 10 April

Titanium-hydrogen peroxIde mteraction: P. Tengvall et al.

1 MPO-system

Radical formation

1 Radical formation H,OZ+e-- OH’+OH-

i

Tissue damage

Lipid peroxidation *

4 - hydoxyalkenals = precursors of chemo - tactic messengers

Figure 1 Smpiified model ot the OH*-generated lipid deterioration. The

product gwes we to precursors of chemotactic messengers (atier M.V.

Torrielh and M.0 D/anzaru, Ref. 1 1)

according to reaction (2). The hydroxyl radicals so formed

cause Injury In biological systems in a number of ways”.

Extracellular fluids containing glucose-aminoglycans, binding

proteins and hyaluronic acid may be degraded by OH-radicals.

Biomembranes containing large amounts of polyunsaturated

fatty acids can undergo peroxidation. Also intracellular

radical generation, with subsequent cell deterioration, can be

caused by aerobic cells switching to anaerobic metabolism

(hypoxia), or through the exposure of aerobic cells to

hyperoxia. The role of oxygen radicals in biomembrane

deterioration through lipid peroxidation is summarized in

Figure 1. This figure emphasizes the influence of hydroxyl

radicals on lipid autodeterioration and the following possible

formation of chemotactic factors. Another possible pathway

for OH-radical formation is the production of radicals via the

myeloperoxidase system. The present study does not,

however, include this possibility.

In a first study, we investigated the behaviour of

titanium immersed in hydrogen peroxide containing saline

solutions. Some of the observations were presented at a

conference in 1 98712 and are summarized in a short

communication’3. Titanium was chosen as a model metal

since it has interesting physical and chemical properties in

saline environments like that of the human body. Its oxide

has a dielectric constant close to that of water, the pK, for the

dissolved oxide in water is greater than 14, i.e. greater than

the pK, for water14, and its oxidation is not affected by

proteins15. Furthermore, Branemark et al. have shown that

titanium is an excellent implantation material in human

bone’6-‘8, and titanium in soft tissue causes no observable

inflammatory reaction”. ‘O. It has been observed that an

oxide (or oxide-like surface layer) grows on titanium implants

in human tissue3.*‘. In preliminary model experiments, a

significant oxide growth on titanium was observed in open-

circuited electrolytes at neutral pH in the presence of

hydrogen peroxide*‘. These observations suggest that

metabolic activity and/or free radical formatton are involved

in the oxidation process. In this paper, we describe m vitro

studies of titanium and TiO,, as well as some other metals,

aimed at the elucidation of the (possiblej role of the metals in

the inflammatory response through the Fenton reaction.

Three experimental techniques have been used. Ellipso-

metryz3 was used to follow the relative rate of oxidation/

corrosion at the titanium surface as a function of the H202

concentration, and electron spin resonance (ES/?) and

spectrophotometry were used together with spin trapsz4-“’

to studv the production of free hydroxyl radicals induced bv

the metals and onginating from H,O,

The rationale for performing the present expenments

can be summarized as follows

(1 ) An oxide like layer grows on titanium implantsin viva.

(2) In model experiments, a growth of an oxide-like layer on

titanium was caused by (small concentrations of) H201.

(3) H,O, occurs during an Inflammatory response

MATERIALS AND METHODS

Materials

Doubly distilled water was used In all the expenments.

Phosphate-buffered saline (PBS) solution was prepared

shortly before use. Crystalline TiO, (anatase) was purchasea

from Fluka AG. Titanium powder was of Specpure” quality

and purchased from Johnson & Matthew Ltd. A Ti gel was

obtained by mixing 1 g of Ti powder and 5- 10 ml 10 M H20;

in a cuvette and then incubating for 24 h at r.t. Hydrogen

peroxide was purchased from Merck Co.

Sample preparation

The titanium substrates for the ellipsometnc experiments

were prepared in the following way. Glass slides were first

washed in a mixture of water, H,O, and NH,OH, rinsed in

distilled water and secondly washed in a mixture of water,

H,O, and HCI. Thereafter, 50 nm Cr, 50 nm Au and

200 nm Ti were evaporated in vacuum on the glass in that

order.

In the spectrophotometric and ESR experiments, all

metal samples were prepared from metal sheets or metal

rods. The metal sample area was 60 mm* + 5%. Before

incubation, the metals were washed in trichloride ethane,

acetone and ethanol, and rinsed in distilled water. The metals

were obtained from Goodfellow Co. and were of at least

99.5% purity.

Ellipsometry

The oxidation/corrosion reaction at the Ti surface In H,02

solutions was studied by ellipsometry. With an ellipsometer,

it is possible to determine the complex ratio p between the

complex optical reflexion coefficients R, and R, of a surface

for s- and p-polarized light, respectivelyz3.

R 0 = p = tan v X elA

RS (3)

Biomaterials 1989. Vol 10 Apni 167

Titanium-hydrogen peroxide interaction: P. TengvaN et ai.

In practice, the settings of the polarizers are measured from which the two angles A and v can be calculated. If the complex refraction index forthe metal surface is known, then one can easily follow the dynamics of the growth of a (oxide) layer on the surface. However, if the refraction index of the surface is unknown, or varying in time, as was most probable in our experiment, it is difficult to calculate the absolute value of the oxide thickness. We have therefore chosen to follow the change in A and use the rate of change in this angle as a measure of the relative oxidation/corrosion rate. In our study, Ti, evaporated on glass in vacuum, was incubated in 0.1 M PBS-buffer, at neutral pH, with hydrogen peroxide concentrations varying between 1 m&r and 0.33 M with incubation times up to one wk. A faster change in the ellipsometer angle A was interpreted as a faster oxidation/ corrosion rate at the Ti surface. A Rudolph Research ellipsometer was used. During the ellipsometric measurements, the hydrogen peroxide concentration was held constant with a Pharmacia P-l peristaltic pump system.

Spectrophotometric experiments

These were performed to make a comparative study of OH-radical formation by a number of metals incubated in H202 solutions. Since the OH-radicals are very short-lived species, it is favourable to trap them in a more stable dye which may be detected in turn. N,N-dimethyl-p-nitro- soaniline2g-32 (RNO) has a very high affinity for OH-radicals (k z 1 X 10” M-’ s-l) and as the RNO-OH product is formed, the RN0 absorption maxima at il = 440 nm will decrease. The bleaching of RN0 through the OH-radical addiion was taken as a measure of the OH0 ~~at~on2g*3’, 33-34. In our experimental set-up, we incubated Ti, Zr, Fe, Cu, Cr, Ni, Au and Al (all of the size 5 X 5 X 3 mm) in 33~~ RNO, 3 InM H202 in 0.1 M PBS at neutral pH up to 92 h. RN0 was purchased from Sigma Chemical Co., and the absorbance measurements were performed with a Perkin Elmer Lambda 2 spectrophotometer.

ESR expe~ments

ESR experiments on the Ti gel were performed to detect oxygen radicals. Suprasil ESR sample tubes containing dried Ti-H202 gel were evacuated on a vacuum line and sealed. Spectra were recorded with a Bruker ER 200D spectrometer operated at X-band. The correct field strength and micro- wave frequency were determined by using an EIP model 54BA frequency counter and Bruker 031 M NMR gauss- meter. To determine the g-values more accurately by increasing the spectral resolution, Q-band (35 GHz) measurements were also performed. The Q-band spectra were recorded using a Varian E-9 spectrometer. Strontium oxide containing Mn*+ was used as a field marker as the hyperfine lines due to Mn2+ serve as an excellent field calibration. Spectra at X-band were recorded both at room temperature and at 7 = 77 K. No significant changes in the spectra were observed at the two temperatures, i.e. the g-values remained unchanged.

ESR spin trapping studies were performed at X-band to detect OH-radicals in the Ti-H,Oz aqueous system. The spin trap experiments with 0.1 M a-(4-pyrridyl-1 -oxide)-N-t- butylnitrone (4-POBN)35.36 were made in 0.01 M PBS, pH = 7, containing 0.3 M H202.

Experiments with 0.1 M 5,5-dimethyl- 1 -pyrroline-l- oxide (DMPO)37-3s were performed in doubly distilled water containing 0.1 M H202. Both 4-PQBN and DMPO form stable, detectable, nitroxide radicals as a result of OH-radical

addition (k =: 1 X IO’ M-’ s-l) and with this technique, one measures indirectly the presence of OH-radicals in the sample tube. A 1 mm flat quartz sample cell was used in all ESR spin trapping experiments. The experiments were made without spin trap purification and de-aeration of the aqueous samples.

RESULTS

Ellipsometric studies

Metal surfaces and metal ions catalyse the H202 decomposition to water and oxygen. The catalysis is explained as a combination of electron-donating and electron-accepting properties of the metal occurring simultaneously4’ as follows:

anodic H,O, --t 0, + 2H+ + 2e-

cathodic H202 + 2H+ + 2e- + HZ0

2H,O, --, H,O + O2 (4)

As H202 also is a strong oxidizer, metals incubated in H202 solutions are readily oxidized to form metal oxides or metal hydroxides. The reaction rates are pH- and [H,O&dependent. The ellipsometric measurements were performed in 0.1 M PBS at r-t. and with a constant flow of H20Z-containing solution over the (200 nm thick) evaporated Ti film. Results obtained show that Ti surfaces are oxidized/corroded by H202 and that with higher H202 concentration, one gets a higher oxidation/corrosion rate (see Figure 2). At high H20z concentrations we observed gas evolution and rapid Ti corrosion. At low H202 concentration, the oxidation/~orrosion rate was lower and there was a significant optical response measured as a change in A when Ti was incubated in the 0.1 M M PBS without addition of H20, (for a long time). The change in A in this experiment was measured to 1.5”/wk compared with a measured electronic drift of 0.05°/wk.

Figure 2 Changa in eljipsometer angle A versus time in 0. f M PBS, PH 7, T = 295 K and constant flow of H 0

‘3 solution. /ej Electronic drift lb1 PBS

alone, (cl 8 x l@~, fd) 1.5 X fO_ M. [a] 1.5 X lO-2M, fr) 3 X 10v2 M

and {gj 0.33 M of /f#&

168 Biomaterials 1989. Vol 10 April

Titanium-hydrogen peroxide interaction: P. Tengvall et al.

10 G I

Figure 3 Uydro~i radical formation in 0.1 M 4-POBN, O&f &? PBS and 0.3 M Hfl2 A spectrum characteristic of hydro~l radicafs appears, aN = 15.0 G, a& = 1.68 G and a,y = 0.36 G. Th a y-splitting decreases if the buffer is changed to deuterated water. The spectrum wes recorded at 3OUK after 5 min

irradiation.

ESR measurements on Ti in H,O,-containing solutions

In an attempt to detect free hydroxyl radicals (OH”) caused by titanium surfaces and/or ions in H202 solutions during dark conditions, ESR spin trapping experiments were performed. Bulk titanium (5 X 3 X 3 mm) was incubated for 1 h in 2 ml fresh 0.1 M 4-POBN in 0.01 M PBS at pH 7 containing 0.3 M

H202. Oxygen evolution was observed during the whole incubation period. Radicals could not be detected at 77 K or at 300 K. The sample was measured within 5 min after the incubation period. When the cell was exposed to UV radiation for 5 min. an ESR signal characteristic of hydroxyl radicals appeared (Figure 3). The triplets of doublets had the coupling constants: aN = 15.0 G, a,.,/3 = 1.576 G and a,y = 0.36 G in accordance with the Iiterature35*36. A similar spectrum was obtained when 5 ~1 FeCI, was added to 1 ml 0.3 M H,O, solution without irradiation. When D20 replaced PBS as a solvent in these experiments, the a,rsplitting disappeared. When 100 mg crystalline TiO, (anatase) was placed in the sample cell together with the H202 solution, no signal was obtained in situ under dark conditions. Oxygen evolution was observed, however, showing a TiO~-mediate catalytic H202 decomposition. UV irradiation for 10 min gave a weak OH. spectrum.

Experiments with 0.1 M DMPO in distilled water and with 0.1 M H202 were also performed. When 100 mg Ti powder was incubated for 2 h in the solution and was measured within 3 min after incubation, no radicals could be detected. The same results were obtained when TiO, replaced the Ti powder. Fe is expected to form OH-radicals readily upon interaction with H202. Therefore, a control

experiment with iron and 0.1 M H202 solution was performed. Fe (60 mm’, 2 X IO4 nm thick) evaporated in vacuum on glass spheres was incubated for 1 h in the solution. A spectrum characteristic of OH-radicals appearedz6 (Figure 4). Addition of ethanol to the solution gave rise to a superimposed a-hydroxyethyl radical spectrumz6 (Figure 5). The result indicates the presence of OH-radicals in the Fe-H20, solution as a-hydroxyethyl radicals are formed when the ethanol-OH* product reacts with DMPOz5. In another experiment‘ OH-radicals were readily generated within 1 min of UV irradiation of the quartz sample cell, containing 0.1 M H,Op in distilled water, in situ in the spectrometer. There was, however, a distinct difference when theTi02-H202 solution was irradiated. After 15 min of UV treatment, only a weak OH0 spectrum was detected.

Comparison of different metals

Spectrophotometric studies using RN0 as a spin trap were made on Ti, Zr, Fe, Cu, Cr, Ni, Au and Al. The experiments were performed to elucidate the behaviour of the metals with respect to hydroxyl radical formation through the Fenton reaction.

Bulk metals with an area of 60 mm2 t 5% were incubated in the H,O,-spin trap solution at neutral pH (Figure 6). Our results show that Ti, Zr, Ni, Cr, Au and Al have a lower bleaching rate, i.e. lower OH-radical formation than Fe and Cu (Figure 7) at low pH values. At higher pH, however, the bleaching of the dye increases for Ni and Cr, indicating a pH-dependent OH-radical formation rate for these metals (Figure 7). A UV-irradiated RNO-H,O, solution

Figure 4 ESR meesoreme~ts on 60 mm’ evaporated Fe ore spherical glass beads, diameter Q, = 3 mm. The beads were incubated for f h in distilled water, 0.1 M H2U2 and 0. f M DMPO. The Off* spectrum has the fo/i~ing coupling consrants: a, = 15.0 G, aHy = 15.0 G.

Biomateriais 1989, Vol t 0 April 169


Figure 5 Control experiment with addition of ethanol in the experiment in Figure 4. Superposition of hydroxyl and a-hydmxyethyl adducts with

aN = aHy = 15 G (OH* trapped) and a,.,, = 16 G and a& = 23.3 G (CHsCHOH trapped).

is rapidly bleached due to the homoi~ic cleavage of the O-O bond in the H202 molecule and the following capture of hydroxyl radicals.

Titanium gel formation and superoxide radical trapping

A most interesting observation was made for titanium incubated in a strong H202 solution. It was found that the metal-H-l,Op interaction gave rise to a gel in the solution if incubated for long enough13.

When 1 g Ti powder and 10 ml 10 M H202 were incubated for 24 h at r.t., a yellow, transparent, Ti(lV)-H,02 complex adduct was formed under gas evolution. The gas evolution slowly ceased during the later part of the incubation time and a non-transparent gel was formed when the gas evolution ceased. Excess water was removed and the gel was dried by heating at = 380 K. A yellow powder was left. After evacuation of trace water on a vacuum line, Q-band ESR measurements were stormed. A spectrum with g-factor anisotropy with g = 2.003.2.009 and 2.023 was displayed. According to the literature, this spectrum was identified as superoxide (02-) on TiOp (Figure 8)4’. A more transparent Ti gel was formed when bulk Ti (5 X 3 X 3 mm) was incubated in 10 M H202 for 6 month. In Figure 9, curve (a) shows theoptical spectrum of 1 OJJM H,O,, (b) shows the

Cr, Ni, Au Ti, Zr Ref.

y--$3==:~ Al

I 4

25 I 1 I

50 75 100

TIME (h)

Figure 6 Change in optical density at h = 440 nm of the solution versus time when Ti, Zr. Fe, Cu, Cr Ni. Au, snd A/ are incubated in 0. I M PBS, 33 PM RN0 and 3 mM HZO, a? neufra/ PH.

spectrum of 10 M H,O, and Ti powder after 4 h of incubation, and (c) the spectrum of the transparent slightly yellow gel formed from 10 M H202 and bulkTi after 6 month of incubation at r.t. (no ESR measurements were performed on this gel).

When 100 mg Ti02 (anatase) was incubated in 10 M

H202, oxygen evolution and light yellow coloration of the solution took place. The incubation time was 1 wk and the oxygen evolution decreased with time. Gel formation was, however, not observed.

DISCUSSION

The oxidation of implanted metals has often considerably exceeded that which could be expected from in vitro experiments using ordinary protein and salt solutions3”‘. It seems to be faster in bone marrow than in muscle tissue, which in turn is faster than in cancellous bone3. This may well be related to either the oxygen tension or the metabolic activity in the tissue. Also, the Ti level found in the tissue, adjacent to the surface, is often found to be higher”’ than the Ti02 solubility in water. This indicates occurence of other events than sole water-surface interaction to take place. From in vitro data it is well known that metals may be oxidized in the presence of hydrogen peroxide, and transition metals may take part in the cyclic Fenton reaction (see reactions (17)-(22)).

The generation of reactive oxygen species has been shown to occur in several situations in vivo. One important case is the superoxide generation by inflammatory ceils, such as polymorphonuclear cells during the infiammato~ process. As the superoxide is dismuted to H202 by the enzyme superoxide dismutase (SOD), the interaction between the implanted metal and H202 may be an important part of the healing process.

ESR spin trapping measurements

The ESR spin-trap results indicate the presence of OH-radicals in the UV-irradiated and Fe-containing samples and absence of OH-radicals for Ti powders and Ti02 powders in aqueous H202 solutions in the dark. The absence of OH-radicals in the titanium-hydr~en peroxide system was unexpected since it is well established thatTi(lll) readi~yforms OH-radicals in the presence of hydrogen peroxide (see reaction (5)) and our expectation was that Ti(IV) complexes would be reduced to Ti(lll) complexes during the H202 decomposition process.

Ti(lll) + H202 + Ti(IV) + OH- + OH* (5)

This reaction is often used to demonstrate OH* generation. Our experiments suggest, however, that no accessible Ti (111)

170 Biomaterials f989, Vol 10 April

Titanium-hydrogen peroxide interaction: P. Tengvall at al.

0

I I I I ) 5 6 7 8

PH

Figure 7 The pH-dependence of the bleaching of 30 JIM RN0 in 0.1 M PBS and 0.1 M H&I,. Fe and Cu induce a large bleaching in the whole pH interval and Ni and Cr higher bleaching rate as pH increases. Ti fr Au, and Al show a small bleaching throughout the whole pH intewal.

occurs during the interaction between Ti and H,02, or the l&O, decomposition takes place inside a complex in such a way that no free OH-radicals are released to the surroundings.

RN0 measurements

To determine the relationship between RN0 bleaching and OH-radical formation bythe metals in H202 solutions, control experiments were carried out with ethanol for the Fe-H,02 and CU-H202 systems at pH 7. The results shown in Tab/e 7 were obtained. The reactions to be considered are as follows3’:

k, RN0 + OH*-+ product (bleaching),

kr = 1.25 X 10” M-j s-’

kz

CH,CH,OH + OH*+ CH,CH~H + ~~0, k2= 1.6x lo”M-‘S-’

(6)

(7)

k3 CHsCHOH + RN0 + CH3CH0 + RNOH (bleaching),

k, = 2.4 X IO’ M-’ s-’ (81

@=2.023

Table 1 Bleaching of RN0 at J_ = 440 nm in the presence of ethanol

Metal Expected bieaching for the Actual bleachtng for the non-deaerated system non-deaerated system

Fe

CU

0.44 0.39 0.44 0.59

Results of experiments with added ethanol in 33 pM RNO, 3 rnM H,O, and respective bulk metal (60 mm*) at neutral pH. The bleaching was set to 1 for the deaerated system.

k, CH,&fOH + O2 -+ CHsCHO + HO,,

ka = 3.6 X IO’M-’ s-’

c-4

IOG

Figure 8 ESR spectrum of dried Tigel prepared fmm metallic Ti powder and 10 M H202

field marker are displa~ad. The spectrum was recorded at 295 K

Addition of 0.5 mM ethanol to the deaerated sample should result in an increase of the bleaching (reactions (7) and (8)) and with no deaeration we expect a decrease (reactions (7)‘ (8) and (9)) in the bleaching3’, because the aldehydeformed is not reactive towards RN03’. This assumes that the OH- radical is the sole initial source of RN0 bleaching. In non-deaerated systems, the O2 concentration in aqueous solution at r.t. and 1 atm (- 101 kPa) was calculated to be 1.3 mM. In the Fe system, the relative bleaching was close to the expected calculated value while the discrepancy is larger

f=35,01 GHz

The principal values of the g-tensor at Q-band obtained with A#+ as

Biomateriats 1989. Vol 10 April 171


f Abs

_-_ 200 300 400 500 nm

Figure 9 Opticalspectra showing fhe change of absorption maxima as Tiis incubated in Hz02 (a) 10~~ H202; [b) 10 M H202 and 0.5~ Ti powder jncubated 4 h: (c) 10 M Hf12 and bulk Ti (5 X 3 X 3 mm) immersed for 6 monfh. Observe that the absorption maximum moves from A = 260 nm to ,I ‘I 33Onm during the gel formation. The f&urn shows that H$z is decomposed and a new Ti species is formed in the sol~i~.

in the&r system.Theoriginof thisdiscrepancyis not known. From the RN0 measurements we conclude that (1) Fe and Cu bleach the spin trap solution through hydroxyl radical formation also at neutral pH, and (2) Ti, Zr, Au and Al give rise to a lower hydroxyl radical formation rate than the other metals compared in the pH range 5-8.

Ti gel fo~ation

Titania gels have recently43’44 been made from a Ti(lll)-Hz02 mixture precipitated with NH40H. In this gel, the superoxide radical is thought to be bound in the inner sphere of the complex45, 46. We have, in our experiments made a Ti gel from both Ti powder and bulk titanium incubated in H,Os. We observed H202 decomposition and Ti(IV)-Hz& complex formation simultaneously without free oxygen radical formation. When all the H202 was decomposed, the yellow solution chelated. The pH of the gel so formed was =: 4. Addition of excess H,O, dissolved the gel and the HzOz decomposition under gas evolution started again. It appears that the yellow Ti(lV) dye chelates only when the H202 has been completely consumed. For the gel produced from the Ti powder, it was possible to obtain enough dry weight to perform ESR experiments to search for the superoxide radical. The 02- was found at both 77 K and 433 K. There were no significant changes in the ESR spectrum (X-band) upon heating to 433 K. We therefore conclude that Os- is tightly bound to the Ti gel. The ESR results in Figure 8 are very similar to those of Ragai46 who recently described the trapping of O2 - in a titania gel. It is interesting to note that a gel could be made from metallic Ti but not from TiOp powder when incubated in 10 M H,02. This indicates that the polymerization was mainly due toTi ions leaking out from the

bulk metal during the interaction with H,O,.The transparent gel formed outside the bulk titanium seems, at least from the optical spectra, to be very similar to the one formed from the Ti powder. The gel structure proposed by Ragai and Symons44r45 is as follows:

0 Ti41/* ’

IoH\ /O Ti4+ Ti4+

‘OH’ ‘0’ ‘OH

It is very similar to the composition suggested for hydrated anodic oxides of titanium. It appears that the oxide formed on and outside the titanium surface due to the interaction with H20, is a hydrogenated polymeric matrix, strongly hydrated, probably with the stoichiometry TiOOH(H20)n or Ti(OHj4 (as.). ESCA studies performed on the dried gel showed the presence of Ti(lV) and oxygen. The spectrum broadening was too large to be explained solely by the 0- and OH-groups, and this may be an indication of some other electronegative species, i.e. 02-coordinated to theTi(lV) ion. The results above may have far-reaching consequences for the bi~ompatibili~ of titanium. If during the inflammato~ response, the superoxide radicals are dismuted to H,O,, we have a situation where the Ti surface may accumulate H202 in TiflVf-H1O;! complexes and form a TiOOH matrix after the H202 decomposition even in viva. The gel may contain the superoxide formed during the H,Oz decomposition. It also binds the TiflV) ions making them less mobile. The Ti(lV)-H*O~ complexes formed in the solution do not create free OH-radicals when the H,Oz decomposes. As the p& > 14 for TiO, (hyd.), it follows that metal-extra~ellular component complexes are not expected14 for thermo- dynamical reasons. However, the dissolved Ti(IV)-H,Oz complex may behave differently and thus an interaction between these and the e~racellular components may take place. The gel, that is the final result, could present an excellent ‘natural’ surrounding for adsorbed extracellular components. Further investigations of the Ti-H,02 formed gel are under way. In this work, we have not managed to form a gel from H202, and the other metals under study.

Oxidation/corrosion of Ti by H202

Detectable amounts of free hydroxyl radicals could not be found during our spin trapping experiments with Ti and TiO, exposed to HzOz, although both a transition metal (Ti) or TiOz and H202 were present in the sample cell. The interaction with H,O1 was, however, associated with gas evolution. This observation supports at first glance the catalytic two-electron model proposed by Haber4’, according to which oxide intermediate formation and reformation takes place simultaneously at the catalytic Ti(TiO*) surface, e.g.:

2Ti0, + H20, + 2e- 3 Ti,Os + Hz0 + O2 (10)

Ti203 + H,O,+ 2Ti02 + Hz0 + 2e- (111

where reactions (10) and (1 1) show a TiO~-mediate catalytic decomposition of H202 to water and oxygen without any oxygen radical formation. On the other hand, a yellow Ti(lV)-H20, complex formation occurred in the test-tube, indicating that the Ti(fV)-HzOs forms a very stable end-product, the TiOOH adduct48.4g, in which the O,-radical may be bound. Therefore, reactions (10) and (1 1) are too simple, and we conclude that they are not completely describing the H202 decomposition mechanism on titanium.

Also when TiOl and H202 were mixed together,

172 Biomaterials r989. Vol IO April


oxygen evolution was observed, indicating HzOz decomposition, and the yellow colour in the dye is again believed to be due to a Ti(lV)-HzOz complex5’. In other TiO, studies, water and hydrogen uptake during low cycling potentials at anodic oxidation of titanium have also been observed47z4s, and the following, not completely reversible4’, mechanism has been proposed when Ti electrodes are cycled between 0 and -0.65 V:

,TiOOH(H,O), (TizOs(HzO),,

TiO,(H,O) + Hf + e- (12)

‘TiOOH(H,O),]Ti(OH),(HzO), _,

We interpret the results obtained in our ellipsometric study as follows. Hydrogen peroxide is decomposed to oxygen and water at the Ti surface. Ti oxidation and Ti corrosion take place simultaneously with water uptake and Ti(lV)-Hz02 complex formation when Ti is incubated in HzO, solutions. The corrosion/oxidation rate is increased with increased [H202]. The H202 concentration in the aqueous solutions, below which the surface reactions cease, appears to be very low. In our experiments, we observe increased surface reactions, i.e. a significantly increased rate of change in the ellipsometer angle A for H,Oz concentrations as low as 1 KIM.

Fenton reaction

Hydrogen peroxide autodecomposition at neutral pH may be initiated through a catalytic HzOz-decomposition step but it is a very slow process. The overall rate coefficient of the decomposition at 300 K (Ref. 33) is = 1 X 1 O-7 M-’ s-’

and the decomposition is a chain reaction involving hydroxyl radical (OH’) and superoxide radical (Oz-) formation. Some important steps in this chain reaction are as follows33:

H,Oz + OH. - 02- + Hz0 + H+, k,z = 3 X lo7 M-' S-'

H,Oz + H0.z -+ Oz + Hz0 + OH’, k ,3 = 0.5 M-l S-’

H,O, + HOP- + Oz- + OH* + H,O, k,4 = 1.8 X 1O-7 Mm’ s-’

HzOz + Oz- + Oz + OH- + OH’, k,5 = 1 X 1O-3 M-’ s-’

(13)

(14)

(15)

(16)

From these equations, it can be seen that the so-called Haber-Weiss reactions (15) and (16) have rate coefficients too low to cause the oxygen radical toxicity as the Oz- is not toxic enough by itself. Transition metal ions catalyse the HzOz decomposition and a mechanism suggested by several authors5’-55 for transition metals (M) in interaction with H,Oz limits the source for the deleterious OH-radical to the reduced metal ion.

M”+ + H,Oz -+ M r” + ‘)+ + OH- + OH* (17)

M”+ + OH’ + M’” + ‘I+ + OH- (18)

H202 + OH*+ Hz0 + HOz (19)

M”+ + H02-M (n+ l)+ + HOP- (20)

H0.z - H+ + Oz- pK, = 4.88 (21)

M(“+‘)+ + 02--+ M”+ + O2 (22)

The reaction rate constants in this chain reaction are considerably higher (=: 1 06-1 0s) than in the spontaneous

HzOz decomposition. The only OH’ source is reaction (17), the Fenton reaction.

At high H,02:Ti(lll) ratios, reactions (19)-(20) should dominate and one would expect Ti(lll) reduction with subsequent free radical generation through reactions (17), (19), (21) and (22). This was, however, not the case. No hydroxyl radicals were detected by spin trapping techniques although the spin traps used have a very high affinity for OH- radicals3’,38. The explanation may be the formation of the very stable TiOOH (HzO), adduct with a Ti(IV) coordinated superoxide radical. If the Ti ions released or dissolved from the Ti surface all possess the (IV+) oxidation state56, and the superoxide radicals formed through reactions (19) and (2 1) are coordinated to the Ti (IV), then the classical Fenton chain reactions (17)-(22) may be quenched. So, it seems that nejther reactions (lo)-(1 1) nor reactions (17)-(22) are effective during H202 decomposition on Ti surfaces. But still, the HzOz is decomposed and TiOOH with a Ti(lV)-coordinated O,-radical is formed. Our interpretation is that the Fenton reactions (17)-(22) are still operating, though in this case reaction (22) (theTi(lV) reductional step) is quenched due to the Oz- trapping. The H202 decomposition may continue internally inside the Ti(lV) complex through reactions (14). (16?), (19) and (2 1) and also through the catalytic reactions (1 0)-( 1 I), eventually with a slight modification. This kind of mechanism would explain the H,O, decomposition, and the absence of the deleterious hydroxyl radical in the aqueous solution. Let us assume that we have 1 X 1 O6 inflammatory cells per cm3 at the titanium implant and that they generate 1 nmol/min H,OP. After 24 h, we would have 20pmol Hz0,/cm3. With a solubility of 3 nmol Ti(lV)/cm3 we find that the H,Oz:Ti ratio is much larger than one. Therefore it is possible that away from the Ti surface, a continuous TiOOH matrix may not be formed, but that particles containing the TiOOH matrix occur together with Ti (IV)-H,O, in solution. At low H,O,:Ti ratios (- 1). it has been observed in flow systems with Ti(lll) and H,Oz that a Ti(lV)-coordinated OH-radical may be formed57. From the preceding discussion, it is seen that we hardly get into this situation with the Ti-H202 system, partly due to the oxidation state of the dissolved Ti ions, and partly due to the superoxide binding in the TiOOH adduct.

Different metals are expected to behave differently with respect to these reactions. Uncomplexed transition metal ions, however, which are dissolved in the solution in reduced form (M”+) may be very effective in forming OH* from HzOz. In this respect, the Fe- and CU-HzOz systems most probably behave according to reactions (17)-(22), thereby readily generating hydroxyl radicals. One generally interesting group of metal ions seems to be the M4+ ions which according to literature58 prefer a polymerized structure with a hydrolysed oxide/hydroxyde. These easily form a macromolecular gel on addition of a suitable base. Several metals in groups IV-VI have low oxide solubilities and pK, > 14 in water. They are not good metal-extracellular component complex formers. Metals with these interesting properties include rhodium, niobium, tantalum, hafnium and zirconium.

The results in Figures 6 and 7 show that several metals have a sustained low OH’ production. It is also interesting to note that metals like Ti, Zr and Al which are known to be biocompatible’3*5g-60 (although to different degrees) appear to keep this property over a large pH range. The noble metal Au has low OH’ formation, most probably because Au ions do not occur in the HzOz solution. Of the three metals Ti, Zr and Al we have investigated Ti in detail due to the greater

Biomaterials 1989, Vol 10 April 173


clinical experience and physical knowledge of this metal’5-20. It is obvious that titanium, like other group IV-VI

cations, and M4+ metal complexes are interesting in this context. The formation of a TiOOH matrix which binds 02- apparently decreases, or perhaps even quenches OH’ production through reaction “* ‘7*22. Other possibilities for a low hydroxyl radical formation rate may be that for some metals, reduced metal ion complexes do not occur at all in the solution, neither through dissolution nor through reduction by the one-electron reducer, 02-. This may be due to complex formation between superoxide and the cation”‘. This could explain the low OH0 production by Au, Zr and Al. Also, Au (unlike Ti) is not actively taking part in a catalytic decomposition of H202. From a biological point of view, as far as an inflammatory response is concerned, a gold surface would then neither decrease nor increase the response.

Tentative model of the surface of a titanium implant

It has been shown that an oxide-like layer grows in vivu on the surface of a titanium implant. It appears that this oxide may be rather complex, having properties which make the interaction between the titanium surface and its biological surroundings very favourable. The fact that an oxide-like layer grows in viva on the titanium surface is not a proof but an indication that the phenomena discussed in this paper may take place also in vivo. Based on our results and experimental findings by others14G3’C4g-51,57 we suggest a tentative model forthetitanium-biological tissue intetfaceas given in Figure 10. In creating the model, we have assumed that the titanium surface behaviour at physiol~ical concentrations of H,Op is similar to that at the concentrations in our model experiments. Our experiments were performed at unrealistically high H202 concentrations to enhance the observed phenomena and to get incubation times on a practical time scale.

We suggest that Ti00H(H20), complexes are formed on and outside the Ti surface as a consequence of the interaction between Ti and oxygen species (e.g. O,- and H202) and that these complexes polymerize as the Hz02 concentration decreases with time. The transition between the oxide layer (‘TiO,‘) and the matrix is very diffuse and interaction between the Ti(lV)-H202 complexes and extracellular components may take place before chelate formation. The matrix formed is hydrated and should possess good ion-exchange properties. It may also be particulate. We believe the matrix is an excellent environment for proteins and living cells. This (thin) gel-like oxide may thus be the environment in which biological integration takes place.

CONCLUSIONS

The main conclusion of this study is that metal ions or l-10, emanating from a titanium surface in a H202 solution seem to be bound in a Ti-H202 complex. This complex, in turn, seems to have the interesting property that it traps the superoxide radical that is formed during H202 decomposition. Such a matrix/gel formation was not observed for any of the other metals studied under similar conditions. This is, however, not proof that titanium is unique in this respect. One important consequence of our obsenrations is that the Fenton type of transition metal-mediated oxygen radical toxicity is diminished (if not totally quenched) at a titanium surface. We made ESR and spectrophotometric spin trapping measurements in an attempt to detect and compare the hydroxyl radical formation rate in Ti-H202 and other

174 Biomateriais r989, Vol 1OAprit

Proteins ,proteoglycans

TiOOH- matrix

t

Ti

Figure 10 Modal proposed for the interface between Ti and its biological surroundings after the infiammato/y reaction. The TiO, layer gradually changes to a hydrated TiOOH matrix with good ion- and protein-exchange pmpetiies. The TiOOHmatrix may be foundin particle-like formations further away fmm the surface.

metal-H202 systems. We found no detectable amounts of OH-radicals formed in TiOp and metallic Ti systems with the ESR technique. In the RN0 measurements we found that Ti, Zr, Au and Al bleached the spin trap solution less than the other metals compared at r-t. and in the pH range 5-8. We believe that cation binding, the ability of the cation to interact with extracellular components (i.e. the pK, of the products released out to the extracellular space), and the fate of 02-/ H,02 at metal surfaces are important properties to consider in future studies of biomaterials. The positive influence on the inflammaton/ response which most probably is a property of titanium, may be one necessary condition for implants which are going to be perfectly incorporated or osseo-integrated into human tissue.

The elucidation of the influence of H,O, and/or free radicals formed during an inflammatory response on the implanted material is of interest not only for metallic implants. It may well be that the reaction of H,02 and/or oxygen radicals with, for example, implanted polymer surfaces may have a deleterious effect, changing the properties of the polymer surface and/or giving rise to soluble species which interact with extracellular materials. Such a scenario may alter the bio~ompatibility of the material in question.

ACKNOWLEDGEMENTS

We are grateful to Dr Anders Lund for useful suggestions regarding the ESR experiments.

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titanium-hydrogen peroxide interaction: model studies of the influence of the inflammatory response...

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