RICERCA ITALIANA     

Localisation with nuclear and atomic techniques of potentially toxic elements released from prosthetic and implant devices

Università degli Studi di Padova

Abstract

Several metals with potential adverse effects are contained in materials which are very used in dentistry, for restorative care, prosthesis, implant and maxillo-facial surgery, i.e chromium, cobalt, titanium, nickel and aluminium, which are present in some prosthetic alloys, and in most endosseous dental implants, as well as mercury, present in the dental restarative amalgam.
The increasing clinical use of dental implants, especially since 80's, with the development of the so-called osseointegrated techniques, drew the attention of many researchers on the possible release of metal components into the host organism, and on a possible distant diffusion and accumulation in target organs. However, the investigations carried out until today with various analytic techniques, led to disagreeing results.
Having the availability of the proton and x-ray microprobes at the Laboratories of Legnaro, of the National Institute for Nuclear Physics, we intend to perform the search, measurement and mapping of titanium, chromium, cobalt and mercury, as they are metals of widespread dental application.
Measurements of mercury and other elements in the oral tissues, saliva, blood and urine of patients bearing amalgam dental restorations, being treated in the Dental Clinic of Padua, will be done.
This study will be carried out also on control subjects, at least two hundred patients in total, during the two forecasted years for this project. Similar experiments will be performed in laboratory animals, in which subcutaneous and intamuscular amalgam will be inserted, in the
Interdipartimental Center of Experimental Surgery of Padua.
As far as titanium and other dental implant metals are concerned, we intend to measure and map them in some bioptic human samples, including implants removed either for inflammation or for the breaking of their metallic components.
Our research groups own some of these specimens, waiting for examination..
Similar analyses and mapping will be done on implants inserted in animals.
The appliances utilized will be the cutting-grinding Precise machine for histologic preparation of hard tissues and body, owned by the Dental Clinic of Padua, and the facilities at the Laboratori Nazionali di Legnaro, of the National Institute of Nuclear Physics.
PIXE micro beam technique will allow to scan the samples, drawing maps with the space distribution of trace elements from the implants dispersed into the tissues.
This will allow to compare the surface morphology of the devices (i.e, roughness, threaded shape) with the possible leakage of their components through the interface, and to draw the spatial distribution of the metals also in the oral biopsies taken from patients with amalgam.
Quantitative measurements will be performed also in saliva, blood and urines with XRF technique,
while other studies based on sonoluminescence will be tuned. This techniques, indeed, promises interesting analytical applications.
Search for ultra trace elements (detection limit below 1ppb) with synchrotrone light will be carried out at Laboratori Nazionali di Frascati (for light elements), whose researchers are already collaborating with the group of Verona from time.
A similar collaboration is forecasted with Sandia Laboratories, Albuquerque, New Mexico, USA where a member of our group went about two years ago and will work in the next months. <<<

Principal Investigator

Pietro PASSI Università degli Studi di PADOVA

Research Objectives

As far as endosseous implants are concerned, the aim is to verify whether, how much and in which conditions a leakage of metallic components may occur. MicroPIXE examination on sections containing implants and the surrounding tissues, obtained with cutting-grinding procedure, will allow a very good study of the tissue-implant interface, drawing the map of the elements present in the examined sample. This evaluation shows the modalities of diffusion of the elements, according to their spatial location, and is to be considered as a valuable aid to study the chemical and physical behavior at the surface of endosseous implants. Indeed, macroscopic and microscopic morphology are main characteristics which differentiate the numerous implants utilized today.
In fact, some implants are smooth, while others underwent roughing treatments of their surface with sand-blasting, acid etching, plasma-spray coating etc. The actual trend is to build implants with rugged surface, that seem to produce a faster and stronger bond with bone.
However, the presence of micro niches at the surface, due to roughness, may lead to differential corrosion because of the different oxygen concentration and increase the leakage also of passivating metals, as titanium.
Hence, it is necessary not only to localize implant components in the tissues, but also to find where chemical attacks to the materials ma take place. Former studies of this research group indicated that PIXE micro analysis, with a beam focalized up to 2.5 micron is a very sensitive instrument, that can detect elements at concentrations of a few ppm.
If a leakage of implant components will not be detected at this threshold, we intend to utilize the synchrotrone light appliance, at the National Laboratories of Frascati, which lowers the detection limit of about 1000 times.
Useful indications may be received from XRF analysis of organs taken from animals with implants, which will be compared to the controls, and will give information about the accumulation of metals at distance, and if some organs may work as reservoirs for titanium, chromium, aluminium, vanadium and other metals contained in the implants.
As far as the problem of a possible leakage of mercury from dental amalgam is concerned, we intend to measure the mercury content in blood. saliva and urine of either patients bearing dental amalgam restorations and control subjects. This study looks as interesting, because silver amalgam is to be considered the best plastic material for dental restorations up today, and the great advantages it offers as duration of restorations must be carefully compared with possible adverse effects, that will be considered as very actual if a noticeable leakage and accumulation of mercury would be confirmed. Mercury, indeed, owns well-known toxic effects..
Mercury content of saliva and urine of patients, measured with XRF techique, will be related with the estimated amount of amalgam present in the mouth, and statistically compared with the control subjects. If the sample will be significant enough ( that is at least 200 subjects + 100 controls), useful indications can be obtained about the leakage of metal.
The collected data will be compared also with the time the dental restoration stayed in the oral cavity. In fact, surface oxidation films may form, and the release of mercury and other metals may decrease with time.
PIXE micro beam examination of gingival biopsies adjacent to amalgam fillings will lead to indications about the modalities of metallic components leakage: an uniform tissue distribution will indicate a diffusion gradient, while localized metal ion accumulation will suggest a micro carrying of metal due to chemical and mechanical wear.
Measurements of metal concentrations in the organs of laboratory animals are to be considered very useful, above all to confirm the existence of accumulation reservoirs of mercury, which was
reported to concentrate in kidneys and testicles. This accumulation, however, was noticed in animals subjected to high doses of Hg, so that it seems necessary to assess whether this phenomenon may occur also in a condition closer to that of a patients with dental fillings.
More, PIXE micro beam analysis of histologic preparations will allow to find mercury in its more typical locations in organs, as the kidney tubules. The presence of mercury in brain and central nervous system of the animal may be considered of primary importance, because of the well-known neurotoxic effects typical of chronic mercury poisoning.
Hence, we intend to work in two groups of research:
1) The unit A in Padua will take saliva and urine in patients with amalgam and control patients. The high number of subjects that are visited and cured in the Dental Clinic, will allow to evaluate at least 200 patients with amalgam and 200 controls, during two years.
We also plan to perform gingival biopsies, in which the presence of trace metals will be evaluated.
The availability of the Centro Interdipartimentale di Chirurgia Sperimentale will allow to carry out experiments in animals, in which endosseous implants of different composition and amalgam samples will be inserted.
Block section of the removed specimens will be prepared with the cutting-grinding appliance of the Dental Clinic. The samples, containing both the inserted metals and the surrounding tissues, will undergo microscopic examination.
2) The unit B in Verona will perform analyses with XRF, PIXE and sonoluminescence either on the samples collected in Padua, or on human implant biopsies, taken in the local Dental Clinic.
We are planning also to utilize the synchrotrone light, a very sensitive analytic appliance, that will allow to lower the detection limits of XRF and PIXE techniques, finding even very small quantities ( less than 1 ppb) of Hg, metal that might be harmful even at this concentration, as tends to accumulate in some tissues and organs, such as kidneys and gonads.
Of great interest will be the availability of PIXE appliances of the Sandia Laboratories, Albuquerque, USA, where a component of our unit will work in the next months, and who is already there from about two years ago. <<<

First Results

From the first phase, a very precise tuning of the targets for PIXE and XRF techniques should be expected, that will be made available by the comparison with the results obtained with spectroscopy.
It must be remebered that spectroscopy, especially with FI-(VG)-ICP-MS tecnique (Flow Injection-(Vapour Generation)-Inductively Coupled Plasma- Mass Spectrometer), that we intend to utilize in third-part aboratories, is very tested and reliable; however, it is expensive and allows to dose just a single element each time, while PIXE and XRF give the simultaneous detection and measuring of all the investigated elements , and their surface distribution (PIXE microbeam).
Thus, these latter two techniques seem to be very versatile at fit also for large-scale investigations
concerning a high number of elements.
We expect to get a first evaluation about the usefulness of the comparison between histologic pictures and elemental maps obtained with PIXE microbeam.
Useful suggestions can be obtained whether saliva is a mean fit for detecting metals released from restoration and dental prostheses, an issue that is still being discussed.We believe to collect enough data to assess:
- which are the best conditions to utilise particles and X-ray beam appliances employed, and their detection limits for the investigated elements.
- whether, and in which extent, a release of metals occurs from the prosthetic/restorative devices
into the surrounding tissues , the organs and the biological fluids.
- according to the obtained results, to establish the biological risk related to the characteristics of toxicity of the investigated metals and their concentations.
- point out, by means of PIXE microbeam maps, whether the possible presence of metals is due to
uniform diffusion or to detachments or microdetachments of material from the surface. This latter indication can be useful to suggest the best coatings and surface shapes for endosseous implants.
- if a correlation between tissue alterations, visible in optical microscopy, and metal accumulation detected with PIXE microbeam exists. <<<

Timescale

24 months

National and international background

Unfavorable biologic effects have been reported or suspected from several metals widely employed in the dental field, especially from mercury contained in amalgam, nickel, chromium, vanadium and aluminium present in numerous prosthetic and implant alloys. Other metals can be diffused into the biologic environment, i.e. titanium, copper, silver, molybdenum, platinum, the possible toxicity of which is suspected.
As far as silver alloy and the possible adverse effects of mercury are concerned, the investigation carried out up today led to disagreeing results.
Kidney lesions were detected in rats which were exposed to inhalation of silver amalgam filings for one hour (coming by grinding an amalgam block), and mercury was found to accumulate in kidneys and testicles (Musajo et al., 1988).
Seidler et al. (1996) studied over 1,000 patients affected by Parkinson's disease in Germany, analyzing their food habits and the possible exposure to toxic compounds. These authors believe there is a significant correlation between amalgam dental fillings and Parkinson's disease, in the group of the patients examined.
Urine content of Hg seems to be a more reliable indicator than blood level, as mercury tends to concentrate in kidneys.
Also the quantitative evaluation of this element in saliva is to be considered very useful. Some studies, indeed, reported a correlation between the number and extension of dental amalgam restorations and Hg content of saliva (Lygre et al., 1999); more, it has been found that the removal of amalgam fillings causes a decrease of Hg content in saliva, after a brief period of increase (Bjorkman et al., 1997.
The daily absorption of mercury from dental fillings has been evaluated in 4-19 mcg/die (Weiner & Nylander, 1995); however, measurements performed in an artificial mouth model pointed out
that the daily intake of Hg should be estimated in about 0.03 mcg/die (Berdouses et. al., 1995).
This value should be considered as harmless, above all considering that the Threshold Limit Value (TLV) for mercury is 82.29 mcg/die, according to U.S. standards.
Halbach (1995) estimates that the daily intake of mercury from amalgam does not exceed approximately 5 mcg/die, and believes that an amount at least equal or higher is assumed from food. The same author, in another article (1994), evaluates that in no case the daily urinary excretion of mercury exceeds one tenth of the value required to cause disturbances.
Schulte et al. (1994) reported that, considering a group of young people between 3 and 15 years, the bearers of amalgam have a Hg urinary excretion of about 0.70 mcg/die, versus approximately 0.17 mcg/die of non-bearers. These results agree with those by Begerow et al. (1994), who found that the removal of amalgam restorations reduces the urinary excretion of mercury to about one fifth after one year.
However, Ulukapi et al. (1994), who measured the urinary outtake of Hg in children with amalgam fillings, conclude that this metal is below the detection limit.
Hence, mercury surely leaks from amalgam restorations, but there are no agreeing conclusions about the possible effects of this phenomenon.
A contact stomatitis can be induced in rats that have been made hypersensitive to mercury chloride (Warfving & Larsson, 1994).
Sometimes, not frequently, a hypersensitivity to mercury of dental fillings exists. A similar case with cutaneous reaction was described by Ulukapi in 1995.
Amalgam was also suspected to be a possible cause of multiple sclerosis, (Ingalls, 1983, 1986). This hypothesis was due to the development, statistically unexpected, of some tenths of cases of this disease in a location of the United States, where pollution from lead and mercury was suspected. Further researches pointed out a worsening of the clinical conditions in patients affected by multiple sclerosis with amalgam fillings, compared to subjects with the same disease but amalgam-free (Siblerud & Kienholz, 1994).
Mercury from amalgam was also suspected to be involved in the cause of lateral amyotrophic sclerosis. Mano et al. (1994) found an increased content of this metal in patients affected by this disease, suggesting that another cause might be a low content of selenium, that reduces the toxicity of mercury. However, they believe that mercury was taken from environment and food (this study was conducted in Japan, where there is a high mercury intake from fish).
An investigation performed by Clausen (1994) on necroscopic specimens of brain could not find
abnormal levels of mercury, but on the contrary in this disease the content of fat-soluble mercury was lower than in normal subjects.
On the other hand, other authors (Pleva, 1994; Lorscheider et al., 1995) assert, after a survey of the literature on this topic, that amalgam can not be regarded as a safe material.
Levy (1995), however, after a similar survey of the literature, asserts that the benefits of amalgam are far greater than its suspected dangers, that according to his opinion could not ever been proved.
Also the diffusion of metal components from dental endosseous implants is under investigation today, but the conclusions are not in agreement.
This topic, besides dentistry, is of high interest also for orthopedics, considering the high number of metal joint prostheses inserted yearly.
Even if titanium, a primary component of dental implants, is considered a metal with a very low toxicity, some reports exist about its possible mutagenic action (Driscoll et al., 1997; Hadfield et al., 1998) and even an inhibition on the growth on fibroblasts and bone cells in vitro was described (Wang et al., 1997).
Other metals frequently contained in implant alloys are cobalt, aluminium, and chromium; this latter owns well-known toxic and oncogenic properties, while aluminium has harmful effects on blood and central nervous system cells (Struys Ponsar et al., 2000; Mahieu et al., 2000). Vanadium is another metal frequently present in titanium alloys, and can have negative effects on osteogenesis, that have been reported also for titanium (Blumenthal & Cosma, 1989).
The results reported in the literature about the diffusion and presence of metals in the tissues and organs of patients and animals with endosseous implants, are very disagreeing. On one side the are authors who believe that this diffusion be unnoticeable (Lugowski et al., 1991; Rodriguez et al., 1999; Bianco et al., 1996); on the other side there are researchers who report an evident presence of Ti and other metals in the tissues and lymph nodes surrounding implants (Jacobs et al., 1998; Ektessabi et al., 1996; Ducheyne et al., 1984).
Of particular interest is the report of a threefold increase of titanium concentration in urine and serum of patients bearing hip prostheses, compared to the normal values; patients who had received similar prostheses in Cr-Co alloys showed the levels of these metals augmented from five to eight times (Jacobs et al., 1998). Studies by Schliephake et al. (1993) pointed out particles of titanium in the tissues adjacent to experimental implants in animals, which had high metal concentrations in the lungs.
However, investigations by Rodriguez et al. (1999), Lugowski et al. (1991), Bianco et al. (1996), carried out in animals, lead to different results, as titanium accumulation was not found in the peri-implant tissues and in the organs, and blood levels of the metal did not increase.
The disagreement of the results obtained by the authors who investigated this topic, and the evident possibility of biological damages from metal diffusion into the organism, together with the continuously increasing clinical employ of dental implants and orthopedic prostheses, make the researches on this subject very actual and interesting.
Some preliminary results of ours, obtained by PIXE microbeam mapping of tissues containing dental implants, showed
the possibility of distinguish the different patterns of the main metals, as far as their release into the tissues is concerned (Passi et al., 2002).
For instance, the following image, taken from a preliminary study of ours, suggests that the presence of giant cells (arrows on the left) might be related to the accumulation of metal (chromium, in the PIXE microbeam map on the right), and that this issue deserves investigation.
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