Abstract
Objective
The aim of this study was to measure titanium (Ti) content in human jawbones and to show that Ti was released from dental implants inserted into these jawbones.
Methods
Seven samples from four human subjects with dental implants were analysed as test group and six bone samples of similar topographical regions from six human subjects without implants served as control. The contents of various elements in human jawbones were detected by inductively coupled plasma optical emission spectrometry. The distributions of various isotopes in human mandibular bone were measured with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Histological analyses of undecalcified, Giemsa-Eosin stained mandible sections were performed by light microscopy and particles were identified in human bone marrow by scanning electron microscope-energy dispersive X-ray analysis.
Results
In test group only Ti content was significantly higher compared to control group. The mean contents of Ti were 1940 μg/kg in test group and 634 μg/kg in control group. The highest Ti content detected in human mandibular bone was 37,700 μg/kg-bone weight. In samples 4–7 (human subjects II–IV), increased Ti intensity was also detected by LA-ICP-MS in human mandibular tissues at a distance of 556–1587 μm from implants, and the intensity increased with decreasing distance from implants. Particles with sizes of 0.5–40 μm were found in human jawbone marrow tissues at distances of 60–700 μm from implants in samples 4–7.
Significance
Ti released from dental implants can be detected in human mandibular bone and bone marrow tissues, and the distribution of Ti in human bone was related to the distance to the implant.
1
Introduction
In the last 50 years titanium (Ti) and its alloys have been widely used for dental and medical applications . Clinically, pure Ti (Ti >99%) and the alloy Ti-6Al-4V are mainly used for endosseous implants . Ti alloys with other metals (e.g. nickel (Ni), chromium (Cr), iron (Fe), and molybdenum (Mo)) are applied for implant devices such as bone plates or screws . Ti is the main component in those implants/implant devices so that a stable TiO 2 layer on the surface can provide Ti based implants/implant devices with a high biocompatibility and resistance to corrosion .
Although Ti based implants have been considered to be biological inert, it has been found that implants in the body can undergo corrosion and release particulate debris over time . It has been reported that the metallic debris from Ti based implants might exist in several forms including particles (micrometer to nanometre size), colloidal and ionic forms (e.g. specific/unspecific protein binding) , organic storage forms (e.g. hemosiderin, as an iron-storage complex), inorganic metal oxides and salts . According to a previous study , the degradation of implants in the human body is primarily induced by wear and corrosion: wear is the mechanical/physical form of implant degradation which produces particles; while corrosion refers to the chemical/electrochemical form of degradation that mainly produces soluble metal ions . Ti particles released from implants have been found in the regenerated bone and peri-implant tissues in animals . It has been shown that also Ti ions can be released from embedded implants in animals . Ti particles/ions are able to enter the circulation of blood and lymph . Ti particles detached from hip, knee and mandible implants have been detected in organs such as liver, spleen, lung and lymph nodes . Increased levels of elementary Ti have also been detected in the blood of patients with poorly functioning implants .
Increased concentrations of metals (e.g. Ti, Cr, Co and Al) derived from implants in body fluids might induce acute or chronic toxicological effects . The long-term effects of Ti derived from implants are still not fully understood, but associated hypersensitivity and allergic reactions in patients have been reported . In a clinical study, 0.6% of 1500 patients were found to exhibit Ti allergic reactions . Additionally, it has been found that detached metal debris from implants might cause marrow fibrosis, necrosis and granulomatosis .
The aim of our study was to measure the release of Ti and other metallic elements from dental implants through detailed post-mortem studies of human subjects with dental implants. In the present study, Ti released from implants inserted into human jawbones was identified and quantified, and the spatial distribution of Ti in human jawbones near implants was also investigated.
2
Materials and methods
2.1
Materials
The test group contained 7 samples from four human subjects with dental implants ( Table 1 ). The control group contained 6 bone samples from similar topographical regions from six human subjects without dental implants.
| Subject number | Age | Gender | Implants | Sample number | |
|---|---|---|---|---|---|
| No. I | 98 | Female | 2 pieces, on the positions of both of the both mandibular canine teeth (FDI System: 33, 43) | No. 1: right mandible segment | No. 2: left mandible segment |
| No. II | 83 | Female | 2 pieces, on the positions of both of the mandibular canine teeth (FDI System: 33, 43) | No. 3: left mandible segment | No. 4: right mandible segment |
| No. III | 84 | Male | 2 pieces, on the positions of both of the mandibular canine teeth (FDI System: 33, 43) | No. 5: right mandible segment | No. 6: left mandible segment |
| No. IV | Unknown | Female | 2 pieces, on the positions of the left first and second molars in the lower jaw (FDI System: 36 and 37) | No. 7: left Mandible segment | |
The bodies were individually donated for medical research. Subjects I–III in the test group and all six of the subjects from the control group were obtained during the dissection course of the anatomical institute of the Ludwig-Maximilian-University Munich. Bone implant sample from subject IV was supplied by the institute of forensic medicine of the Ludwig-Maximilian-University Munich. Subjects I–III carried two implants (one on each side of the mandible) replacing the mandibular canine teeth (FDI System: 33, 43). Subject IV carried two implants that replaced the left first and second molars in the mandible (FDI System: 36 and 37). All the lower jawbones in the test group contained no additional natural teeth. Four out of the six control subjects were toothless as well. The other two control subjects were partially edentulous. The average age of the subjects in the test group (excluding subject IV) and control group were 88 and 85 years, respectively.
For subjects I–III, each of the two implants with the adjacent jawbone was taken as a single sample; for subjects IV, two implants with adjacent jawbone on each side were taken as a single sample. Therefore, there were seven implant samples in our experiment ( Table 1 ). Implants used in samples 5–6 were produced by Astra Tech implant system (Mannheim, Germany), and the implants used for sample 4 and sample 7 were produced by Straumann AG (Basel, Switzerland). The manufacturers of the implants of samples 1–3 were unknown. Due to ethical consideration, it is not possible to get more information about the implants, such as the “age” of the implants (for how many years it has been loaded), the type of the implants and the surface treatment on the implants. Ti dental implants have no identification number to provide detail information (e.g. for age determination).
2.2
Sample analysis by inductively coupled plasma optical emission spectrometry (ICP-OES)
2.2.1
Sample preparation
All 7 samples of test group and all 6 samples of control group had been fixed in 4% buffered formalin (Merck, Darmstadt, Germany). The soft tissue was cautiously removed with Feather ® disposable stainless steel scalpels and then immersed in 100% methanol (Carl Roth, Karlsruhe, Germany).
2.2.1.1
Test group
Jawbone slices in the sagittal plane were prepared from samples 1–7 by cutting with a diamond band saw (cut-grinder macro, patho-service GmbH, Oststeinbek, Germany). The samples were cut at intervals of 1 mm in a direction moving toward the implants, and cutting was stopped immediately adjacent to the implant ( Fig. 1 ). From each sample, at least 5 jawbone slices with an approximate thickness of 500 μm were obtained. For analysis the 5 jawbone slices closest to the implant were used.
Each jawbone slice was further divided into 4 quadrants (A–D) ( Fig. 1 ). Each quadrant was weighed and dissolved in 1 ml sub-boiled distilled nitric acid at 170 °C for 12 h. The solution was subsequently diluted (1:5) in Milli-Q Water. The contents of the elements Ti, Al, Cd, Cr, Co, Cu, Fe, Mn, Mo, Ni and V in each quadrant were analysed by ICP-OES (Optima 7300 DV, Perkin Elmer, Rodgau-Jügesheim, Germany). The remaining specimens containing the implant and adjacent bone were reserved for further analysis (Sections 2.3 and 2.4 ).
2.2.1.2
Control group
Control samples were taken from mandibular regions topographically comparable to those bearing the implants. Again 5 adjacent jawbone slices were cut and each of the slices was divided into 4 quadrants. For analysis by ICP-OES, same procedure as samples 1–7 was performed.
2.2.2
Analytical procedure
An ICP-OES, Optima 7300 DV system (Perkin Elmer, Rodgau-Jügesheim,Germany) was used for element determination. Sample introduction was carried out using a peristaltic pump connected to a Seaspray nebulizer with a cyclon spray chamber. The measured spectral element lines were (in nm): Al: 167.078, Cd: 214.438, Cr: 267.716, Co: 228.616, Cu: 324.754, Fe: 259.941, Mn: 257.611, Mo: 202.030, Ni: 231.604, V: 292.464, Ti: 334.941.
The RF power was set to 1400 W; the plasma gas was 13 L Ar/min, whereas the nebulizer gas was approximately 0.6 L Ar/min after daily optimization.
Routinely each ten measurements, three blank determinations and a control determination of a certified standard for all mentioned elements were performed. Calculation of results was carried out on a computerized lab-data management system, relating the sample measurements to calibration curves, blank determinations, control standards and the weight of the digested sample.
The volumes of sample digests were sufficient for duplicate measurements ( n = 2).
2.2.3
Calculations and statistics
For a total jaw bone slice the content (μg/kg) of each investigated element was calculated by dividing the summed concentrations of the 4 quadrants with their summed masses.
The results are presented as mean ± standard error of the mean (SEM). Independent two-sample t -test and a one-way ANOVA analysis followed by post hoc Bonferroni adjustment were performed for statistical analysis. Differences were considered statistically significant only when the p -value was less than 0.05 ( p < 0.05) .
2.3
Content distribution measured by Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS)
2.3.1
Sample preparation
The remaining parts of samples 4–7 (the bone surrounding the implants) and bone samples from the control group were dehydrated in ascending ethanol fractions (70%, 80%, 90% and 100%), defatted in xylene (Merck, Darmstadt, Germany), and embedded in methylmethacrylate (Fluka, Switzerland). Samples 4–7 were analysed because in these samples significantly higher Ti contents (compared to controls) were detected with ICP-OES measurement in Section 2.2 .
Details of the embedding and cutting process can be found in a previous study . The polymerized methacrylate blocks were cut at a thickness of approximately 300 μm parallel to the long axis of the implants in the mesio-distal plane using a Leica SP 1600 saw-microtome (Leica, Wetzlar, Germany). The sections were used for spectroscopy and for histological analysis.
The distribution of the isotopes 47 Ti, 27 Al, 43 Ca, 52 Cr, 59 Co, 63 Cu, 57 Fe, 39 K, 55 Mn, 60 Ni, 51 V, 64 Zn and 66 Zn in each section were analysed by Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) – NWR-213 ® (New Wave Research Co. Ltd.) coupled to a NexION ® 300 ICP-MS (PerkinElmer).
2.3.2
Analytical procedure
The laser ablations started from the bone region at a distance of 5 mm from the implant and ended in the implant region. In this manner, sample-specific backgrounds could be measured by moving from low to high contents , and memory effects were avoided. Two to three parallel ablation lines with each time 6 measurements into the deep (10 μm depth ablation per measurement) were performed for each jawbone slice. The average value of 2–6 measurements was calculated for each line (the first measurement was discarded in order to eliminate possible contamination on the surface of the bone sections which could have occurred during the cutting process). Instrument settings and parameters are shown in Table 2 .
| Laser | Energy: 0.930 mJ |
| Power: 100% | |
| Pulse repetition rate: 10 Hz | |
| Scan speed: 35 μm/s | |
| Spot size: 50 μm | |
| Ablation pattern: line | |
| Depth: approx. 10 μm | |
| ICP | Plasma power: 1200 W |
| Transport gas: 1.2 L/min Ar | |
| Auxiliary gas: 0.8 L/min Ar | |
| Cool gas: 17 L/min Ar | |
| MS | Registered isotopes: 47 Ti, 27 Al, 43 Ca, 52 Cr, 59 Co, 63 Cu, 57 Fe, 39 K, 55 Mn, 60 Ni, 51 V, 64 Zn and 66 Zn |
| Dwell time/isotope: 25 ms |
After the laser ablations, contact radiographs (Faxitron X-ray Corporation, Lincolnshire, IL, USA) for each section were taken on Agfa Strukturix X-ray sensitive film (Agfa-Gevaert, Mortsel, Belgium). The laser lines were clearly visible on the X-ray film and were photographed with an Axiophot microscope (Zeiss, Goettingen, Germany), equipped with Zeiss Plan-Neofluar objectives (5× and 10×) in transmitted light mode.
2.4
Histological analysis
2.4.1
Sample preparation
From each of the samples 4–7, one of the sections (cutting procedure described in Section 2.3.1 ) was glued (Cyanolit 201, Panacol LTD., Zürich, Switzerland) on opaque plastic slides, ground thinner, polished (EXAKT ® 400CS grinding system, EXAKT Vertriebs GmbH, Norderstedt, Germany) and stained with Giemsa-Eosin stain (Sigma Aldrich, Steinheim, Germany).
The same procedure was applied for the control samples.
2.4.2
Light microscopy observation and scanning electron microscope-energy dispersive X-ray (SEM-EDX) measurements
The stained sections were examined with an Axiophot microscope (Zeiss, Goettingen, Germany) that was equipped with Zeiss Plan-Neofluar objectives (5× and 10×) in transmitted light mode. Images were recorded with an Axciocam HRc digital camera (Zeiss, Goettingen, Germany).
The sections that contained visible dark particles were coated with gold and investigated with a SEM (EVD MA 15) equipped with secondary electron (SE) and back-scattered electron (BSE) detectors. The energy dispersive X-ray (EDX) analyses were performed using an Oxford INCA Penta Fetx 3.
The samples of the control group were also histologically investigated, coated with gold and analysed by SEM-EDX.
2
Materials and methods
2.1
Materials
The test group contained 7 samples from four human subjects with dental implants ( Table 1 ). The control group contained 6 bone samples from similar topographical regions from six human subjects without dental implants.
| Subject number | Age | Gender | Implants | Sample number | |
|---|---|---|---|---|---|
| No. I | 98 | Female | 2 pieces, on the positions of both of the both mandibular canine teeth (FDI System: 33, 43) | No. 1: right mandible segment | No. 2: left mandible segment |
| No. II | 83 | Female | 2 pieces, on the positions of both of the mandibular canine teeth (FDI System: 33, 43) | No. 3: left mandible segment | No. 4: right mandible segment |
| No. III | 84 | Male | 2 pieces, on the positions of both of the mandibular canine teeth (FDI System: 33, 43) | No. 5: right mandible segment | No. 6: left mandible segment |
| No. IV | Unknown | Female | 2 pieces, on the positions of the left first and second molars in the lower jaw (FDI System: 36 and 37) | No. 7: left Mandible segment | |
The bodies were individually donated for medical research. Subjects I–III in the test group and all six of the subjects from the control group were obtained during the dissection course of the anatomical institute of the Ludwig-Maximilian-University Munich. Bone implant sample from subject IV was supplied by the institute of forensic medicine of the Ludwig-Maximilian-University Munich. Subjects I–III carried two implants (one on each side of the mandible) replacing the mandibular canine teeth (FDI System: 33, 43). Subject IV carried two implants that replaced the left first and second molars in the mandible (FDI System: 36 and 37). All the lower jawbones in the test group contained no additional natural teeth. Four out of the six control subjects were toothless as well. The other two control subjects were partially edentulous. The average age of the subjects in the test group (excluding subject IV) and control group were 88 and 85 years, respectively.
For subjects I–III, each of the two implants with the adjacent jawbone was taken as a single sample; for subjects IV, two implants with adjacent jawbone on each side were taken as a single sample. Therefore, there were seven implant samples in our experiment ( Table 1 ). Implants used in samples 5–6 were produced by Astra Tech implant system (Mannheim, Germany), and the implants used for sample 4 and sample 7 were produced by Straumann AG (Basel, Switzerland). The manufacturers of the implants of samples 1–3 were unknown. Due to ethical consideration, it is not possible to get more information about the implants, such as the “age” of the implants (for how many years it has been loaded), the type of the implants and the surface treatment on the implants. Ti dental implants have no identification number to provide detail information (e.g. for age determination).
2.2
Sample analysis by inductively coupled plasma optical emission spectrometry (ICP-OES)
2.2.1
Sample preparation
All 7 samples of test group and all 6 samples of control group had been fixed in 4% buffered formalin (Merck, Darmstadt, Germany). The soft tissue was cautiously removed with Feather ® disposable stainless steel scalpels and then immersed in 100% methanol (Carl Roth, Karlsruhe, Germany).
2.2.1.1
Test group
Jawbone slices in the sagittal plane were prepared from samples 1–7 by cutting with a diamond band saw (cut-grinder macro, patho-service GmbH, Oststeinbek, Germany). The samples were cut at intervals of 1 mm in a direction moving toward the implants, and cutting was stopped immediately adjacent to the implant ( Fig. 1 ). From each sample, at least 5 jawbone slices with an approximate thickness of 500 μm were obtained. For analysis the 5 jawbone slices closest to the implant were used.
