Accumulation of aluminium in lamellar bone after implantation of titanium plates, Ti-6Al-4V screws, hydroxyapatite granules

Davide Zaffe ^{, , a}, Carlo Bertoldi ^b and Ugo Consolo <sup>b

a</sup> Departments of Anatomy and Histology, Section of Human Anatomy, University of Modena and Reggio Emilia, Via del Pozzo 71, Policlinico, 41100, Modena, MO, Italy
^b Department of Neurosciences, Head-Neck and Rehabilitation, Section of Dentistry, University of Modena and Reggio Emilia, Via del Pozzo 71, Policlinico, 41100, Modena, MO, Italy

Received 18 June 2003;  accepted 7 October 2003.  Available online 26 November 2003.
Biomaterials
Volume 25, Issue 17 , August 2004, Pages 3837-3844

1. Abstract

Titanium plates, Ti6Al4V screws and surrounding tissues, and biopsies of hydroxyapatite (Osprovit^®) grafts of maxillary sinus lifting were investigated to evaluate the release and accumulation of ions. Optical microscopy, SEM and X-ray microanalysis were carried out to evaluate the plates and screws removed from patients presenting inflammation and biopsies. Ions release from metallic appliances or leaching from granules towards soft tissues was observed. An accumulation of aluminium but not titanium was found in soft tissues. A peculiar accumulation of aluminium in the dense lamella of newly formed bone was recorded. The results seem to indicate that biological perturbations may be related to aluminium release from the tested biomaterials. The aluminium content of these biomaterials, its diffusion and accumulation are discussed. Further studies on ion release from biomaterials and aluminium fate in skeletal tissues are suggested.

Author Keywords: Author Keywords: Titanium; Hydroxyapatite; EDS; Connective tissue; Bone

2. Article Outline

1. Introduction

2. Materials and methods

3. Results

3.1. Titanium appliances

3.2. Hydroxyapatite granules

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

Biomaterials currently in use are primary chosen because well tolerated by biological systems. Titanium is a widely used metal in appliances, and hydroxyapatite (natural or synthetic) is the most common ceramic applied to promote bone defect healing. Sometimes, appliances made of these materials produce unexpected clinical results or mild side effects. We retain that the molecular composition of the appliance might play a role in these outcomes.

Some materials are known to produce inflammatory responses (chromium, cobalt, copper, nickel, palladium, titanium and zinc alloys) in in vitro studies [1, 2 and 3] and allergic reactions (chromium, cobalt, nickel, titanium and Ni/Ti) in vivo [4, 5 and 6]. Several studies highlight that elements can diffuse from appliances and accumulate in tissues [7, 8 and 9]. Due to increased particle surface, metallic wear debris [10] may enhance the above mentioned effects. Optical (OM) and scanning electron (SEM) microscopy [11] or ultrastructural analysis of tissues and analysis of metal ions of the system [12, 13 and 14] have been performed in many studies to analyse opaque particles (wear debris). Instead, few studies focus on the elements released from biomaterials, without wear, and their subsequent fate in the surrounding tissues [8 and 15].

In this study we analysed the skeletal tissues surrounding two types of biomaterials without wear debris formation: titanium plates and screws and hydroxyapatite granules. The study was performed on material retrieved from patients with untoward effects from implants, such as swelling, inflammation or clinical deficiencies. Tissue morphology was studied using a light microscope and SEM, whereas elemental analysis was performed by means of an Energy Dispersive X-ray Spectroscopy (EDS). This work aims to contribute to better our knowledge on biomaterial ion diffusion in periprosthetic tissues.

4. 2. Materials and methods

The study was performed on (A) titanium appliances and (B) hydroxyapatite granules used in human beings.

(A) A total of 12 plates and 51 titanium screws (Gebrüder Martin GmbH & Co. KG, Tuttlingen, D), 4 or more screws necessary to fix a plate, were removed 1-6 years after surgery from 6 trauma (facial bone fractured) patients (aged from 25 to 65 years). Patients had previously undergone maxillofacial surgery and fixing of bone segments by titanium plates (internal rigid fixation). The metallic appliances and surrounding tissue were harvested from selected patients who presented a marked local inflammation without dark pigmentation of the mucosa. This excluded exposed devices or devices producing metallic particles that led to mucosal pigmentation. Plates and screws and the soft and hard tissues surrounding them were fixed in 4% paraformaldehyde in 0.1 
phosphate buffer, pH 7.2 for 4 h at room temperature, then dehydrated through an ethanol series. Part of the connective tissue adjacent to the appliance was detached and embedded in paraffin and in methyl-methacrylate (PMMA). Plates, screws and the adjacent soft tissue were desiccated in a critical point dryer (CPD030, Bal-Tec, FL) at a temperature of 40°C and pressure of 7.4 MPa, using CO₂ as the intermediate agent. A small fragment of bone in contact with the plate was harvested with the appliances, fixed in paraformaldehyde, dehydrated and embedded in PMMA without decalcification. Titanium plates and screws and the surrounding connective tissue used to study the titanium release [16] were used as controls.

(B) Biopsies were performed 3-6 months after surgery in 6 additional patients (ranging in age from 42 to 67 years) with atrophic maxillary bone. Patients had previously undergone lifting of the sinus floor to achieve adequate bone volume for titanium implant (implant-based prosthetic rehabilitation). Sinus lift was performed using a 50/50 (v/v) mixture of autologous bone/Osprovit^® (porous hydroxyapatite granule, Feldmühle A.G., Plochingen, D), externally covered by the vestibular periosteum. Osprovit^® is a trade hydroxyapatite material commonly used in oral and maxillo-facial treatments of bone defects. Biopsies were executed after radiographies showed anomalous radiotransparent areas inside the graft. Biopsies were fixed in 4% paraformaldehyde in 0.1 
phosphate buffer, pH 7.2 for 1 h at room temperature and then at 4°C, dehydrated through an ethanol series and embedded in PMMA without decalcification.

The PMMA blocks containing soft tissues or bone were sectioned using a bone microtome (Autocut1150, Reichert-Jung GmbH, Nußloc, D) to obtain 5 micron-thick slices. The sections were stained with Hematoxylin-Eosin, Toluidine Blue or histochemically treated [17] to detect total alkaline phosphatase (TALP) and tartrate resistant acid phosphatase (TRAP). The remaining part of the PMMA block containing bone was sectioned using a diamond saw microtome (1600, Leica, Wetzlar, D) to obtain a 1 mm-thick slice. The surface of each section was polished with sandpaper and diamond compound (3 
m). The polished sections and appliances surrounded by fibrous tissue were carbon coated (CED 020, Bal-Tec, FL), examined by SEM using a back-scattered electron detector (XL40, Philips, NL) and analysed with an X-ray microprobe, EDS system (EDAX9900, Philips, Heindhoven, NL), at 25 kV, 0° tilt, 31° take-off, 0.08 
m spot. Each specimen was submitted to 10-30 X-ray analyses at different sites. Semiquantitative analysis was subsequently performed after the appropriate ZAF (Z, atomic number; A, absorption; F, secondary fluorescence) correction [18]. X-ray maps (128×100 pixel) were drawn up under the same analysis conditions, but with 0.1 
m spot and 0.2 s dwell-time. Embedded bone, not mixed with Osprovit^®, and connective tissue from each patient were used as controls. The hydroxyapatite (produced by Fin-Ceramica Faenza s.r.l., Faenza RA, Italy) and the surrounding tissues of a previous study [19] were used as controls.

5. 3. Results

3.1. Titanium appliances

No substantial differences were observed among the biopsies despite the different implant time (1-6 years). Histology of the connective tissue showed a feeble lymphoplasmacytic reaction and polymorphonuclear infiltrate with many mast cells. Scattered mastocytes were found inside the fibrous tissue. SEM displayed a nearly smooth surface of plates and screws, with no scratches or other alterations (Fig. 1).

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Fig. 1. Back-scattered SEM images showing the fibrous tissue surrounding a titanium plate (A) and a Ti6Al4V screwhead (B). Note the tissue density. The numbers indicate the sites that correspond to the EDS analysis reported in Fig. 2. Field width: A=4.4 mm; B=1.75 mm.

EDS confirmed the Ti/Al/V composition (90/6/4 wt%), i.e. Ti6Al4V alloy, of the screws and the pure titanium composition of all titanium plates (Fig. 2, graphs
and
). EDS analysis on the fibrous tissue enveloping plates and screws revealed titanium in proximity of the metal parts. In the connective tissue layer (100-150 
m thickness) adjacent to the titanium appliance, titanium release followed a decreasing concentration gradient, whereas no titanium was found at a distance greater than 300-500 
m. On the contrary, aluminium was found in connective tissue in proximity of the Ti6Al4V screws and adjacent to plates (Fig. 1), even though the aluminium content was low in the latter. Instead, the aluminium content detected in the fibrous tissue near Ti6Al4V screws was very high ( Fig. 2, graph
), and aluminium was also found at a distance greater than 300-500 
m.

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Fig. 2. Graphs showing the EDS (full area at ×5000 SEM magnification) of a Ti6Al4V screw (
) and titanium plate (
), plotted on the same scale, and the fibrous tissue (
) bounded to a Ti4Al6V screw (see Fig. 1). Note in
how fibrous tissue reveals the usual elements such as carbon, oxygen, phosphorous and calcium, in addition to titanium (small amounts) and aluminium (great amounts).

Titanium was not detected in lamellar bone surrounding titanium plates. On the contrary, aluminium was almost always detected in lamellar bone (Fig. 3, bs). EDS analysis revealed aluminium in lamellar bone, both near the screw surfaces but also at a distance greater than 1-2 mm. The dense lamella, named also acellular lamella (Fig. 3), showed a very high aluminium content whereas only traces of aluminium were found in the loose lamella, also named cellular lamella ( Fig. 3,
). No aluminium was detected in woven bone.

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Fig. 3. Back-scattered SEM image (bs) and X-ray maps for aluminium (Al) and phosphorus (P) showing three dense lamellae (asterisks) and an osteocyte lacunae (inside a large loose lamella) of lamellar bone formed at a distance from the titanium plate. The dense lamellae (darker in bs and P) have a low phosphorus (P) but high aluminium content (lighter in Al). The opposite is seen in the loose lamella (lighter in bs and P). Field WIDTH=32.5 
m.

Moreover, no aluminium was detected in the fibrous control tissues.

3.2. Hydroxyapatite granules

Histology confirmed an adequate amount of newly formed bone, residues of autologous bone and fragmented Osprovit^® granules in 3-month biopsies (Fig. 4). Autologous bone presented a highly indented surface (Howship lacunae) due to osteoclast aggression. New bone, woven but with lamellar structure in many parts, was more often located at a distance from granules, whereas fragmented bone was found in the proximity of fragmented granules (Fig. 4). In 6-month biopsies, the amount of bone and granules were lower than previously recorded. Hydroxyapatite granules were highly fragmented and surrounded by large zones of fibrous tissue.

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Fig. 4. Back-scattered SEM image showing a 3-month Osprovit^® biopsy. Note on the right how granules appear highly fragmented and surrounded by bone fragments. The arrows point to autologous bone residues (light gray), the result of osteoclast erosion, on which the osteoblasts have formed the new bone (NFB). Field WIDTH=1200 
m.

In 3-month biopsies, the TRAP reaction detected several osteoclast-like cells eroding the Osprovit^® porous granules (Fig. 5A). Few TRAP reactive cells were found on the bone surrounding the granules whereas many TRAP reactive cells were located close to the outer surface and on the surface of the internal cavities of granules (Fig. 5A, arrows). In 6-month biopsies, the number of granules was low and, accordingly, few TRAP reactive cells were found.

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Fig. 5. Optical microscope images showing the result of TRAP (A) and TALP (B) histochemical reactions performed on a 3-month Osprovit^® biopsy. The black arrows in A point to osteoclast-like cells (red in slide) which erode the granule (O). An osteoclast in the fibrous tissue is identified by a white arrow; no osteoclasts are eroding the bone (b). In B note the osteoblast lamina, identified by the positive reaction for alkaline phosphatase (blue in slide). Osteoblasts form new bone in apposition on autologous bone (b), confirmed microradiographically, at a distance from the Osprovit^® granules. Field width: A=B=350 
m.

The TALP reaction was strongly expressed only in 3-month biopsies, on autologous bone located far from the hydroxyapatite granules (Fig. 5B). No evidence of TALP was found both around the granules and its surrounding bone fragments. In the latter, the bone surface was highly indented subsequent to osteoclast activity. TALP was poorly expressed around bone in 6-month biopsies.

EDS revealed aluminium in skeletal tissue surrounding hydroxyapatite granules (Fig. 6). The amount of aluminium was higher in the connective tissue containing large amounts of phosphorous and calcium (Fig. 6, Al and P maps; Fig. 7, graph
). Nevertheless, a remarkable aluminium content was also found in sites with fibrous tissue poor in phosphorous and calcium (Fig. 6, Al and P maps; Fig. 7, graph
).

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Fig. 6. Back-scattered SEM image (bs) and X-ray maps for oxygen (O), aluminium (Al) and phosphorus (P) showing the topographic composition of a 3-month Osprovit^® biopsy. The greater the element content, the lighter the pixel. The numbers indicate the sites that correspond to the EDS analysis reported in Fig. 7. Field WIDTH=250 
m.

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Fig. 7. Graphs (all plotted on the same scale) showing the EDS (full area at ×5000 SEM magnification) of Osprovit^® granule (
), and of sites rich (
) and poor (
) in P and Ca of fibrous tissue (see Fig. 6). Note how the aluminium peak (1.49 keV) is the highest on both sites of fibrous tissue.

Semiquantitative X-ray analyses of unimplanted and embedded Osprovit^® revealed an uneven aluminium content in granules. The mean aluminium content of each unimplanted granule ranged between 0% and 0.5%.

EDS detected aluminium in bone adjacent to the granules and also in newly formed bone located far from the granules. No aluminium was found in lamellar autologous bone (evaluated on microradiographs and with the back-scattered SEM probe) and in newly formed woven bone at a distance from hydroxyapatite. On the contrary, newly formed lamellar bone (Fig. 8) was peculiarly rich in aluminium: loose lamella contained little aluminium whereas dense lamella contained greater amounts of aluminium (Fig. 9).

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Fig. 8. Back-scattered SEM image showing the bone, formed at a distance from Osprovit^® granules, in a 3-month biopsy. The new bone was formed in apposition to a residue of autologous bone (AB—lighter in the image), presenting an indented surface due to osteoclast erosion. The numbers indicate the sites that correspond to the EDS analysis reported in Fig. 9. Field WIDTH=205 
m.

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Fig. 9. Graphs (all plotted on the same scale) showing the EDS (full area at ×10,000 SEM magnification) of a dense (
) and loose (
) lamella of bone, formed at a distance from the Osprovit^® granules. Note how the aluminium peak (1.49 keV) is hardly plotted in the loose lamella whereas a strong peak can be observed in the dense lamella.

No aluminium was detected in control bone and connective tissues. Moreover, no aluminium was detected in hydroxyapatite (produced by Fin-Ceramica) and in the surrounding newly formed hard and soft tissue inside a hydroxyapatite chamber implanted in rabbit [19].

6. 4. Discussion

Titanium and hydroxyapatite are considered good biomaterials. They normally give excellent results [19 and 20] and are widely used in implantology due to their high biocompatibility. The observed interference of these materials with oral tissue is probably due to different reasons for the metallic and ceramic material tested.

EDS reveals titanium release from the surfaces of the tested metallic appliances. In a recent work [16] we showed that titanium produces small amounts of ions which are rapidly drained through skeletal tissues. Ti6Al4V releases titanium but also aluminium and vanadium. We were not able to follow the fate of vanadium ions because in the EDS method applied vanadium K
emission (4.95 keV) is covered by titanium K
emission (4.93 keV). On the contrary we can easily study the aluminium, whose release from Ti6Al4V screws can probably be ascribed to the activity of cells and biological fluids. Operation processes and fretting between plate and screw might be an additional cause of ion release. We did not find metallic debris at the plate removal. A debris dissolution probably occurred over the extended period from positioning to removal.

Hydroxyapatite does not contain aluminium, but small amounts of aluminium were detected in Osprovit^® granules. We cannot ascribe the aluminium content of the tested granules to impurities in the original material or to the manufacturing processes. Granules release large amount of aluminium which is not drained off like titanium [16] but stored locally. This high aluminium concentration may produce biological perturbations: not only is bone erosion enhanced, but osteoclast-like cells erode the granules reducing them to small fragments. Moreover, granule erosion increases the surface-bulk rate and perpetuates aluminium release. This boosted erosion is probably not the first event which occurs after grafting. Erosion of autologous bone is normal after a sinus lift, but new bone is soon formed in apposition to the residues of autologous bone. At the same time, new bone is formed in proximity of the granule before hydroxyapatite dissolution. The last event blocked bone deposition near the granules and produced initial bone erosion, transforming bone trabeculae into small fragments, after which erosion activity focused on the granule surfaces. This is the typical situation found 3 months after implant; 6 months after implant, bone mass was reduced. Hydroxyapatite is used to avoid this event, which normally occurs after bone grafting (subsidence). We believe that the hydroxyapatite tested not only obviates subsidence but prompts severe osteoclasia.

Despite different biomaterials, skeletal tissues show the same behaviour towards aluminium. In line with Shahgaldi et al. [21], our results indicate that aluminium is locally accumulated in soft tissue. A long period of aluminium draining follows, and aluminium accumulation in soft tissues will eventually cease if new release does not occur. This draining and diffusion of aluminium towards the surrounding soft tissues may explain the peculiar accumulation of aluminium in bone in sites distant from the biomaterials. In fact, newly formed lamellar bone had marked aluminium accumulation, and a large amount of aluminium was found inside the dense lamella.

Lamellar bone is not a variety of parallel-fibred bone [22 and 23] but, instead, is made up of alternating collagen-rich (dense lamellae) and collagen-poor (loose lamellae) layers, all having an interwoven arrangement of fibres [22, 23 and 24]. The loose lamella is also named cellular lamella because it contains osteocyte lacunae, whereas the dense lamella is also named acellular lamella due to the absence of lacunae. The lamellae have a relatively constant size in osteons [23 and 24]: the loose lamella is thinner (about 1.5-2 
m) and the dense lamella is broader (about 3-6 and up
m). It has been hypothesized that the alternation of dense and loose lamellae depends on osteocyte recruitment from osteogenic laminae in successive layers [23]. The loose lamella contains fewer fibres but more bone matrix, the dense lamella contains more fibres but less bone matrix. Due to the high affinity for calcium salt of the bone matrix, the loose lamella is more mineralized whereas the dense lamella has the same apatitic structure (Ca/P=2.15 w/w) but is less mineralized (about 2-4% in Ca content) [25].

We can make two speculations: (A) collagen has a higher affinity for aluminium than bone matrix, so that the dense lamella is richer in this element; (B) if collagen production by the osteoblast remains constant over the time required to form two adjacent lamellae, the difference between the loose and dense lamella may depend on matrix production but not on the time of formation. Osteocyte recruitment constitutes a signal for osteogenic lamina: osteoblasts and preosteocytes become active in abundant matrix production, and so loose lamella is formed. If the aluminium concentration of soft tissue surrounding the osteogenic lamina remains substantially constant, the dense and loose lamella should contain an equal amount of aluminium. However, the lower the lamella thickness, the higher the aluminium density. Both events may occur at the same time. Further studies are needed to confirm our hypothesis.

Aluminium is an element involved in severe neurological, e.g. Alzheimer's disease [26], and metabolic bone diseases, e.g. osteomalacia [27]. Aluminium accumulation in bone is only a temporal soft tissue deprivation. Bone remodeling will produce further aluminium release, even after the appliances no longer leach out aluminium, perpetrating the dangerous side effects of this element [26 and 27].

7. 5. Conclusions

Our results highlight that biocompatible materials may lead to problems due to impurities contained in biomaterial (as in the examined hydroxyapatite) or to aluminium alloys (Ti4Al6V). Our results show that aluminium is able to perturb the biological system causing the local inflammation. We do not know if the small amount of aluminium released from the tested appliances can have a systemic impact or promote severe neurological and metabolic bone diseases. This aspect will need additional quantitative studies, as also underlined by Chang et al. [28], in terms of elements known to be dangerous but also for titanium which we advance might be active in bone processes [16].

Our results document a slow drainage of aluminium by fibrous tissue and a peculiar accumulation in lamellar bone. We do not know the fate and meaning of these effects, what happens when bone is remodeled, and if aluminium is accumulated in the same way in all situations. Since our work was performed in man, we do not know the relationship between aluminium-bone accumulation and the amount of aluminium release from biomaterial, in particular if all the skeletal segments are concerned. Another question may concern the aluminium introduced "per os": is aluminium accumulated in bone in the same way? Only further more in depth qualitative and quantitative studies with experimental animals and different types of biomaterials containing aluminium or diet treatments will be able to answer these questions.

8. Acknowledgements

The authors wish to thank Dr. John Pradelli, M.D. for assistance in manuscript revision and the "Centro Interdipartimentale Grandi Strumenti" (CIGS) of the University of Modena and Reggio Emilia for software, SEM and EDS availability and assistance. The Research Fund of the Department of Anatomy and Histology, University of Modena and Reggio Emilia supported this investigation.

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