Detachment of titanium and fluorohydroxyapatite particles in unloaded endosseous implants
D. Martini, , a, M. Finib, M. Franchia, V. De Pasqualea, B. Bacchellia, M. Gamberinia, A. Tintic, P. Taddeic, G. Giavaresib, V. Ottania, M. Raspantid, S. Guizzardie and A. Ruggeria
a Istituto di Anatomia Umana Normale, Via Irnerio 48, 40126, Bologna, Italy
b Serv. di Chirurgia Sperim., Ist. di Ric. Codivilla-Putti, IOR Via di Barbiano 1/10, Bologna, Italy
c Dip. di Biochimica "G. Moruzzi", Via Belmeloro 8/2, 40126, Bologna, Italy
d Lab.di Morfologia Umana "Luigi Cattaneo, Via Montegeneroso 71, 21100, Varese, Italy
e Dip.Medicina Sperimentale- Sez. di Istologia, Via Volturno 39, 43100, Parma, Italy
Received 23 April 2002; accepted 28 August 2002. ; Available online 7 January 2003.
Abstract
The shape, surface composition and morphology of orthopaedic and endosseous dental titanium implants are key factors to achieve post-surgical and long-term mechanical stability and enhance implant osteointegration.
In this study a comparison was made between 12 titanium screws, plasma-spray-coated with titanium powders (TPS), and 12 screws with an additional coating of fluorohydroxyapatite (FHA-Ti). Screws were implanted in the femoral and tibial diaphyses of two mongrel sheep and removed with peri-implant tissues 12 weeks after surgery.
The vibrational spectroscopic, ultrastructural and morphological analyses showed good osteointegration for both types of implants in host cortical bone. The portion of the FHA-Ti implants in contact with the medullary canal showed a wider area of newly formed peri-implant bone than that of the TPS implants.
Morphological and EDAX analyses demonstrated the presence of small titanium debris in the bone medullary spaces near the TPS surface, presumably due to the friction between the host bone and the implant during insertion. Few traces of titanium were detected around FHA-Ti implants, even if smaller FHA debris were present.
The present findings suggest that the FHA coating may act as a barrier against the detachment of titanium debris stored in the medullary spaces near the implant surface.
Author Keywords: Titanium implants; Coating; Fluorohydroxyapatite; Scanning electron microscopy; Vibrational spectroscopy
Article Outline
1. Introduction
Post-surgical and long-term mechanical stability is the fundamental requirement for the osteointegration of orthopaedic and endosseous dental implants. Therefore, the shape, chemical composition and surface morphology of the implants, as well as the surgical techniques to achieve bone anchoring, are being increasingly studied.
Of the various implant materials, titanium is a particularly suitable metal for orthopaedic and endosseous dental implants on account of its good mechanical properties and biocompatibility [1]. The host bone tissue, in fact, grows in close contact to the metal, thus ensuring implant osteointegration.
The role played by the surface morphology in terms of implant stability has been highlighted by many studies [2 and 3]. A rough implant surface appears to be particularly suitable for primary implant stability as compared to a smooth implant surface. Moreover, the microcavities on the titanium implant surface are quickly colonised by blood cells that promote the ingrowth of new bone tissue [4].
To enhance and accelerate osteointegration, titanium implants can also be coated with other similar materials, such as bioapatites, which are similar to natural bone in terms of mineral content. These coatings have been reported to increase bone apposition and implant fixation rates by enhancing chemical bonding at the implant/new bone interface [3, 5, 6 and 7]. Among bioactive apatites, fluorinated apatites have shown in vivo the best chemical stability, good integration with bone and negligible long-term degradation [8 and 9].
Some authors have investigated the biological interaction between peri-implant tissues and implant surface in relation to the material used. Attention has been mainly focussed on the mechanisms of dissolution of an implant material in the host tissues. Regarding titanium, nitric acid passivation has been demonstrated to lead to titanium oxide dissolution and to result in a thinner and less stable oxide layer [10 and 11]. A porous-like structure provides more interstitial spaces for higher serum attack and would account for the increased levels of ion release over the polished samples [1].
The dissolution of metal ions decreases by 50% upon coating with hydroxyapatite (HA), thus providing a kind of physical barrier to metal dissolution [12]. Moreover, an increase in HA coating thickness has been suggested to reduce metal ion release [13]. However, HA dissolution has also been observed in aqueous and Ringer's solutions [14]. HA and fluoroapatite coating dissolution has been demonstrated to occur in the presence of bovine serum albumine solution, although fluoroapatite coating has shown a slightly lower dissolution rate [3].
Titanium screws with different coatings (plasma-sprayed powders of titanium (TPS) and the same TPS with adjunctive fluorohydroxyapatite coating (FHA-Ti)) were implanted in the tibia and femur of mongrel sheep to study the biological interactions between peri-implant tissues and implant surface 12 weeks after surgery.
2. Materials and methods
Twenty-four tapered cylindrical titanium screws, 4.5 mm in outer diameter and 28 mm in length (Biocoatings, Fornovo Taro, Italy), were coated with different powders by plasma spraying: 12 implants were coated with TPS (granule size ranging from 290 to 310
m) (TPS) and another 12 with TPS+FHA powder (granule size ranging from 3 to 80
m) (FHA-Ti). All of the procedures involving the sheep were performed according to the ethical guidelines on animal experimentation by the University of Bologna.
The implants were implanted bilaterally in the femoral and tibial diaphyses of two mongrel sheep (3-4 years old) anaesthetised according to a standardised protocol: premedication with intramuscular injection of 10 mg/kg b.w. ketamine (Ketavet 100, Farmaceutici Gellini, SpA, Aprilia, Italy), 0.3 mg/kg b.w. xylazine (Rompun, Bayer AG, Leverkusen, Germany) and subcutaneous injection of 0.0125 mg/kg b.w. atropine sulphate; induction with intravenous injection of 6 mg/kg sodium thiopentone (2.5% solution, Pentothal, Hoechst AG, Germany); maintenance with O2, N2O and 1-2.5% halothane under assisted ventilation (Servo Ventilator 900 D, Siemens, Germany). A 3.5 mm-diameter drill was used to predrill three holes in each diaphysis, which were tapped with a 4.5 mm device. Three screws were then inserted into the diaphyseal cortex of the tibiae and femora and tightened to the final insertion torque of 2.2±0.1 N m. The TPS and FHA-Ti screws were implanted on the right and on the left side of each animal, respectively.
Antibiotics (cefalosporin, 1 g/day for 5 days) and analgesics (ketoprofen 500 mg/day for 3 days) were administered post-operatively. The sheep were maintained in single boxes for 20 days after each surgery, and were then returned to external housing conditions. After 12 weeks, the animals were euthanised with intravenous administration of Tanax (Hoechst, Frankfurt am Main, Germany) under general anaesthesia. The implants with the surrounding peri-implant tissues were then removed and specimens containing implants were sawed for histological, morphological and spectroscopic analyses.
The diaphyseal bone segments containing the screws were isolated and fixed in 10% formalin-buffered solution (pH 7.2). Some samples were then dehydrated in ethanol and embedded in methyl methacrylate. Thirty-fifty
m thick sections were obtained by sawing and grinding operations (Saw and Grinding, Remet, Bologna, Italy), stained with toluidine blue and acid fuchsin and finally observed with a light microscope.
Some unstained methyl methacrylate-embedded sections were also mounted on stubs with carbon bioadhesive film, carbon-coated with an Emitech sputter-coater and observed with a Philips XL-30 FEG scanning electron microscope (Philips XL30FEG, Eindhoven, Holland) fitted with secondary electron (SE) and back-scattered electron (BSE) probes, and with X-ray dispersive spectroscopy (EDAX), at voltages of 10-12 kV.
Vibrational Raman and infrared (IR) spectroscopies were used for the chemico-physical analysis of the implanted TPS- and FHA-Ti-coated screws. Micro-Raman spectra were obtained in a non-destructive way using a Jasco NRS-2000C instrument with a ×20 magnification microscope. All the spectra were recorded in backscattering conditions with 5 cm−1 spectral resolution using the 488 nm line (Innova Coherent 70) with power of ca. 20 mW. The detector was a 160 K frozen CCD from Princeton Instruments Inc. IR spectra were performed using a Jasco Model FTIR 300E Fourier transform spectrophotometer with spectral resolution of 4 cm−1. The spectra were obtained from KBr pellets (about 0.5% w/w) containing finely ground powders removed from the surface of the implanted screws.
3. Results
All animals showed no evidence of complication during surgery and survived the whole post-surgical period of 12 weeks without any infection. All screws were used for investigations after explantation.
3.1. Vibrational spectroscopic analysis
The micro-Raman spectra recorded after 12 weeks in a non-destructive way on the screws implanted in the sheep tibia (Fig. 1) were exactly the same as those recorded on the corresponding screws in the femur (data not reported).
Fig. 1. Micro-Raman spectra of: (a) the material on the implant surface of a TPS-coated screw; (b) the material on the implant surface of a FHA-Ti-coated screw; and (c) FHA coating before implantation.
The micro-Raman spectrum of the material in contact with the TPS screw (Fig. 1a) showed the typical bone tissue bands, and therefore bands from organic (1660 cm−1 =Amide I of collagen; 1450 cm−1=
CH2; 1250 cm−1=Amide III of collagen; 1003 cm−1=partially derived from phosphorylated amino acids) [15] and inorganic (1003 cm−1=partially derived from HPO42− ions; 961, 592, 584, 450 and 434 cm−1=PO43− ions; 1070 and 1045 cm−1=PO43− and CO32− ions) [16, 17 and 18] bone components could be observed. The micro-Raman spectrum of the material in contact with the FHA-Ti-coated screws ( Fig. 1b) revealed the typical bands of the FHA coating before implantation (in particular, the 964 cm−1 band) (Fig. 1c), in addition to those previously mentioned for organic and inorganic bone components.
The IR spectra (Fig. 2) of the material removed from the implanted screws showed a trend similar to that of the Raman spectra. The spectrum of the material removed from the TPS-coated screw (Fig. 2a) revealed the typical bone tissue bands, and therefore bands from organic (1650 cm−1=Amide I of collagen; 1540 cm−1=Amide II of collagen; 1455 cm−1=
CH2) [19] and inorganic (1155, 1110, 575 cm−1=HPO42− ions; 1035, 962, 604, 564 cm−1=PO43− ions [16 and 17], 1417, 874, 712 cm−1=CO32− ions of a B-type carbonate apatite) [20] bone components could be observed. The IR spectrum of the material removed from the FHA-Ti-coated screw ( Fig. 2b) showed the typical coating bands (in particular, those at 1092, 1045 and 570 cm−1) (Fig. 2c), in addition to those previously mentioned for organic and inorganic bone components.
Fig. 2. IR spectra of: (a) the material removed from the implant surface of a TPS-coated screw; (b) the material removed from the implant surface of a FHA-Ti-coated screw; and (c) FHA coating before implantation.
It is interesting to note that the IR spectra in Figs. 2a and b show a band at 1385 cm−1. Such a band is not attributable to bone components and has been observed in the spectra of carbonate apatites containing impurities of the NO3− ions [17, 21 and 22]. The presence of the 1385 cm−1 band in the spectra of Fig. 2 is therefore ascribable to the NO3− component released by the nitrurised microtome saw used for histological samples preparation.
3.2. Histological and ultrastructural analysis
Twelve weeks after surgery, histological and ultrastructural analysis of the methyl methacrylate-embedded samples showed evidence of newly formed bone in both coatings.
Both types of implants appeared to be osteointegrated and showed a newly-mineralised tissue in close contact with almost the entire implant surface, thus filling the space between the TPS and FHA-Ti implant surfaces and the host cortical bone (Fig. 3 and Fig. 4). The portion of TPS surface crossing the whole medullary canal showed a small amount of primary new bone (Fig. 5), while the analogous portion of FHA-Ti surface was covered by a thicker well-distributed layer of new bone (Fig. 6).
Fig. 3. Light microscopy: A TPS implant in the sheep tibia after 12 weeks. A particle of titanium is detectable inside the newly formed bone (black arrow). Some titanium debris are present in the medullary spaces near the titanium surface (grey arrows). Scale bar 100
m.
Fig. 4. Light microscopy: An FHA-Ti implant in the sheep tibia after 12 weeks. Some particles of FHA and titanium are visible in the medullary spaces near the implant surface (arrows). Scale bar 100
m.
Fig. 5. Light microscopy: A TPS implant in the sheep tibia after 12 weeks. The portion of TPS surface crossing the whole medullary canal shows a small amount of primary new bone. Scale bar 500
m.
Fig. 6. Light microscopy: An FHA-Ti implant in the sheep tibia after 12 weeks. The portion of FHA-Ti surface is covered by a well-distributed layer of new bone. Scale bar 500
m.
The ultrastructural analysis of the TPS samples implanted in the cortical tibia and femur showed the newly formed peri-implant bone to be in tight contact with the Ti surface with no spaces at the interface (Fig. 7a). The newly formed bone grew in the coating micropores of the FHA-Ti implants and appeared to be in continuity with the implant surface (Fig. 8a). No evidence of detachment from the bulk titanium screws was found in either the titanium or FHA coatings.
Fig. 7. (a) TPS implants in the sheep tibia after 12 weeks. BSE analysis. Titanium debris are observable in the medullary spaces near the implant surface (arrows). Scale bar 500
m. (b) The EDAX analysis of (a) (Ti=light grey; Ca=dark grey).
Fig. 8. (a) FHA-Ti implants in the sheep tibia after 12 weeks. Titanium (white arrow) and FHA (grey arrow) debris are detectable in the medullary spaces. Scale bar 500
m. (b) The EDAX analysis reveals FHA and titanium debris (Ti=light grey; Ca=dark grey).
Detachment of titanium particles was observable at the TPS surface and inside the newly formed peri-implant bone (Fig. 3). Moreover, small titanium debris were sometimes detectable in the bone medullary spaces near the TPS surface. On the contrary, titanium particles were not seen in the medullary spaces far from the implant ( Fig. 3). Small particles of FHA and a small amount of titanium particles were evident in the medullary spaces near the FHA-Ti implant surface ( Fig. 4). In TPS implants, titanium debris were mostly found within 200-250
m from the metal surface, although some debris could occasionally be observed at 500
m and even at greater distances. On the other hand, FHA particles had a wider distribution and could be seen at greater distances in FHA-Ti implants. However, the greater the distance, the smaller the particles became, until being undetectable. They appeared in some way to crumble and disintegrate as their distance from the implant surface increased. BSE imaging and EDAX analysis confirmed the presence of titanium and FHA debris in the medullary spaces near the implant surface (FFig. 7 and Fig. 8).
No signs of detachment were detectable at the titanium coating-bulk titanium and titanium coating-FHA coating interfaces, and only some fractures, probably due to sample manipulation, were observed in the FHA coating thickness.
4. Discussion
Osteointegration is the fundamental requirement for the long-term success of orthopaedic or dental implants, and primary stability is a key factor to achieve osteointegration. An extensive and close contact between the implant and the host bone surfaces is the condition that maintains primary stability and avoids excessive interfacial micromotion during bone healing that may be detrimental to the osteointegration process [23]. Some authors, however, have observed that the presence of adequate spaces to allow bone remodelling is also useful to enhance and accelerate osteointegration [24], provided that implant mobilisation is avoided. Therefore, clinicians may obtain satisfactory primary stability through the friction occurring at the implant surface-host bone interface, thus avoiding implant micromotion. Such friction, however, could increase stress concentrations on the implant coating and alter its morphology and integrity, resulting in the detachment of metal particles. Some studies have reported the long-term presence of metallic wear particles from endosseous implants in the liver, spleen, small aggregates of macrophages and even in para-aortic lymph nodes [25]. Metal ions released from implants may arise from dissolution, fretting and wear, and may be a source of concern due to their potentially harmful local and systemic carcinogenic effects [1 and 26]. However, local and systemic adverse effects of titanium ion release are not universally recognised. Some authors have in fact observed that titanium-alloy dental implants with and without plasma-sprayed HA coating did not show any toxic effects on cells in dogs [27]. In any case, a reduction in metal ion release is also preferable on account of the adverse effect that they may have on the sensitive differentiation processes necessary for normal bone formation [1]: an excessive metal ion release can inhibit cell function and apatite formation [28 and 29].
In this study TPS- and FHA-Ti-coated screws were implanted in the femoral and tibial diaphyses of two mongrel sheep. Histological observations showed good osteointegration of both implants in host bone 12 weeks after surgery. No significant differences in osteointegration were found between the two implants in terms of diaphyseal compact bone. However, a different response of the host tissue to TPS and FHA-Ti coatings was observed in the implant portion crossing the medullary canal. On the TPS implant surfaces, a small amount of primary new bone was observed around some nucleation sites, while on the analogous portion of the FHA-Ti-coated implants there was a thicker and more homogeneously distributed layer of new bone near the implant. These observations confirm the stimulating effect of apatites on osteogenesis. The new bone appears as a fairly continuous layer overlapped with a well-preserved FHA-Ti coating, whose in vivo level of stability is implicitly confirmed [8 and 9].
The spectroscopic analysis of the material on the implant surface of TPS and FHA-Ti screws showed the presence of bone tissue, as revealed by the typical bands of its organic and inorganic components. The characteristic bands of HPO42− ions (at 1003 cm−1 in the Raman spectra and at 1155, 1110 and 575 cm−1 in the IR spectra) indicated the presence of newly formed bone, since they have been reported to decrease during the aging and maturation of the mineral phase [15, 30 and 31]. In vivo and in vitro [32] bone deposition occurs by means of amorphous precursors, such as tricalcium phosphate and octacalcium phosphate, which contain HPO42− ions. Successively, these amorphous metastable phases are transformed into a crystalline octacalcium phosphate-like phase which, in turn, is transformed into a crystalline apatitic phase by taking up OH− and HPO42− ions. With maturation, this apatitic phase is transformed into non-stoichiometric HA. Termine et al. [30] have observed that the mineral component of young rat deproteinated bone shows another band, in addition to the typical bands of PO43− ion, which, seen at ~1100 cm−1, is absent in the spectrum of mature bone and is typical of the HPO42− ion.
It is interesting to note that in the FHA-Ti-coated screws both IR and Raman spectra showed the presence of bands attributable to bone in addition to the coating bands. These results suggest that bone grows in close contact with the FHA coating and confirm a high level of osteointegration.
Ultrastructural observations confirmed a high level of biointegration since the newly formed bone can grow inside the FHA coating porosity in the same way as in other HA coatings. The strong adhesion between the coating and new bone was confirmed by some artificial fractures due to the mechanical stress developed during sawing (Saw and Grinding System) that developed on the coating thickness but not at the bone-coating interface.
The morphological analysis showed the presence of medullary spaces near the TPS surface. Small titanium debris were detectable inside these areas and in the newly formed peri-implant bone. On the contrary, titanium particles were never found in the medullary spaces located far from the implant surface. The size and position of these particles suggest that they may be due to the friction between the implant and the host bone during surgical implantation. The screws were in fact implanted in diaphyseal compact bone and should be considered unloaded. Consequently, no fretting or wear may have led to the detachment of metal particles from the implant coatings after surgery.
The EDAX analysis demonstrated that the chemical nature of the released debris depends on the composition of the implant surface. In FHA-Ti implants only a few traces of titanium were seen together with some FHA granules dispersed in the bone medullary spaces near the implant surface. The lower amount of titanium debris in the present samples may be due to the FHA coating that could protect titanium during the insertion or healing process. The FHA debris were in any case always present in the medullary spaces of these samples but represent biocompatible material [8]. Moreover, FHA resorption may be expected to occur according to the results of a study on more stable fluoroapatite coatings by Overgaard et al. [33], who have demonstrated that osteoclast-like cells, osteocytes, macrophage-like cells and fibroblasts phagocytize fluoroapatite fragments, thus indicating cell-mediated coating resorption.
On the contrary, evident titanium dispersion inside the medullary spaces was observed when TPS was implanted. These titanium particles decreased in size at great distances from the implant surface. This reduction in size of the particles may be due to mechanical effects, but also to a gradual and passive dissolution. Such a mechanism has been reported to always occur on any metal surface interacting with surrounding fluids and tissues [34].
In conclusion, the current findings suggest that:
the medullary spaces not only represent areas promoting bone turnover [35], but can also act as a sort of "door" to connect the implant surface to the systemic compartment,
among the porous surfaces of titanium implants, the FHA coating not only offers the positive factor of providing a substrate to enhance new bone formation, but also represents a protective coating to limit detachment of titanium particles.
Acknowledgements
The authors thank Biocoatings for providing the samples. This research project was supported by grants from CNR (Progetto finalizzato CNR/MSTA II, Sottoprogetto Biomateriali) and MIUR (60%).
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