Letter pubs.acs.org/NanoLett
Atomically Resolved Tissue Integration Johan Karlsson,† Gustav Sundell,‡ Mattias Thuvander,‡ and Martin Andersson*,† †
Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10 SE-412 96 Gothenburg, Sweden ‡ Department of Applied Physics, Chalmers University of Technology, Fysikgränd 3 SE-412 96 Gothenburg, Sweden S Supporting Information *
ABSTRACT: In the field of biomedical technology, a critical aspect is the ability to control and understand the integration of an implantable device in living tissue. Despite the technical advances in the development of biomaterials, the elaborate interplay encompassing materials science and biology on the atomic level is not very well understood. Within implantology, anchoring a biomaterial device into bone tissue is termed osseointegration. In the most accepted theory, osseointegration is defined as an interfacial bonding between implant and bone; however, there is lack of experimental evidence to confirm this. Here we show that atom probe tomography can be used to study the implant−tissue interaction, allowing for three-dimensional atomic mapping of the interface region. Interestingly, our analyses demonstrated that direct contact between Ca atoms and the implanted titanium oxide surface is formed without the presence of a protein interlayer, which means that a pure inorganic interface is created, hence giving experimental support to the current theory of osseointegration. We foresee that this result will be of importance in the development of future biomaterials as well as in the design of in vitro evaluation techniques. KEYWORDS: Biomedical implants, osseointegration, atom probe tomography, in vivo, biomaterials, nanotopography iomedical research is a truly interdisciplinary field, where the key for successful implantation is the ability of inserted biomaterials to induce a desired host tissue response. A successful implantation is obtained when good control of the healing at the interface of the implant and the surrounding tissue is achieved.1 For bone-anchored implants, Branemark found in the 1950s that bone tissue could form direct contact with inserted titanium implants. He termed the phenomenon osseointegration and defined it as the formation of intimate contact between a load-carrying implant and living bone.2 Since this definition is based on observations made using optical microscopy, it provides no detailed explanation of the phenomenon on length scales smaller than a few hundred nanometers. The treatment of osseointegrating implants is used worldwide, and in the United States alone over 300 000 hip and knee implants and between 100 000 and 300 000 dental implants are placed annually.3 Despite progress within implant technology,4 further development is sought in order to enable treatment of patients suffering from bone diseases as well as allowing for accelerated wound healing, thus permitting earlier or immediate loading of the device. Moreover, bone formation ability decreases with age, and as higher life expectancies are anticipated in the future, the demand for treatment will increase.5 As mentioned above, the original description of osseointegration was based on observations made using optical
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© XXXX American Chemical Society
microscopy. Today, the most widely accepted theory to describe this phenomenon invokes the presence of interfacial bonding, that is, an atomic continuity between the implant and the bone.9 The development in electron microscopy techniques has enabled the examination of bone−implant interfaces with relatively high resolution, thus revealing some details of such interfaces. The results demonstrate the presence of a bone− mineral interface being formed in the gap between the implant and the bone.6−8 To date, however, no experimental data providing atomic information on the interface between implants and living tissue exist. 10 Here we examine osseointegration with atomic resolution using atom probe tomography (APT) to provide experimental data to verify the current theory describing osseointegration. Such increased knowledge would give insights that may prove instrumental in the development of strategies for optimal tissue healing of biomedical devices for bone anchorage. In addition, such information would be crucial in the development of improved in vitro evaluation techniques to decrease the need for animal testing. APT is an analytical technique based on mass spectrometry that has traditionally been used to characterize hard conducting Received: January 29, 2014 Revised: June 25, 2014
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Figure 1. Conceptual illustration of the mesoporous implant serving for local drug delivery. (left) SEM micrograph of a mesoporous titanium oxide -coated implant. (right) SEM micrograph depicting the mesopores, showing that the pores are facing out from the surface. At the bottom, the chemical structure of the active pharmaceutical ingredient RLX is shown.
Figure 2. SEM images of the in vivo healing and of the tip geometry together with the APT analysis. (a) Backscattered SEM image of the implant in bone retrieved after 4 weeks of healing. The arrow indicates the interface between the implant and the surrounding bone tissue from where the liftout was extracted. (b) SEM image of the sharpened tip, together with the APT reconstruction, in which the Ti-containing ions are displayed in dark green, Ca-containing ions are gray, and C-containing ions are brown.
To examine the implant−bone tissue interface, a sample with fully developed bone anchoring to the implant must be used. The strategy in this study was to use implants with an inbuilt drug delivery system. In previous work, mesoporous implants serving for local drug delivery of the osteoporotic drug raloxifene (RLX) exhibited superior osseointegrating ability when evaluated in a rat model.17 This novel implant design was therefore selected for the present study, where mesoporous titanium oxide was formed using the evaporation-induced selfassembly (EISA) method. Figure 1 shows our approach of using mesoporous implants with an inbuilt drug delivery function. The prepared mesoporous matrix possesses a wellordered porous structure, having an average pore size of 6 nm with a narrow pore size distribution.18 Mesoporous titanium oxide was deposited onto titanium implants as a thin film. The pores were loaded with RLX prior to insertion in order to serve for local drug delivery in vivo. The samples implanted in rat tibia were harvested together with the surrounding bone tissue after 4 weeks of healing. Since the probed volumes in APT are extremely small (typically 50
materials. The development of fast laser-pulsed atom probes over the past decade has broadened the application areas to include also semiconductors11 and oxides.12 The method has previously been identified as a promising technique for biological materials.13 Recently, Gordon and co-workers successfully analyzed a chiton tooth14 and some synthetic biominerals.15 These studies yielded new insights into mineral growth and helped to describe the chemical hierarchy of common apatite through utilization of APT. However, until now no APT experiments have been reported where the interface between a biomedical device and tissue has been analyzed. The modus operandi of the instrument is based on field evaporation of surface atoms on a needle-shaped specimen by the application of very strong electric fields.16 As atomic or molecular species are ionized and ejected from the surface, they are accelerated toward a position-sensitive detector, and the flight time is recorded to reveal their chemical identity. Spatial coordinates along with the time of flight for each individual ion allow for three-dimensional (3D) reconstruction of the specimen. B
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nm × 50 nm × 200 nm), high precision in the sample preparation is required. Therefore, specimens were prepared using a focused ion beam lift-out technique with subsequent ion beam tip sharpening in accordance with established procedures.19,20 Lift-outs were performed from polished cross sections of the bone−implant interface, as shown in Figure 2a. In order to improve accuracy in the spatial atomic distributions, the 3D reconstructions were based on scanning electron microscopy (SEM) images of the tip geometry. Figure 2b shows a 3D reconstruction of an APT analysis, beginning in the outermost 60 nm of the coating layer and continuing across the interface into the surrounding bone tissue. Figure 3 displays an enlarged volume within the
Figure 4. 3D reconstruction of the implant−tissue interface and 1D concentration profiles. (a) APT image displaying the direct contact between Ca of the bone mineral and Ti of the coating surface. (b) 1D concentration profiles of Ti, C, Ca, and P from the coating and across the interface into the bone tissue, obtained from the reconstruction in (a).
titanium oxide surface and appears next to the Ca. Carbon is randomly distributed throughout the bone tissue, but no “larger” complexes originating from, for example, collagen protein fibers are observed in the region close to the implant that was examined in this study. It should be noted that bone apatite is partly substituted with carbonate, which also would give rise to the presence of carbon in the mineral. In conclusion, our study demonstrates that the APT technique is capable of determining the distribution of different atoms at the interface between biomedical devices and living tissue, something that has never been demonstrated before. APT analysis of the in vivo healing showed a direct contact between the titanium oxide surface of the implant and Ca of the bone mineral. This result gives experimental authenticity to the current theory of osseointegration, that is, an atomic continuum of inorganic bone mineral between the implant and the bone. In the context of biomedical applications, this unique information with atomic-scale resolution may prove crucial for the development of the next generation of biomedical devices. Especially, the role of nanotopography, which has been demonstrated to strongly affect osseointegration, could be understood further with these new insights.23 This will assist engineers in designing refined products with optimized tissue integration functionality. Moreover, it indicates that the use of truly inorganic in vitro methods, such as the ones based on simulated body fluids are highly relevant in evaluating a material’s in vivo performance. This may result in reduced need for animal testing.24
Figure 3. Enlarged reconstruction of the outermost region of the mesoporous titanium oxide and connecting bone tissue below. Isoconcentration surfaces of TiO (green) and Ti3O2 (red) are displayed along with individual P atoms (pink), C atoms (brown), and Ca atoms (gray).
mesoporous titanium oxide region to clearly visualize how the porous structure enables diffusion and subsequent incorporation of mineral ions as well as the presence of organic matter inside the coating. The organic matter is believed to originate from the embedded RLX. The mesoporous material was found to be inhomogeneous, consisting of two TixOy phases, one with a stoichiometry close to TiO and another appearing as Ti3O2. The inhomogeneous nature of mesoporous titania is well-known, as it is a mixture of amorphous oxide coexisting with crystalline anatase,21 which in the APT analysis results in different stoichiometries.12 Figure 4a shows a volume containing the interface between the titanium oxide and the bone tissue. In Figure 4b, 1D concentration profiles are presented for Ti, Ca, C, and P that were retrieved from a region stretching from the titanium oxide matrix into the surrounding bone tissue. Interestingly, it emerges that a Ca-enriched layer of approximately 5 nm thickness is located directly on the outer surface of the titanium oxide. The same Ca enrichment can also be observed in the bone tissue below the mesopore presented in Figure 3. This suggests that Ca has a high affinity for the implant, which likely is the result of the titanium oxide being slightly negatively charged under the physiological conditions, thus attracting positive Ca counterions to the surface.22 Phosphorus, on the other hand, is not enriched in the immediate vicinity of the
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ASSOCIATED CONTENT
S Supporting Information *
Material characterization, in vivo study, and APT analysis. This material is available free of charge via the Internet at http:// pubs.acs.org. C
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(19) Miller, M. K.; Russell, K. F.; Thompson, G. B. Ultramicroscopy 2005, 102 (4), 287−298. (20) Thompson, K.; Lawrence, D.; Larson, D. J.; Olson, J. D.; Kelly, T. F.; Gorman, B. Ultramicroscopy 2007, 107 (2−3), 131−139. (21) Andersson, M.; Birkdal, H.; Franklin, N.; Ostomel, T.; Buecher, S.; Palmqvist, A. E. C.; Stucky, G. D. Chem. Mater. 2005, 17 (6), 1409−1415. (22) Kokubo, T.; Kim, H.-M.; Kawashita, M. Biomaterials 2003, 24 (13), 2161−2175. (23) Le Guehennec, L.; Soueidan, A.; Layrolle, P.; Amouriq, Y. Dent. Mater. 2007, 23 (7), 844−854. (24) Kokubo, T.; Takadama, H. Biomaterials 2006, 27 (15), 2907− 2915.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
M.A. conceived the present project. M.A. and M.T. designed the project plan. J.K. performed the material preparation and the material characterization. G.S. and J.K. performed the FIBSEM lift-outs with subsequent tip polishing. G.S. carried out the APT analyses. G.S. and M.T. made the APT reconstructions. J.K. and M.A. elucidated the tissue integration from the APT data. J.K. and G.S. prepared the manuscript, and all authors edited the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Materials Area of Advance at Chalmers University of Technology. We thank N. Harmankaya, A. Palmquist, P. Tengvall, and M. Halvarsson for their input to this work.
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ABBREVIATIONS APT, atom probe tomography; RLX, raloxifene; EISA, evaporation-induced self-assembly; SEM, scanning electron microscopy.
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REFERENCES
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