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Osseointegrated implants are frequently used in reconstructive surgery, both in the dental and orthopedic ﬁeld, restoring physical function and improving the quality of life for the patients. The bone anchorage is typically evaluated at micrometer resolution, while bone tissue is a dynamic composite material composed of nanoscale collagen ﬁbrils and apatite crystals, with deﬁned hierarchical levels at different length scales. In order to understand the bone formation and the ultrastructure of the interfacial tissue, analytical strategies needs to be implemented enabling multiscale and multimodal analyses of the intact interface. This paper describes a sample preparation route for successive analyses allowing assessment of the different hierarchical levels of interest, going from macro to nano scale and could be implemented on single samples. Examples of resulting analyses of different techniques on one type of implant surface is given, with emphasis on correlating the length scale between the different techniques. The bone-implant interface shows an intimate contact between mineralized collagen bundles and the outermost surface of the oxide layer, while bone mineral is found in the nanoscale surface features creating a functionally graded interface. Osteocytes exhibit a direct contact with the implant surface via canaliculi that house their dendritic processes. Blood vessels are frequently found in close proximity to the implant surface either within the mineralized bone matrix or at regions of remodeling. 1 Introduction Transmission electron microscopy (TEM) was used exten- sively, and demonstrated intimate contact between bone and The term osseointegration, meaning a direct structural the implant surface, however, often separated by an electron connection between living bone and an implant surface, was lucent or electron dense layer in the range of 20–50 nm, coined by Professor P.I. Brånemark . The pioneering followed by mineralized collagenous bone, despite the fre- development of the osseointegrated dental implant during quent use of demineralization (summarized in ). An the 60 and 70s  led to their global introduction in the 80s alternative theory, especially for roughened implant sur- and is currently a routine treatment modality in dentistry. faces was the contact osteogenesis theory , where a µm Since then, other clinical applications have emerged such as thick cement line is formed at the immediate implant sur- osseointegrated facial prostheses, bone anchored hearing face , similar to the cement line separating osteons of aid, and major limb amputation prosthesis . different ages, characterized as a collagen deﬁcient hyper- The healing around commercially pure titanium (cp-Ti) mineralized zone . implants was originally described as being very similar to Recently, it has been proposed that bone healing around natural bone healing , hence being biocompatible and implants is a “foreign body reaction in equilibrium” with a avoiding the formation of ﬁbrous encapsulation. bony encapsulation, characterized as a poorly vascularized dense tissue interfacing the implant surface . However, no experimental data has been shown to support this latter theory and standardized protocols for the evaluation of Electronic supplementary material The online version of this article osseointegration at different length-scales and resolutions (https://doi.org/10.1007/s10856-018-6068-y) contains supplementary are needed in order to characterize the bone tissue interfa- material, which is available to authorized users. cing an implant. * Anders Palmquist The aim of this article is to describe an analytical strategy email@example.com to probe the different length-scales of osseointegration going from macro to nano. The focus is to have a platform Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden enabling comprehensive, multiscale and multimodal 1234567890();,: 1234567890();,: 60 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:60 analysis of single samples, allowing detailed characteriza- responsible for adaptive remodeling of bone, hence main- tion of clinical retrieved samples where the limited amount tenance of structural integrity and long-term performance. of material poses a major challenge. This review is limited The mechanics of bone (in the context of bone formation to structural and compositional analyses and does not cover around metal implants, mechanical load-transfer from the the cellular and molecular biology underlying osseointe- metal into the surrounding bone, and adaptive remodeling gration, which may be found elsewhere [10–14]. etc.) are strongly dependent on the nanoscale organization and composition. It is therefore important to understand how bone forms on an implant surface, generally, and 2 Bone tissue whether this nanoscale organization varies with respect to implant surface modiﬁcation. Technical challenges in the Bone tissue, a highly hierarchical biocomposite material preparation of TEM samples of the interfacial bone led to (Fig. 1) mainly composed of an organic matrix (collagen the development of strategies involving elimination of the type I) and an inorganic reinforcement phase (apatite), is an bulk properties of the implant in the sample preparation. adaptive tissue constantly undergoing remodeling. This was achieved either through the use of polymer Bone formation occurs through secretion of tropocolla- implants with a thin coating of titanium (restricted to gen by the osteoblasts (bone forming cells) which is later experimental studies) , mechanically separating the mineralized. The early healing is characterized by the rapid implant from the embedded bone tissue , or by elec- formation of woven bone, an unorganized bone tissue trochemically dissolving bulk titanium while the surface which is later remodeled into more ordered bone through oxide layer was left intact . All these techniques had the coupled action of osteoclasts (bone resorbing cells) and their shortcomings and the focused ion beam (FIB) milling osteoblasts. In this process, the osteoblasts start a coordi- technique for the preparation of intact bone-metal interface nated action of bone formation and it is believed that they samples for TEM was introduced in 2006 . can orient the collagen matrix  leading to the higher hierarchical levels through a bottom-up approach. In the bone formation process, some osteoblasts stop producing 3 Ex vivo tissue preservation and handling extracellular matrix and instead start undergoing differ- entiation into osteocytes and become entrapped within the Tissue-implant samples are retrieved from the biological matrix produced by their neighboring osteoblasts. The milieu, either from designed experimental models in ani- osteocyte resides within lacunae (ellipsoidal shaped cavities mals or clinically retrieved from patients. The tissues in the bone) and the osteocyte is connected with the undergo chemical ﬁxation by immersion in aldehyde solu- neighboring osteocytes, the blood circulation, and to the tions, dehydration in a graded series of ethanol, and suc- osteoblast (in a bone formation site) and lining cells (in a cessive resin inﬁltrated prior to polymerization. This steady state) via the lacuno-canalicular network (LCN). It is procedure was developed in order to be able to prepare believed that the osteocyte is the mechanosensing cell  undecalciﬁed ground-sections for histology . Fixation, Fig. 1 Bone hierarchy: Illustration of the different hierarchical levels of bone tissue with corresponding length scales, from macro to nano scale. Reprinted with permission from Springer Nature  Journal of Materials Science: Materials in Medicine (2018) 29:60 Page 3 of 10 60 Fig. 2 Multiscale and multimodal analysis: The work-ﬂow from retrieval and sample processing to sequential evaluation using a range of imaging and complementary spectroscopic techniques allowing comprehensive analysis of the same specimen Fig. 3 Macro level of osseointegration: Different views of a micro-CT image stack. e A three-dimensional surface rendering of the segmented analysis starting with a X-ray imaging. b Typical reconstruction of a data set representing the implant (grey) and bone (yellow) which has low-resolution scan, 12 µm voxel size (typically 15 min). c Typical been made semi-transparent. f Data set rotated to locate the slice reconstruction of a high-resolution scan, 2.5 µm voxel size (typically obtained as the histological ground-section. g Overview image of the 8 h). d A three-dimensional volume rendering of the reconstructed ground-section stained by toluidine blue dehydration, and resin embedding tend to induce dimen- 4.1 Micro-focused X-ray computed tomography sional changes and distort the ultrastructure [22, 23]. (micro-CT) However, it is possible to perform high-resolution analyses in combination with histology , and therefore such Micro-CT is the least destructive technique, where minimal sample preparation methods are suitable for a successive sample preparation is needed and is emerging in the ﬁeld of analytical strategy even for clinically retrieved implants osseointegration to study the bone-implant interface. The where limited volume of biological tissue and/or number of technique offers a rapid data acquisition, generating results samples are obtained. In Fig. 2, the sample preparation without the need for tedious sample preparation that is chain is shown to illustrate the correlative approach generally necessary for histology. This means that samples enabling multiscale and multimodal analysis of the intact can be scanned at any stage from the time of retrieval to bone-implant interface. post-embedment. The biggest advantage of micro-CT over histomorphometry is that morphometric measurements can be performed in 3D rather than having one representative 4 Successive analytical approach and histological section from each sample. Furthermore, micro- examples CT imaging can be directly correlated to histology by locating the histological section in the micro-CT data set To exemplify the successive analytical approach, exam- (Fig. 3). ples are shown of a hierarchically structured implant Nevertheless, the technique is associated with artifacts surface having a distinct microtopography and super- introduced during data acquisition, many of which may be imposed nanostructure (Supplementary Figure 1), from corrected for during reconstruction. However, widely dif- several experimental and clinical retrieval studies [24–30] fering densities of the metal implant and the biological in combination with the development of different analy- tissue compromise the analysis close to the implant surface tical techniques adapted for the bone-implant interface [35, 36]. The technique has been validated to assess bone [30–34]. volume accurately by correlating with histological 60 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:60 measurements [37, 38] even at fast/low resolution scans osteocyte lacunae as well as the presence of osteocytes in (e.g., 14 µm). While some investigators have reported a the lacunae could be evaluated (Fig. 3). Bone remodeling is correlation between micro-CT and BSE-SEM based mea- frequently observed both close to and at a distance from the surements of bone-implant contact , it is advisable that implant surface where osteoblasts are often co-localized measurements be correlated with histology and with osteoclasts as well as blood supply. The mineralization histomorphometry. front (and therefore the direction of bone formation) and the embedment of differentiating osteocytes can be observed 4.2 Light optical microscopy (LM) (Supplementary Figure 2) (Figs. 4 and 5). The gold standard in biomaterials research is to evaluate 4.3 Raman spectroscopy tissue response to biomaterials using simple optical micro- scopy, where both quantitative histomorphometry and Raman spectroscopy is becoming increasingly used in the qualitative histology can be performed. Qualitative histol- ﬁeld of biomaterials, as a versatile tool requiring minimal ogy enables a detailed description of the state of the tissues, sample preparation and is quick and minimally destructive. the maturity (woven, lamellar, or osteonal) and the presence It has been used for material characterization , evalua- of cells such as different bone cells (osteoblasts, osteoclasts tion of in vitro formed extracellular matrix  as well as and osteocytes), adipose cells, blood cells, inﬂammatory the molecular composition of bone tissue before  and cells and multinucleated giant cells as well as the general after resin embedding  and is considered to give an morphology and to some extent the structure and alignment assessment of the bone quality . Site-speciﬁc analyses of the tissues. A limiting factor is the sample preparation, of interfaces such as natural bone interfaces, i.e., cement where a section of single-cell thickness is preferable. lines  or the bone-implant interface can be carried out Moreover, the ﬁxation and dehydration steps must be [48, 49]. Confocal Raman imaging allows 2D and 3D optimized in order to avoid large artifacts from shrinkage mapping of composition at submicron resolution, and such especially in unmineralized areas, i.e., the marrow com- data can be easily correlated with other imaging techniques. partment. It has been shown that quantiﬁcation of bone- In composites such as bone, the relative amounts of the implant contact is highly dependent on the sectioning individual organic and inorganic constituents can be deter- direction as well as the thickness of the ground-section mined. Raman spectroscopy is particularly useful in iden- [40, 41]. tiﬁcation of different calcium phosphate phases found in The morphology of the tissue in association with the bone . Furthermore, in experiments involving selective implant surface, bone lamellar orientation and alignment of removal of tissue components for structural analysis, Fig. 4 Microscale osseointegration: Light optical microscopy com- the threads. e Correlative elemental mapping of the two threads, bined with BSE-SEM, EDS, Raman spectroscopy and SE-SEM of showing calcium (green), titanium (blue) and carbon (red). f Single resin cast etched samples, showing the successive increase in magni- thread in BSE-SEM showing a biomechanical testing induced frac- ﬁcation to follow the bone growth adjacent to the implant. a Overview tured zone at the thread valley as well as osteocytes and blood vessels histological image, showing the amount of bone tissue around the throughout the tissue. g Corresponding thread after resin cast etching, implant. b Closer view of two threads almost completely ﬁlled with showing the plastic surrounding osteocytes and blood vessels pro- mature bone tissue. c A closer view at the implant surface showing a truding from the etched bone surface. h and i Raman spectra from the remodeling zone with active bone formation, blood supply. Osteocytes corresponding spots marked in f, showing the molecular composition are visible in the bone with stained nuclei. d BSE-SEM image of two of the tissue. Images modiﬁed and reprinted with permission from threads showing a similar picture as the histology, mature bone ﬁlling John Wiley & Sons and Public Library of Science [25, 27] Journal of Materials Science: Materials in Medicine (2018) 29:60 Page 5 of 10 60 Fig. 5 Osteocytes at the implant surface: The osteocyte connection to lacuna and the implant surface showing a uniform directionality, the implant surface. a Histological image of osteocytes close to the indicating that the former osteoblast, now osteocyte produced the bone implant surface, the cell nuclei stained in blue. b BSE-SEM image of in a contact osteogenesis fashion. Collagen banding is observed per- an osteocyte close to the implant surface. c Osteocyte close to the pendicular to the implant surface as well as the lacuna indicating implant surface shown after resin cast etching where the canaliculi can collagen parallel to the surface, the morphology further indicate a be observed reaching the implant surface (Osteocyte and canaliculi mature bone with ﬁbril bundles of 1–2 µm in diameter. h STEM image highlighted in red). d Canaliculi making intimate contact with the of canaliculi directly interfacing the implant surface. i Corresponding implant surface, a top view showing the network of canaliculi. e FIB electron tomography volume rendering of h (Courtesy of Assistant section during TEM sample preparation made across the osteocyte in Professor Kathryn Grandﬁeld). Images reprinted and modiﬁed with b. A canaliculi is seen running in the 8 o’clock direction. f STEM permission from the American Chemical Society and Public Library of image of the thin sample, the canaliculi was removed during the Science [27, 33] thinning process. g Closer view of the bone between the osteocyte Raman spectroscopy can be used to identify and char- Secondary electron imaging generates contrast from acterize the remaining component . surface irregularities and is hence used for evaluating osteocyte lacunae and the canaliculi network after resin cast 4.4 Scanning electron microscopy (SEM) etching  where the resin ﬁlled voids in bone could be highlighted, showing the intimate contact between the SEM is a versatile and easy to use technique that enables canalicular network and the implant surface as well as the high-resolution imaging in conjunction with chemical ana- interconnectivity of the LCN with the blood vessels and lyses. Different contrast phenomena can be used to high- bone marrow . light different aspects. The most frequently used are In a successive manner, BSE-SEM is performed on secondary electron (SE-SEM) and backscattered electron polished surfaces without the use of a conductive coating. (BSE-SEM) modes, where the former gives contrast from The sample is subsequently processed for resin cast etching, the surface texture and the latter gives a Z-(atomic number) coated with a conductive coating for high vacuum SE-SEM, contrast. The Z-contrast is particularly useful for studying to be able to evaluate the same region of interest. bone, since the degree of mineralization is directly resolved and could be calibrated using standard materials to deter- 4.5 Focused ion beam-scanning electron microscopy mine the absolute wt. % calcium content . In many (FIB-SEM) ways, BSE-SEM provides a similar picture of the miner- alized tissue as histology, where the morphology of the FIB-SEM is a useful tool in biomaterials research, where tissue, degree of mineralization and alignment of osteocytes both 3D reconstructions could be performed using a serial could be directly visualized, in addition to details in slice-and-view (SSV) function. Much work has been done remodeling zones with embedment of osteocytes and the to study the ultrastructure of lamellar bone where imaging granular appearance of the mineralization front (Supple- was performed through successive lamellae and recon- mentary Figure 2). Electron bombardment onto the sample structing the lacuno-canalicular network, using a dec- surface induces charging artifacts, which are typically cir- alciﬁcation and staining protocol in order to visualize cumvented by the addition of a thin conductive coating. the collagen mesh  Without decalciﬁcation, the meth- Another solution is the use of an environmental SEM odology has been used to visualize bone ingrowth operated at low vacuum with the presence of water vapor to in microporous titanium oxide, however without being minimize (or completely eliminate) the charging artifacts, able to resolve the unique ultrastructural characteristics of without the need of an electrically conductive coating. bone . 60 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:60 Another application of FIB-SEM is preparation of TEM 4.7 Electron tomography (ET) samples. Thin lamellae or needle-shaped samples are cut out with sub-micrometer precision, readily cutting through Electron tomography uses similar principle as micro-CT, both metal and undecalciﬁed bone. The dimensions of the where a rotation series of images is acquired, aligned, and resulting sample are limited, and typically in the 25 × 1 µm reconstructed by back-projection. When performed in range, while being able to obtain a thickness of roughly HAADF-STEM, similar contrast is obtained as in micro- 100 nm. For more information on TEM sample preparation, CT and difﬁculties in reconstruction due to diffraction the interested reader is referred elsewhere [56, 57]. contrast as in regular TEM can be avoided. It has suc- cessfully been performed on the bone-implant interface 4.6 Transmission electron microscopy (TEM) enabling a greater understanding of apatite ingrowth into the nanostructured surface oxide layer [26, 30, 31, 33], The TEM is a very powerful instrument with not only high however, limitations in the reconstruction due to lack of magnifying power but also multiple in-built analytical images originating from limited tilt angles creates artifacts techniques, such as diffraction, energy dispersive X-ray in the images. The use of needle-shaped samples and spectroscopy (EDS), and electron energy loss spectroscopy special sample holders enables complete rotation and (EELS). For bone-implant interface analysis, during the 80 improved 3D reconstruction . As image acquisition is and 90s, the TEM was used mainly for imaging but no performed with high Z-contrast, contrast based segmenta- analytical techniques were implemented. The ﬁrst chemical tion protocols similar to micro-CT can be applied, thus mapping was reported in 2006  and much work has permitting quantiﬁcation and enhanced visualization of been done since. Furthermore, the use of scanning trans- collagen structure and orientation. Furthermore, with the mission electron microscopy (STEM) and the use of a use of needle-shaped sample, electron tomography could nanoprobe allow elemental analysis with high spatial reso- may be complemented by EELS tomography (chemical 3D lution as compared to the SEM where typically µm reso- mapping with nanometer resolution) and atom probe lution is obtained. The contrast phenomena typically used tomography (APT). are either bright-ﬁeld TEM or high-angle annular dark ﬁeld (HAADF-) STEM, where the latter gives Z-contrast and is 4.8 Complementary analytical techniques preferred for the bone-implant interface. The bone-implant interface has been shown to be com- Additional complementary tools may be utilized at different posed of bundles of collagen ﬁbrils running parallel to the stages of the sequential analytical approach and will further implant surface in very close proximity to the oxide layer. bridge the different length scales and bring new dimensions In the case of nanostructured implant surfaces, apatite to the analysis. ingrowth into surface irregularities has been shown both by Synchrotron radiation at large scale facilities is of high EDS [25, 28] and EELS [30, 33, 34]. The presence of interest, where multiple different analyses could be car- features smaller than the thickness of the sample pose ried out, using different sample sizes obtaining various considerable difﬁculties in resolving the actual interface due ﬁelds of view and spatial resolution levels. Particularly for an overlap in information, necessitating the use of high- tomographic techniques, higher ﬂux enables improved resolution 3D imaging techniques. resolution and contrast than conventional methods . For the osteocyte lacuno-canalicular network, as the Phase contrast tomography can be used for improved sample is sectioned from only a top view, it is difﬁcult to resolution and complement lab-based micro-CT, where selectively have canaliculi in the sample, but due to their improved resolution at the implant interface could be large presence in the bone, often transversely cut circular obtained, as well as observing and distinguish low con- features (typically 200 nm, in agreement with canaliculi trast components in the bone tissue in 3D . The ) are visible in the TEM section, while part of osteo- average alignment of the mineralized collagen bundles cytes could be sectioned readily . and crystal thickness could be obtained in 2D [60, 61]and The structure and alignment of the tissue components, for 3D  by scanning small-angle X-ray scattering example the size and orientation of apatite crystallites, can (sSAXS) tomography at comparable low resolution with be evaluated, thus revealing details of the smallest hier- larger ﬁeld of view. At higher resolution, the direction of archical level of mineralized tissue in osseointegration . individual collagen bundles can be investigated [63, 64] The ultrastructural pattern of bone is highly dependent on however in smaller sample volumes of a few hundreds of the cutting direction, where mineralized collagen ﬁbers may micrometers. 2D sSAXS has been applied to characterize be cut longitudinally or transversally. In longitudinal sec- bone healing around degradable metal implants [65, 66]. tioning, characteristic 67-nm striations are seen while holes By ptychographic tomography, the osteocyte canaliculi and circular patterns are observed in transversal sectioning. network could be resolved in very small samples in the Journal of Materials Science: Materials in Medicine (2018) 29:60 Page 7 of 10 60 Fig. 6 Nano-osseointegration: The nano-scale characteristics of the series of images of electron tomography of a needle-shaped implant bone-implant interface could be visualized and analyzed using the where full rotation could be performed for improved reconstruction. In transmission electron microscope. a A 3D rendering of the bone the perpendicular slices the bone structure with darker features (carbon implant interface where the collagen bundles are observed parallel to rich collagen ﬁbrils) and aligned apatite could be visualized while at the implant surface running in the plane of the section, collagen the interface to the implant surface, the structure of the oxide layer banding is observed perpendicular to the implant surface. The indi- with nanoscale features could be seen ﬁlled with apatite. Two 3D vidual mineralized collagen ﬁbrils which bundles up to make the surface rendering of contrast-based segmented data set with implant bundles could be observed in the tomogram very close to the outer- (grey), collagen ﬁbrils (red) and with/without apatite (yellow), show- most surface of the oxide layer. b STEM image of the interface ing the individual collagen ﬁbrils seemingly being from two different showing the typical collagen banding and parallel fashion of the col- collagen bundles (bundles typically in the range of 1–2 µm in dia- lagen to the implant surface. Site speciﬁc chemical analysis by EDS meter) with slightly different alignment, both however in a rather across the interface show a zone of overlapping information indicating parallel to the implant surface. Images reprinted and modiﬁed with apatite formation in the nanostructures formed by the surface oxide. c permission from the Royal Society (UK), the American Chemical TEM image of the interface with corresponding EFTEM image ﬁltered Society and the Royal Society of Chemistry [26, 30, 33, 34] for the calcium showing the ingrowth into the nanostructures. d A Acknowledgements The author would like to thank Dr. Furqan Ali range of tens of micrometers . It is possible to apply Shah and Professor Peter Thomsen at the University of Gothenburg most of these techniques to plastic embedded samples at and Assistant Professor Kathryn Grandﬁeld at McMaster University successive stages depending on their need for smaller for all the fruitful scientiﬁc discussions during the years. Ms Lena samples sizes, and could hence be integrated in the pro- Emanuelsson, laboratory technician at the University of Gothenburg for her enormous expertise and long-term experience in sample pre- posed analytical approach. Following STEM tomography servation and preparation. Financial support was received from the of needle-shaped samples preparing using the FIB (Fig. 6) BIOMATCELL VINN Excellence Center of Biomaterials and Cell , the tip can be thinned down further for EELS Therapy, the Region Västra Götaland, an ALF/LUA grant, the tomography and APT. APT is also becoming popular in IngaBritt and Arne Lundberg Foundation, Swedish Research Council, the Dr. Felix Neubergh Foundation, Promobilia, the Hjalmar Svensson the ﬁeld of biomaterials  and bone research , Foundation, and the Materials Science Area of Advance at Chalmers whereatomicresolutioncan be obtained in 3D. For fur- and the Department of Biomaterials, University of Gothenburg. ther reading on high-resolution techniques, the interested reader is referred elsewhere . Compliance with ethical standards Conﬂict of interest The authors declare that they have no conﬂict of interest. 5 Concluding remarks Open Access This article is distributed under the terms of the Creative Through the use of correlative and complementary imaging Commons Attribution 4.0 International License (http://crea and analytical techniques, osseointegration can be probed tivecommons.org/licenses/by/4.0/), which permits unrestricted use, and evaluated at relevant hierarchical scales. The bone- distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a implant interface shows an intimate contact between link to the Creative Commons license, and indicate if changes were mineralized collagen bundles and the outermost surface of made. the oxide layer, while within the nanoscale surface features only bone mineral is found, thus creating a functionally References graded interface. Osteocytes exhibit a direct contact with the implant surface via canaliculi that house their dendritic 1. Brånemark PI, Hansson BO, Adell R, Breine U, Lindström J, processes. Blood vessels are frequently found in close Hallen O, et al. Osseointegrated implants in the treatment of the edentulous jaw. Experience from a 10-year period. 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