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The possibility of decreasing the dissolution rate of SiN coatings using Fe and C is promising and the released ions were demonstrated to be compatible with microglia viability, in both 2D cultures and 3D collagen hydrogels.

The last decade has witnessed rapid advancements in manufacturing technologies for biomedical implants. Additive manufacturing (or 3D printing) has broken down major barriers in the way of producing complex 3D geometries. Electron beam melting (EBM) is one such 3D printing process applicable to metals and alloys. EBM offers build rates up to two orders of magnitude greater than comparable laser-based technologies and a high vacuum environment to prevent accumulation of trace elements. These features make EBM particularly advantageous for materials susceptible to spontaneous oxidation and nitrogen pick-up when exposed to air (e.g., titanium and titanium-based alloys). For skeletal reconstruction(s), anatomical mimickry and integrated macro-porous architecture to facilitate bone ingrowth are undoubtedly the key features of EBM manufactured implants. Using finite element modelling of physiological loading conditions, the design of a prosthesis may be further personalised. This review looks at the many unique clinical applications of EBM in skeletal repair and the ground-breaking innovations in prosthetic rehabilitation. From a simple acetabular cup to the fifth toe, from the hand-wrist complex to the shoulder, and from vertebral replacement to cranio-maxillofacial reconstruction, EBM has experienced it all. While sternocostal reconstructions might be rare, the repair of long bones using EBM manufactured implants is becoming exceedingly frequent. Despite the various merits, several challenges remain yet untackled. Nevertheless, with the capability to produce osseointegrating implants of any conceivable shape/size, and permissive of bone ingrowth and functional loading, EBM can pave the way for numerous fascinating and novel applications in skeletal repair, regeneration, and rehabilitation. STATEMENT OF SIGNIFICANCE: Electron beam melting (EBM) offers unparalleled possibilities in producing contaminant-free, complex and intricate geometries from alloys of biomedical interest, including Ti6Al4V and CoCr. We review the diverse range of clinical applications of EBM in skeletal repair, both as mass produced off-the-shelf implants and personalised, patient-specific prostheses. From replacing large volumes of disease-affected bone to complex, multi-material reconstructions, almost every part of the human skeleton has been replaced with an EBM manufactured analog to achieve macroscopic anatomical-mimickry. However, various questions regarding long-term performance of patient-specific implants remain unaddressed. Directions for further development include designing personalised implants and prostheses based on simulated loading conditions and accounting for trabecular bone microstructure with respect to physiological factors such as patient's age and disease status.

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with a diameter up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: i) low-contrast nanosized circular spaces (“holes”) observed in sections of bone oriented perpendicular to the long axis of a long bone; and ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most “holes” are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signature (Ca and P) within the gap zones, but the signal appears weaker than that associated to the extra-fibrillar mineral, pointing towards the existence of dissimilarities between the two types of mineralization.

Significance Large cranial reconstructions are increasingly performed worldwide and still represent a substantial clinical challenge. The gold standard, autologous bone, has limited availability and high donor-site morbidity. Current alloplastic materials are associated with high complication and failure rates. This study shows the capacity of a customized, purely synthetic, 3D-manufactured bioceramic implant to regenerate and restore large cranial defects with mature, well-vascularized bone, with a morphology, ultrastructure, and composition similar to those of native skull bone. This approach triggers the regenerative potential of host tissue by tailoring the implant composition and design. The regeneration of large defects using purely synthetic material without adjunct cell therapy or growth factors represents a major advancement for rehabilitating patients in need of large cranial reconstructions., The repair of large cranial defects with bone is a major clinical challenge that necessitates novel materials and engineering solutions. Three-dimensionally (3D) printed bioceramic (BioCer) implants consisting of additively manufactured titanium frames enveloped with CaP BioCer or titanium control implants with similar designs were implanted in the ovine skull and at s.c. sites and retrieved after 12 and 3 mo, respectively. Samples were collected for morphological, ultrastructural, and compositional analyses using histology, electron microscopy, and Raman spectroscopy. Here, we show that BioCer implants provide osteoinductive and microarchitectural cues that promote in situ bone regeneration at locations distant from existing host bone, whereas bone regeneration with inert titanium implants was confined to ingrowth from the defect boundaries. The BioCer implant promoted bone regeneration at nonosseous sites, and bone bonding to the implant was demonstrated at the ultrastructural level. BioCer transformed to carbonated apatite in vivo, and the regenerated bone displayed a molecular composition indistinguishable from that of native bone. Proof-of-principle that this approach may represent a shift from mere reconstruction to in situ regeneration was provided by a retrieved human specimen, showing that the BioCer was transformed into well-vascularized osteonal bone, with a morphology, ultrastructure, and composition similar to those of native human skull bone.

Bone-anchored attachment of amputation limb prostheses is increasingly becoming a clinically accepted alternative to conventional socket suspension. The direct transfer of loads demands that the percutaneous implant system and the residual bone withstand all forces and moments transferred from the prosthesis. This study presents load measurements recorded at the bone-anchored attachment in 20 individuals with unilateral transfemoral amputation performing the everyday ambulatory activities: level ground walking, stairs ascent/descent and slope ascent/descent. Mean peak values for the sample populations across activities ranged from 498–684 N for the resultant force, 26.5–39.8 Nm for the bending moment, and 3.1–5.5 Nm for the longitudinal moment. Significant differences with respect to level walking were found for the resultant force during stairs ascent, (higher, p = 0.002), and stairs descent, (lower, p = 0.005). Using a crutch reduced the peak resultant forces and the peak bending moments with averages ranging from 5.5–12.6 % and 13.2–15.6 %, respectively. Large inter-participant variations were observed and no single activity resulted in consistently higher loading of the bone-anchored attachment across the participants. Results from this study can guide future development of percutaneous osseointegrated implant systems for limb prostheses and their rehabilitation protocols.

The surface of bone tells a story – one that is worth a thousand words – of how it is built and how it is repaired. Chemical (i.e., composition) and physical (i.e., morphology) characteristics of the bone surface are analogous to a historical record of osteogenesis and provide key insights into bone quality. Analysis of bone chemistry is of particular relevance to the advancement of human health, cell biology, anthropology/archaeology, and biomedical engineering. Although scanning electron microscopy remains a popular and versatile technique to image bone across multiple length scales, limited chemical information can be obtained. Micro-Raman spectroscopy is a valuable tool for nondestructive chemical/compositional analysis of bone. However, signal integrity losses occur frequently during wide-field mapping of non-planar surfaces. Samples for conventional Raman imaging are, therefore, rendered planar through polishing or sectioning to ensure uniform signal quality. Here, we demonstrate ν1 PO43- and ν1 CO32- peak intensity losses where the sample surface and the plane of focus are offset by over 1–2 μm when underfocused and 2–3 μm when overfocused at 0.5–1 s integration time (15 mW, 633 nm laser). A technique is described for mapping the composition of the inherently irregular/non-planar surface of bone. The challenge posed by the native topology characteristic of this unique biological system is circumvented via real-time focus-tracking based on laser focus optimization by continuous closed-loop feedback. At the surface of deproteinized and decellularized/defatted sheep tibial cortical bone, regions of interest up to 1 mm2 were scanned at micrometer and submicrometer resolution. Despite surface height deviations exceeding 100 μm, it is possible to seamlessly probe local gradients in organic and inorganic constituents of the extracellular matrix as markers of bone metabolism and bone turnover, blood vessels and osteocyte lacunae, and the rope-like mineralized bundles that comprise the mineral phase at the bone surface.

Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (similar to 75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.

Much debate still revolves around bone architecture, especially at the nano- and microscale. Bone is a remarkable material where high strength and toughness coexist thanks to an optimized composition of mineral and protein and their hierarchical organization across several distinct length scales. At the nanoscale, mineralized collagen fibrils act as building block units. Despite their key role in biological and mechanical functions, the mechanisms of collagen mineralization and the precise arrangement of the organic and inorganic constituents in the fibrils remains not fully elucidated. Advances in three-dimensional (3D) characterization of mineralized bone tissue by focused ion beam-scanning electron microscopy (FIB-SEM) revealed mineral-rich regions geometrically approximated as prolate ellipsoids, much larger than single collagen fibrils. These structures have yet to become prominently recognized, studied, or adopted into biomechanical models of bone. However, they closely resemble the circular to elliptical features previously identified by scanning transmission electron microscopy (STEM) in two-dimensions (2D). Herein, we review the presence of mineral ellipsoids in bone as observed with electron-based imaging techniques in both 2D and 3D with particular focus on different species, anatomical locations, and in proximity to natural and synthetic biomaterial interfaces. This review reveals that mineral ellipsoids are a ubiquitous structure in all the bones and bone-implant interfaces analyzed. This largely overlooked hierarchical level is expected to bring different perspectives to our understanding of bone mineralization and mechanical properties, in turn shedding light on structure-function relationships in bone. STATEMENT OF SIGNIFICANCE: In bone, the hierarchical organization of organic (mainly collagen type I) and inorganic (calcium-phosphate mineral) components across several length scales contributes to a unique combination of strength and toughness. However, aspects related to the collagen-mineral organization and to mineralization mechanisms remain unclear. Here, we review the presence of mineral prolate ellipsoids across a variety of species, anatomical locations, and interfaces, both natural and with synthetic biomaterials. These mineral ellipsoids represent a largely unstudied feature in the organization of bone at the mesoscale, i.e., at a level connecting nano- and microscale. Thorough understanding of their origin, development, and structure can provide valuable insights into bone architecture and mineralization, assisting the treatment of bone diseases and the design of bio-inspired materials.

Within the dental alveolar socket, the sequence of events following tooth extraction involves deposition of a provisional connective tissue matrix that is later replaced by woven bone and eventually by lamellar bone. Bone regeneration within the dental alveolar socket is unique since the space occupied by the root(s) of a tooth does not originally contain any bone. However, extracellular matrix composition of the healing alveolar socket has not previously been investigated. Here, alveolar bone biopsies representing early (7-46 months,4y) and late (48-60 months; 4-5y) healing periods were investigated using Raman spectroscopy, X-ray micro-computed tomography and backscattered electron scanning electron microscopy. Partially or completely edentulous individuals and those with a smoking habit were not excluded. Between4y and 4-5y, mineral crystallinity and bone mineral density increase, phenylalanine, proline/hydroxyproline, and bone surface-to-volume ratio decrease, while the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, and the collagen crosslink ratio remain relatively unchanged. Observed exclusively at 4-5y, hypermineralised osteocyte lacunae contain spherical and rhomboidal mineral nodules. Spearman correlation analysis reveals several significant, high (ρ = 0.7-0.9; p ≤ 0.01) and moderate (ρ = 0.5-0.7; p ≤ 0.01) correlations. Mineral crystallinity and proline/hydroxyproline, the carbonate-to-phosphate ratio and phenylalanine, mineral crystallinity and bone surface-to-volume ratio, the carbonate-to-phosphate ratio and bone surface-to-volume ratio, proline/hydroxyproline and bone mineral density, and bone mineral density and bone surface-to-volume ratio are negatively correlated. Mineral crystallinity and bone mineral density, and proline/hydroxyproline and bone surface-to-volume ratio are positively correlated. Although bone regeneration in the dental alveolar socket follows typical bone healing patterns, the compositional and microstructural patterns reveal mature bone at4y with indications of better mechanical competence at 4-5y.

Accelerated bone grow (osseointegration) can be achieved by modifying the surface of medical implants. For this purpose, pulsed lasers can be used to successfully texture such beneficial surfaces on titanium, e.g. a BioHelix™ structure. This surface typically includes ridges and droplets with a size range between 1 and 20 μm. This paper presents the results of an experimental program where a range of laser parameters was used to create different surface textures on titanium substrates, using pulsed laser spattering. The resultant surfaces are analysed by scanning electron microscope and X-ray Micro Computer Tomography. It is shown that optimisation of the laser parameters results in a robust process which produces a surface that has proven to be beneficial for osseointegration. The results are also deeper analysed, explaining how different types of surface are created by the laser-material interaction under different conditions. Further, droplet flight distances and the formation of the spongeous nano-scale surface that characterizes the surface structure depends on very fast cooling and is also evaluated.

In healthy conditions, prepubertal growth follows an individual specific growth channel. Growth hormone (GH) is undoubtedly the major regulator of growth. However, the homeostatic regulation to maintain the individual specific growth channel during growth is unclear. We recently hypothesized a body weight sensing homeostatic regulation of body weight during adulthood, the gravitostat. We now investigated if sensing of body weight also contributes to the strict homeostatic regulation to maintain the individual specific growth channel during prepubertal growth. To evaluate the effect of increased artificial loading on prepubertal growth, we implanted heavy (20% of body weight) or light (2% of the body weight) capsules into the abdomen of 26-day-old male rats. The body growth, as determined by change in biological body weight and growth of the long bones and the axial skeleton, was reduced in rats bearing a heavy load compared with light load. Removal of the increased load resulted in a catch-up growth and a normalization of body weight. Loading decreased hypothalamic growth hormone releasing hormone mRNA, liver insulin-like growth factor (IGF)-1 mRNA, and serum IGF-1, suggesting that the reduced body growth was caused by a negative feedback regulation on the somatotropic axis and this notion was supported by the fact that increased loading did not reduce body growth in GH-treated rats. Based on these data, we propose the gravitostat hypothesis for the regulation of prepubertal growth. This states that there is a homeostatic regulation to maintain the individual specific growth channel via body weight sensing, regulating the somatotropic axis and explaining catch-up growth.

Osseointegration is controlled by a number of cellular mechanisms. Although factors governing bone formation are well-understood, the maintenance of bone at the bone-implant interface is less clear. Of some interest is the role of osteocytes, which via mechanotransduction are believed to be responsible for mechanical loading-based remodelling events in bone. Using a resin cast etching technique, we investigated the osteocyte lacuno-canalicular network adjacent to nanostructured titanium human dental implants after four years in clinical function. Correlative electron microscopy showed nanoscale osteocyte processes extending directly onto the implant surface. Calcium signal mapping via electron energy loss spectroscopy (EELS) showed apatite ingrowth into the nanotextured surface, while the apatite platelet c-axis was oriented approximately parallel to the collagen fibril direction. Furthermore, Z-contrast electron tomography demonstrated that natural bone-osteocyte and engineered bone-implant interfaces ar...

The initial cellular and molecular activities at the bone interface of implants with controlled nanoscale topography and microscale roughness have previously been reported. However, the effects of such surface modifications on the development of osseointegration have not yet been determined. This study investigated the molecular events and the histological and biomechanical development of the bone interface in implants with nanoscale topography, microscale roughness or a combination of both. Polished and machined titanium implants with and without controlled nanopatterning (75 nm protrusions) were produced using colloidal lithography and coated with a thin titanium layer to unify the chemistry. The implants were inserted in rat tibiae and subjected to removal torque (RTQ) measurements, molecular analyses and histological analyses after 6, 21 and 28 days. The results showed that nanotopography superimposed on microrough, machined, surfaces promoted an early increase in RTQ and hence produced greater implant stability at 6 and 21 days. Two-way MANOVA revealed that the increased RTQ was influenced by microscale roughness and the combination of nanoscale and microscale topographies. Furthermore, increased bone-implant contact (BIC) was observed with the combined nanopatterned machined surface, although MANOVA results implied that the increased BIC was mainly dependent on microscale roughness. At the molecular level, the nanotopography, per se, and in synergy with microscale roughness, downregulated the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In conclusion, controlled nanotopography superimposed on microrough machined implants promoted implant stability during osseointegration. Nanoscale-driven mechanisms may involve attenuation of the inflammatory response at the titanium implant site. Statement of Significance The role of combined implant microscale and nanotopography features for osseointegration is incompletely understood. Using colloidal lithography technique, we created an ordered nanotopography pattern superimposed on screwshaped implants with microscale topography. The midterm and late molecular, bone-implant contact and removal torque responses were analysed in vivo. Nanotopography superimposed on microrough, machined, surfaces promoted the implant stability, influenced by microscale topography and the combination of nanoscale and microscale topographies. Increased bone-implant contact was mainly dependent on microscale roughness whereas the nanotopography, per se, and in synergy with microscale roughness, attenuated the proinflammatory tumor necrosis factor alpha (TNF-alpha) expression. It is concluded that microscale and nanopatterns provide individual as well as synergistic effects on molecular, morphological and biomechanical implant-tissue processes in vivo. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-02891]; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy; Vastra Gotaland Region; Swedish government [ALFGBG725641]; Swedish county councils, the ALF agreement [ALFGBG725641]; TUA/Region Vastra Gotaland research grant; Stiftelsen Handlanden Hjalmar Svensson; IngaBritt and Arne Lundberg Foundation; Eivind o Elsa K: son Sylvan Foundation; Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative; Svenska Sallskapet for Medicinsk Forskning (SSMF)

Upon placement of an implant into living bone, an interface is formed through which various biochemical, biological, physical, and mechanical interactions take place. This interface evolves over time as the mechanical properties of peri-implant bone increase. Owing to the multifactorial nature of interfacial processes, it is challenging to devise a comprehensive model for predicting the mechanical behavior of the bone-implant interface. We propose a simple spatio-temporally evolving mechanical model – from an elementary unit cell comprising randomly oriented mineralized collagen fibrils having randomly assigned stiffness all the way up to a macroscopic bone-implant interface in a gap healing scenario. Each unit cell has an assigned Young's modulus value between 1.62 GPa and 25.73 GPa corresponding to minimum (i.e., 0) and maximum (i.e., 0.4) limits of mineral volume fraction, respectively, in the overlap region of the mineralized collagen fibril. Gap closure and subsequent stiffening are modeled to reflect the two main directions of peri-implant bone formation, i.e., contact osteogenesis and distance osteogenesis. The linear elastic stochastic finite element model reveals highly nonlinear temporal evolution of bone-implant interface stiffness, strongly dictated by the specific kinetics of contact osteogenesis and distance osteogenesis. The bone-implant interface possesses a small stiffness until gap closure, which subsequently evolves into a much higher stiffness, and this transition is reminiscent of a percolation transition whose threshold corresponds to gap closure. The model presented here, albeit preliminary, can be incorporated into future calculations of the bone-implant system where the interface is well-defined mechanically. Statement of significance A simple, physically informed model for the mechanical characteristics of the bone-implant interface is still missing. Here, we start by extending the reported mechanical characteristics of a one cubic micrometre unit cell to a 250 µm long interface made of 1 µm thick layers. The stiffness of each cell (based on mineral content) is assigned randomly to mimic bone micro-heterogeneity. The numerical study of this interface representative structure allows for the simultaneous determination of the spatio-temporal evolution of the mechanical response at local (discrete element) and global (overall model) scales. The proposed model is the first of this kind that can easily be incorporated into realistic future models of bone-implant interaction with emphasis on implant stability and different loading conditions.

Augmentation materials, such as ceramic and polymeric bone cements, have been frequently used to improve the physical engagement of screws inserted into bone. While ceramic, degradable cements may ultimately improve fixation stability, reports regarding their effect on early fixation stability have been inconsistent. On the other hand, a newly developed degradable ceramic adhesive that can bond with tissues surrounding the screw, may improve the pullout performance, ensure early stability, and subsequent bony integration. The aim of this study was to investigate failure mechanisms of screw/trabecular bone constructs by comparing non-augmented screws with screws augmented with a calcium phosphate cement or an adhesive, i.e. a phosphoserine-modified calcium phosphate. Pullout tests were performed on screws inserted into trabecular cylinders extracted from human femoral bone. Continuous and stepwise pullout loading was applied with and without real-time imaging in a synchrotron radiation micro-computed tomograph, respectively. Statistical analysis that took the bone morphology into account confirmed that augmentation with the adhesive supported significantly higher pullout loads compared to cement-augmented, or non-augmented screws. However, the adhesive also allowed for a higher injection volume compared to the cement. In-situ imaging showed cracks in the vicinity of the screw threads in all groups, and detachment of the augmentation materials from the trabecular bone in the augmented specimens. Additional cracks at the periphery of the augmentation and the bone-material interfaces were only observed in the adhesive-augmented specimen, indicating a contribution of surface bonding to the pullout resistance. An adhesive that has potential for bonding with tissues, displayed superior pullout resistance, compared to a brushite cement, and may be a promising material for cementation or augmentation of implants., Journal of the Mechanical Behavior of Biomedical Materials, 110, ISSN:1751-6161, ISSN:1878-0180

Continual bone apposition at the cranial sutures provides the unique opportunity to understand how bone is built. Bone harvested from 16-week-old Sprague Dawley rat calvaria was either (i) deproteinised to isolate the inorganic phase (i.e., bone mineral) for secondary electron scanning electron microscopy or (ii) resin embedded for X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and micro-Raman spectroscopy. Interdigitated finger-like projections form the interface between frontal and parietal bones. Viewed from the surface, bone mineral at the mineralisation front is comprised of nanoscale mineral platelets arranged into discrete, ~0.6–3.5 μm high and ~0.2–1.5 μm wide, marquise-shaped motifs that gradually evolve into a continuous interwoven mesh of mineralised bundles. Marquise-shaped motifs also contribute to the burial of osteoblastic–osteocytes by contributing to the roof over the lacunae. In cross-section, apices of the finger-like projections resemble islands of mineralised tissue, where new bone apposition at the surface is evident as low mineral density areas, while the marquise-shaped motifs appear as near-equiaxed assemblies of mineral platelets. Carbonated apatite content is higher towards the internal surface of the cranial vault. Up to 4 μm from the bone surface, strong Amide III, Pro, Hyp, and Phe signals, distinct PO43− bands, but negligible CO32– signal indicate recent bone formation and/or delayed maturation of the mineral. We show, for the first time, that the extracellular matrix of bone is assembled into micrometre-sized units, revealing a superstructure above the mineralised collagen fibril level, which has significant implications for function and mechanical competence of bone., Graphical abstract Unlabelled Image, Highlights • The mineralisation front at cranial sutures of 16-week-old rats was investigated • Interdigitated finger-like projections extend between frontal and parietal bones • Micrometre-sized, marquise-shaped motifs of bone apatite at the mineralisation front • Distinct motifs evolve into interwoven mesh of mineralised bundles • Cranial bones are more mineralised at the internal surface (towards the dura mater)

This chapter provides an overview of the cellular interactions and the genetic regulations at the bone-implant interface, based on experimental in vivo studies and the available studies in humans. The first part discusses the current knowledge on the cellular and molecular events governing the initial cell recruitment, early inflammation, and the transition from inflammation to bone formation and remodeling during the phases of osseointegration. The modulation of these events, by different implant surfaces, and their relationship with the structural and functional development of the interface, are emphasized. A subsequent section is focused on selected key biological factors, potentially involved in the osteogenic differentiation of mesenchymal stem cells or in coupling of bone formation and remodeling at the interface. Further, possible phenotypic polarizations of macrophages at the interface, in vivo, is discussed. Finally, some insights on possible dysregulations of the molecular activities at the interface, under selected bone-compromising conditions, are provided.

This chapter discusses recent applications and findings in additive manufacturing (AM), or 3D printing, applied in oral and maxillofacial surgery. The reader will get an introduction to the basics of AM technology followed by oral and maxillofacial applications like printing of anatomical models and the design and manufacturing of customised implants. Recent research on the biological response of some AM metal alloys is also discussed at the end of the chapter.

Osteoporosis is a major cause of age-related fractures. Healing complications in osteoporotic patients are often associated with increased mortality and morbidity. Stimulation of the implant-adjacent bone could be beneficial in terms of the surgical outcome. Over the past decade, numerous investigations have implicated insulin in normal bone growth, and recent studies have described the advantages of administering insulin locally to increase bone formation. Therefore, we hypothesized that insulin-coated titanium implants would increase bone formation in osteoporotic animals. The aim of this study was to evaluate the effects of insulin delivered from an implant surface on bone-related gene expression and bone formation in osteoporotic rats. Characterizations of the surfaces of insulin-coated and control implants were performed using ellipsometry and interferometry. Forty ovariectomized and four healthy Sprague Dawley rats were used and implants were inserted in the tibias. The systemic effect of insulin was assessed by measuring the blood glucose levels and total body weight. The animals were sacrificed either 1 day or 3 weeks postimplantation. Implant-adherent cells were analyzed by quantitative real-time PCR, and the bone adjacent to the implants was examined by microcomputed tomography and histomorphometry. The insulin-coated implants had no systemic effects. The insulin-coated samples demonstrated significantly lower expression of the gene for interleukin 1β (p = 0.019) at 1 day, and significantly exhibited more periosteal callus (p = 0.029) at 3 weeks. Locally delivered insulin has potential for promoting bone formation and it exerts potentially anti-inflammatory effects in osteoporotic rats. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2472-2480, 2018.

Electron beam melting (EBM) is a three-dimensional (3D) printing technique for the production of metal structures where complex geometries with interconnected porosities can be built. Incorporation of as little as 0.04% Zr into the CoCr alloy can significantly improve the biomechanical anchorage of constructs fabricated by EBM. Here we investigate bone material properties, including microstructure and composition, adjacent to 3D printed CoCr implants with and without addition of 0.04% Zr, after 8 weeks of healing in the rabbit femur. In low amounts, zirconium addition does not alter the microstructure and extracellular matrix composition of bone formed adjacent to the surface of EBM manufactured implants. Bone ingrowth into surface irregularities of 3D printed CoCr and CoCr + Zr implants is seen. Extensive remodeling is also evident. Osteocytes attach directly on to the implant surface. The interfacial tissue at CoCr and CoCr + Zr has similar mineral crystallinity, apatite-to-collagen ratio, carbonate-to-phosphate ratio, Ca/P ratio, bone-implant contact, percentage porosity, and osteocyte density (N.Ot/B.Ar). Compared to the native bone, the mineral crystallinity of the interfacial tissue was lower while N.Ot/B.Ar was higher for both CoCr and CoCr + Zr. Overall, the results indicate that bone tissue adjacent to CoCr and CoCr + Zr implants is highly mature and exhibits comparable healing kinetics. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1655-1663, 2018.

Cortical bone plays a vital role in determining overall bone strength. We investigate the structural, compositional, and nanomechanical properties of cortical bone following ovariectomy (OVX) of 12 ...

Autogenous bone fragments generated during surgery (e.g. implant site preparation) accelerate bone formation by the release of a large variety of growth factors from the extracellular matrix and the cells contained within. Osteocytes, whether viable or apoptotic, within such fragments are able to recruit osteoclasts to a site of bone remodelling. Here, using correlative scanning electron microscopy, we provide compelling evidence that at one week healing in the Sprague Dawley rat tibia, following surgery (and/or the placement of a bone-anchored implant), autogenous bone fragments support bone formation on their surface. Furthermore, osteocytes within the autogenous fragments are frequently able to repair the disrupted canalicular networks and appear to connect with osteocytes (or osteoblastic-osteocytes) in the de novo formed bone on the surface of the fragment.

This paper investigates the application of X-ray micro-computed tomography (micro-CT) to accurately evaluate bone formation within 3D printed, porous Ti6Al4V implants manufactured using Electron Beam Melting (EBM), retrieved after six months of healing in sheep femur and tibia. All samples were scanned twice (i.e., before and after resin embedding), using fast, low-resolution scans (Skyscan 1172; Bruker micro-CT, Kontich, Belgium), and were analysed by 2D and 3D morphometry. The main questions posed were: (i) Can low resolution, fast scans provide morphometric data of bone formed inside (and around) metal implants with a complex, open-pore architecture?, (ii) Can micro-CT be used to accurately quantify both the bone area (BA) and bone-implant contact (BIC)?, (iii) What degree of error is introduced in the quantitative data by varying the threshold values?, and (iv) Does resin embedding influence the accuracy of the analysis? To validate the accuracy of micro-CT measurements, each data set was correlated with a corresponding centrally cut histological section. The results show that quantitative histomorphometry corresponds strongly with 3D measurements made by micro-CT, where a high correlation exists between the two techniques for bone area/volume measurements around and inside the porous network. On the contrary, the direct bone-implant contact is challenging to estimate accurately or reproducibly. Large errors may be introduced in micro-CT measurements when segmentation is performed without calibrating the data set against a corresponding histological section. Generally, the bone area measurement is strongly influenced by the lower threshold limit, while the upper threshold limit has little or no effect. Resin embedding does not compromise the accuracy of micro-CT measurements, although there is a change in the contrast distributions and optimisation of the threshold ranges is required.

Mesenchymal stem cells (MSCs) have important roles during osseointegration. This study determined (i) if MSC-derived extracellular vesicles (EVs)/exosomes can be immobilized on titanium (Ti) surfaces and influence the behavior of MSCs, (ii) if the response is differentially affected by EVs from expanded vs differentiated MSCs and (iii) if the EV protein cargos predict the functional features of the exosomes. EVs secreted by human adipose-derived MSCs were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis, Western blotting and relative quantitative mass spectrometry. Fluorescence microscopy, scanning electron microscopy, cell counting assay and quantitative polymerase chain reaction were used to analyze MSC adhesion, proliferation and differentiation. Exosome immobilization on Ti promoted MSC adhesion and spreading after 24 h and proliferation after 3 and 6 days, irrespective of whether the exosomes were obtained from expansion or differentiation conditions. Immobilized exosomes upregulated stromal cell-derived factor (SDF-1α) gene expression. Cell adhesion molecules and signaling molecules were abundant in the exosomal proteome. The predicted functions of the equally-abundant proteins in both exosome types were in line with the observed biological effects mediated by the exosomes. Thus, exosomes derived from MSCs and immobilized on Ti surfaces interact with MSCs and rapidly promote MSC adhesion and proliferation. These findings provide a novel route for modification of titanium implant surfaces.

Smoking is a major risk factor for dental implant failure. In addition to higher marginal bone loss around implants, the cellular and molecular responses to injury and implant physicochemical properties are also differentially affected in smokers. The purpose of this work is to determine if smoking impairs bone microstructure and extracellular matrix composition within the dental alveolar socket after tooth extraction. Alveolar bone biopsies obtained from Smokers (> 10 cigarettes per day for at least 10 years) and Ctrl (never-smokers), 7–146 months after tooth extraction, were investigated using X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and Raman spectroscopy. Both Smokers and Ctrl exhibited high inter- and intra-individual heterogeneity in bone microstructure, which varied between dense cortical and porous trabecular architecture. Regions of disorganised/woven bone were more prevalent during early healing. Remodelled lamellar bone was predominant at longer healing periods. Bone mineral density, bone surface-to-volume ratio, mineral crystallinity, the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, the collagen crosslink ratio, and the amounts of amino acids phenylalanine and proline/hydroxyproline were also comparable between Smokers and Ctrl. Bone microstructure and composition within the healing dental alveolar socket are not significantly affected by moderate-to-heavy smoking.

Bone is an architecturally complex system that constantly undergoes structural and functional optimisation through renewal and repair. The scanning electron microscope (SEM) is among the most frequently used instruments for examining bone. It offers the key advantage of very high spatial resolution coupled with a large depth of field and wide field of view. Interactions between incident electrons and atoms on the sample surface generate backscattered electrons, secondary electrons, and various other signals including X-rays that relay compositional and topographical information. Through selective removal or preservation of specific tissue components (organic, inorganic, cellular, vascular), their individual contribution(s) to the overall functional competence can be elucidated. With few restrictions on sample geometry and a variety of applicable sample-processing routes, a given sample may be conveniently adapted for multiple analytical methods. While a conventional SEM operates at high vacuum conditions that demand clean, dry, and electrically conductive samples, non-conductive materials (e.g., bone) can be imaged without significant modification from the natural state using an environmental scanning electron microscope. This review highlights important insights gained into bone microstructure and pathophysiology, bone response to implanted biomaterials, elemental analysis, SEM in paleoarchaeology, 3D imaging using focused ion beam techniques, correlative microscopy and in situ experiments. The capacity to image seamlessly across multiple length scales within the meso-micro-nano-continuum, the SEM lends itself to many unique and diverse applications, which attest to the versatility and user-friendly nature of this instrument for studying bone. Significant technological developments are anticipated for analysing bone using the SEM.

Biomaterials are frequently used in various clinical applications, improving function, mobility, and the overall quality of life for patients. The clinical performance of bone-anchored implants is typically assessed by X-ray-based analytical technologies and various methods to evaluate the stability of the implant–bone unit. For eventual clinical translation of preclinical knowledge of tissue response to various biomaterials, it is essential to evaluate implants retrieved with associated biological tissues from humans. Information pertaining to causes of failure mechanisms and factors contributing to successful tissue integration can thus be obtained. This chapter highlights the process of implant retrieval analysis, with a focus on (1) reasons that necessitate implant retrieval, (2) recommended sample handling and preparation procedures, and (3) state-of-the-art ex vivo assessment. Furthermore, typical analyses of three different clinical applications are exemplified where microstructural and ultrastructural analysis and microbiological evaluation are performed on the same bone–implant specimen. While valuable knowledge could be gained, often only a limited amount of retrieved material is available. Therefore a carefully planned sample preparation and analytical approach is needed.

Three-dimensional (3D) visualization of bone-implant interfaces via electron tomography (ET) has contributed to the novel perspective of nano-osseointegration and offers evidential support for nanoscaled biomaterial surface modification. Conventional single-axis ET provides a relatively large field of view of the human bone to titanium implant interface showing bone structure arrangement near the interface. However, the "missing wedge" associated with conventional single-axis ET leads to artifacts and elongation in the reconstruction, limiting the resolution and fidelity of reconstructions, as well as the ability to extract quantitative information from nanostructured interfaces. On-axis ET, performed by 180° rotation of a needle-shaped sample, is a promising method to solve this problem. In this work, we present the first application of on-axis ET for investigation of human bone and laser-modified titanium implant interfaces without the missing wedge. This work demonstrates a near artifact-free 3D visualization of the nanotopographies of the implant surface oxide layer and bone growth into these features. Complementary electron energy-loss spectroscopy (EELS) mapping was used to illustrate the gradual intermixing of carbon and calcium (characteristic elements of bone) with the nanoscaled oxide layer of the implant surface. Ultimately, this approach serves as direct evidence of nano-osseointegration and as a potential platform to evaluate differently structured implant surfaces.

Osseointegrated transfemoral amputation prostheses have proven successful as an alternative method to the conventional socket-type prostheses. The method improves prosthetic use and thus increases the demands imposed on the bone-implant system. The hypothesis of the present study was that the loads applied to the bone-anchored implant system of amputees would result in locations of high stress and strain transfer to the bone tissue and thus contribute to complications such as unfavourable bone remodeling and/or elevated inflammatory response and/or compromised sealing function at the tissue-abutment interface. In the study, site-specific loading measurements were made on amputees and used as input data in finite element analyses to predict the stress and strain distribution in the bone tissue. Furthermore, a tissue sample retrieved from a patient undergoing implant revision was characterized in order to evaluate the long-term tissue response around the abutment. Within the limit of the evaluated bone properties in the present experiments, it is concluded that the loads applied to the implant system may compromise the sealing function between the bone and the abutment, contributing to resorption of the bone in direct contact with the abutment at the most distal end. This was supported by observations in the retrieved clinical sample of bone resorption and the formation of a soft tissue lining along the abutment interface. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1113-1122, 2017.

Triggering of the early healing events, including the recruitment of progenitor cells, has been suggested to promote bone regeneration. In implantology, local drug release technologies could provide an attractive approach to promote tissue regeneration. In this study, we targeted the chemotactic SDF-1α/CXCR4 axis that is responsible e.g. for the homing of stem cells to trauma sites. This was achieved by local delivery of plerixafor, an antagonist to CXCR4, and/or SDF-1α, from titanium implants coated with mesoporous titania thin films with a pore size of 7.5 nm. In vitro drug delivery experiments demonstrated that the mesoporous coating provided a high drug loading capacity and controlled release. The subsequent in vivo study in rat tibia showed beneficial effects with respect to bone-implant anchorage and bone-formation along the surface of the implants when plerixafor and SDF-1α were delivered locally. The effect was most prominent by the finding that the combination of the drugs significantly improved the mechanical bone anchorage. These observations suggest that titanium implants with local delivery of drugs for enhanced local recruitment of progenitor cells have the ability to promote osseointegration. This approach may provide a potential strategy for the development of novel implant treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2466-2475, 2016.

In orthopaedic surgery, cobalt chromium (CoCr) based alloys are used extensively for their high strength and wear properties, but with concerns over stress shielding and bone resorption due to the high stiffness of CoCr. The structural stiffness, principally related to the bulk and the elastic modulus of the material, may be lowered by appropriate design modifications, to reduce the stiffness mismatch between metal/alloy implants and the adjacent bone. Here, 3D printed CoCr and Ti6Al4V implants of similar macro-geometry and interconnected open-pore architecture prepared by electron beam melting (EBM) were evaluated following 26week implantation in adult sheep femora. Despite higher total bone-implant contact for Ti6Al4V (39±4%) than CoCr (27±4%), bone formation patterns were similar, e.g., densification around the implant, and gradual ingrowth into the porous network, with more bone in the outer half (periphery) than the inner half (centre). Raman spectroscopy revealed no major differences in mineral crystallinity, the apatite-to-collagen ratio, or the carbonate-to-phosphate ratio. Energy dispersive X-ray spectroscopy showed similar Ca/P ratio of the interfacial tissue adjacent to both materials. Osteocytes made direct contact with CoCr and Ti6Al4V. While osteocyte density and distribution in the new-formed bone were largely similar for the two alloys, higher osteocyte density was observed at the periphery of the porous network for CoCr, attributable to slower remodelling and a different biomechanical environment. The results demonstrate the possibility to achieve bone ingrowth into open-pore CoCr constructs, and attest to the potential for fabricating customised osseointegrated CoCr implants for load-bearing applications.Although cobalt chromium (CoCr) based alloys are used extensively in orthopaedic surgery, stress shielding due to the high stiffness of CoCr is of concern. To reduce the stiffness mismatch between CoCr and bone, CoCr and Ti6Al4V implants having an interconnected open-pore architecture were prepared by electron beam melting (EBM). After six months of submerged healing in sheep, both alloys showed similar patterns of bone formation, with densification around the implant and gradual ingrowth into the porous network. The molecular and elemental composition of the interfacial tissue was similar for both alloys. Osteocytes made direct contact with both alloys, with similar overall osteocyte density and distribution. The work attests to the potential for achieving osseointegration of EBM manufactured porous CoCr implants.