Systemically administered zoledronic acid activates locally implanted synthetic hydroxyapatite particles enhancing peri-implant bone formation: A regenerative medicine approach to improve fracture fixation.

Fracture fixation in an ageing population is challenging and fixation failure increases mortality and societal costs. We report a novel fracture fixation treatment by applying a hydroxyapatite (HA) based biomaterial at the bone-implant interface and biologically activating the biomaterial by systemic administration of a bisphosphonate (zoledronic acid, ZA). We first used an animal model of implant integration and applied a calcium sulphate (CaS)/HA biomaterial around a metallic screw in the tibia of osteoporotic rats. Using systemic ZA administration at 2-weeks post-surgery, we demonstrated that the implant surrounded by HA particles showed significantly higher peri­implant bone formation compared to the unaugmented implants at 6-weeks. We then evaluated the optimal timing (day 1, 3, 7 and 14) of ZA administration to achieve a robust effect on peri­implant bone formation. Using fluorescent ZA, we demonstrated that the uptake of ZA in the CaS/HA material was the highest at 3- and 7-days post-implantation and the uptake kinetics had a profound effect on the eventual peri­implant bone formation. We furthered our concept in a feasibility study on trochanteric fracture patients randomized to either CaS/HA augmentation or no augmentation followed by systemic ZA treatment. Radiographically, the CaS/HA group showed signs of increased peri­implant bone formation compared with the controls. Finally, apart from HA, we demonstrated that the concept of biologically activating a ceramic material by ZA could also be applied to ß-tricalcium phosphate. This novel approach for fracture treatment that enhances immediate and long-term fracture fixation in osteoporotic bone could potentially reduce reoperations, morbidity and mortality. STATEMENT OF SIGNIFICANCE: • Fracture fixation in an ageing population is challenging. Biomaterial-based augmentation of fracture fixation devices has been attempted but lack of satisfactory biological response limits their widespread use. • We report the biological activation of locally implanted microparticulate hydroxyapatite (HA) particles placed around an implant by systemic administration of the bisphosphonate zoledronic acid (ZA). The biological activation of HA by ZA enhances peri­implant bone formation. •Timing of ZA administration after HA implantation is critical for optimal ZA uptake and consequently determines the extent of peri­implant bone formation. • We translate the developed concept from small animal models of implant integration to a proof-of-concept clinical study on osteoporotic trochanteric fracture patients. • ZA based biological activation can also be applied to other calcium phosphate biomaterials.

Exploratory neutron tomography of articular cartilage.

OBJECTIVE: To investigate the feasibility of using neutron tomography to gain new knowledge of human articular cartilage degeneration in osteoarthritis (OA). Different sample preparation techniques were evaluated to identify maximum intra-tissue contrast. DESIGN: Human articular cartilage samples from 14 deceased donors (18-75 years, 9 males, 5 females) and 4 patients undergoing total knee replacement due to known OA (all female, 61-75 years) were prepared using different techniques: control in saline, treated with heavy water saline, fixed and treated in heavy water saline, and fixed and dehydrated with ethanol. Neutron tomographic imaging (isotropic voxel sizes from 7.5 to 13.5 µm) was performed at two large scale facilities. The 3D images were evaluated for gradients in hydrogen attenuation as well as compared to images from absorption X-ray tomography, magnetic resonance imaging, and histology. RESULTS: Cartilage was distinguishable from background and other tissues in neutron tomographs. Intra-tissue contrast was highest in heavy water-treated samples, which showed a clear gradient from the cartilage surface to the bone interface. Increased neutron flux or exposure time improved image quality but did not affect the ability to detect gradients. Samples from older donors showed high variation in gradient profile, especially from donors with known OA. CONCLUSIONS: Neutron tomography is a viable technique for specialized studies of cartilage, particularly for quantifying properties relating to the hydrogen density of the tissue matrix or water movement in the tissue.

Nanoscale characterization of collagen structural responses to in situ loading in rat Achilles tendons.

The specific viscoelastic mechanical properties of Achilles tendons are highly dependent on the structural characteristics of collagen at and between all hierarchical levels. Research has been conducted on the deformation mechanisms of positional tendons and single fibrils, but knowledge about the coupling between the whole tendon and nanoscale deformation mechanisms of more commonly injured energy-storing tendons, such as Achilles tendons, remains sparse. By exploiting the highly periodic arrangement of tendons at the nanoscale, in situ loading of rat Achilles tendons during small-angle X-ray scattering acquisition was used to investigate the collagen structural response during load to rupture, cyclic loading and stress relaxation. The fibril strain was substantially lower than the applied tissue strain. The fibrils strained linearly in the elastic region of the tissue, but also exhibited viscoelastic properties, such as an increased stretchability and recovery during cyclic loading and fibril strain relaxation during tissue stress relaxation. We demonstrate that the changes in the width of the collagen reflections could be attributed to strain heterogeneity and not changes in size of the coherently diffracting domains. Fibril strain heterogeneity increased with applied loads and after the toe region, fibrils also became increasingly disordered. Additionally, a thorough evaluation of radiation damage was performed. In conclusion, this study clearly displays the simultaneous structural response and adaption of the collagen fibrils to the applied tissue loads and provide novel information about the transition of loads between length scales in the Achilles tendon.

The Hydration State of Bone Tissue Affects Contrast in Neutron Tomographic Images.

Neutron tomography has emerged as a promising imaging technique for specific applications in bone research. Neutrons have a strong interaction with hydrogen, which is abundant in biological tissues, and they can penetrate through dense materials such as metallic implants. However, in addition to long imaging times, two factors have led to challenges in running in situ mechanical characterization experiments on bone tissue using neutron tomography: 1) the high water content in specimens reduces the visibility of internal trabecular structures; 2) the mechanical properties of bone are dependent on the hydration state of the tissue, with drying being reported to cause increased stiffness and brittleness. This study investigates the possibility of improving image quality in terms of neutron transmission and contrast between material phases by drying and rehydrating in heavy water. Rat tibiae and trabecular bovine bone plugs were imaged with neutron tomography at different hydration states and mechanical testing of the bone plugs was carried out to assess effects of drying and rehydration on the mechanical properties of bone. From analysis of image histograms, it was found that drying reduced the contrast between bone and soft tissue, but the contrast was restored with rehydration. Contrast-to-noise ratios and line profiles revealed that the contrast between bone tissue and background was reduced with increasing rehydration duration but remained sufficient for identifying internal structures as long as no free liquid was present inside the specimen. The mechanical analysis indicated that the proposed fluid exchange protocol had no adverse effects on the mechanical properties.

Fracture behavior of a composite of bone and calcium sulfate/hydroxyapatite.

Calcium sulfate/hydroxyapatite (CaS/HA) biomaterials have been investigated for use in several orthopedic applications. However, the mechanical interactions between the composite of CaS/HA and bone at the microscale are still unknown. The aim of this study was to determine if and how augmentation with CaS/HA alters the fracture behavior of bone. Eleven cylinders of trabecular bone were drilled from human femoral heads and cleaned from bone marrow. Among them, five cylinders were injected with CaS/HA to generate composite specimens, while the others were kept intact. One extra specimen of pure CaS/HA was prepared. All specimens were compressed in situ using synchrotron X-ray tomography and imaged at ∼2% strain intervals. Structural properties were calculated from the images in unloaded state and mechanical properties were determined from the load-curves. CaS/HA alone displayed the highest peak force and stiffness and the lowest strain at fracture. All composite specimens had a higher peak force than the pure bone specimens and the composite specimens had higher toughness than the pure CaS/HA specimen. Furthermore, the fracture behavior was analyzed further to characterize the local deformations. The pure bone specimens presented damage in multiple trabeculae and the CaS/HA specimen displayed sharp transition in strains, with low strain in one load step and large cracks in the next. The composite specimens deformed uniformly, with the CaS/HA preventing tissue damage and the bone preventing cracks in the CaS/HA from propagating through the specimen. In conclusion, using tomography with in situ loading, it was possible to show how CaS/HA can help prevent bone tissue damage before global failure.

A quality optimization approach to image Achilles tendon microstructure by phase-contrast enhanced synchrotron micro-tomography.

Achilles tendons are mechanosensitive, and their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles. To fully understand how their microstructure responds to mechanical loading a non-invasive approach for 3D high resolution imaging suitable for soft tissue is required. Here we propose a protocol that can capture the complex 3D organization of the Achilles tendon microstructure, using phase-contrast enhanced synchrotron micro-tomography (SR-PhC-µCT). We investigate the effects that sample preparation and imaging conditions have on the resulting image quality, by considering four types of sample preparations and two imaging setups (sub-micrometric and micrometric final pixel sizes). The image quality is assessed using four quantitative parameters. The results show that for studying tendon collagen fibers, conventional invasive sample preparations such as fixation and embedding are not necessary or advantageous. Instead, fresh frozen samples result in high-quality images that capture the complex 3D organization of tendon fibers in conditions as close as possible to natural. The comprehensive nature of this innovative study by SR-PhC-µCT breaks ground for future studies of soft complex biological tissue in 3D with high resolution in close to natural conditions, which could be further used for in situ characterization of how soft tissue responds to mechanical stimuli on a microscopic level.

Dual modality neutron and x-ray tomography for enhanced image analysis of the bone-metal interface.

The bone tissue formed at the contact interface with metallic implants, particularly its 3D microstructure, plays a pivotal role for the structural integrity of implant fixation. X-ray tomography is the classical imaging technique used for accessing microstructural information from bone tissue. However, neutron tomography has shown promise for visualising the immediate bone-metal implant interface, something which is highly challenging with x-rays due to large differences in attenuation between metal and biological tissue causing image artefacts. To highlight and explore the complementary nature of neutron and x-ray tomography, proximal rat tibiae with titanium-based implants were imaged with both modalities. The two techniques were compared in terms of visualisation of different material phases and by comparing the properties of the individual images, such as the contrast-to-noise ratio. After superimposing the images using a dedicated image registration algorithm, the complementarity was further investigated via analysis of the dual modality histogram, joining the neutron and x-ray data. From these joint histograms, peaks with well-defined grey value intervals corresponding to the different material phases observed in the specimens were identified and compared. The results highlight differences in how neutrons and x-rays interact with biological tissues and metallic implants, as well as the benefits of combining both modalities. Future refinement of the joint histogram analysis could improve the segmentation of structures and tissues, and yield novel information about specimen-specific properties such as moisture content.

Neutron microtomography to investigate the bone-implant interface-comparison with histological analysis.

Bone properties and especially its microstructure around implants are crucial to evaluate the osseointegration of prostheses in orthopaedic, maxillofacial and dental surgeries. Given the intrinsic heterogeneous nature of the bone microstructure, an ideal probing tool to understand and quantify bone formation must be spatially resolved. X-ray imaging has often been employed, but is limited in the presence of metallic implants, where severe artifacts generally arise from the high attenuation of metals to x-rays. Neutron tomography has recently been proposed as a promising technique to study bone-implant interfaces, thanks to its lower interaction with metals. The aim of this study is to assess the potential of neutron tomography for the characterisation of bone tissue in the vicinity of a metallic implant. A standardised implant with a bone chamber was implanted in rabbit bone. Four specimens were imaged with neutron tomography and subsequently compared to non-decalcified histology to stain soft and mineralised bone tissues, used here as a ground-truth reference. An intensity-based image registration procedure was performed to place the 12 histological slices within the corresponding 3D neutron volume. Significant correlations (p < 0.01) were obtained between the two modalities for the bone-implant contact (BIC) ratio (R = 0.77) and the bone content inside the chamber (R = 0.89). The results indicate that mineralised bone tissue can be reliably detected by neutron tomography. However, theBICratio and bone content were found to be overestimated with neutron imaging, which may be explained by its sensitivity to non-mineralised soft tissues, as revealed by histological staining. This study highlights the suitability of neutron tomography for the analysis of the bone-implant interface. Future work will focus on further distinguishing soft tissues from bone tissue, which could be aided by the adoption of contrast agents.

Spatio-temporal evolution of hydroxyapatite crystal thickness at the bone-implant interface.

A better understanding of bone nanostructure around the bone-implant interface is essential to improve longevity of clinical implants and decrease failure risks. This study investigates the spatio-temporal evolution of mineral crystal thickness and plate orientation in newly formed bone around the surface of a metallic implant. Standardized coin-shaped titanium implants designed with a bone chamber were inserted into rabbit tibiae for 7 and 13 weeks. Scanning measurements with micro-focused small-angle X-ray scattering (SAXS) were carried out on newly formed bone close to the implant and in control mature cortical bone. Mineral crystals were thinner close to the implant (1.8 ± 0.45 nm at 7 weeks and 2.4 ± 0.57 nm at 13 weeks) than in the control mature bone tissue (2.5 ± 0.21 nm at 7 weeks and 2.8 ± 0.35 nm at 13 weeks), with increasing thickness over healing time (+30 % in 6 weeks). These results are explained by younger bone close to the implant, which matures during osseointegration. Thinner mineral crystals parallel to the implant surface within the first 100 µm indicate that the implant affects the ultrastructure of neighbouring bone , potentially due to heterogeneous interfacial stresses, and suggest a longer maturation process of bone tissue and difficulty in binding to the metal. The bone growth kinetics within the bone chamber was derived from the spatio-temporal evolution of bone tissue's nanostructure, coupled with microtomographic imaging. The findings indicate that understanding mineral crystal thickness or plate orientation can improve our knowledge of osseointegration.

Comparison of small-angle neutron and X-ray scattering for studying cortical bone nanostructure.

In this study, we present a combined small-angle neutron and X-ray scattering (SANS and SAXS) study of the nanoscale structure of cortical bone specimens from three different species. The variation of the scattering cross section of elements across the periodic table is very different for neutrons and X-rays. For X-rays, it is proportional to the electron density while for neutrons it varies irregularly with the atomic number. Hence, combining the two techniques on the same specimens allows for a more detailed interpretation of the scattering patterns as compared to a single-contrast experiment. The current study was performed on bovine, porcine and ovine specimens, obtained in two perpendicular directions with respect to the main axis of the bone (longitudinal and radial) in order to maximise the understanding of the nanostructural organisation. The specimens were also imaged with high resolution micro-computed tomography (micro-CT), yielding tissue mineral density and microstructural orientation as reference. We show that the SANS and SAXS patterns from the same specimen are effectively identical, suggesting that these bone specimens can be approximated as a two-component composite material. Hence, the observed small-angle scattering results mainly from the mineral-collagen contrast, apart from minor features associated with the internal collagen structure.

Mineralization of cortical bone during maturation and growth in rabbits.

INTRODUCTION: The composite nature of bone as a material governs its structure and mechanical behavior. How the collagenous matrix mineralizes, in terms of both mineral deposition and structure of the mineral crystals, is highly interesting when trying to elucidate the complex structural changes that occur during bone growth and maturation. We have previously looked at mineral deposition and structural evolution of the collagenous matrix, linking both to changes in mechanics. The purpose of this study was to provide specific information on changes in crystal size and organization as a function of growth and maturation. MATERIALS AND METHODS: Using micro-computed tomography (µCT) and micro-focused scanning small-angle X-ray scattering (SAXS) we investigated cortical bone in two orthogonal directions relative to the long axis of the humeri of New Zealand White rabbits spanning from new-born to 6-months of age. We also investigated the changes with tissue age by looking at radial profiles of osteonal structures in the 6-months old rabbits. The findings were compared to our previous compositional, structural and mechanical data on the same sample cohort. RESULTS: µCT showed a continuous mineral deposition up until 3-months of age, whilst the SAXS data showed an increase in both crystal thickness and degree of orientation up until 6-months of age. The osteonal profiles showed no statistically significant changes in crystal thickness. CONCLUSIONS: Comparison to previously collected mechanical data suggests that changes are not only explained by amount of mineral in the tissue but also by the crystal dimensions.