Advaced Microtomographic Techniques for in vitro and in vivo evaluation of Biomaterials in Orthopedic Research

Micro-CT is a very useful non-destructive technique for the 3D study of bone, biomaterials and their interactions because it is able to supply structural and densitometric information by obtaining images of the internal structure of a small object with a high spatial resolution. Besides, Micro-CT is an important tool to study the interactions between biomaterials and bone tissue from a 3D point of view. Typical uses in biomedical fields include the study of in vitro bone samples and biomaterials such as hydroxyapatite (HA), bioactive glasses, pharmaceutical granules, metals and composites (polymers + HA or calcium phosphate). Usually, the most evaluated material parameter is porosity because it plays a dominant role in the biomechanical characteristics, the initial cell attachment and thus the subsequent tissue regeneration. Due to the linear attenuation coefficient specific for every material, is important to set X-ray source voltage and image reconstruction corrections depending on involved materials. More specifically, bone is a connective tissue composed by an average of about 2/3 of inorganic substances and 1/3 of organic substances. The main constituents of the inorganic matrix of bone is a mixture of HA and TCP while biomaterials in orthopedics can be enclosed within two categories:- biomaterials used as replacement of bone to stimulate tissue regeneration and that can be completely absorbed and degraded (mainly ceramic and polymers); - biomaterials for prosthetic implants that have mechanical properties that ensure stabilization of fractures or damaged joints (mainly metals, ceramics or high density polymers). Moreover, micro-CT, deriving from clinical CT, allow studying the interactions between biomaterials and bone tissue also in a longitudinal way. The statistical power of longitudinal studies compared to cross-sectional studies has a great meaning principally because the effect of an implanted biomaterial can be evaluated over time in the same sample. In fact, an in vivo micro-CT analysis allows an assessment and quantification of the development over time in healing processes due to the application of engineered medical devices. The ability to study the time evolution of anatomical changes occurring in the course of the experiment could exclude the use of different experimental groups and, thus, has a significant ethical meaning that should not be underestimated. Limitations of the in vivo micro-CT image acquisition are: the complexity of displaying small structures that are moving dynamically due principally to breathing and heart beating; the limitation in administered X-ray dose distribution per animal due to the destructive effects on living organisms and cells; and the linear attenuation coefficient specific for every material including animal soft and hard tissues. The evaluation of the mineral content is another important and peculiar task of micro-CT. The variations in mineral content are determinable due to the principle that the gray levels of every micro-CT section give a map of the distribution of the absorption coefficients of the X-rays related to the analyzed sample. Such coefficients depend on the material density, from the atomic number of the elements that constitute it and from the incidental energy used. In orthopedic preclinical studies, usually two different densitometric parameters are evaluated: Bone Mineral Density (BMD) and Tissue Mineral Density (TMD). Mineralization is an important aspect in several bone disease evolution such as, for example, osteoporosis or osteoarthritis.The first year of the research was dedicated to the micro-CT analysis of different kinds of biomaterials in the pre-implantation phase studying new procedures to widen the acquisition possibilities and the kinds of quantitative analytical methods. In particular the project followed 3 different research lines: a) the study of thixotropic carboxymethylcellulose (CMC) hydrogels added with iron magnetic-nanoparticles (CMC-NPs) examining the differences in magnetic particles distribution and using a micro-CT freezing chamber to overcome the limitation of movements during the acquisition; b) the study of the 3D cell (MG63) distribution seeded onto a polymeric scaffold using osmium tetroxide as contrast agent and developing a new micro-CT segmentation protocol; c) the study of dimensional metrology establishing an approach for the quantification of wear in ZrO2 head prosthesis components using micro-CT and to validate the method comparing it with the gold standard, i.e. the gravimetric analysis. The second year was dedicated to the study of the interactions between different types of biomaterials implanted in bone tissue. The micro-CT analyses were performed as a result of in vivo preclinical studies and clinical retrieved studies. During this year, the project was divided in the following research lines: a) the evaluation of the in vivo behaviour of ceramic custom made prosthesis in a suitable animal model (adult sheep) at 6 and 12 months from surgical cranioplasty; b) the evaluation of the characteristics of bone quality and its microarchitecture in retrieved metal-on-metal Metal-on-metal HR; c) the analysis of granules characteristics using a new injectable multiphasic bone substitutes based on gel-coated OsproLife® HA/TTCP. During the third year, the non “functional”, i.e. non quantitative, information obtainable from a micro-CT analysis was deepened, testing the most important computer algorithms for 3D visualization and modelling: maximum intensity projection (MIP), shaded surface display (SSD) and volume rendering (VR). Moreover, the Micro-CT analyses performed were divided in the following research lines: a) the evaluation of the in vivo osteoinductive behaviour of three-dimensional interconnected porous scaffolds of gelatin with or without contents of nanocrystalline HA over time; b) the evaluation of bone quality in terms of mineral content in a study of osteoarthritis treatment in a large animal model with engineered hyaluronic acid scaffolds. The main objective of this PhD research was to develop innovative techniques and procedures of 3D image analysis for the characterization of polymeric, ceramic and metal biomaterials used in various fields of bone tissue preclinical research. In detail the aims can be summarized in: – assessing micro-CT procedures applicable to pre-implanted biomaterials through both metrological studies and in vitro studies of 3D cell scaffold colonization, with the definition of effective segmentation techniques; – developing micro-CT techniques to evaluate different kinds of implanted biomaterials both ex vivo and in vivo establishing standard test protocols and identifying the mechanisms of material resorption and degradation in physiological environment and the mechanisms of bone remodeling; – exploring the densitometric analysis in relation to the contribution of mineralization in healing process at peri-implant site. These program objectives have been achieved through the development of reliable experimental procedures for the morphological and mechanical evaluation of implantation biomaterials (scaffold and prostheses); the investigation of a large number of possible applications of biomaterials in orthopedic preclinical studies through in vitro, ex-vivo and in vivo micro-CT analysis; the elaboration of new interpretative models of the bone regeneration mechanisms through 3D morphometric parameters; and the extension of the knowledge and the expertise in 3D biomaterial evaluations used in orthopedic research

Three-dimensional imaging and reconstruction of the whole ovary and testis: a new frontier for the reproductive scientist.

AbstractThe 3D functional reconstruction of a whole organ or organism down to the single cell level and to the subcellular components and molecules is a major future scientific challenge. The recent convergence of advanced imaging techniques with an impressively increased computing power allowed early attempts to translate and combine 2D images and functional data to obtain in-silico organ 3D models. This review first describes the experimental pipeline required for organ 3D reconstruction: from the collection of 2D serial images obtained with light, confocal, light-sheet microscopy or tomography, followed by their registration, segmentation and subsequent 3D rendering. Then, we summarise the results of investigations performed so far by applying these 3D image analyses to the study of the female and male mammalian gonads. These studies highlight the importance of working towards a 3D in-silico model of the ovary and testis as a tool to gain insights into their biology during the phases of differentiation or adulthood, in normal or pathological conditions. Furthermore, the use of 3D imaging approaches opens to key technical improvements, ranging from image acquisition to optimisation and development of new processing tools, and unfolds novel possibilities for multidisciplinary research

Quantitative Evaluation of the 3D Anatomy of the Human Osseous Spiral Lamina Using MicroCT.

PURPOSE The osseous spiral lamina (OSL) is an inner cochlear bony structure that projects from the modiolus from base to apex, separating the cochlear canal into the scala vestibuli and scala tympani. The porosity of the OSL has recently attracted the attention of scientists due to its potential impact on the overall sound transduction. The bony pillars between the vestibular and tympanic plates of the OSL are not always visible in conventional histopathological studies, so imaging of such structures is usually lacking or incomplete. With this pilot study, we aimed, for the first time, to anatomically demonstrate the OSL in great detail and in 3D. METHODS We measured width, thickness, and porosity of the human OSL by microCT using increasing nominal resolutions up to 2.5-µm voxel size. Additionally, 3D models of the individual plates at the basal and middle turns and the apex were created from the CT datasets. RESULTS We found a constant presence of porosity in both tympanic plate and vestibular plate from basal turn to the apex. The tympanic plate appears to be more porous than vestibular plate in the basal and middle turns, while it is less porous in the apex. Furthermore, the 3D reconstruction allowed the bony pillars that lie between the OSL plates to be observed in great detail. CONCLUSION By enhancing our comprehension of the OSL, we can advance our comprehension of hearing mechanisms and enhance the accuracy and effectiveness of cochlear models

Multiscale and multimodal X-ray analysis: Quantifying phase orientation and morphology of mineralized turkey leg tendons

Fibrous biocomposites like bone and tendons exhibit a hierarchical arrangement of their components ranging from the macroscale down to the molecular level. The multiscale complex morphology, together with the correlated orientation of their constituents, contributes significantly to the outstanding mechanical properties of these biomaterials. In this study, a systematic road map is provided to quantify the hierarchical structure of a mineralized turkey leg tendon (MTLT) in a holistic multiscale evaluation by combining micro-Computed Tomography (micro-CT), small-angle X-ray scattering (SAXS), and wide-angle X-ray diffraction (WAXD). We quantify the interplay of the main MTLT components with respect to highly ordered organic parts such as fibrous collagen integrating inorganic components like hydroxyapatite (HA). The microscale fibrous morphology revealing different types of porous features and their orientation was quantified based on micro-CT investigations. The quantitative analysis of the alignment of collagen fibrils and HA crystallites was established from the streak-like signal in SAXS using the Ruland approach and the broadening of azimuthal profiles of the small and wide-angle diffraction peaks. It has been in general agreement that HA crystallites are co-aligned with the nanostructure of mineralized tissue. However, we observe relatively lower degree of orientation of HA crystallites compared to the collagen fibrils, which supports the recent findings of the structural interrelations within mineralized tissues. The generic multiscale characterization approach of this study is relevant to any hierarchically structured biomaterials or bioinspired materials from the μm-nm-Å scale. Hence, it gives the basis for future structure-property relationship investigations and simulations for a wide range of hierarchically structured materials

Otosclerosis under microCT: New insights into the disease and its anatomy

Purpose: Otospongiotic plaques can be seen on conventional computed tomography (CT) as focal lesions around the cochlea. However, the resolution remains insufficient to enable evaluation of intracochlear damage. MicroCT technology provides resolution at the single micron level, offering an exceptional amplified view of the otosclerotic cochlea. In this study, a non-decalcified otosclerotic cochlea was analyzed and reconstructed in three dimensions for the first time, using microCT technology. The pre-clinical relevance of this study is the demonstration of extensive pro-inflammatory buildup inside the cochlea which cannot be seen with conventional cone-beam CT (CBCT) investigation. Materials and Methods: A radiological and a three-dimensional (3D) anatomical study of an otosclerotic cochlea using microCT technology is presented here for the first time. 3D-segmentation of the human cochlea was performed, providing an unprecedented view of the diseased area without the need for decalcification, sectioning, or staining. Results: Using microCT at single micron resolution and geometric reconstructions, it was possible to visualize the disease's effects. These included intensive tissue remodeling and highly vascularized areas with dilated capillaries around the spongiotic foci seen on the pericochlear bone. The cochlea's architecture as a morphological correlate of the otosclerosis was also seen. With a sagittal cut of the 3D mesh, it was possible to visualize intense ossification of the cochlear apex, as well as the internal auditory canal, the modiolus, the spiral ligament, and a large cochleolith over the osseous spiral lamina. In addition, the oval and round windows showed intense fibrotic tissue formation and spongiotic bone with increased vascularization. Given the recently described importance of the osseous spiral lamina in hearing mechanics and that, clinically, one of the signs of otosclerosis is the Carhart notch observed on the audiogram, a tonotopic map using the osseous spiral lamina as region of interest is presented. An additional quantitative study of the porosity and width of the osseous spiral lamina is reported. Conclusion: In this study, structural anatomical alterations of the otosclerotic cochlea were visualized in 3D for the first time. MicroCT suggested that even though the disease may not appear to be advanced in standard clinical CT scans, intense tissue remodeling is already ongoing inside the cochlea. That knowledge will have a great impact on further treatment of patients presenting with sensorineural hearing loss

Multibody Computer Model of the Entire Equine Forelimb Simulates Forces Causing Catastrophic Fractures of the Carpus during a Traditional Race

Simple Summary Palios are traditional horseraces held in the main square of few Italian cities. Due to peculiar features of such circuits, adapted to the square architecture and thus characterized by tight curves and unconventional footing surface, horses involved are at particular risk of accidents. Prevention of catastrophic musculoskeletal injuries is a significant issue and matter of debate during these events. In particular, the negotiation of the curves in the city circuits is a significative concern. An experiment was set up to build a model of entire forelimb at the point of failure in the context of a turn comparable to that in a Palio circuit. The model was informed by live data and the output compared to post-mortem findings obtained from a horse that sustained a catastrophic fracture of the carpus during this competition. The objective of this study is to determine the magnitude and distribution of internal forces generated across the carpus under which the catastrophic injury has occurred and describe related post-mortem findings. A catastrophic fracture of the radial carpal bone experienced by a racehorse during a Palio race was analyzed. Computational modelling of the carpal joint at the point of failure informed by live data was generated using a multibody code for dynamics simulation. The circuit design in a turn, the speed of the animal and the surface characteristics were considered in the model. A macroscopic examination of the cartilage, micro-CT and histology were performed on the radio-carpal joint of the limb that sustained the fracture. The model predicted the points of contact forces generated at the level of the radio-carpal joint where the fracture occurred. Articular surfaces of the distal radius, together with the proximal articular surface of small carpal bones, exhibited diffuse wear lines, erosions of the articular cartilage and subchondral bone exposure. Even though the data in this study originated from a single fracture and further work will be required to validate this approach, this study highlights the potential correlation between elevated impact forces generated at the level of contact surfaces of the carpal joint during a turn and cartilage breakdown in the absence of pre-existing pathology. Computer modelling resulted in a useful tool to inversely calculate internal forces generated during specific conditions that cannot be reproduced in-vivo because of ethical concerns

Multipotent adult rat, thyroid stem cells can be differentiated to follicular thyrocyte, and hepatocyte- like cells in 2D and 3D culture systems

We have recently characterized and differentiated towards endodermal and mesoder- mal lineages progenitor cells of the adult rat thyroid, expressing multipotency markers [1]. We have now assessed their clonogenicity, extent of side population, consistency of stem cell marker expression, and commitment to either follicular or hepatocyte-like lineages when in monolayer (2D), and suspension or Matrigel (3D). Colony forming unit (CFU)-like cultures were obtained by long-term subcultures of primary rat thyroid cells, under starvation conditions. CFU-like cultures seeded in Petri dishes by limiting dilution (1 cell / cm2) were observed to give rise to toluidine blue-positive, individual clones. In these cultures, quantitative densitometric analysis of immunoblotted Oct-3/4, Sca1, and GATA4 revealed an increase in stem cell markers ranging from 95% to 270% with respect to standard, primary thyroid cultures. In addition, using three different analytical techniques including DyeCycle Violet staining by flow cytometry, ABCG2 immunocytochemistry, and Hoechst 33342 histochemistry + the ABCG2 inhibitor, verapamil a side population involving 1-2% of CFU-like cultures was detected. Then, CFU-like cultures were differentiated using TSH, either in 2D or in 3D. Differentiated adherent cells resulted immunopositive for thyrocyte markers including thyroglobulin (TG), sodium-iodide symporter (NIS), and thyroperoxidase (TPO). Differentiation in suspension and in Matrigel gave rise to follicles with cells having ultrastructural features consistent with thyrocytes, and immunoreactivity (IR) for TG, NIS, and TPO. Finally, CFU-like cultures were differentiated in adherence to hepatocyte-like cells, resulting in pre-hepatocyte morphology, high periodic acid-Schiff reaction, and IR for α-fetoprotein and albumin. We conclude that our CFU-like thyroid cultures are enriched with a multipotent, stem cell population whose hepatic differentiation capacity has been revealed for the first time

Magnetic Forces And Magnetized Biomaterials Provide Dynamic Flux Information During Bone Regeneration

The fascinating prospect to direct tissue regeneration by magnetic activation has been recently explored. In this study we investigate the possibility to boost bone regeneration in an experimental defect in rabbit femoral condyle by combining static magnetic fields and magnetic biomaterials. NdFeB permanent magnets are implanted close to biomimetic collagen/hydroxyapatite resorbable scaffolds magnetized according to two different protocols. Permanent magnet only or non-magnetic scaffolds are used as controls. Bone tissue regeneration is evaluated at 12 weeks from surgery from a histological, histomorphometric and biomechanical point of view. The reorganization of the magnetized collagen fibers under the effect of the static magnetic field generated by the permanent magnet produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. In contrast, only partial defect healing is achieved within the control groups. We ascribe the peculiar bone regeneration to the transfer of micro-environmental information, mediated by collagen fibrils magnetized by magnetic nanoparticles, under the effect of the static magnetic field. These results open new perspectives on the possibility to improve implant fixation and control the morphology and maturity of regenerated bone providing “in site” forces by synergically combining static magnetic fields and biomaterials

Three-dimensional imaging and reconstruction of the whole ovary and testis: a new frontier for the reproductive scientist

The 3D functional reconstruction of a whole organ or organism down to the single cell level and to the subcellular components and molecules is a major future scientific challenge. The recent convergence of advanced imaging techniques with an impressively increased computing power allowed early attempts to translate and combine 2D images and functional data to obtain in-silico organ 3D models. This review first describes the experimental pipeline required for organ 3D reconstruction: from the collection of 2D serial images obtained with light, confocal, light-sheet microscopy or tomography, followed by their registration, segmentation and subsequent 3D rendering. Then, we summarise the results of investigations performed so far by applying these 3D image analyses to the study of the female and male mammalian gonads. These studies highlight the importance of working towards a 3D in-silico model of the ovary and testis as a tool to gain insights into their biology during the phases of differentiation or adulthood, in normal or pathological conditions. Furthermore, the use of 3D imaging approaches opens to key technical improvements, ranging from image acquisition to optimisation and development of new processing tools, and unfolds novel possibilities for multidisciplinary research

Tensile and Impact Toughness Properties of a Zr-Based Bulk Metallic Glass Fabricated via Laser Powder-Bed Fusion

In the past few years, laser powder-bed fusion (LPBF) of bulk metallic glasses (BMGs) has gained significant interest because of the high heating and cooling rates inherent to the process, providing the means to bypass the crystallization threshold. In this study, (for the first time) the tensile and Charpy impact toughness properties of a Zr-based BMG fabricated via LPBF were investigated. The presence of defects and lack of fusion (LoF) in the near-surface region of the samples resulted in low properties. Increasing the laser power at the borders mitigated LoF formation in the near-surface region, leading to an almost 27% increase in tensile yield strength and impact toughness. Comparatively, increasing the core laser power did not have a significant influence. It was therefore confirmed that, for BMGs like for crystalline alloys, near-surface LoFs are more detrimental than core LoFs. Although increasing the border and core laser power resulted in a higher crystallized fraction, detrimental to the mechanical properties, reducing the formation of LoF defects (confirmed using micro-computed tomography, Micro-CT) was comparatively more important