Resolving trabecular metaphyseal bone profiles downstream of the growth plate adds value to bone histomorphometry in mouse models.

Introduction: Histomorphometry of rodent metaphyseal trabecular bone, by histology or microCT, is generally restricted to the mature secondary spongiosa, excluding the primary spongiosa nearest the growth plate by imposing an 'offset'. This analyses the bulk static properties of a defined segment of secondary spongiosa, usually regardless of proximity to the growth plate. Here we assess the value of trabecular morphometry that is spatially resolved according to the distance 'downstream' of-and thus time since formation at-the growth plate. Pursuant to this, we also investigate the validity of including mixed primary-secondary spongiosal trabecular bone, extending the analysed volume 'upstream' by reducing the offset. Both the addition of spatiotemporal resolution and the extension of the analysed volume have potential to enhance the sensitivity of detection of trabecular changes and to resolve changes occurring at different times and locations. Method: Two experimental mouse studies of trabecular bone are used as examples of different factors influencing metaphyseal trabecular bone: (1) ovariectomy (OVX) and pharmacological prevention of osteopenia and (2) limb disuse induced by sciatic neurectomy (SN). In a third study into offset rescaling, we also examine the relationship between age, tibia length, and primary spongiosal thickness. Results: Bone changes induced by either OVX or SN that were early or weak and marginal were more pronounced in the mixed primary-secondary upstream spongiosal region than in the downstream secondary spongiosa. A spatially resolved evaluation of the entire trabecular region found that significant differences between experimental and control bones remained undiminished either right up to or to within 100 µm from the growth plate. Intriguingly, our data revealed a remarkably linear downstream profile for fractal dimension in trabecular bone, arguing for an underlying homogeneity of the (re)modelling process throughout the entire metaphysis and against strict anatomical categorization into primary and secondary spongiosal regions. Finally, we find that a correlation between tibia length and primary spongiosal depth is well conserved except in very early and late life. Conclusions: These data indicate that the spatially resolved analysis of metaphyseal trabecular bone at different distances from the growth plate and/or times since formation adds a valuable dimension to histomorphometric analysis. They also question any rationale for rejecting primary spongiosal bone, in principle, from metaphyseal trabecular morphometry.

Subchondral involvement in osteonecrosis of the femoral head: insight on local composition, microstructure and vascularization.

OBJECTIVE: To determine changes of subchondral bone composition, micro-structure, bone marrow adiposity and micro-vascular perfusion in end-stage osteonecrosis of the femoral head (ONFH) compared to osteoarthritis (OA) using a combined in vivo and ex vivo approach. DESIGN: Male patients up to 70 years old referred for total hip replacement surgery for end-stage ONFH were included (n = 14). Fifteen patients with OA were controls. Pre-operative MRI was used to assess bone perfusion (dynamic contrast-enhanced (DCE) sequences) and marrow fat content (chemical shift imaging). Three distinct zones of femoral head subchondral bone - necrotic, sclerotic, distant - were compared between groups. After surgery, plugs were sampled in these zones and Raman spectroscopy was applied to characterize bone mineral and organic components (old and newly-formed), and contrast-enhanced micro-computed tomography (CE-µCT) to determine bone micro-structural parameters and volume of bone marrow adipocytes, using conventional 2D histology as a reference. RESULTS: In the necrotic zone of ONFH patients compared to OA patients: 1) the subchondral plate did not exhibit significant changes in composition nor structure; 2) the volume fraction of subchondral trabecular bone was significantly lower; 3) type-B carbonate substitution was less pronounced, 4) collagen maturity was more pronounced; and 5) bone marrow adipocytes were significantly depleted. The sclerotic zone from the ONFH group showed greater trabecular thickness, and higher DCE-MRI AUC and Ktrans. Volume fraction of subchondral bone, trabecular number, and Kep were significantly lower in the distant zone of the ONFH group. CONCLUSIONS: This study demonstrated alterations of subchondral bone microstructure, composition, perfusion and/or adipose content in all zones of the femoral head.

Human periosteal-derived cell expansion in a perfusion bioreactor system: proliferation, differentiation and extracellular matrix formation.

Perfusion bioreactor systems have shown to be a valuable tool for the in vitro development of three-dimensional (3D) cell-carrier constructs. Their use for cell expansion, however, has been much less explored. Since maintenance of the initial cell phenotype is essential in this process, it is imperative to obtain insight into the bioreactor-related variables determining cell fate. Therefore, this study investigated the influence of fluid flow-induced shear stress on the proliferation, differentiation and matrix deposition of human periosteal-derived cells in the absence of additional differentiation-inducing stimuli; 120 000 cells were seeded on additive manufactured 3D Ti6Al4V scaffolds and cultured for up to 28 days at different flow rates in the range 0.04-6 ml/min. DNA measurements showed, on average, a three-fold increase in cell content for all perfused conditions in comparison to static controls, whereas the magnitude of the flow rate did not have an influence. Contrast-enhanced nanofocus X-ray computed tomography showed substantial formation of an engineered neotissue in all perfused conditions, resulting in a filling (up to 70%) of the total internal void volume, and no flow rate-dependent differences were observed. The expression of key osteogenic markers, such as RunX2, OCN, OPN and Col1, did not show any significant changes in comparison to static controls after 28 days of culture, with the exception of OSX at high flow rates. We therefore concluded that, in the absence of additional osteogenic stimuli, the investigated perfusion conditions increased cell proliferation but did not significantly enhance osteogenic differentiation, thus allowing for this process to be used for cell expansion. Copyright © 2014 John Wiley & Sons, Ltd.

Changes in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography.

High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 µm scale, no change was observed in the intracortical structure. At 1 µm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging.

Combining microCT-based characterization with empirical modelling as a robust screening approach for the design of optimized CaP-containing scaffolds for progenitor cell-mediated bone formation.

Biomaterials are a key ingredient to the success of bone tissue engineering (TE), which focuses on the healing of bone defects by combining scaffolds with cells and/or growth factors. Due to the widely variable material characteristics and patient-specificities, however, current bone TE strategies still suffer from low repeatability and lack of robustness, which hamper clinical translation. Hence, optimal TE construct (i.e. cells and scaffold) characteristics are still under debate. This study aimed to reduce the material-specific variability for cell-based construct design, avoiding trial-and-error, by combining microCT characterization and empirical modelling as an innovative and robust screening approach. Via microCT characterization we have built a quantitative construct library of morphological and compositional properties of six CE approved CaP-based scaffolds (CopiOs®, BioOss™, Integra Mozaik™, chronOS Vivify, MBCP™ and ReproBone™), and of their bone forming capacity and in vivo scaffold degradation when combined with human periosteal derived cells (hPDCs). The empirical model, based on the construct library, allowed identification of the construct characteristics driving optimized bone formation, i.e. (a) the percentage of ß-TCP and dibasic calcium phosphate, (b) the concavity of the CaP structure, (c) the average CaP structure thickness and (d) the seeded cell amount (taking into account the seeding efficiency). Additionally, the model allowed to quantitatively predict the bone forming response of different hPDC-CaP scaffold combinations, thus providing input for a more robust design of optimized constructs and avoiding trial-and error. This could improve and facilitate clinical translation. STATEMENT OF SIGNIFICANCE: Biomaterials that support regenerative processes are a key ingredient for successful bone tissue engineering (TE). However, the optimal scaffold structure is still under debate. In this study, we have provided a useful innovative approach for robust screening of potential biomaterials or constructs (i.e. scaffolds seeded with cells and/or growth factors) by combining microCT characterization with empirical modelling. This novel approach leads to a better insight in the scaffold parameters influencing progenitor cell-mediated bone formation. Additionally, it serves as input for more controlled and robust design of optimized CaP-containing bone TE scaffolds. Hence, this novel approach could improve and facilitate clinical translation.

A novel multimodular methodology to investigate external cervical tooth resorption.

AIM: To introduce a multimodular combination of techniques as a novel minimal invasive approach to investigate efficiently and accurately external cervical resorption (ECR). METHODOLOGY: One case of a central incisor with extensive external cervical resorption was selected to demonstrate the potential of a comparative novel study methodology. ECR diagnosis was based on clinical inspection, digital radiography and cone-beam computed tomography (CBCT). After extraction, the tooth was investigated using microfocus computed tomography (micro-CT), nano-CT and hard tissue histology. These techniques were compared for their accuracy and applicability to highlight their advantages and disadvantages. RESULTS: Nano-CT was more effective than micro-CT and CBCT for detailed ex vivo exploration of ECR. The reparative tissue, pericanalar resorption resistant sheet (PRRS), pulp tissue reactions, resorption channels and their interconnection with the periodontal ligament space were accurately visualized by detailed processing and analysis of the nano-CT data set with Dataviewer and CTAn software. Nano-CT analysis provided better insight in the true extent of the resorption, based on quantitative measurements and 3D visualization of the tooth structure. Nano-CT imaging results were similar to hard tissue histology at the mineralized tissue level. To clarify the dynamic phenomenon of reparative tissue formation and substitution of the resorbed tissues, nano-CT needed to be associated with hard tissue histology. CONCLUSION: Nano-CT is a fast and minimal invasive technique for the ex vivo analysis and understanding of ECR and is complementary with hard tissue histology. A combined approach of clinical and CBCT examination, with nano-CT and histological mapping measurements, can provide an ideal platform for future ECR imaging and exploration studies.

Contrast-enhanced nanofocus computed tomography images the cartilage subtissue architecture in three dimensions.

We describe a non-destructive imaging method, named contrast-enhanced nanofocus X-ray computed tomography (CE-nanoCT), that permits simultaneously imaging and quantifying in 3D the (sub)tissue architecture and (biochemical) composition of cartilage and bone in small animal models at a novel contrast and spatial resolution. To demonstrate the potential of this novel methodology, a newborn mouse was scanned using CE-nanoCT. This allowed simultaneously visualising the bone and cartilage structure much like the traditional alcian blue-alizarin red skeletal stain. Additionally, it enabled a 3D visualisation at such a high spatial image resolution that internal, micro-scale structures could be digitally dissected and evaluated for size, structure and composition. Ex vivo treatment with papain, that is known to specifically remove the non-calcified cartilage layer but keep the calcified cartilage intact, proved CE-nanoCT to be applicable to visualise the subdivisions within the hyaline cartilage of the articular joint of mice. The quantitative power of CE-nanoCT in vivo was evaluated using a mouse model for osteoarthritis (OA), where OA-like cartilage lesions are induced by meniscus destabilisation surgery. The thickness of both the non-calcified and calcified cartilage layer in the knee joint of such mice was visualised and quantified in 3D and compared to unaffected mice. Finally, to show that different forms of cartilage and tissue combinations can be distinguished using CE-nanoCT, different cartilaginous body parts of the mouse were imaged. In conclusion, CE-nanoCT can provide novel insights in preclinical research by quantifying in a non-destructive 3D manner pathological differences, in particular in developing mice, newborns or adults.

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds.

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 µm and 1000 µm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 µm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.

Prediction of permeability of regular scaffolds for skeletal tissue engineering: a combined computational and experimental study.

Scaffold permeability is a key parameter combining geometrical features such as pore shape, size and interconnectivity, porosity and specific surface area. It can influence the success of bone tissue engineering scaffolds, by affecting oxygen and nutrient transport, cell seeding efficiency, in vitro three-dimensional (3D) cell culture and, ultimately, the amount of bone formation. An accurate and efficient prediction of scaffold permeability would be highly useful as part of a scaffold design process. The aim of this study was (i) to determine the accuracy of computational fluid dynamics (CFD) models for prediction of the permeability coefficient of three different regular Ti6Al4V scaffolds (each having a different porosity) by comparison with experimentally measured values and (ii) to verify the validity of the semi-empirical Kozeny equation to calculate the permeability analytically. To do so, five CFD geometrical models per scaffold porosity were built, based on different geometrical inputs: either based on the scaffold's computer-aided design (CAD) or derived from 3D microfocus X-ray computed tomography (micro-CT) data of the additive manufactured (AM) scaffolds. For the latter the influence of the spatial image resolution and the image analysis algorithm used to determine the scaffold's architectural features on the predicted permeability was analysed. CFD models based on high-resolution micro-CT images could predict the permeability coefficients of the studied scaffolds: depending on scaffold porosity and image analysis algorithm, relative differences between measured and predicted permeability values were between 2% and 27%. Finally, the analytical Kozeny equation was found to be valid. A linear correlation between the ratio Φ(3)/S(s)(2) and the permeability coefficient k was found for the predicted (by means of CFD) as well as measured values (relative difference of 16.4% between respective Kozeny coefficients), thus resulting in accurate and efficient calculation of the permeability of regular AM scaffolds.

Validation of x-ray microfocus computed tomography as an imaging tool for porous structures.

X-ray microfocus computed tomography (micro-CT) is recently put forward to qualitatively and quantitatively characterize the internal structure of porous materials. However, it is known that artifacts such as the partial volume effect are inherently present in micro-CT images, thus resulting in a visualization error with respect to reality. This study proposes a validation protocol that in the future can be used to quantify this error for porous structures in general by matching micro-CT tomograms to microscopic sections. One of the innovations of the protocol is the opportunity to reconstruct an interpolated micro-CT image under the same angle as the physical cutting angle of the microscopic sections. Also, a novel thresholding method is developed based on matching micro-CT and microscopic images. In this study, titanium porous structures are assessed as proof of principle. It is concluded for these structures that micro-CT visualizes 89% of the total amount of voxels (solid and pore) correctly. However, 8% represents an overestimation of the real structure and 3% are real structural features not visualized by micro-CT. When exclusively focusing on the solid fraction in both the micro-CT and microscopic images, only an overestimation of about 5% is found.