3D histopathology of stenotic aortic valve cusps using ex vivo microfocus computed tomography

BackgroundCalcific aortic stenosis (AS) is the most prevalent heart valve disease in developed countries. The aortic valve cusps progressively thicken and the valve does not open fully due to the presence of calcifications. In vivo imaging, usually used for diagnosis, does not allow the visualization of the microstructural changes associated with AS.MethodsEx vivo high-resolution microfocus computed tomography (microCT) was used to quantitatively describe the microstructure of calcified aortic valve cusps in full 3D. As case study in our work, this quantitative analysis was applied to normal-flow low-gradient severe AS (NF-LG-SAS), for which the medical prognostic is still highly debated in the current literature, and high-gradient severe AS (HG-SAS).ResultsThe volume proportion of calcification, the size and number of calcified particles and their density composition was quantified. A new size-based classification considering small-sized particles that are not detected with in vivo imaging was defined for macro-, meso- and microscale calcifications. Volume and thickness of aortic valve cusps, including the complete thickness distribution, were also determined. Moreover, changes in the cusp soft tissues were also visualized with microCT and confirmed by scanning electron microscopy images of the same sample. NF-LG-SAS cusps contained lower relative amount of calcifications than HG-SAS. Moreover, the number and size of calcified objects and the volume and thickness of the cusps were also lower in NF-LG-SAS cusps than in HG-SAS.ConclusionsThe application of high-resolution ex vivo microCT to stenotic aortic valve cusps provided a quantitative description of the general structure of the cusps and of the calcifications present in the cusp soft tissues. This detailed description could help in the future to better understand the mechanisms of AS

Three dimensional characterization of tissue-engineered constructs by contrast enhanced nanofocus computed tomography.

peer reviewedIn order to successfully implement tissue engineered (TE) constructs as part of a clinical therapy, it is necessary to first develop and validate quality control tools that will ensure accurate and consistent TE construct release specifications. Hence advanced methods to monitor TE construct properties need to be further developed. In this study we showed proof of concept for contrast enhanced nanofocus computed tomography (CE-nanoCT) as a 'whole-construct' imaging technique with non-invasive potential that enables 3D visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular we performed a 3D quantitative and qualitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely Hexabrix(R) and phosphotungstic acid (PTA). CE-nanoCT image data were validated by comparison to Live/Dead viability/cytotoxicity and picrosirius red staining data, and to the net dry weight of the TE constructs. When using Hexabrix(R) as contrast agent, ECM volume fitted linearly with net dry ECM weight independent from the flow rate used. When using PTA as contrast agent, CE-nanoCT data showed pronounced distinction between flow conditions when compared to both net dry weight and picrosirius red staining data although linearity was maintained, indicating culture-specific structural ECM differences. This was attributed to the binding specificity of this contrast agent. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of 'TE constructs' in well characterized 'TE products'.I.P. is funded by the ENDEAVOUR project G.0982.11N of the FWO;M.S. is supported by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT/111457). G.K. and L.G. acknowledge support by the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC grant agreement n°279100

Exploring polyoxometalates as non-destructive staining agents for contrast-enhanced computed tomography

Due to the high complexity and heterogeneous structure of biological tissues, imaging techniques that allow for precise and quantitative structural analyses of such materials are of high importance. These techniques could also advance clinical translation of regenerative medicine by providing better insights in tissue development and disease. The standard techniques for evaluating biological tissues such as histological sectioning and staining, have a high discriminative power, but allow assessment of the tissue distribution only in two dimensions. Due to restricted sectioning orientation and limited depth resolution, this leads to loss of information in three dimensions (3D), and therefore, more precise imaging of the 3D microstructure and spatial interrelationships of the different tissues within organs is crucial. MicroCT can provide full 3D structural information of mineralized tissues and dense biomaterials. However, the intrinsic low X-ray absorption of soft tissues requires contrast-enhancing staining agents (CESAs) to be used. We have shown that a range of polyoxometalate clusters (POMs) can be excellent non-destructive staining agents for high-resolution contrast-enhanced microCT (CE-CT) visualization of various tissues, of bone and its marrow vascularization and adiposity. A range of Wells-Dawson POMs, differing in structure and overall charge, has been synthesized and evaluated for their potential as soft tissue CESAs. We have shown that hafnium-substituted POM (Hf-POM) allows for simultaneous contrast-enhanced microCT (CE-CT) visualization of bone and its marrow vascularization and adiposity. Monolacunary Wells-Dawson POM (Mono-WD POM) showed similar soft tissue enhancement as Hf-WD POM and phosphotungstic acid (PTA), a frequently used but destructive CESA. However, compared to PTA, the POMs are much less destructive and show a better diffusion. The solubility of Mono-WD POM can be improved by simple addition of lithium chloride to the staining solution, leading to further enhancement of the soft tissue contrast. In vivo toxicity of Wells-Dawson POM has been also evaluated according to standard toxicological protocols using Wistar albino rats, which is the first and important step in evaluating side effects of these polyoxometalate nanoclusters that show large potential as therapeutics and contrast agents.Simpozijum „Stremljenja i novine u medicini“ Medicinskog fakulteta u Beogradu, Beograd, 04-08. decembra, 2023

Characterization of the porous structure of biodegradable scaffolds obtained with supercritical CO2 as foaming agent

Poly(ε-caprolactone) foams were prepared, via a batch process, by using supercritical CO2 as foaming agent. Their porous structure was characterized through mercury porosimetry, helium and mercury pycnometry, scanning electron microscopy (SEM) and X-ray microtomography observations coupled with image analysis. The pore size distributions obtained by these two latter techniques show that the pore structure is more homogeneous when the foaming process is performed under a high CO2 saturation pressure (higher than 250 bars)

Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects.

peer reviewedBACKGROUND: Bones have a remarkable capacity to heal upon fracture. Yet, in large defects or compromised conditions healing processes become impaired, resulting in delayed or non-union. Current therapeutic approaches often utilize autologous or allogeneic bone grafts for bone augmentation. However, limited availability of these tissues and lack of predictive biological response result in limitations for clinical demands. Tissue engineering using viable cell-based implants is a strategic approach to address these unmet medical needs. METHODS: Herein, the in vitro and in vivo cartilage and bone tissue formation potencies of human pluripotent stem cells were investigated. The induced pluripotent stem cells were specified towards the mesodermal lineage and differentiated towards chondrocytes, which subsequently self-assembled into cartilaginous organoids. The tissue formation capacity of these organoids was then challenged in an ectopic and orthotopic bone formation model. RESULTS: The derived chondrocytes expressed similar levels of collagen type II as primary human articular chondrocytes and produced stable cartilage when implanted ectopically in vivo. Upon targeted promotion towards hypertrophy and priming with a proinflammatory mediator, the organoids mediated successful bridging of critical size long bone defects in immunocompromised mice. CONCLUSIONS: These results highlight the promise of induced pluripotent stem cell technology for the creation of functional cartilage tissue intermediates that can be explored for novel bone healing strategies

A new era of virtual histology: contrast-enhanced microCT to simultaneously visualize and quantify in 3D soft and mineralized biological tissues

status: publishe

Morphological and Mechanical Quantification of Porous Structures by Means of Micro-CT (Morfologische en mechanische kwantificatie van poreuze structuren met behulp van micro-CT)

Suitable characterization techniques for porous structures are required to (i) understand and to be able to simulate, via finite elements (FE), the structure-properties relationships and (ii) understand the relationship between the morphology and mechanical behaviour on one hand, and the failure mechanisms on the other hand. X-ray microfocus computed tomography (micro-CT) offers a solution as it provides a means to acquire a complete 3D set of images of the structure visualizing the internal architecture at the microscopic level in a non-destructive way. Additionally, the micro-CT images enable subsequent image analysis, resulting in an extensive 3D quantitative description of the morphology that cannot be obtained by other methods. But, one has to be aware of the fact that micro-CT images are inherently subjected to artefacts and that the image quality and accuracy depend on multiple factors. For example, the acquisition settings (target material, tube voltage and filter material) influence the X-ray spectra and hence also the image quality. The spatial resolution strongly influences the accuracy of the micro-CT images and as a result also the morphological analysis. Closely related to the latter, the material architecture has a significant influence on the micro-CT image accuracy, which is also affected by the material type. In a first part of this study, systematic, fast and user-independent protocols have been developed both for acquisition parameter optimization and for image accuracy validation, and the influence of the different factors mentioned above on the image accuracy has been investigated. As a result, when applying these protocols, the micro-CT user should know, for different material types and architectures, what the capabilities and limitations of micro-CT are for morphological assessment of porous structures. In a second part, this knowledge has been applied and the use of micro-CT has been expanded to the mechanical characterization of porous structures by combining micro-CT imaging and 3D image analysis with in-situ mechanical loading, FE analysis and local strain mapping as this combination allows to (i) provide in-situ and experimentally the mechanical properties, (ii) link the mechanical properties to the morphology, (iii) investigate the morphological changes under compressive loading, (iv) feed and validate a FE model which can be applied for the prediction of the mechanical properties that cannot readily be determined experimentally and (v) predict the failure modes by using experimental local strain mapping

MICRO-CT AND CONTRAST-ENHANCED CT AS INNOVATIVE 3D CHARACTERIZATION TOOLS FOR ENGINEERED TISSUES: AN OVERVIEW

status: publishe

A combinatory approach of advanced 3D imaging, mechanical testing and computational modelling to improve insight into the decreased biomechanical performance of type 2 diabetes mouse bones: an overview

status: publishe