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Introduction: Bone tissue engineering (TE) aims to develop biomaterials that replicate the specific mechanical strength essential for the functional characteristics of the bone (1). Osteoblastic spheroids provide a three-dimensional (3D) culture model that better mimics in vivo conditions than traditional two-dimensional (2D) cultures. Amorphous calcium phosphate (ACP), derived from eggshell waste, has shown enhanced osteogenic potential, making it a promising biomaterial for TE applications (2). However, due to limited penetration depth and structural distortion, conventional imaging techniques such as confocal microscopy and histology pose challenges in visualizing 3D constructs. This study evaluates contrast-enhanced micro-computed tomography (micro-CT) as a non-invasive alternative for 3D morphological analysis of ACP-engineered osteoblastic spheroids.
Methods: Osteoblastic spheroids were cultivated using flat-bottom, U-bottom, and rotary flask techniques. Spheroids were supplemented with ACP derived from eggshell waste and synthesized ‘control’ ACP to assess its effects on osteogenic differentiation. Contrast-enhanced micro-CT imaging was performed using phosphotungstic acid (PTA) and iodine-based contrast agents. Scans were performed with a laboratory nano-CT (SkyScan 2211 Multiscale x-ray Nano-CT System, Bruker) and at the Synchrotron Radiation for Medical Physics (SYRMEP) beamline in the synchrotron laboratory ELETTRA (Trieste, Italy). Imaging results were compared with confocal laser scanning microscopy, scanning electron microscopy, and classical histology.
Results: The cultivation method significantly influenced spheroid morphology, with rotary flask cultivation producing the most structurally uniform spheroids. ACP incorporation enhanced osteogenic activity and spheroid integrity and altered the spheroids’ morphology in volume and roundness. Contrast-enhanced micro-CT provided superior overall visualization of the spheroid and of internal architecture, allowing detailed analysis of cell and ACP distribution and matrix deposition. Unlike traditional imaging techniques, micro-CT enabled virtual histology without sectioning artifacts or depth limitations (figure in attachment applying pseudo-colors).
Discussion: This study underscores the need for advanced imaging modalities to evaluate engineered tissues effectively. Contrast-enhanced micro-CT offers a non-invasive, high-resolution approach for visualizing cellular interactions within biomaterials, addressing key limitations of conventional imaging techniques. The findings support the integration of ACP into bone TE strategies and demonstrate the potential of micro-CT for enhancing the assessment of biofabricated constructs, particularly for large cell models including a mineralized portion.
References:
1. Qu H, Fu H, Han Z, Sun Y. Biomaterials for bone tissue engineering scaffolds: a review. RSC Advances. 2019;9(45):26252-62.
2. Ma Q, Rubenis K, Sigurjónsson ÓE, Hildebrand T, Standal T, Zemjane S, et al. Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model. Smart Materials in Medicine. 2023;4:522-37.
Acknowledgments: We acknowledge financial support from the Baltic Research Programme Project No. EEARESEARCH-85 ‘Waste-to-resource: eggshells as a source for next generation biomaterials for bone regeneration (EGGSHELL)’ under the EEA Grant of Iceland, Liechtenstein and Norway No. EEZ/BPP/VIAA/2021/1 and access to the infrastructure and expertise of the BBCE—Baltic Biomaterials Centre of Excellence (European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 857287). We also acknowledge ELETTRA Syncrotrone Trieste for the beamtime provided at the SYRMEP beamline under Project Number 20225228.
Disclosure Information: The authors declare no conflicts of interest related to this study.
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