Name: Longitudinal Micro-Computed Tomography Image Analysis for User-Defined Region of Interest in Critical-Sized Bone Defects (Video) | JoVE Journal
Uploaded: 2025-06-24T13:20:00.000+00:00
Duration: 8 min 39 s
Description: Learn longitudinal micro-CT analysis for tracking bone regeneration in critical-sized rat radial defects using user-defined regions of interest. This method enables precise scaffold comparison across time points.

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5 minutes

We want to thank Mark Kenney for training on current processes and discussion in the development of this process as well as Lindsay Schnur from the Biomechanics and Multimodal Tissue Imaging Core at the University of Rochester. This study was supported by grants from NIH/NIAMS: H.A.A. (R01AR07061, P50AR072000, and P30AR069655) and V.Z.Z (T32GM007356, T32GM152318, and T32AR076950).

<ol><li>ASTM Standard F-2721-09. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Standard+Guide+for+Pre-clinical+in+vivo+Evaluation+in+Critical+Size+Segmental+Bone+Defects%22">Standard Guide for Pre-clinical in vivo Evaluation in Critical Size Segmental Bone Defects</a>. , ASTM International. West Conshohocken, PA. (2023).</li>
<li>Amini, A. R., Laurencin, C. T., Nukavarapu, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+tissue+engineering%3A+recent+advances+and+challenges%5BTitle%5D)+AND+%22Crit+Rev+Biomed+Eng%22%5BJournal%5D)">Bone tissue engineering: recent advances and challenges.</a> Crit Rev Biomed Eng. 40 (5), 363-408 (2012).</li>
<li>Ghassemi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+concepts+in+scaffolding+for+bone+tissue+engineering%5BTitle%5D)+AND+%22Arch+Bone+Jt+Surg%22%5BJournal%5D)">Current concepts in scaffolding for bone tissue engineering.</a> Arch Bone Jt Surg. 6 (2), 90-99 (2018).</li>
<li>Tang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+trends+in+the+development+of+bone+regenerative+biomaterials%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Recent trends in the development of bone regenerative biomaterials.</a> Front Cell Dev Biol. 9, 665813(2021).</li>
<li>Battafarano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+bone+regeneration%3A+from+graft+to+tissue+engineering%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Strategies for bone regeneration: from graft to tissue engineering.</a> Int J Mol Sci. 22 (3), 1128(2021).</li>
<li>Ghelich, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%28Bio%29manufactured+solutions+for+treatment+of+bone+defects+with+emphasis+on+US-FDA+regulatory+science+perspective%5BTitle%5D)+AND+%22Adv+Nanobiomed+Res%22%5BJournal%5D)">(Bio)manufactured solutions for treatment of bone defects with emphasis on US-FDA regulatory science perspective.</a> Adv Nanobiomed Res. 2 (4), 2100073(2022).</li>
<li>Kim, Y., Brodt, M. D., Tang, S. Y., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroCT+for+scanning+and+analysis+of+mouse+bones%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">MicroCT for scanning and analysis of mouse bones.</a> Methods Mol Biol. 2230, 169-198 (2021).</li>
<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+bone+quality+assessment+in+human+bone+tissue%3A+a+systematic+review%5BTitle%5D)+AND+%22J+Orthop+Surg+Res%22%5BJournal%5D)">Methods for bone quality assessment in human bone tissue: a systematic review.</a> J Orthop Surg Res. 17, 174(2022).</li>
<li>Bouxsein, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Miner Res. 25, 1468-1486 (2010).</li>
<li>Wee, H., Khajuria, D. K., Kamal, F., Lewis, G. S., Elbarbary, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+bone+fracture+healing+using+micro-computed+tomography%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Assessment of bone fracture healing using micro-computed tomography.</a> J Vis Exp. (190), e64262(2022).</li>
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The authors have no conflict of interest to disclose.

Quantifying bone volume change is essential for investigating bone regeneration potential and outcomes in longitudinal in vivo studies. This protocol builds upon established &#181;CT image segmentation methods11, providing a systematic approach for specifying a user-defined region of interest (ROI). This technique has been critical in the analysis of bone scaffold implant effectiveness for in vivo rat studies. By expanding upon established protocols11, thi...

Critical-sized bone defects pose significant clinical challenges in orthopedic treatment management. Per ASTM F2721, a critical-sized defect is characterized as a defect with a length 1.5 to 2 times the diameter of the bone of interest1. Repair of these defects has traditionally been through the use of autologous and allogeneic transplantations limited by the procedural expenses, associated risks of secondary surgeries, and bone graft volume required2. Current bone regeneration techniques focus on the use of allogeneic and xenogeneic scaffolds designed to produce both osteoconductive and osteoinductive effects through optimizing their mechanical properties, biocompatibility, bioactivity, angiogenic potential, and degradation profiles3,4,5. Biomaterials investigated broadly range from bioceramics and biopolymers to metals and other composite materials6. Variations of these biomaterials are tested both in vitro and in vivo to interrogate their potential as bone regeneration scaffolds.
&#181;CT is the gold standard for non-invasive, high-accuracy imaging for the assessment of bone morphology, structure, and microstructure in rodent models7,8,9. This imaging modality has been described to assess the longitudinal, in vivo progression of bone regeneration in fracture healing models10. Methods have been developed to standardize the quantification of cortical and trabecular bone from &#181;CT scans9. Semi-automated segmentation workflows have been developed utilizing commercially available visualization software for whole bone segmentation with complex anatomical structures11. These methods allow for simplified, approachable methods for users across varying experience levels to produce standardized, reproducible results. However, these methods remain limited in abilities to investigate user-defined ROI.
Here, we present a protocol that expands upon current methods to permit user-defined ROI bone volume analysis surrounding a critical-sized bone defect for longitudinal in vivo rat models using visualization software. Establishing a consistent alignment and ROI selection method between weeks of the longitudinal study was essential for the development of a robust protocol. An initial timepoint is used as the baseline for the alignment of subsequent weeks to ensure consistent orientation of solid models. Provided this alignment, corresponding &#181;CT image slices from the overlayed solid models can be selected, encompassing the critical-sized defect. Consistent ROI is verified not only through slice location but also through comparison of the number of slices within the region. The selected ROI from the baseline model can then be replicated on subsequent weeks, allowing for comparative, quantitative analysis.

<table><tbody><tr><td>Amira 3D</td><td>FEI SAS, a part of Thermo Fisher Scientific</td><td>v2024.1</td><td>Program used for segmentation of microCT images.</td></tr><tr><td>Graph Pad Prism</td><td>GraphPad Software LLC</td><td>v10.0.3 (217)</td><td>Program used for graph development.</td></tr><tr><td>R Statistical Software</td><td>The R Foundation for Statistical Computing</td><td>v4.4.0 (2024-04-24)</td><td>Program used to perform ICC analysis.</td></tr><tr><td>Scanco Medical VivaCT 40</td><td>Scanco Medical</td><td>NA</td><td>microCT scanner used for collection of microCT images.</td></tr></tbody></table>

longitudinal micro-computed tomography image analysis for user-defined region of interest in critical-sized bone defects

Longitudinal Micro-Computed Tomography Image Analysis for User-Defined Region of Interest in Critical-Sized Bone Defects

Micro-computed tomography (&#181;CT) imaging analysis of bone volume is a necessary quantitative tool for investigating bone regeneration potential and outcomes within longitudinal in vivo studies. Established methods for bone segmentation utilize visualization software for whole bone &#181;CT segmentation and alignment of complex anatomical structures. These segmentation protocols provide a robust, high-accuracy method for segmentation, alignment, and analysis but are limited in abilities of user-defined region of interest (ROI) analysis. We present a protocol expanding upon these methods to permit user-defined ROI bone volume analysis surrounding a critical-sized bone defect. The user-defined ROI surrounding the defect can be analyzed over time for in vivo longitudinal studies. Herein, we investigate &#181;CT images of three unique rat specimens each implanted with a polycaprolactone (PCL) control scaffold. Models are analyzed by three users (2 experienced and 1 novice) at time points of 0 and 6 weeks to illustrate the ability to measure an ROI surrounding a critical-sized defect throughout a longitudinal study.

µCT images of three unique rat models, each treated with a polycaprolactone (PCL) scaffold, were investigated to illustrate positive results. Analysis of a longitudinal study across time points requires the collected solid models to be aligned prior to selecting and cropping to an ROI. To illustrate this capability over multiple weeks, solid models collected at weeks 0 and 6 were aligned using common regions (Figure 2).
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We present a method for analyzing a user-defined region of interest (ROI) in a longitudinal in vivo rat radial defect model. This method enables comparative analysis between different scaffolds previously limited by variations in microcomputed tomography (&#181;CT) scan field of view, specimen orientation, and baseline presence of scaffold.

Micro-computed tomography (&#181;CT) imaging analysis of bone volume is a necessary quantitative tool for investigating bone regeneration potential and ...

longitudinal-micro-computed-tomography-image-analysis-for-user

Research

JoVE Journal

Bioengineering

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

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Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

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Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SRµCT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Micro-computed tomography (µCT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on µCT procedures for analyzing the bone healing callus. This study provides a step-by-step µCT protocol that allows the monitoring of fracture healing.

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

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The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

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Multimodal Approach to Assess Bone Regeneration and Scaffold Performance

Learn longitudinal micro-CT analysis for tracking bone regeneration in critical-sized rat radial defects using user-defined regions of interest. This method enables precise scaffold comparison across time points.

Longitudinal Micro-Computed Tomography Image Analysis for User-Defined Region of Interest in Critical-Sized Bone Defects

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