Method Article

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

DOI:

10.3791/67904

⸱

June 24th, 2025

 ,  ,  ,  , 
1Department of Biomedical Engineering, University of Rochester, 2Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester Medical Center, 3Department of Translational Biomedical Science, University of Rochester Medical Center, 4Department of Orthopaedics, University of Rochester Medical Center

In This Article

Summary

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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 (µCT) scan field of view, specimen orientation, and baseline presence of scaffold.

Abstract

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Micro-computed tomography (µCT) 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 µCT 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 µCT 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.

Introduction

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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 op....

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Protocol

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Longitudinal µCT images for this study were collected at weeks 0 and 6 from 3 mm critical radial defects in adult female Charles River SASCO-SD rats treated with a polycaprolactone (PCL)-based scaffold. All animal use was performed in accordance with protocols approved by the University of Rochester's Committee on Animal Resources (UCAR). µCT image collection was performed using Scanco Medical VivaCT 40.

NOTE: The primary steps in this protocol are divided into µCT image segmentation, alignment, ROI selection and cropping, and analysis and visualization (Figure 1). The protocol for µCT image segmentation is ada....

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Results

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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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Discussion

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Quantifying bone volume change is essential for investigating bone regeneration potential and outcomes in longitudinal in vivo studies. This protocol builds upon established µCT 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.......

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Disclosures

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The authors have no conflict of interest to disclose.

Acknowledgements

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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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Amira 3DFEI SAS, a part of Thermo Fisher Scientificv2024.1Program used for segmentation of microCT images.
Graph Pad PrismGraphPad Software LLCv10.0.3 (217)Program used for graph development.
R Statistical SoftwareThe R Foundation for Statistical Computingv4.4.0 (2024-04-24)Program used to perform ICC analysis.
Scanco Medical VivaCT 40Scanco MedicalNAmicroCT scanner used for collection of microCT images.

References

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  1. ASTM Standard F-2721-09. Standard Guide for Pre-clinical in vivo Evaluation in Critical Size Segmental Bone Defects. , ASTM International. West Conshohocken, PA. (2023).
  2. Amini, A. R., Laurencin, C. T., Nukavarapu, S. P. Bone tissue engineering: recent advances and challenges. Crit Rev Biomed Eng.

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Tags

Micro Computed TomographyBone Volume AnalysisLongitudinal ImagingRegion Of InterestCritical Sized DefectBone RegenerationImage RegistrationPolycaprolactone ScaffoldVolume QuantificationRat Bone Model

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