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Biomedical Imaging and Instrumentation

Visualization of Tendon Organization of Rat Tails and Hindlimbs in 3D Using Micro-CT

(E-161) Visualization of Tendon Organization of Rat Tails and Hindlimbs in 3D Using Micro-CT

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row E - Poster # 161

Presenting Author(s)

DD

Destinee Ditton

Undergraduate Student
University of Idaho
Moscow, Idaho, United States

Co-Author(s)

CM

Colin R. Marchus

Graduate Student
University of Idaho
Rathdrum, Idaho, United States
MB

Michele Brumley

Professor
Idaho State University, Idaho, United States

Primary Investigator(s)

NS

Nathan Schiele

Associate Professor
University of Idaho, Idaho, United States

Introduction:: Tendons are connective, collagenous tissues that transmit mechanical forces to allow for body movement, but tendons injure easily and have poor healing capabilities. New treatment methods are needed, requiring a better understanding of multiscale structure-function relationships through comprehensive imaging. Micro-computed tomography (CT) is an imaging modality that produces three-dimensional (3D), high-resolution images, but is limited to high density objects as the x-rays render low-density soft tissues transparent. To address this issue, we explored a contrast-enhanced technique using a serial dehydration in ethanol and employing a chemical stain, specifically phosphotungstic acid (PTA), to image tendon using 3D micro-CT imaging. Utilizing this new contrast enhancing method, 3D images from tissues were analyzed and processed to identify and trace tendon fascicles and analyze the anatomical organization of immature (postnatal day 21) rat tails and hindlimbs (1). From this analysis, we found that tail tendon fascicles appeared to cross between fascicle bundles, whereas it was previously assumed that the fascicle bundles maintain specific tracks within the rat tail tendons. Based on these initial findings, the objective of this study was to visualize the tendons in the tails and hindlimbs of both younger and older, sexually mature rats to further validate our imaging technique and evaluate fascicle organization.

Materials and Methods:: Postnatal day (P) 21 and P50 Sprague Dawley rat hindlimbs and tails were collected from an IACUC-approved study at Idaho State University and stored frozen. After thawing and chemical fixation for 7 days in 10% formalin and serial dehydration to 70% ethanol, the whole hindlimbs (n=4: P21 and n=3: P50) and whole tails (n=4: P21 and n=3: P50) were stained with 2.5% PTA in 70% ethanol for 10-11 days. They were then imaged in a micro-CT scanner (Bruker Skyscan 1275) with a voltage of 70 kV, and a current of 40-63 µA. Images were reconstructed with Skyscan’s NRecon reconstruction software and viewed using CTvox and DataViewer.

Results, Conclusions, and Discussions:: The dehydrated and PTA-stained P21 (Fig 1.1 and 1.3) and P50 tails and hindlimbs (Fig 1.2 and 1.4) have clearly visible tendons and other soft tissues, providing additional evidence that this staining and imaging technique is applicable to younger and older (sexually mature P50) rats. Even with their much larger size and increased tissue density with age, PTA staining appears to be effective and penetrated the tissues and enhanced soft tissue contrast. Tendon fascicle organization was examined in both P21 (Fig 2.1) and P50 (Fig 2.1) tails. When viewing the P21 tails and moving from a more proximal to distal location, individual tendon sub-fascicles appear to move from one fascicle bundle to a neighboring fascicle bundle (outlined in red and yellow in Fig 2.1). However, the P50 tails appear to have tendon fascicles that divide into smaller sub-fascicles (outlined in pink and blue in Fig 2.2), moving from a more proximal to distal location. Tendon insertions in P50 tissue (Fig 3) appear to follow a specific pattern of outer fascicle bundles moving to insert at the proximal side of the tail vertebra. Overall, validation of this imaging technique in both young and mature rats has allowed for tracking changes in tendon organization and structure with development. Coupling this contrast-enhancing technique with micro-CT scanning enables clear visualization of tendon and its organization and insertion sites, which advances future studies to determine tendon structure and organization in situ. Ultimately, better understanding of tendon structure-function relationships, including how they transmit mechanical forces, will improve treatment methods for restoring tendon function after an injury.

Acknowledgements (Optional): : This project was supported by the M.J. Murdock Charitable Trust (micro-CT scanner), the University of Idaho Office of Undergraduate Research – S.U.R.F and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408.

References (Optional): : 1. Ditton D, Marchus CR, Brumley MR, Schiele NR. Contrast-enhancing method to visualize tendon in 3D using micro-CT. Biomedical Engineering Society Annual Meeting. October 12-15, 2022. San Antonio, TX. Poster Presentation.