Abstract
A good understanding of fibre arrangement at tendon-bone junctions (entheses) is essential for improvement of tendon rupture repair surgery and successful tissue engineering of tendon entheses. Currently, destructive histological analysis is the commonest approach to visualise fibre arrangements at the entheses. This study aims to compare and optimise contrast-enhancing staining protocols for microCT scanning of the human Achilles tendon enthesis to achieve visualisation of the fibre arrangement and allow for subsequent histological sectioning and staining.
Samples of Achilles tendon entheses were acquired from human cadaveric specimens and stained with 5% sodium tungstate (Na2WO4), 10% Lugol’s solution (I2KI), 0.3, 0.7 and 2.5% phosphotungstic acid (PTA) and a 3.5% hafnium-substituted Wells Dawson polyoxometalate (Hf-WD POM). Conventional X-ray imaging was used for a preliminary test of staining results. Hf-WD POM- and PTA-stained samples were selected for microCT scanning. Scans were performed at several time points (from 1 week to 4 weeks of staining) to monitor penetration of the stains into the tissue and image processing of the microCT datasets was performed with ImageJ, Avizo and Drishti. Samples used for further histological processing were destained with PBS after microCT scanning and histological sections were stained with Van Gieson stain and visualised with light microscopy.
In the PTA-stained samples, the fibre arrangement within the unmineralised tissues (tendon and unmineralised fibrocartilage) was visible, but contrast within the bone and mineralised fibrocartilage was reduced. 3D rendering of the scan showed the general direction of fibres. The Hf-WD POM-stained sample did show a clearly identifiable tidemark and cement line within the bone and mineralised fibrocartilage, but tendon fibres were less visible. Destaining allowed for successful histological staining of fibres with Van Gieson for both PTA- and Hf-WD POM-treated samples.
Visualisation of key features of the human Achilles tendon enthesis, including tendon fibres, was possible using PTA and Hf-WD POM staining. 3D fibre visualisation showed promising results, but further image processing is needed to achieve higher quality images required for quantification of structural parameters. Future research could also include combined staining with PTA and Hf WD POM of the same sample.
Samples of Achilles tendon entheses were acquired from human cadaveric specimens and stained with 5% sodium tungstate (Na2WO4), 10% Lugol’s solution (I2KI), 0.3, 0.7 and 2.5% phosphotungstic acid (PTA) and a 3.5% hafnium-substituted Wells Dawson polyoxometalate (Hf-WD POM). Conventional X-ray imaging was used for a preliminary test of staining results. Hf-WD POM- and PTA-stained samples were selected for microCT scanning. Scans were performed at several time points (from 1 week to 4 weeks of staining) to monitor penetration of the stains into the tissue and image processing of the microCT datasets was performed with ImageJ, Avizo and Drishti. Samples used for further histological processing were destained with PBS after microCT scanning and histological sections were stained with Van Gieson stain and visualised with light microscopy.
In the PTA-stained samples, the fibre arrangement within the unmineralised tissues (tendon and unmineralised fibrocartilage) was visible, but contrast within the bone and mineralised fibrocartilage was reduced. 3D rendering of the scan showed the general direction of fibres. The Hf-WD POM-stained sample did show a clearly identifiable tidemark and cement line within the bone and mineralised fibrocartilage, but tendon fibres were less visible. Destaining allowed for successful histological staining of fibres with Van Gieson for both PTA- and Hf-WD POM-treated samples.
Visualisation of key features of the human Achilles tendon enthesis, including tendon fibres, was possible using PTA and Hf-WD POM staining. 3D fibre visualisation showed promising results, but further image processing is needed to achieve higher quality images required for quantification of structural parameters. Future research could also include combined staining with PTA and Hf WD POM of the same sample.
| Original language | English |
|---|---|
| Publication status | Published - 3 Dec 2019 |
| Event | The Scottish Microscopy Society’s Symposium - Roslin Institute, Easter Bush Veterinary Campus, EH25 9RG, Edinburgh, United Kingdom Duration: 3 Dec 2019 → 3 Dec 2019 Conference number: 47 http://scottishmicroscopygroup.org.uk/events/2019-symposium |
Conference
| Conference | The Scottish Microscopy Society’s Symposium |
|---|---|
| Country/Territory | United Kingdom |
| City | Edinburgh |
| Period | 3/12/19 → 3/12/19 |
| Internet address |