A platelet aggregation-inducing factor podoplanin is highly expressed in metastatic lesions of osteosarcoma

Hiroharu Oki, Mika Kaneko, Satoshi Ogasawara, Yuta Tujimoto, Xing Liu, Masato Sugawara, Yuya Takakubo, Takashi Tuchiya, Yukinari Kato, Michiaki Takagi
2016 Journal of Orthopaedic Translation  
Calcium hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 accounts for 60% to 70% of the bone tissue and contributes to the hardness of bone [1] . Decalcification is a process to dissolution the hydroxyapatite complex in bone matrix to soften the bone. After decalcification, the bone samples would be compatible with routine paraffin histologic preparation. The traditional decalcification method requires long incubation time and can lead to tissue swelling and hydrolysis of the bone matrix [2] . Ultrasound
more » ... ix [2] . Ultrasound (US) is believed to enhance the decalcification by a cavitation mechanism [3] . We hypothesized that the application of US during bone decalcification would accelerate the decalcification compare with the traditional decalcification method without damaging bone tissue for histological assessments. Methods: A human femur was obtained from Science Care (Arizona, USA) and was sectioned into 5mm thick transverse sections. The bone slices (nZ6) were fixed in 4% phosphate buffered paraformaldehyde for 24 hours and were divided into two groups: Ultrasound Decalcification group (US DeCal) and Normal Decalcification group (Normal DeCal). For US DeCal, the bone sections were place in the US decalcifier (DeCa DX100, Pro-Cure Medical Technology Co. Ltd, Hong Kong, 50W at a frequency of 40kHz) with 300mL of 0.5M EDTA solution. The temperature of the EDTA solution was maintained at 30e45 C. For the Normal DeCal, bone slices were placed in a container with 300mL of 0.5M EDTA and maintained at 37 C. All EDTA solution was refreshed daily. The mineral content of the bone slices was measured by micro-CT and Dual-energy X-ray absorptiometry (DXA) at different time points. Calcium concentration of EDTA was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). After decalcification had been completed, the samples were processed, embedded in paraffin, sectioned at 5mm, and stained with hematoxylin and eosin for analysis. Results: For the US DeCal, the bone samples retained less than 7% of the mineral content at Day 6 and they were completely decalcified by Day 8. For Normal DeCal, the mineral content of these bone samples was 36.32AE5.09% and 24.30AE4.80% at Day 6 and Day 8 respectively. In addition, Normal DeCal samples took over 50 Days to complete decalcification. The concentration of calcium ions in the EDTA solution of the US DeCal group was 80% higher than the Normal DeCal (p<0.05). Histological analysis showed that there was no significant difference between the sections of the two groups. Discussion and Conclusion: Ultrasound decalcification accelerated the decalcification process of human femoral bone slices compared with the traditional decalcification method. Our results suggested that decalcification by US shorten the duration by six times compare with the normal decalcification. Others reported that US shortened the decalcification process by 4e12 times depending on the thickness of the sample and the type of tissues [2] . The application of ultrasound technology would be suitable for the routine sample decalcification process for bone histology in both basic and clinical research. Acknowledgements: This project was supported by the Public Sector Trial Scheme of the Innovation and Technology Commission of the Hong Kong SAR (Ref. ITT/003/ 13GP) and the SMART Program,
doi:10.1016/j.jot.2016.06.144 fatcat:ztwrv5zmyndktbkhhfqbazz4yq