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Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM

The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of b...

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Autores principales: Sato, Chikara, Yamazaki, Daiju, Sato, Mari, Takeshima, Hiroshi, Memtily, Nassirhadjy, Hatano, Yuri, Tsukuba, Takayuki, Sakai, Eiko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517404/
https://www.ncbi.nlm.nih.gov/pubmed/31089159
http://dx.doi.org/10.1038/s41598-019-43608-6
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author Sato, Chikara
Yamazaki, Daiju
Sato, Mari
Takeshima, Hiroshi
Memtily, Nassirhadjy
Hatano, Yuri
Tsukuba, Takayuki
Sakai, Eiko
author_facet Sato, Chikara
Yamazaki, Daiju
Sato, Mari
Takeshima, Hiroshi
Memtily, Nassirhadjy
Hatano, Yuri
Tsukuba, Takayuki
Sakai, Eiko
author_sort Sato, Chikara
collection PubMed
description The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of bone mineralization surrounded by many cells and matrices in natural aqueous buffer, decreasing the risk of changes. In osteoblast primary cultures, mineralization was visible without staining. Correlative energy dispersive X-ray spectrometry indicated the formation of calcium phosphate mineral. Fixed bone was sectioned, and the section surface was inspected by ASEM. Mineralized trabeculae of talus spongy bone were directly visible. Associated large and small cells were revealed by phosphotungstic acid staining, suggesting remodeling by bone-absorbing osteoclasts and bone-rebuilding osteoblasts. In tibia, cortical bone layer including dense grains, was bordered by many cells with protrusions. Tissue immuno-EM performed in solution for the first time and anti-cathepsin-K antibody, successfully identified osteoclasts in femur spongy bone. A microfluidics chamber fabricated on the silicon nitride film window of an ASEM dish allowed mineralization to be monitored in vitro; calcium phosphate crystals as small as 50 nm were imaged. ASEM is expected to be widely applied to study bio-mineralization and bone-remodeling, and to help diagnose bone-related diseases.
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spelling pubmed-65174042019-05-24 Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM Sato, Chikara Yamazaki, Daiju Sato, Mari Takeshima, Hiroshi Memtily, Nassirhadjy Hatano, Yuri Tsukuba, Takayuki Sakai, Eiko Sci Rep Article The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of bone mineralization surrounded by many cells and matrices in natural aqueous buffer, decreasing the risk of changes. In osteoblast primary cultures, mineralization was visible without staining. Correlative energy dispersive X-ray spectrometry indicated the formation of calcium phosphate mineral. Fixed bone was sectioned, and the section surface was inspected by ASEM. Mineralized trabeculae of talus spongy bone were directly visible. Associated large and small cells were revealed by phosphotungstic acid staining, suggesting remodeling by bone-absorbing osteoclasts and bone-rebuilding osteoblasts. In tibia, cortical bone layer including dense grains, was bordered by many cells with protrusions. Tissue immuno-EM performed in solution for the first time and anti-cathepsin-K antibody, successfully identified osteoclasts in femur spongy bone. A microfluidics chamber fabricated on the silicon nitride film window of an ASEM dish allowed mineralization to be monitored in vitro; calcium phosphate crystals as small as 50 nm were imaged. ASEM is expected to be widely applied to study bio-mineralization and bone-remodeling, and to help diagnose bone-related diseases. Nature Publishing Group UK 2019-05-14 /pmc/articles/PMC6517404/ /pubmed/31089159 http://dx.doi.org/10.1038/s41598-019-43608-6 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Sato, Chikara
Yamazaki, Daiju
Sato, Mari
Takeshima, Hiroshi
Memtily, Nassirhadjy
Hatano, Yuri
Tsukuba, Takayuki
Sakai, Eiko
Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title_full Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title_fullStr Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title_full_unstemmed Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title_short Calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric SEM (ASEM) without staining: microfluidics crystallization chamber and immuno-EM
title_sort calcium phosphate mineralization in bone tissues directly observed in aqueous liquid by atmospheric sem (asem) without staining: microfluidics crystallization chamber and immuno-em
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517404/
https://www.ncbi.nlm.nih.gov/pubmed/31089159
http://dx.doi.org/10.1038/s41598-019-43608-6
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