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

Chikara Sato,Daiju Yamazaki,Hiroshi Takeshima,Eiko Sakai,Mari Sato,Yuri Hatano,Nassirhadjy Memtily,Takayuki Tsukuba

doi:10.1038/s41598-019-43608-6

Abstract

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.

Highlights

Mineralization plays a crucial role in life
Based on protocols developed for other samples including neurons[23,24,25], bacteria biofilm[26], Langerhans islet in pancreas[27] and cardiac- and skeletal- muscles[28] bearing end plates[29], the cells and tissues were aldehyde fixed, immersed in aqueous solution containing a radical scavenger - to minimize the effect of radicals formed by electron radiation at low-accelerating voltage (30 kV) suppressing knock-on damage - and imaged by atmospheric scanning EM (SEM) (ASEM) (Fig. 1)
Mineralization onset varied from area to area on the same dish and from dish to dish: almost half of the ASEM dishes were not mineralized at this time-point

Summary

Introduction

Mineralization plays a crucial role in life. As animals evolved, the formation of extracellular collagen conferred mechanical strength to multicellular organisms, allowing the size of their bodies to increase. Calcium phosphate (CaP) precipitation followed by oxidization formed hydroxylapatite (HA) on crosslinked collagen fibers, leading to bone formation. This resulted in further body enlargement, especially during the evolution of air-breathing vertebrates. Environmental capsules were developed to image wet samples for TEM11–13 and SEM14 Such systems have been successfully employed to image hydrophilic molecular complexes[15], cells[16] and tissues[17,18]. This development led to the first visualization of biological mineralization in wet conditions. To identify osteoclasts, immuno-EM of tissue in solution was realized for the first time using anti-cathepsin K protease antibody

Methods

Results

Discussion

Conclusion