Contributions of Resin Cast Etching to Visualising the Osteocyte Lacuno-Canalicular Network Architecture in Bone Biology and Tissue Engineering

Abstract

Recent years have witnessed an evolution of imaging technologies towards sophisticated approaches for visualising cells within their natural environment(s) and for investigating their interactions with other cells, with adjacent anatomical structures, and with implanted biomaterials. Resin cast etching (RCE) is an uncomplicated technique involving sequential acid etching and alkali digestion of resin embedded bone to observe the osteocyte lacuno-canalicular network using scanning electron microscopy. This review summarises the applicability of RCE to bone and the bone-implant interface. Quantitative parameters such as osteocyte size, osteocyte density, and number of canaliculi per osteocyte, and qualitative metrics including osteocyte shape, disturbances in the arrangement of osteocytes and canaliculi, and physical communication between osteocytes and implant surfaces can be investigated. Ageing, osteoporosis, long-term immobilisation, spinal cord injury, osteoarthritis, irradiation, and chronic kidney disease have been shown to impact osteocyte lacuno-canalicular network morphology. In addition to titanium, calcium phosphates, and bioactive glass, observation of direct connectivity between osteocytes and cobalt chromium provides new insights into the osseointegration potential of materials conventionally viewed as non-osseointegrating. Other applications include in vivo and in vitro testing of polymer-based tissue engineering scaffolds and tissue-engineered ossicles, validation of ectopic osteochondral defect models, ex vivo organ culture of whole bones, and observing the effects of gene dysfunction/deletion on the osteocyte lacuno-canalicular network. Without additional contrast staining, any resin embedded specimen (including clinical biopsies) can be used for RCE. The multitude of applications described here attest to the versatility of RCE for routine use within correlative analytical workflows, particularly in biomaterials science.

Mesoscale characterization of osseointegration around an additively manufactured genistein-coated implant

“…The formation of an interconnected network of osteocytes was visualized by resin cast etching (Fig. 4 A), which is a facile method to expose the LCN and directly visualize it by SEM 37 – 39 . However, despite the 3D-like appearance of SEM images post-resin cast etching, such images are inherently 2D.…”

Bone hierarchical organization through the lens of materials science: Present opportunities and future challenges

“…SEs are generated from the first few tens of nanometers below the sample surface, hence the SE signal is mostly affected by surface topography ( Goldstein et al, 2018 ). While SE-SEM is of little use for imaging embedded and polished bone surfaces, it is the imaging mode of choice when inspecting bone samples rich in topographical cues, for example after treatments such as acid etching to reveal the resin-infiltrated LCN ( Sato and Shah, 2023 ) ( Fig. 4 B), or deproteinization, where mineral organization at the osteocyte lacuna floor ( Shah et al, 2016d ) or marquise-shaped mineral agglomerates ( Shah et al, 2020 ) can be imaged ( Fig.…”

“…This review discusses several techniques used in such a multimodal and multiscale characterization platform of bone, covering basic working principles and example applications of both conventional and advanced techniques. The interested reader may further refer to existing technique-specific reviews on X-ray tomography ( Müller, 2009 ; Peyrin et al, 2014 ) and X-ray scattering ( Paris, 2008 ), scanning electron microscopy (SEM) ( Shah et al, 2019b ), including resin cast etching ( Sato and Shah, 2023 ), focused ion beam (FIB)-SEM ( Weiner et al, 2021 ), Fourier transform infrared (FTIR) and Raman spectroscopy ( Taylor and Donnelly, 2020 ), and atom probe tomography (APT) ( Grandfield et al, 2022 ). Instead, we aim to complement existing reviews on multiscale characterization of bone ( Grandfield, 2015 ; Georgiadis et al, 2016 ; Binkley and Grandfield, 2017 ; Palmquist, 2018 ), specifically discussing current opportunities offered by this materials science-inspired characterization approach, highlighting the limitations, and addressing future challenges.…”