Towards quantitative 3D imaging of the osteocyte lacuno-canalicular network

Abstract

Osteocytes are the most abundant cells in bone and the only cells embedded in the bone mineral matrix. They form an extended, three-dimensional (3D) network, whose processes interconnecting the cell bodies reside in thin canals, the canaliculi. Together with the osteocyte lacunae, the canaliculi form the lacuno-canalicular network (LCN). As the negative imprint of the cellular network within bone tissue, the LCN morphology is considered to play a central role for bone mechanosensation and mechanotransduction. However, the LCN has neither been visualized nor quantified in an adequate way up to now. On this account, this article summarizes the current state of knowledge of the LCN morphology and then reviews different imaging methods regarding the quantitative 3D assessment of bone tissue in general and of the LCN in particular. These imaging methods will provide new insights in the field of bone mechanosensation and mechanotransduction and thus, into processes of strain sensation and transduction, which are tightly associated with osteocyte viability and bone quality.

Introduction

Bone is a hierarchically organized tissue, which is able to repair and adapt itself dynamically to its mechanical environment. The different aspects of bone adaptation, such as osteogenesis, bone modeling, and bone remodeling processes, are typically observed at the whole organ and tissue level. But it is at the cellular level (1–10 μm) where bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts) alter the local structure of the bone tissue. Consequently, it is of vital importance to examine bone at the cellular level.

The most abundant bone cells are osteocytes. They are the only cells embedded in the calcified bone matrix and they occupy small cavities called osteocyte lacunae. Osteocytes are connected with neighboring cells by means of dendritic cell processes. In this way, osteocyte cell bodies and osteocyte processes form a three-dimensional (3D) cellular network, called the osteocyte network. The processes interconnecting the cell bodies within the mineral matrix reside in thin canals, which are called canaliculi. Accordingly, the osteocyte lacunae and the canaliculi represent the negative imprint of the osteocyte network within the bone matrix, which we will refer to as lacuno-canalicular network (LCN) in this article.

Contrary to osteoblasts and osteoclasts, the many functions attributed to osteocytes are not yet fully understood [1]. In particular, osteocytes are thought to sense and to respond to mechanical stimuli and to send signals triggering resorption and formation to osteoclasts and osteoblasts, respectively. Consequently, they are thought to play a pivotal role for bone mechanosensation and mechanotransduction processes, which are essential for bone modeling and remodeling [2], [3], [4], [5]. Moreover, osteocytes are believed to be involved in the regulation of bone phosphate metabolism [6], [7] and their viability is thought to be closely associated to the many aspects of bone quality [8], [9]. It is primarily due to the abundance of osteocytes throughout the bone matrix and their vast interconnectivity, which provide the requisites for strain sensing and cellular communication involved in bone mechanosensation and mechanotransduction. Signals may be transmitted through osteocyte processes or via bone fluid within the LCN. However, up to now, there is no conclusive answer to the questions how strain is sensed by osteocytes or how potential signals are conveyed to other cells [1]. Possible strain sensing mechanisms include direct strain sensation by the osteocyte due to matrix deformations or indirect strain sensation mediated by fluid flow [10], [11], [12], [13], [14], [15]. In any case, all these strain-sensing mechanisms imply that the signal sensed by the osteocytes depends on the geometry of the LCN.

To clarify the paradox that the magnitude of macroscopic in vivo load magnitudes is too small to cause a cellular response in bone cells in vitro [11], [16], [17], [18], [19] strain-sensing mechanisms need closer attention. Hence, local matrix strains or fluid flow-induced shear stresses should ideally be measured in response to macroscopic strains in situ. This could further elucidate the role of the osteocytes for bone mechanosensation and mechanotransduction. Unfortunately, the direct strain measurements in situ pose an intricate problem because the LCN is inaccessible for direct observation. For this reason, different computational models, such as finite element (FE) and computational fluid dynamic (CFD) models, have been proposed to investigate local matrix strains around osteocyte lacunae and canaliculi [20], [21], [22] and to study wall shear stresses at the cell–fluid interface [23], [24], respectively. Calculated strain values were found to be several times higher than the imposed macroscopic strains. Additionally, it was shown that geometric idealization of the LCN may not be appropriate to calculate local strains [20] and further, cause a profound underestimation of forces transduced to osteocytes via fluid flow-induced shear stresses [24]. Consequently, refined computational models should be created, which are derived directly from 3D image data of the LCN at the cellular and sub-cellular level. The 3D images will then facilitate both morphometric assessment of the LCN and form a basis for direct conversion of these images to 3D microstructural models to be used for simulations. Nevertheless, the relative inaccessibility of the LCN, which is buried deeply within the bone matrix, did not allow for a true 3D mapping of the LCN so far.

Due to the absence of an established strategy to assess the 3D nature of the LCN, this article comprises two sections: First, the current state of knowledge relating to the LCN morphology is reviewed. This review includes findings in respect of shape, size, orientation, and distribution of osteocyte lacunae and canaliculi as well as a survey on the influence of age and different pathological states on the LCN. Within the second section, potential imaging methods for the assessment of bone tissue in general, and for the LCN in particular are discussed, where the focus is on the 3D imaging capabilities of these methods.