Elsevier

Bone

Volume 54, Issue 2, June 2013, Pages 296-306
Bone

Review
In vitro and in vivo approaches to study osteocyte biology

https://doi.org/10.1016/j.bone.2012.09.040Get rights and content

Abstract

Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models.

We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed.

In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes.

This article is part of a Special Issue entitled "The Osteocyte".

Introduction

Osteocytes represent more than 95% of the cellular component of mature adult bone [1], [2], [3]. They are stellate shaped cells enclosed within the skeletal lacuno-canalicular network. Morphologically they are characterized by numerous elongated cell processes and dendrites extending into channels in the matrix called canaliculi (~ 250–300 nm in diameter) [4]. The shape of embedded osteocytes is dependent on their location with cells present in trabecular bone exhibiting rounded morphology whereas osteocytes from cortical bone are elongated [5]. These cells display polarity in terms of the distribution of their cell processes with the majority coming from the cell membrane facing the bone surface [6]. Osteocytes are connected, via gap junctions, to neighboring osteocytes, to cells on the bone surface such as lining cells, osteoclasts and osteoblasts [7] and to pericytes of capillaries, which supply nutrients and oxygen to osteocytes and other bone cells [8]. Furthermore, Kamioka et al., suggest the existence of a direct signaling system between the osteocytes and the bone marrow compartment without involvement of the osteoblast/lining cells [9]. Dynamic imaging studies showed that the dendritic connections between cells in the bone matrix, and into marrow spaces, appear to be able to extend and retract [10], [11]. Osteocytes also appear to show undulating motion of their cell bodies within their lacunae, suggesting that they are not inactive cells, as previously thought.

During the transition from osteoblast to osteocyte, the cell undergoes a dramatic transformation that requires extensive restructuring of the cytoskeletal and intracellular machineries. Cells at the early stages of osteocyte differentiation are larger than mature osteocytes and have numerous ribosomes, a well-developed endoplasmic reticulum and a wide Golgi complex [3]. Once the osteoid mineralizes, they decrease protein synthesis and secretion [12]. Moreover, osteocytes lose the apical and the basolateral plasma membrane polarization normally seen in osteoblasts [13]. The study of different markers shows that osteocyte differentiation is accompanied by the progressive reduction of numerous bone markers (bone sialoprotein, collagen type I, alkaline phosphatase (ALP), osteocalcin, Runx2), the maintenance of some others (osteopontin and E11/gp38 antigen), and the appearance of new markers (dentin matrix protein 1 (DMP1), CD44, matrix extracellular phosphoglycoprotein (MEPE), phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), sclerostin (SOST) and fibroblast growth factor 23 (FGF23)) [3], [12], [14]. Osteocytes have proved difficult to isolate and study due to their localization within the mineralized matrix, and the fact that they are terminally differentiated post-mitotic cells. This review will discuss in vitro and in vivo techniques and tools that have enabled great advances in our understanding of osteocyte biology in recent years.

Section snippets

Primary culture of osteocytic cells

Over the last 20 years a number of methods have been developed to study osteocytes in vitro and this has greatly expanded our knowledge on the autocrine and paracrine functions of these inaccessible cells. Van der Plas and Nijweide [15] were the first to report a method to isolate and culture osteocytes from embryonic chicken calvaria. After releasing cells by serial collagenase/EDTA digestion, they purified osteocytes by immunomagnetic separation with a monoclonal antibody MAb OB 7.3 later

Osteocyte-like cell lines

Development of osteocyte-like cell lines has greatly aided investigations of osteocyte biology. A summary of the sources and phenotypic characteristics of cell lines that have been reported is shown in Table 1. The most widely used cell lines, MLO-Y4 and MLO-A5, were developed by Lynda Bonewald's group [23], [24]. These are immortalized cells and undergo proliferation which is not a characteristic of osteocytes, however they show stellate morphology and expression of selected osteocyte marker

Use of osteocyte-like cell cultures to understand mechanobiology in bone

In healthy individuals bone accrues in response to a weight bearing activity, and is lost in the absence of mechanical stimuli. The osteocyte network is thought to be the main mechanosensor orchestrating this process, most likely by sensing changes in fluid flow that occur in the canalicular network in response to loading [52], [53]. It is not clear how this occurs, but it may involve the dendritic processes [54], [55], the cell body and/or the primary cilium [56], [57]. Modeling in vivo

Transgenic approaches to identify and characterize osteocytes

Osteoprogenitor cell differentiation is a process that is much easier to document in an in vitro than in an in vivo context [70]. Becoming an osteocyte represents terminal differentiation of the osteoprogenitor lineage. Until recently, osteocyte isolation was performed using only a few osteocyte-specific antibodies such as monoclonal antibodies MAb OB7.3 [71], MAb SB5 [72] and MAb OB37.11 [73], specific for avian osteocytes. Breakthroughs in osteocyte isolation were made, thanks to the

New transgenic reporter mice for studying osteocyte biology

New recombineering approaches have been used to make transgenic mice for studying the complete cis-regulatory regions of genes expressed in osteocytes. Patients with Van Buchem disease lack mutations in SOST coding regions, but they have a homozygous 52 kb noncoding deletion downstream of SOST gene and upstream of the MEOX1 gene. The first study to utilize the human SOST BAC sequences confirmed that this deletion affected a SOST-specific regulatory element resulting in a phenotype with

The osteoblast versus the osteocyte

The mechanism of transition of osteoblasts into osteocytes remains poorly defined. To better understand this process and define the gene expression changes between osteoblasts and osteocytes, dual transgenic mice expressing Dmp1-GFP (topaz) and Col2.3CFP (cyan, excitation 433 nm, emission 475 nm) were utilized. Osteocytes were identified as Dmp1-GFP+ cells, while osteoblasts are represented by the Col2.3CFP+/Dmp1-GFP population [89]. This combinatorial approach allows for more detailed

Models to evaluate the effects of osteocyte-targeted gene deletion or overexpression

In the past decade the use of the Cre/loxP technology became a popular method to evaluate gene deletion or to target genes to tissues or particular stages within specific cell lineages. The efficacy and specificity of the recombination depend on the promoter utilized as well as the particular gene locus that is targeted. To direct recombination to the osteoprogenitor lineage, multiple promoter-Cre transgenic mice have been generated. Cre has been utilized to target earlier stages of

Use of osteocyte enrichment by serial digestion of bone

To evaluate the specificity of gene deletion in osteocytes, Kramer et al. utilized serial digestions of long bones [100]. The third digestion presented an osteoblastic phenotype, while the sixth digestion contains cells that expressed Dmp1 and Sost, defining it as an osteocyte population [100], [101]. This approach can be utilized to evaluate the effects of Cre recombination on the expression of a targeted gene within osteocyte and osteoblast populations.

Breeding Dmp1-GFP into the Dmp1-Cre targeted deletion model

To confirm that Dmp1-Cre deletion was

Strategies to target overexpression or gene deletion in osteocytes

Deleting genes in a lineage-specific manner using the Cre/loxP system has provided insights into numerous aspects in bone biology. Following reports using Oc-Cre and Col2.3 kb-Cre models that act on osteoblasts and osteocytes, the Dmp1 promoter has become the main tool to target the expression or deletion of genes to the most mature stages of the osteoblast lineage (Table 2). Transgenic mice expressing a constitutively active PTH receptor (caPTH1R) under the control of the 8 kb Dmp1 (8 kb

Summary

We have described the in vitro and in vivo models that are currently available to study osteocyte biology. The identification of several osteocyte specific genes has allowed the generation of a plethora of new data on osteocyte functions. Researchers have shown that the Dmp1 promoter can be successfully utilized to overexpress or delete genes in the most mature cells of the osteoblast lineage. Currently, a DMP1-GFP model is used to isolate osteocyte cell populations, while few models are

Acknowledgments

We would like to thank Dr. Jerry Feng for providing us with 10 kb Dmp1-Cre mice. The loading of bone tubes is a result of collaboration with Dan Nicollela at Southwest Research Institute in San Antonio, TX. We thank Dr. Teresita Bellido and Dr. Lilian Plotkin for their important comments and suggestions. This work was supported by NIH/NIAMS grant AR059315 to IK.

References (118)

  • L. You et al.

    Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading

    Bone

    (2008)
  • T.J. Heino et al.

    Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

    Exp Cell Res

    (2004)
  • J. Klein-Nulend et al.

    Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts–correlation with prostaglandin upregulation

    Biochem Biophys Res Commun

    (1995)
  • N.E. Ajubi et al.

    Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes—a cytoskeleton-dependent process

    Biochem Biophys Res Commun

    (1996)
  • M.A. Kamel et al.

    Activation of beta-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE(2): implications for the study of mechanosensation in bone

    Bone

    (2010)
  • R.G. Bacabac et al.

    Round versus flat: bone cell morphology, elasticity, and mechanosensing

    J Biomech

    (2008)
  • A. Miyauchi et al.

    Parathyroid hormone-activated volume-sensitive calcium influx pathways in mechanically loaded osteocytes

    J Biol Chem

    (2000)
  • S.D. Tan et al.

    Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption

    Bone

    (2007)
  • P.S. Vezeridis et al.

    Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

    Biochem Biophys Res Commun

    (2006)
  • A. Bakker et al.

    Shear stress inhibits while disuse promotes osteocyte apoptosis

    Biochem Biophys Res Commun

    (2004)
  • S.P. Bruder et al.

    Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes

    Bone

    (1990)
  • X. Li et al.

    Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling

    J Biol Chem

    (2005)
  • O. Leupin et al.

    Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function

    J Biol Chem

    (2011)
  • Z. Kalajzic et al.

    Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters

    Bone

    (2002)
  • I. Kalajzic et al.

    Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene

    Bone

    (2004)
  • W. Yang et al.

    Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo

    J Biol Chem

    (2005)
  • D.P. Nicolella et al.

    Osteocyte lacunae tissue strain in cortical bone

    J Biomech

    (2006)
  • D.P. Nicolella et al.

    Machine vision photogrammetry: a technique for measurement of microstructural strain in cortical bone

    J Biomech

    (2001)
  • F. Paic et al.

    Identification of differentially expressed genes between osteoblasts and osteocytes

    Bone

    (2009)
  • C. Maes et al.

    Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels

    Dev Cell

    (2010)
  • M. Zhang et al.

    Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization

    J Biol Chem

    (2002)
  • G. Marotti

    The structure of bone tissues and the cellular control of their deposition

    Ital J Anat Embryol

    (1996)
  • A.M. Parfitt

    Bone forming cells in clinical conditions

  • T.A. Franz-Odendaal et al.

    Buried alive: how osteoblasts become osteocytes

    Dev Dyn

    (2006)
  • C. Palumbo et al.

    Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes

    Acta Anat. (Basel)

    (1990)
  • L.F. Bonewald

    Generation and function of osteocyte dendritic processes

    J Musculoskelet Neuronal Interact

    (2005)
  • P. Veno et al.

    Live imaging of osteocytes within their lacunae reveals cell body and dendrite motions

    J Bone Miner Res

    (2006)
  • S.L. Dallas et al.

    Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5

    Cells Tissues Organs

    (2009)
  • D.A. Cameron et al.

    Changes in the fine structure of bone cells after the administration of parathyroid extract

    J Cell Biol

    (1967)
  • G. Gu et al.

    Isolated primary osteocytes express functional gap junctions in vitro

    Cell Tissue Res

    (2006)
  • K.E. Poole et al.

    Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation

    FASEB J

    (2005)
  • A. van der Plas et al.

    Isolation and purification of osteocytes

    J Bone Miner Res

    (1992)
  • I. Westbroek et al.

    Osteocyte-specific monoclonal antibody MAb OB7.3 is directed against phex protein

    J Bone Miner Res

    (2002)
  • A. van der Plas et al.

    Characteristics and properties of osteocytes in culture

    J Bone Miner Res

    (1994)
  • E.M. Aarden et al.

    Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

    Histochem Cell Biol

    (1996)
  • Y. Mikuni-Takagaki et al.

    Matrix mineralization and the differentiation of osteocyte-like cells in culture

    J Bone Miner Res

    (1995)
  • A.R. Stern et al.

    Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

    Biotechniques

    (2012)
  • Y. Kato et al.

    Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture

    J Bone Miner Res

    (2001)
  • Y. Kato et al.

    Establishment of an osteocyte-like cell line, MLO-Y4

    J Bone Miner Res

    (1997)
  • J. Rosser et al.

    Studying osteocyte function using the cell lines MLO-Y4 and MLO-A5

  • Cited by (129)

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