A MICROFLUIDIC SYSTEM TO STUDY THE EFFECTS OF
MECHANICALLY LOADED OSTEOCYTES ON OSTEOCLAST
RECRUITMENT AND FORMATION
K. Middleton* and L. You
University of Toronto, CANADA
ABSTRACT
Balanced bone remodeling is critical for maintaining healthy bone, and is led by osteoclasts that
remove old bone. To date, there is no effective in vitro platform to study osteocyte-osteoclast
communication in real-time, specifically how it pertains to bone remodeling. We have developed a
microfluidic co-culture system that shows that unloaded osteocytes promote osteoclast migration and
differentiation.
KEYWORDS: Osteocytes, Osteoclasts, Bone Remodeling, Basic Multicellular Unit, Fluid Shear Stress,
Mechanotransduction, Microfluidics, Receptor Activator Of Nuclear Factor Kappa-β Ligand,
Osteoprotegerin.
INTRODUCTION
It has been well established that bone remodels to accommodate the loads under which it is placed
[1]. This remodeling occurs through the action of the basic multicellular unit (BMU), where osteoclasts
remove old bone and osteoblasts secrete new bone matrix [2]. In healthy bone, a balance exists between
bone removal and formation; however, in many bone mass diseases, such as osteoporosis, this balance
breaks down. By better understanding the underlying mechanisms that links bone loading to bone
remodeling and net bone formation, it will be possible to develop drugs to target specific pathways to
reduce the impact of these diseases. A third bone cell type, the osteocyte, is believed to play a significant
role in providing this link. Osteocytes are well distributed throughout the bone, sense mechanical
stimuli, and produce a variety of signals that are critical to bone remodeling, such as Receptor Activator
of Nuclear Factor kappa-β Ligand (RANKL) and Osteoprotegerin (OPG) [3].
Current in vitro bone mechanotransduction studies typically occur within macro-scaled parallel
plate flow chambers. To study how the different bone cells interact with one another, conditioned
medium from the flow chamber is added to a different cell population [4]. This methodology, however,
is severely limited in that it doesn’t allow for real-time dynamic signaling, as well as preventing crosstalk between these cell populations within physiologically-relevant distances.
To mitigate these issues and investigate how osteocytes play a role in initiating and guiding bone
remodeling in terms of communicating to osteoclast precursors, we have developed a novel microfluidic
system. This system promotes dynamic cell-signaling and real-time evaluation of cellular response in
multiple directions.
EXPERIMENTAL
We developed a Polydimethylsiloxane (PDMS) device using soft lithography. This system consists
of three parallel cell seeding channels (60μm x 1mm). These channels are separated by 200μm long
high-resistance side channels (60μm x 20μm), which prevent convective fluid flow between channels
while still allowing for solute diffusion.
In this device, osteoclast precursors (RAW264.7 cells) are seeded within the central channel, and an
osteocyte model (MLO-Y4 cells, a gift from Lynda Bonewald) are seeded within the two outer channels
as shown in Figure 1. For the duration of this experiment, all cells receive a perfusion flow (1μl/min) of
their respective culture media (RAW264.7 – 87% DMEM, 10% FBS, 2% L-Glutamine, 1% P/S; (MLOY4 (low proliferation) – 96.5% α-MEM, 2.5% FBS, 1% P/S). For one hour once per day, one channel of
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18th International Conference on Miniaturized
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osteocytes receives a stimulatory steady flow (1Pa) while the other two channels are blocked and
completely denied of flow.
To analyze the degree of migration within the central channel because of the cell generated signal
gradient, we imaged the central channel once per day. Images of the central channel were divided into 5
columns of 200μm each. Cells were counted within each column and normalized to the original seeded
cell density.
After 7 days, the cells were also quantified in terms of osteoclastogenesis as can be observed in
Figure 1. Multinucleated cells (>3 nuclei) were identified as osteoclasts and spatially counted across the
width of the channel. Additionally, cells were stained for tartrate resistant acid phosphatase (TRAP), a
known marker of osteoclasts.
Figure 1: (A) Stitched together image of cells seeded within device, with MLO-Y4 cells (OCY) in outer channels and
RAW264.7 cells (OCL) in central channel. (B) Experimental layout of device; one channel undergoes stimulatory
steady flow (SF), and the other channels receive a perfusion flow (PF). (C) Phase contrast image of multinucleated
osteoclasts. (D) TRAP stain for osteoclasts; TRAP is identified by a reddish hue.
RESULTS AND DISCUSSION
COMSOL modeling (not shown) successfully showed that we would be able to generate a significant
shear stress within the fluid flow channel while producing no significant flow within the other two
channels. Further COMSOL modeling showed that a chemical gradient of RANKL and OPG produced
within an osteocyte channel would be able to diffuse up to 400μm into an adjacent channel. Validation
of this result is shown through fluorescent dye experiments (not shown), where under a specific perfusion
flow (chosen so the diffusion of the dye would be similar to either protein) diffusion of the dye was
observed up to 350μm into the channel.
In the co-culture system we observed a significant increase in normalized cell density in the vicinity
of the fluid shear stimulated osteocytes compared to unstimulated osteocytes. Additionally, preliminary
osteoclastogenesis results showed an increase in density closer to unloaded osteocytes.
These results (shown in Figure 2) first and foremost suggest that signals produced from osteocytes
support the recruitment of osteoclast precursors, and also promote their differentiation into osteoclasts,
which is required for the removal of old and damaged bone.
Future work with this system will try to better investigate the role of the variety of signals involved in
recruiting precursor cells as well as osteoclastogenesis. We will do this through the systematic blocking
of some of the signals produced by the MLO-Y4 cells, such as RANKL and Nitric Oxide. Additionally,
we will investigate the cross-talk that is occurring back from the osteoclasts to the osteocytes.
Specifically, we will study any change in the response of osteocytes to mechanical stimulation in terms
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of [Ca2+]i release due to the presence of osteoclasts. This could present us with a unique mechanism in
which osteocytes become more sensitive to mechanical loading, and thus are better prepared to promote
the recruitment of osteoprogenitors and their differentiation into bone secreting osteoblasts.
Figure 2: (A) Cell density change after 5 days normalized to original seeding density, N=2. (B) Osteoclast density
across width of channel after 7 days, N=1. Different letters signify a significant difference (P<0.05).
CONCLUSION
We have successfully developed a device to study cross-communication between osteocytes and osteoclasts. Specifically, we have shown that unstimulated osteocytes, such as those found at the head of
the BMU, aid in the recruitment of osteoclast precursors to the resorption site. Additionally, we have
shown that these same osteocytes also facilitate the formation of osteoclasts to begin resorbing the old
bone in which they are placed. This unique device can similarly be used to study cross-talk between the
many cell types involved in bone remodeling, and could be used to study osteoclast-osteoblast, osteocyteosteoblast, or osteocyte-endothelial communication. This will provide a physiologically relevant method
to better understand the mechanisms involved within bone remodeling, and potentially aid in discovering
novel drug targets for combating a variety of bone mass diseases, such as osteoporosis.
ACKNOWLEDGEMENTS
The authors would like to thank NSERC, CIHR and the TMC for providing funding for this research.
Additionally, the authors would like to thank Xueting Mei, Chao Liu, and Mike Borrett for their assistance with this work.
REFERENCES
[1] J. H. Chen, C. Liu, L. You, and C. A. Simmons, "Boning up on Wolff's Law: Mechanical regulation
of the cells that make and maintain bone," Journal of Biomechanics, vol. 43, pp. 108-118, 2010.
[2] D. J. Hadjidakis and Androulakis, II, "Bone remodeling," Ann N Y Acad Sci, vol. 1092, pp. 385-96,
Dec 2006.
[3] L. F. Bonewald, "The amazing osteocyte," J Bone Miner Res, vol. 26, pp. 229-38, Feb 2011.
[4] S. D. Tan, T. J. de Vries, A. M. Kuijpers-Jagtman, C. M. Semeins, V. Everts, and J. Klein-Nulend,
"Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption," Bone, vol. 41,
pp. 745-51, Nov 2007.
CONTACT
* K. Middleton; phone: 416-697-4640; [email protected]
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