THE ROLE OF OSTEOCYTES IN DISUSE
AND MICROGRAVITY-INDUCED BONE LOSS
byMASCUETINTTT
AF
TCHNOG
Jordan Matthew Spatz
B.S., M.S. University of Colorado at Boulder
SEP 2 4 2015
Submitted to the
Harvard-MIT Program in Health Sciences and Technology
in Partial Fulfillment of the Requirements for the Degree of
LIBRARIES
DOCTOR OF PHILOSOPHY IN HEALTH SCIENCES AND TECHNOLOGY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 2015
2015 Massachusetts Institute of Technology. All rights reserved.
Signature of Author:
Signature redacted
Harvard-MIT Program in Health Sciences and Technology
September 1 st, 2015
Certified by:
Signature redacted
Mary L. Bouxsein, Ph.D.
Associate Professor of Orthopedic Surgery, Harvard Medical School
Thesis Supervisor
A
Certified by:
A
Signature redacted
___
i aola Divieti Pajevic, MD, Ph.D.
Ass ciate Professor of Iolecular and Cell, Boston University
Thesiy Supervisor
Accepted byr.
Signature redacted
Emery N. Brown, MD, Ph.D.
Director, Harv d-MIT Program in Health Sciences and Technology
Professor of Computational Neuroscience & Health Sciences and Technology
I
2
The Role of Osteocytes in Disuse
and Microgravity-Induced Bone Loss
by
Jordan Matthew Spatz
B.S., M.S. University of Colorado at Boulder
Submitted to the
Harvard-MIT Health Sciences and Technology
September 2015, in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy in Health Sciences and Technology
Abstract
A human mission to Mars will be physically demanding and presents a variety of
medical risks to crewmembers. It has been recognized for over a century that
loading is fundamental for bone health, and that reduced loading, as in prolonged
bed rest or space flight, leads to bone loss. Osteocytes, the most abundant bone
cell type, are thought to be key mechanical sensors in bone, yet the molecular
mechanism of this action remains poorly understood. Improved understanding of
how osteocytes regulate skeletal responses to mechanical loading and unloading
could have significant implications for treatment of bone disorders related to
disuse or immobilization. Thus, we conducted in vitro and in vivo studies on
osteocytes exposed to unloading to investigate their role in disuse and
microgravity-induced bone loss. Specifically, we generated and characterized a
novel osteocytic cell line that recapitulates the response to hormonal and
mechanical stimuli of osteocytes in vivo. This novel cell line provided the first
evidence of a cell-autonomous increase in sclerostin, a potent inhibitor of Wntsignaling, following exposure to simulated microgravity. These cells were also
used for a spaceflight mission after demonstrating their ability to maintain an
osteocytic phenotype when cultured in a fully automated flight-certified system.
Finally, we utilized murine models of unloading to show that pharmacologic
inhibition of sclerostin induces bone formation and prevents disuse-induced bone
loss.
Thesis Supervisor: Mary L. Bouxsein, Ph.D.
Associate Professor of Orthopedic Surgery, Harvard Medical School
Thesis Supervisor: Paola Divieti Pajevic, MD, Ph.D.
Associate Professor of Molecular and Cell Biology, Boston University
3
Thesis Committee
Laurence R. Young, Sc.D (Chair)
Apollo Program Professor of Astronautics and Professor of Health Sciences and
Technology, Director of HST PhD Program in Bioastronautics
Mary L. Bouxsein, Ph.D. (Thesis Supervisor)
Associate Professor of Orthopedic Surgery, Harvard Medical School
Associate Biologist, Endocrine Division, Massachusetts General Hospital
Adjunct Assistant Professor, Department of Mechanical Engineering,
Boston University
Faculty Member, Bioastronautics Program,
Harvard-MIT Division of Health Sciences and Technology
Paola Divieti Pajevic, MD, Ph.D. (Thesis Supervisor)
Associate Professor of Molecular and Cell Biology, Goldman School of Dental
Medicine, Boston University
Associate Biologist, Endocrine Unit, Massachusetts General Hospital
Jeffrey M Karp, Ph.D. (Thesis Reader)
Associate Professor, Harvard Medical School
Co-Director, Center for Regenerative Therapeutics, Brigham and Women's
Hospital
4
Acknowledgments
In an effort that spans so many years and milestones there are many to be
thanked. My graduate school years would be nothing were it not for those who
encouraged me to follow my dreams, supported my development academically,
personally, and as a scientist throughout this process.
To my advisors: for always having an open-door, putting my career at the top of
your list, and answering my endless questions no matter your other priorities.
Mary Bouxsein: I will always remember our afternoon advisor conversations and
hope to live up to your passion for science to do my part to make the world a
better place. Your dedication to always have my back, my career development as
a person, and a scientist has been something I will carry forward with me
throughout my life and career.
Paola Divieti Pajevic: I could not have known from coming to your lab as
engineer for help to learn to grow osteocytes how much I would learn from you. I
cannot thank you enough for passionately teaching me the amazing science of
biology that I gained from working side-by-side with you at the bench and how to
always do the best science. Thank you for the courage to allow me to try crazy
ideas and for sticking with me throughout this learning journey.
Jeff Karp: Thank you for imparting to me your forward thinking and progressive
perspective on advancing science throughout my M.I.T. graduate studies. I will
carry with, throughout my career, your advice to change the calculus when
presented with an otherwise intractable problem and remember your advice on
how to think outside the box.
5
Larry Young: My passion for bioastronautics would not have led to me to M.I.T.
without your foresight to believe in me. Thank you for enriching and enhancing
my passion for aerospace medicine and the human exploration of the universe.
To my funding sources: Northrop Grumman Aerospace Systems Ph.D. Training
Fellowship, M.I.T.
Hugh Hampton Young Fellowship, the National Space
Biomedical Research Institute through NASA NCC 9-58, Beth Israel Deaconess
Medical Center Translational Research in Aging Training Program, and the U.S.
Army Institute for Environmental Medicine Oak Ridge Science Institute for
Science and Education fellowship program.
To my NASA mentors: Jean Sibonga, Scott Smith, Zara Smith, and Honglu Wu
thank you for mentorship and hosting my research at the NASA Johnson
Spaceflight Center.
To my U.S. Army Institute of Environmental Medicine mentors: Julie Hughes and
Wayne Matheny thank you for the mentorship, friendship, and support.
To my colleagues: that helped make this thesis a reality by conducting
experiments with me throughout my graduate work, but especially Rachel
Ellman, Keertik Fulzele, Yili Qu, Shawn Liu, Chris Dedic, Forest Lai, Jonathan
Gooi, Alison Cloutier, Leeann Louis, Miranda Van Vliet, Daniel Brooks, and
Jenna Garr thank you from the bottom of my heart for the long hours and tireless
dedication.
To Hank Kronenberg: thank you for always spending time to provide deep insight
and teachings, shadowing in clinic, and for advice all things endocrine over the
many years at M.G.H.
To Marc Wein: your starting to work with the osteocyte cell line during the midstretch of my thesis studies was like having an all-star baseball reliever in
6
baseball coming out of the bullpen with the bases loaded and nobody out. Thank
you for all the career mentorship, opportunity to work with you both at the bench
and the bedside, and always spending time to help make me a better scientist.
To Chris Adamson, Lowell Misener, Margaret Eberle, and the rest of the Calm
Technologies team: thank for just being awesome for many years of dedication
and answering of my endless questions on the Osteo-4 project.
To my fellow bioastronautics and M.I.T. manned vehicle colleagues: Dava
Newman, Alan Natapoff, Leia Sterling, Alexander Bruno, Dan Buckland, Katelyn
Burkhart, Dustin Kendrick, Conor Culliane, Nikhil Vadhavkar, Justin Kaderka,
Aaron Johnson, Allie Anderson, Torin Clark, and Erika Wagner thank you for
your friendship and long conversations about human space exploration and
aerospace medicine.
To my M.G.H. endocrine unit colleagues: Lynn Moulton, Julia Maclaughlin,
Latanya Turner, Leslie Johnson, Marie Demay, Tatsuya Kobayashi, John Potts,
Henry Keutmann, Tom Gardella, Harald Jueppner, Kelly Lauter, Melissa Putnam,
Paula Cohen at Beth Israel Deaconess Medical Center, and Elizabeth Zotos at
M.I.T. this thesis would not have occurred without your tireless efforts of support
over the many years of my graduate studies.
To my Northrop Grumman colleagues: thank you for the continued dedicated
support throughout my graduate school years at M.I.T.
To the National Space Biomedical Research Institute (NSBRI): thanks to
educational support and encouragement from Dr. Jeff Sutton, Dr. John Clark,
and Amanda Hackler.
And last but first in my heart, to my friends and family: for all your love and
support!
7
8
Biographical Note
Jordan Matthew Spatz was born in Fontana, California and raised in Los
Angeles. From 2001 to 2006, he attended the University of Colorado at Boulder,
earning Masters and Bachelors degrees in Aerospace Engineering Sciences. In
2006, Jordan joined Northrop Grumman Corporation as an aerospace systems
engineer for two years prior to starting his doctoral studies at the Massachusetts
Institute of Technology. During his tenure at M.I.T. Jordan also lead an Engineers
Without Borders project to build a preschool in the Palaung hill tribe village, Ban
Nor Lae, in the province of Chiang Mai, Thailand. He held fellowships from
Northrop Grumman Corporation, M.I.T. Hugh Hampton Young, the National
Space Biomedical Research Institute, Beth Israel Deaconess Medical Center
Translational Research in Aging Training Program, and the U.S. Army Institute
for Environmental Medicine Oak Ridge Science Institute for Science and
Education.
9
"We found ourselves bidding goodbye to the old learn-by-heart method, and
beginning the study of observing the facts and laws of nature. We learned from
experiment and experience what might be expected to happen if a given set of
forces started to act.
In short, our feet were set at last in the way of real knowledge. We learned,
perhaps falteringly at the outset, the four steps that mark the only route to true
science: how to observe, how to record, how to collate, and how to conclude."
Robert Hallowell Richards, M.I.T., 1868
10
1.1
BACKGROUND
15
1.2
HYPOTHESIS AND RESEARCH OBJECTIVES
THESIS OUTLINE
16
OSTEOCYTE'S ROLE IN SKELETAL BIOLOGY
27
1.3
2
16
28
29
30
30
31
2.6
LIFE OF AN OSTEOCYTE
OSTEOCYTE ORCHESTRATION OF BONE HOMEOSTASIS
OSTEOCYTE AS BONE'S MECHANO-SENSOR
OSTEOCYTE ORCHESTRATION OF MINERAL HOMEOSTASIS
OSTEOCYTE ORCHESTRATION OF HEMATPOIESIS
OSTEOCYTE ORCHESTRATION OF IMMUNE FUNCTION AND FAT METABOLISM
2.7
SUMMARY
32
2.1
2.2
2.3
2.4
2.5
OSTEOCYTES: MICROGRAVITY AND DISUSE INDUCE BONE LOSS
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.7.1
3.7.2
SKELETAL HEALTH IN LONG DURATION SPACEFLIGHT
OSTEOCYTE ORCHESTRATION OF MECHANOTRANSDUCTION
OSTEOCYTE ORCHESTRATION OF OSTEOBLASTS IN MECHANICAL UNLOADING
OSTEOCYTE ORCHESTRATION OF OSTEOCLASTS IN MECHANICAL UNLOADING
OSTEOCYTE DEPLETED MICE ARE RESISTANT TO DISUSE-INDUCED BONE LOSS
OSTEOCYTE OSTEOLYSIS IN DISUSE-INDUCED BONE LOSS
GROUND BASED MODELS OF MICROGRAVITY AND DISUSE BONE LOSS
In-vivo (rodent) ground based models of mechanical unloading
In-vitro models of mechanical unloading
31
37
37
38
39
40
41
41
42
42
42
DEVELOPMENT AND CHARACTERIZATION OF A NOVEL OSTEOCYTIC CELL LINE
OCY454)
49
4
4.1
RATIONALE
50
4.2
INTRODUCTION
50
4.3
MATERIALS AND METHODS
51
4.3.1
4.3.2
4.3.3
4.3.4
4.3.5
4.4
4.4.1
4.4.2
4.5
5
15
INTRODUCTION
1
Osteocytic cell line
Quantitative real time pcr
Western blot
Sclerostin immunohistochemistry
Sclerostin elisa
51
52
53
53
54
RESULTS
55
Osteocytic cell line basal and hormonal characterization
Three-dimensional culture enhances osteocytic phenotype
CONCLUSION
55
63
65
OSTEOCYTES AS BONE'S GRAVITY SENSOR: CELL AUTONOMOUS INCREASES IN
67
SCLEROSTIN IN MECHANICAL UNLOADING
68
5.1
RATIONALE
68
INTRODUCTION
5.2
5.3
5.3.1
5.3.2
5.3.3
5.3.4
5.4
5.4.1
5.4.2
5.4.3
5.4.4
5.5
5.6
70
MATERIALS AND METHODS
Simulated microgravity
Two dimensional laminar fluid shear stress
Three dimensional laminar fluid shear stress
Statistical Analysis
70
71
71
71
72
RESULTS
Fluid shear stress regulation of Ocy454 in two-dimensional culture
Simulated microgravity increases SOST/sclerostin and Rankl
GPCR responsiveness: SOST/Sclerostin in simulated microgravity
Long Term fluid shear stress regulation of Ocy454
72
72
75
76
79
DISCUSSION
CONCLUSION
82
11
6
PREPARATION FOR AN OSTEOCYTE CELL LINE EXPERIMENT TO THE
INTERNATIONAL SPACE STATION
6.1
6.2
6.3
6.4
6.4.1
6.5
6.5.1
6.5.1
6.6
6.6.1
6.6.2
6.6.3
6.7
7
RATIONALE
KEY FINDINGS
FLIGHT HARDWARE FOR MICROGRAVITY BONE BIOLOGY STUDIES
OSTEo-4 UPGRADES FOR ISS COMPATIBILITY
Osteo-4 Fluid Pathway
MATERIAL AND METHODS
Cell Culture
Quantitative Real Time PCR
RESULTS
Osteo-4 spaceflight bioreactors
Osteocytic response to random launch vibration
Preservation of Osteo-4 bioreactors in space flight environment_
CONCLUSIONS
87
88
88
89
90
93
94
94
95
95
95
97
98
100
SCLEROSTIN ANTIBODY INHIBITS SKELETAL DETERIORATION DUE TO REDUCED
MECHANICAL LOADING
101
7.1
INTRODUCTION AND RATIONALE
102
7.2
7.3
7.4
7.5
7.5.1
7.5.2
7.5.3
7.5.4
7.5.5
7.5.6
7.5.7
7.6
76.1
7.6.2
7.6.3
7.6.4
7.6.5
7.6.6
7.6.7
7.7
7.8
8
PHARMACOLOGIC PREVENTION OF BONE LOSS
KEY FINDINGS
BACKGROUND
MATERIAL AND METHODS
Overview of study design _
Bone mineral density and body composition
Specimen harvesting and preparation
Bone turnover markers
Histology and quantitative histomorphometry
Mechanical testing
Statistical analysis
RESULTS
Bndvmas_
102
103
104
105
105
106
106
106
106
107
108
109
109
Muscle mass
Bone mineral density
Bone microarchitecture
Mid-femoral biomechanics and pFEA of the distal femur metaphysis
Serum sclerostin and bone turnover markers
Histomorphometry
109
109
110
115
117
118
119
122
DISCUSSION
CONCLUSION
SCLEROSTIN ANTIBODY INHIBITS SKELETAL DETERIORATION IN MICE EXPOSED
TO PARTIAL WEIGHT-BEARING IN MICE
127
8.1
8.2
8.3
8.4
127
RATIONALE
KEY FINDINGS
INTRODUCTION
MATERIAL AND METHODS
128
128
129
8.4.1
Overview of study design _
8.4.2
Partial weight-bearing (PWB) model
8.4.3
Bone mineral density and muscle mass
8.4.4
Specimen harvesting and preparation
8.4.5
Serum markers of bone metabolism
8.4.6
Bone microarchitecture
8.4.7
Mechanical testing _131
8.4.8 Statistical analysis
8.5
RESULTS
12
129
129
130
130
130
130
131
132
8.5.1
Body mass and muscle mass
8.5.2
Bone mineral density
8.5.3
Bone volume and microarchitecture
8.5.4
Femoral strength
8.5.5
Bone turnover markers
8.6
DISCUSSION
8.7
CONCLUSION
9
SERUM SCLEROSTIN INCREASES IN HEALTHY ADULT MEN IN BED REST
132
132
133
136
137
137
139
141
9.1
RATIONALE
142
9.2
INTRODUCTION
142
9.3
SUBJECTS AND MATERIALS
143
9.3.1
9.3.2
9.3.3
9.3.4
9.4
10
Subjects
Serum sclerostin and bone turnover markers
Bone mineral density
Statistical analysis
RESULTS
143
144
144
145
145
9.4.1
Serum sclerostin and PTH
9.4.2
Bone mineral density
9.4.3
Serum and urinary markers of bone turnover
9.5
DISCUSSION
9.6
CONCLUSION
145
146
146
147
149
SUMMARY AND CONCLUSIONS
10.1
SUMMARYOF HYPOTHESES
153
153
10.2
10.3
154
155
FUTURE WORK
CONCLUSIONS
13
List of Abbreviations
BMD
Bone Mineral Density
eOSTEO
Enhanced OSTeoporosis Experiments on Orbit
HLU
Hind limb unloading
ISS
International Space Station
NASA
National Aeronautics and Space Administration
NIH
National Institute of Health
Ocy454
Osteocyte cell line
PWB
Partial Weight Bearing
PGE 2
Prostaglandin E2
PTH
Parathyroid hormone
SclAblI
Sclerostin antibody
SpaceX
Space Exploration Technologies Corporation
STS
Space Transportation System
pFEA
Micro-finite element analysis
14
Chapter 1
1
Introduction
1.1
Background
It has been recognized for over a century that mechanical loading is fundamental
for the proper development and maintenance of the musculoskeletal system'.
Reduced loading in the setting of spinal cord injury, prolonged bed rest, aging or
experienced by astronauts in space flight is invariability associated with bone
Ioss2-5.
Although it is known that bone responds to its mechanical environment,
.
the mechanisms underlying this response are poorly understood 6 - 6
Osteocyte cells are the most abundant, 90% of all bone cells, yet least
understood bone cell type in the human body. Recent discoveries ascribe
osteocytes as the mechanostat of bone1 3, 1 4 5, 7 -6 0 , yet the biological mechanism of
this action
remains elusive. Osteocyte's dendritic morphology connected
regularly throughout the mineralized matrix of bone has been shown to act as a
key mechanical sensor of bone, orchestrating the action of bone forming
osteoblasts and bone resorbing osteoclasts 3 6'61-64 in response to mechanical
stimuli. Improved understanding of the mechanisms by which osteocytes sense
and regulate the skeletal response to mechanical loading at the cellular level
could have significant implications for the treatment of bone disorders ranging
from osteoporosis, fracture healing, disuse-induced bone loss, and microgravityinduced bone loss. Our overall hypothesis is that disuse- and microgravityinduced bone losses are regulated by osteocytes and can be mitigated by
modulating their functions.
15
1.2
Hypothesis and Research Objectives
Mechanical loading is required for proper development and maintenance of the
musculoskeletal system. The hypothesis and research development focus of this
thesis is centered on the role of osteocytes in the skeleton's response to
mechanical unloading. In addition, the thesis provides preliminary data for the
preparation of the first spaceflight experiment evaluating the in-vitro effects of
microgravity on isolated osteocytic cells. Four primary hypotheses were explored
in this work:
Hypothesis 1: Conditionally immortalized osteocytic cell lines, derived from
long bones, can be established that express markers of mature osteocytes
and follow the hormonal responses of in-vivo osteocytes.
Hypothesis 2: Osteocytic cells directly sense mechanical unloading to
increase SOST and sclerostin in simulated microgravity.
Hypothesis 3: Pharmacologic inhibition of sclerostin prevents disuseinduced bone loss and promotes bone formation in adult mice subjected to
hind limb unloading and partial weight bearing.
Hypothesis 4: The osteocyte secreted protein sclerostin, is elevated in
healthy adult men subjected to 90 days of controlled bed rest.
1.3
Thesis Outline
Chapter 2 provides a review of bone biology with an emphasis on the emerging
role for osteocytes in a variety of skeletal and non-skeletal functions. Chapter 3
reviews disuse-induced and microgravity-induced bone loss in the context of
osteocyte biology and the implications for developing targeted pharmaceutical
therapies for both earth and long-duration space applications.
16
The precise mechanisms of how osteocytes respond to and convert mechanical
stimuli to biochemical signals remain elusive because their relative inaccessibility
has resulted in a lack of available in vitro models. For example, in vitro studies of
osteocytes have relied upon chicken primary osteocytes6 5,66, primary murine
osteocytes, or immortalized murine osteocytic cell lines6 7 -69 that have some, but
not all of the hallmark characteristics of in-vivo osteocytes. In particular, mature
osteocytes are one of the few cells that produce the protein sclerostin, the
product of the gene SOST47' 0'61 '70 78 . Importantly, the SOST/sclerostin pathway
has been implicated in the response of bone to mechanical loading in murine
models. Increased skeletal loading reduces SOST expression79 , whereas
decreased mechanical loading increases it7 9 , and sclerostin is potent negative
regulator of bone mass27,36,62,72,73,75,80-83. Thus, we have focused a significant
portion of this thesis on understanding the role of SOST/sclerostin regulation by
osteocytes in mechanical unloading and the use of an emerging biologic, antisclerostin antibody, to prevent disuse and microgravity-induced bone loss.
To investigate if osteocytes have an endogenous and direct response to
mechanical
unloading as postulated by Wolf's Law,
SOST/sclerostin
are a consequence
or if increases in
of changes in systemic endocrine
regulators, such as PTH 36 6, 1,6 3,67 ,74,84 ,85, we developed a novel osteocytic cell line
(Ocy454) to study the effects of mechanical unloading on osteocytes in an
isolated in-vitro model. Importantly, at the onset of this work, available osteocyte
cell lines lacked significant expression of sclerostin 9 , thus limiting their use for
investigating
this
important
osteocyte-mediated
mechanical-to-biochemical
signaling pathway.
Chapter 4 addresses this shortcoming in understanding osteocyte research with
the development and validation of a novel, long bone-derived osteocytic cell line
(Ocy454) that better recapitulates the in vivo response of osteocytes to hormonal
and mechanical stimuli. Our cell line's osteocytic phenotype is exemplified by a
rapid, high-level expression of SOST/sclerostin that is responsive to hormonal
17
(PTH), cytokine (PGE 2) and mechanical stimuli. In Chapter 5 we utilize an in vitro
ground-based model 8 6 of microgravity to show our novel osteocytic cell line
increased
SOST/sclerostin
levels
in a
cell-autonomous
manner
that
is
independent, but responsive, to changes in PTH. However, earth-based cell
culture unloading analogs for the study of in-vitro osteocytes cannot separate
effects of fluid flow shear stress from the effects of simulated mechanical
unloading. Thus, to evaluate the osteocytic response to true microgravity, our
osteocyte cell line conducted an experiment onboard International Space Station
(ISS) and SpaceX Dragon Crew Resupply Mission-6 (launched April 14th 2015).
This set of experiments, for the first time, investigated the effect of microgravity
on osteocytes cultured in-vitro in a three-dimensional scaffold. In Chapter 6, we
describe the flight hardware (Osteo-4) and the development of optimized
culturing conditions for maintaining an osteocytic phenotype in spaceflight
bioreactors suitable for up to 7-day experiments in microgravity.
Our in vitro osteocyte experiments are important first steps towards a better
understanding of the role of osteocytes in regulating the bone's response to
mechanical forces. We expand upon the role of osteocyte secreted sclerostin in
mechanical unloading, in Chapter 7, by showing that serum sclerostin is
increased in the murine hind limb unloading (HLU) model via tail suspension.
Next, we show the ability of a murine sclerostin antibody (SclAblI, Amgen, Inc.) to
prevent bone loss in adult mice subjected to hind limb unloading (HLU) via tail
suspension for 21 days. Interestingly, the anabolic effects of sclerostin inhibition
on some bone outcomes appeared to be enhanced by normal mechanical
loading.
Future missions to the Moon and Mars will also have astronauts spending a
considerable amount of time in partial gravity environments. Despite the profound
effects of reduced unloading on muscle atrophy and skeletal fragility in humans,
there has been little investigation into the physiological effects of partial reduction
of weight bearing87' 88 or the ability of emerging therapeutics to diminish the bone
18
loss in partial-gravity environments. Thus, our laboratory developed a novel
model for providing chronic, titrated (i.e., 20%, 40%, 70% of normal) quadrupedal
loading in mice 7 '88 . We used this model to show for the first time that bone loss
is linearly proportional to the changes in the reduction of mechanical loads 8 8 . To
further investigate the response to sclerostin inhibition and partial mechanical
unloading, we tested the ability of sclerostin antibody to inhibit skeletal
deterioration during exposure to prolonged (21-day) partial weight bearing at
20%, 40%, and 70% of normal loading. In Chapter 8, we show greater weight
bearing lead to even greater benefit of SclAblI, particularly in the trabecular
compartment.
Chapter 9 highlights a study in which we show serum sclerostin levels increases
in humans in the setting of disuse by measuring longitudinal changes in serum
sclerostin in healthy young men exposed to 90 days of bed rest8 3 demonstrating
osteocyte physiology in unloaded humans. A conclusion chapter 10 provides a
summary of key findings of this thesis.
19
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2
3
4
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26
Chapter 2
2
Osteocyte's role in skeletal biology
Bone is a dynamic metabolically active tissue with diverse functions. It provides
the
body's protection
of vital organs and
enables the lever arms for
musculoskeletal movements. In addition, bone has well established endocrine
roles, such as coordinating the body's calcium-phosphate mineral stores, growth
factor storage, helping to maintain the body's acid-base balance, as well as
providing the niche environment for critical functions of the immune system,
hematopoiesis, and regulation of fat metabolism.
Classically, bone is known as a mechanically responsive tissue actively
remodeling to align its underlying cortical structure along the axis of maximum
stress (Wolff's Law)' 2 . However, the molecular mechanisms underlying bone's
response to mechanical unloading are not completely understood and are a
focus of this thesis work. Bone is comprised of both inorganic and organic matrix
as well as cells derived from the mesoderm (osteoclasts, osteoblasts, and
osteocytes) and endoderm (i.e., endothelial) germ layers. The matrix is primarily
comprised of inorganic hydroxyapatite and organic proteins comprised of type I
collagen,
non-collagenous
proteins
(e.g.
osteocalcin,
osteopontin),
and
proteoglycans.
Osteoclasts, derived from the monocyte lineage, are multinucleated cells
responsible for bone resorption secreting such enzymes as tartrate-resistant acid
phosphatase (TRAP).
Osteoblasts, derived from mesenchymal stem cells
(MSCs), are bone-forming cells that produce the non-mineralized organic
portions of bone, known as osteoid, which is subsequently mineralized.
Osteoblasts are thought to have three known fates: 1) differentiation into bone
lining cells, 2) differentiation into osteocytes, or 3) apoptosis. The molecular cues
determining which fate an individual osteoblast takes are largely unknown.
27
Osteocytes are the most abundant cell type in bone (90-95% of all bone cells),
outnumbering osteoblasts by approximately a 10:1 ratio and osteoclasts by a
100:1 ratio in adult bone. Osteocytes are post mitotic, terminally differentiated
osteoblasts that, during the process of bone formation, assume a more
differentiated morphology and become entrapped in individual lacuna within the
matrix that they are actively synthesizing. The morphology of the osteoblast, a
plump polygonal cell, changes dramatically as it becomes an osteocyte with
reduced cytoplasm and numerous dendritic processes
The dendritic processes
3.
of osteocytes travel within a canaliculi network to allow for extensive connections
between osteocytes, establishing an osteocytic network within bone. The
osteocyte network
osteoclasts
connects osteocytes to each
resident
on
bone
surfaces,
and
other, osteoblasts
to
the
bone
and
marrow
microenvironment. Over the past two decades, our understanding of osteocytes
has exponentially expanded and it is now appreciated these cells are key
regulators of skeletal metabolism, mineral homeostasis, hematopoiesis, and
bone's response to mechanical loading and unloading 4.
The remainder of this
chapter will review the diverse role of osteocytes in skeletal biology and Chapter
3I
'.,
IAl
vvIuI
p~roiden
tjlviI..I,
-n rev;InAAew
cl
IuVIqUVV
f d~qisue%induced and-%
UI0U0
-ii IUUt-,u
aI iu
'.JI
mL-grv
b -IIdUVICIVILY UUI I I
loss
in
U
II
the
UlI
context of osteocyte biology.
2.1
Life of an osteocyte
Osteocytes are evolutionary highly conserved and the organized structure of
these cells within a mineralized matrix is present in bone specimens from
Tyrannosaurus rex, dating back more than 80 million of years ago, clearly
indicating an important role for these cells in skeletal metabolism. The life of an
osteocyte is thought to be split into four stages: an early stage (young osteocyte)
in which the cell is smaller in size, reside into the osteoid and do not express
alkaline phosphatase, an actively mineralizing stage, a late stage (mature
osteocyte) in which the larger cell re-express alkaline phosphatase and is deeply
embedded in the mineralized bone8 , and if it occurs, apoptosis which is thought
to be an initiation signal for bone remodeling by osteoclast resorption. The young
28
osteocyte within the osteoid, is characterized by its proximity to the endosteal or
periosteal bone surfaces and biochemically by the expression of transcripts, such
as
El1/gp38/podoplain,
matrix extracellular phosphoglycoprotein
(MEPE),
dentin-matrix protein-1 (DMP1), and phosphate-regulating gene with homologies
to endopeptidases on the X chromosome (Phex), and the absence of expression
of small-leucine rich proteoglycans (i.e. Keratin) produced by osteoblasts9 -.
Mature osteocytes are characterized by their deeply embedded position in bone
and biochemically by their expression of the gene SOST, which produces the
protein sclerostin, or their expression of the fibroblast growth factor-23 (FGF-23),
a protein involved in the body's regulation of phosphate1 '-.
2.2
Osteocyte orchestration of bone homeostasis
Osteocytes are thought to regulate both osteoblasts and osteoclast differentiation
and function. For example, osteocytes are the major source in the adult of the
protein sclerostin 18-25 which inhibits bone formation, both in vitro and in vivo, by
directly reducing proliferation and differentiation of osteoblasts via the canonical
Wnt signaling pathway. Sclerostin is thought to act by binding the low-density
lipoprotein receptor 5 and 6 (LRP5 and 6) to inhibit Wnt-pcatenin-signaling
2631
Moreover, sclerostin appears central to the bone's response to mechanical
loading and will be further discussed in Chapter 3 and throughout this thesis.
More recently, osteocytes have been shown to be central regulators of bone
resorption through their interaction with osteoclasts. For example, the major
factors which govern osteoclast differentiation from osteoclast progenitor cells
are: 1) the cytokine receptor activator of NFkB ligand (RANKL), which is essential
for osteoclast formation, function and survival; and 2) osteoprotegerin (OPG), a
.
decoy receptor for RANKL that prevents its binding to osteoclast progenitors 32
Many different cell types, including bone marrow stromal cells, osteoblasts at
various
stages
of
differentiation,
T-
and
B-lymphocytes,
hypertrophic
chondrocytes and synovial fibroblasts, express RANKL and were thought to
contribute to osteoclastogenesis .
However, recent paradigm-shifting work
29
reports that osteocyte-derived RANKL is a significant source of RANKL involved
in osteoclast formation and remodeling in cancellous bone33 3 4 . In these studies,
mice with osteocyte-specific deletion of RANKL developed normally, but had
slightly increased bone mass with increasing age and dramatically reduced
osteoclast numbers and serum CTX, a marker of bone resorption.
2.3
Osteocyte as bone's mechano-sensor
Owing to their evolutionary conservation, sheer number, dendritic processes, and
the lacunar network, osteocytes have been investigated for more than fifty years
.
as the major cell type in bone responsible for sensing mechanical loads 35
Numerous studies have established osteocytes role in both mechano-sensation
and transduction 3 3 -4 3 . However, the exact mechanisms and pathways responsible
for load sensation, intracellular signal and extracellular transduction, remain
active areas of research and will be discussed in Chapter 3.
2.4
Osteocyte orchestration of mineral homeostasis
Osteocytes are thought to be the main source of FGF-231 ' 4
44
, a key
regulator, together with parathyroid hormone (PTH) of the body's phosphate
homeostasis
12.
Secreted FGF-23 from osteocytes acts on the kidneys, where it
decreases the expression of NPT2, a sodium-phosphate cotransporter in the
proximal tubule decreasing the reabsorption
of phosphate.
Evidence for
supporting this role of osteocytes comes from DMP1-null mice and patients with
inactivating mutation of DMP1 who have autosomal dominant hypophosphatemic
rickets with osteomalacia as a consequence of abnormally high levels of FGF-23
12,13,17,45-47. Conversely, mice lacking FGF-23 are hyperphosphatemic
and are
osteopenic.
30
2.5
Osteocyte orchestration of hematopoiesis
Recently emerging evidences points to the osteocyte networks' paracrine
interactions with the bone marrow microenvironment niche where hematopoiesis
occurs in the adult. Osteoblasts and osteocytes express several G-protein
coupled receptors (GPCRs) and signaling trough these receptors has been
shown to control the bone marrow niche7 . Evidence for this role of osteocytes
comes
from
mice
lacking
Gsa
in
osteocytes
displaying
a
profound
myeloproliferative phenotype characterized by a dramatic increase in myeloid
cells in bone marrow, spleen, and peripheral blood with granulocyte colonystimulating factor secreted by osteocytes identified as the principal cytokine
.
regulating granulopoiesis in these mice7
2.6
Osteocyte orchestration of immune function and fat
metabolism
Recent evidence also suggests osteocytes play a significant role in the regulation
of immune function and fat metabolism. For example, the targeted ablation of
osteocytes utilizing a transgenic mouse with expression of diphtheria toxin
receptor (DTR) under the promoter of DMP-1
resulted in impaired B-cell
lymphopoiesis and marked reductions in double-positive CD4/CD8 T-cells in the
thymus 4 8 '4 9. Furthermore, SOST/sclerostin has been shown to have a role in the
fate of B-cells, as recently reported5 . Cain et al. 5 showed SOST knockout mice,
despite the high bone mass and increased number of osteoblasts, have no
differences in the frequency or absolute number of hematopoietic stem cells
(HSCs),
common
lymphoid
progenitors,
common
myeloid/megakaryocyte
erythroid progenitors, or granulocyte/monocyte progenitors, confirming the
findings in mice over-expressing the constitutive PTH/PTHrP
osteocytes
5.
receptor in
However, in these SOST null mice, B cells are significantly
reduced in both their frequency and cell number in the bone marrow and the
reduction was a consequence of increased apoptosis due to a reduction in
.
Cxcl12 expression in stromal cells 50
31
Furthermore, Sato et al.4
using the targeted osteocyte ablation model also
provide evidence that osteocytes are required in the maintenance of fat
metabolism and cooperate with the hypothalamus in the regulation of fat in the
liver. For example, osteocyte ablated mice lacked visible white adipose tissue
(WAT) including subcutaneous, mesenteric, and retroperitoneal fat tissue and
.
epididymal fat pad mass along with decreased serum leptin levels 4 9
2.7
Summary
In summary, osteocytes have been shown to be critically essential for diverse
roles in human physiology acting both to control bone homeostasis and as an
endocrine cell7 , 12 ,13 ,15,48,49.
Importantly, these emerging
roles of osteocyte
functions are still to be explored in the context of microgravity physiology and
disuse bone loss, with the primary focus of this thesis on osteocytes'
orchestration of the bone's response to mechanical unloading.
32
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36
Chapter 3
3
Osteocytes: microgravity and disuse induce bone loss
3.1
Skeletal health in long duration spaceflight
Bone loss in spacefaring humans has been noted since the early days of the
Gemini program. For the most part, despite rigorous exercise protocols, bone
loss in weight bearing bones (-0.5-1.6% per month) has been nearly 10-fold
higher than the rate seen in postmenopausal women. Further, femoral strength
predictions by finite element analysis of QCT data declined 2.6% per month in
astronauts who spent 4 to 6 months on the International Space Station (ISS) 1
using the interim resistance exercise device (IRED) used on early ISS increment
missions. The estimated increased fracture risk that accompanies this bone loss
may be substantial, as a 20% decrease in femoral neck bone mineral density
(BMD) during a year of spaceflight would correspond to 30 years of age-related
bone loss in a postmenopausal woman, equating to an unacceptable potential
20-40% increase in fracture risk
2.
Notably, two recent studies reported effective mitigation of spaceflight-induced
bone loss. In the first study, bisphosphonate treatment inhibited bone loss due to
spaceflight'. Despite this apparent success, bisphosphonate treatment may not
be an adequate solution as several of the study subjects dropped out of the study
due to drug-related side effects. Moreover there remain concerns about use of
bisphosphonates in young astronauts due to the long-term retention of
bisphosphonates in the skeleton and concerns about the negative effects of
prolonged suppression of bone turnover 4. Furthermore, no pharmaceutical
therapies exist that mimics the effects of gravity on osteocytes as current
therapies modulate osteoblast (e.g. PTH) and osteoclast (bisphosphonates, antiRANKL antibodies) functions.
37
A second study reported that in five astronauts, use of the advanced resistive
exercise device (ARED) coupled with adequate energy and nutritional intake was
successful in maintaining bone mineral density (BMD) in some astronauts, as
.
assessed by dual-energy x-ray absorptiometry (DXA) 5
Despite these promising reports, it is clear that issues related to skeletal health in
long duration spaceflight are not solved. First, in the ARED exercise and nutrition
study, urinary measures of bone resorption markers remained 2 -3 fold higher
than baseline, indicative of negative skeletal effects, such as microarchitectural
deterioration, that were likely not captured by the BMD measurements5 . Second,
large inter-subject differences in rates of bone loss remain unexplained, and of
concern, as one of the astronauts who exercised with the ARED still lost BMD at
a rate of 1.5% per month 6 . Third, exploration missions of long duration will likely
require exercise equipment of minimal mass and size, which may not be able to
achieve loading magnitudes optimal for musculoskeletal health, and alternative
countermeasures in the case of exercise hardware failure 7. Finally, while most
physiological systems reach an adaptive plateau during exposure to spaceflight,
bone loss shows no clear signs of slowing, and bone mass exhibits a slow and
inconsistent recovery upon return to 1-g6 ,8 . Thus, there is strong rationale for
further investigations to better understand the mechanisms regulating the
skeletal response to spaceflight and mechanical unloading for mitigating bone
loss in disuse and microgravity.
3.2
Osteocyte orchestration of mechanotransduction
Osteocytes have been recognized for over 30 years as the mechanosensor of
bone 9-1 3 . Osteocytes location, deep within the mineralized matrix, and their
structural organization of a cellular network, make them ideal to sense
mechanical stimuli and to coordinate the action of the other cell types in bone.
This complex communication network between osteocytes, osteoblasts, and
osteoclasts is thought to guarantee the health and function of the skeleton.
38
Krempein et al. 14 first demonstrated the effects of reduced mechanical loading on
osteocyte morphology. Rats were immobilized by spinal cord severing, plaster
cast, or nerve dissection. Three weeks of immobilization caused a significant
decrease in the percentage of small metabolically inactive osteocytes (spinal
cord severing, -20%, plaster cast, -15.4%) and a corresponding increase in the
percentage of mature enlarged osteocytes (spinal cord severing, +12.6%, plaster
cast, +14.6%). Recent murine hind limb tail suspension experiments showed
increased osteocyte apoptosis in both trabecular and cortical bone 5 . It has also
been postulated that osteocyte cell death is an initiator signal for osteoclast
remodeling activity or that loaded osteocytes release an osteoclast-inhibiting
factor1 6-18
While evidence from both animals and humans indicates that bone loss due to
microgravity results from reduced osteoblastic bone formation, increased
osteoclast-mediated bone resorption, as well as osteocyte-mediated osteolysis
19-22, the relative contribution of each process and the molecular
mechanisms by
which osteocytes influence these processes in response to mechanical unloading
are poorly understood. Yet, progress has been made towards identifying some of
the osteocyte-specific mechanisms that contribute to mechanotransduction in
bone, including: i) regulation of bone formation via the Wnt/P-catenin signaling
pathway, ii) regulation of osteoclast activity via the RANKL pathway, and iii)
regulation of osteocyte osteolysis.
3.3
Osteocyte orchestration of osteoblasts in mechanical
unloading
Mature osteocytes are one of the only cells that express, postnatally, the protein
sclerostin, a product of the gene SOST2 3 3 2 . Mutations in the SOST'
34
exons or
a distal conserved regulatory region 35 are causative of human high bone mass
disorders, sclerosteosis and van Buchem disease, respectfully 36 . Consistent with
the negative effect of sclerostin on bone mass, transgenic mice over expressing
SOST show low bone mass 37 whereas SOST-null animals have high bone
39
mass36 . Specifically, murine studies demonstrated that increased skeletal loading
dramatically reduces SOST/Sclerostin levels, whereas SOST/Sclerostin levels
were up-regulated by unloading 38. Furthermore, mice constitutively lacking
sclerostin have increased bone mass, but are subsequently resistant to disuseinduced bone loss 39 . In addition, mice lacking an evolutionary conserved region
.
(ECR5) that is known to control the expression of SOST/Sclerostin also have a
high bone mass phenotype 0
In addition, in a cross-sectional study in postmenopausal women with recent
immobilization due to stroke, serum sclerostin levels were 3-fold higher than
.
those in age matched postmenopausal women41
Furthermore, it has been reported that osteocytes are also the major source of
insulin-like growth factor IGF-1. For example, deletion of IGF-1 in osteocytes
impairs skeletal growth, reduces the periosteal circumference, and inhibits bone's
response to mechanical forces 42
43
. When osteocyte IGF-1 conditional knockout
mice were subjected to mechanical loading, there was a significant reduction in
bone formation indicating that osteocyte derived IGF-1 is also an important
.
determinant of bone's response to mechanical loading 43
3.4
Osteocyte orchestration of osteoclasts in mechanical
unloading
Osteocytes are a major source of the osteoclast differentiation cytokine NFkB
ligand (RANKL), which is essential for osteoclast formation, function and survival;
and 2) osteoprotegerin (OPG), a decoy receptor for RANKL that prevents its
binding to osteoclast progenitors 44 . Importantly, mice lacking RANKL in
osteocytes, have increased bone mass and are resistant to bone loss after tail
suspension4 5' 46. However, these studies used the DMP1 promoter, which is also
expressed in a subset of osteoblasts. Thus, additional studies with alternative
osteocyte promoters driving the loss of RANKL is required to validate the role of
osteocyte derived RANKL to the bone's response to mechanical unloading.
40
3.5
Osteocyte depleted mice are resistant to disuse-induced
bone loss
A mouse model in which osteocytes are selectively ablated upon diphtheria toxin
administration demonstrated not only that osteocytes are key regulators of
skeletal homeostasis, as these mice have severe osteopenia, but also that
without osteocytes bone is resistant to unloading induced bone loss4
3.6
Osteocyte osteolysis in disuse-induced bone loss
Microgravity-induced bone loss in humans has been historically thought of as
increased bone resorption via osteoclast activity and, in some studies, a
decrease in osteoblast function6 . However,
recent evidence shows that
osteocytes can directly resorb their perilacunar/canalicular matrix through a
process termed 'osteocytic osteolysis'
48*.
To our knowledge, the only spaceflight study of osteocyte biology comes from a
Russian Bion-1 1 biosatellite launched in 1996 carrying two Macaca mullatta
monkeys for a 14-day mission. Examination of iliac crest biopsies of the flight
animals showed mature osteocytes had increased specific volume of the Golgi
complex and increased osteolytic activity 49. Evidence for osteocytic osteolysis as
a contributor to microgravity-induced bone loss is supported by iliac crest
biopsies of the Macaca mullatta monkeys (Bion 11, 1996) that showed mature
osteocytes had increased osteolytic activity 49 . Additional evidence of osteocytic
osteolysis (e.g., increased lacunar area and perimeter) following microgravity
exposure was recently reported in adult mice exposed to 15 days of microgravity
on STS-131 20 . However, the mechanisms that drive the increase in osteocytic
osteolysis in microgravity and disuse-induced bone loss are still largely unknown.
Importantly, a recent ground-based in vitro study reported that sclerostin
promotes the release of osteocytic perilacunar bone mineral by inducing
osteocyte expression of carbonic anhydrase 2 (Car2), cathepsin K (Ctsk), and
.
tartrate-resistant acid phosphatase (Acp5)2 5
41
3.7
Ground based models of microgravity and disuse bone
loss
Given the financial expense and limited nature of spaceflight experiments,
several models of ground (earth) based in-vivo and in-vitro mechanical unloading
models have been developed. Several of these model systems are utilized in this
thesis and are reviewed, herein.
3.7.1
In-vivo (rodent) ground based models of mechanical unloading
Various models of disuse-induced bone and muscle loss have been developed,
including, but not limited to cast immobilization 14, spinal cord injury 50, muscle
paralysis (e.g. Botulinum toxin) 51 , and the tail suspension hind limb unloading
model52-54 . The tail suspension hind limb unloading model, developed by National
Aeronautics and Space Administration (NASA), is unique in its ability to mimic
the cephalad-fluid shift that occurs in microgravity while avoiding weight bearing
by the hindquarters 5 2-5 4. More recently, to investigate the physiological effects
and response to partial unloading environments, Wagner et al. 55 developed a
rodent model that allows for chronic exposure to various
IPx/Pv
rf nrtial weight-
bearing. For this thesis, we employed the hind limb unloading (Chapter 7) and
partial weight bearing model (Chapter 8) to study the musculoskeletal effects of
sclerostin inhibition in rodents.
3.7.2
In-vitro models of mechanical unloading
Rotating wall vessels (RWVs) and three-dimensional clinostats, also called
random positioning machines (RPMs), are two systems commonly used to
simulate microgravity5-58.
The
RPM consists of two
frames that rotate
independently and randomly about orthogonal axes resulting in a gravity vector
56 8
that is in constant motion and can approach a residual force as low as 10-5 g -5
force. NASA developed the RWV bioreactor as a model for the low-shear, lowturbulence conditions of microgravity cell culture. Cells, tissues, or scaffolds are
rotated synchronously in the bioreactor vessel such that the fluid dynamic effect
42
on them mimics a particle allowed to free fall with the time-averaged gravitational
vector on individual cells modeled as 10-3 g force56 . At the commencement of this
thesis, neither the RPM or RWV models had been used to study isolated
osteocytes in simulated microgravity. The RWV bioreactors have been broadly
published as models of simulated microgravity for a wide variety of cell types 5965.
Of particular note, is the recently performed study by Martinez, et al. that show
the NASA bioreactors reproduce key findings of actual spaceflight microgravity
conditions in immunologic cells5 6 . For this thesis, we have chosen to utilize the
RWV bioreactors to study the effects of simulated microgravity in osteocytes
(Chapter 5).
43
1
2
3
4
5
6
7
8
9)
10
11
12
13
14
15
Leblanc, A. et al. Bisphosphonates as a supplement to exercise to protect
bone during long-duration spaceflight. Osteoporos Int 24, 2105-2114,
doi:10.1 007/sOO1 98-012-2243-z (2013).
Looker, A. C. et al. Updated data on proximal femur bone mineral levels of
US adults. Osteoporos Int 8, 468-489 (1998).
Sibonga, J. D. Spaceflight-induced bone loss: is there an osteoporosis
risk? Curr Osteoporos Rep 11, 92-98, doi:10.1007/s11914-013-0136-5
(2013).
Khosla, S. et al. Benefits and risks of bisphosphonate therapy for
osteoporosis. J Clin Endocrinol Metab 97, 2272-2282, doi:jc.2012-1027
[pii] 10.1210/jc.2012-1027 (2012).
Smith, S. M. et al. Benefits for bone from resistance exercise and nutrition
in long-duration spaceflight: Evidence from
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48
Chapter 4
4
Development and characterization of a novel osteocytic
cell line (Ocy454)
This thesis chapter, in part, previously published as the manuscript: Spatz, et al.,
The Wnt-inhibitor Sclerostin is Up-regulated
Osteocytes in-vitro, JBC, 2015.
49
by Mechanical
Unloading in
4.1
Rationale
Currently available osteocytic cell lines'
2
express low level of key osteocytic
proteins (e.g. Sclerostin, FGF23) and require high cell density with extended time
in
culture
investigate
under differentiation
osteocyte
responses
conditions to
to
become
unloading,
we
osteocyte-like.
have
isolated
To
and
characterized a novel osteocytic cell line (Ocy454), reported herein, which
faithfully recapitulates the in vivo response of osteocytes to mechanical stimuli
(Chapter 5). Ocy454 cells show rapid, high-level expression of SOST/sclerostin
that is responsive to hormonal (PTH), cytokine (PGE 2), and mechanical stimuli.
Furthermore, Gsa knockdown in Ocy454 led to significant increases in SOST
expression matching known osteocyte in-vivo regulation 3 demonstrating the
broad utility of this new osteocytic cell line for studying SOST/sclerostin
regulation, as we have recently reported4 . Ocy454 also showed an enhanced
osteocytic phenotype when cultured on a three dimensional (3D) biomaterial, by
increasing FGF23 expression upon PTH stimulation highlighting the importance
of optimizing in-vitro culture conditions for studying certain aspects of osteocyte
biology.
4.2
Introduction
Studies of osteocyte biology have been hampered by their inaccessibility and by
the lack of techniques to generate cell lines that faithfully characterize this cell
population5 . Selective cell lines, HOB-01-C1, MLO-Y4, and IDG-SW3 exhibit
osteocytic characteristics' 2 , but require differentiation factors and extended time
in culture. While, primary cell cultures can be differentiated to express a mature
osteocytic phenotype, these cultures have limited passage capability. The lack of
mature osteocytic cell models and the limited passage ability of primary cells
highlight the need for additional osteocyte cellular models.
50
4.3
Materials and methods
4.3.1
Osteocytic cell line
Mice expressing the green fluorescent protein (GFP) under the control of the 8Kb
of Dentin Matrix-Protein 1 (8KbDMP1-GFP) (kindly provided by Dr. Ivo Kalajzic,
University of Connecticut Health Center)5 were mated with mice carrying a
ubiquitously expressed SV4OAg (Immortomouse Charles River) and osteocytes
were isolated from the long bones of 4-week old double transgenic mice. Long
bones were cut at the epiphysis, flushed with medium (aMEM) (Gibco, Grand
Island, NY) supplemented with 0.1% of bovine serum albumin, 25mM HEPES
(pH 7.4) and containing 1 mg/ml collagenase Type 1:11 (ratio 1:3) (Worthington,
Lakewood, NJ) subjected to 4 sequential collagenase digestions, one EDTA
digestion, a final sixth collagenase digestion, and minced bone fragments placed
in collagen coated 100 mm tissue discs. Cells were allowed to reach confluence
at 33 'C and then grown for an additional 10-12 days at 37 'C prior to FACS
sorting
for
DMP1-GFP
expression.
Bulk-sorted
GFP-positive
cells were
maintained on collagen coated flasks grown in aMEM supplemented with 10%
FBS
(Gibco,
Grand
Island,
NY)
and
1%
antibiotic-antimycotic
(Gibco).
Subsequently, two criteria were selected for further identification of a mature
osteocytic cell line: 1) sorted GFP-positive were required to have high levels of
production of known osteocytic genes (SOST, DMP1) at early time point of 14
days at the semi-permissive temperature, 37 'C, in the absence of differentiation,
and 2) respond to the known effects of PTH stimulation by suppression of SOST
and increased expression of RANKL. This method provided a heterogeneous
population of DMP1-GFP positive cells that more faithful resemble osteocytes in
vivo, which are known to be a mix of cells with various degrees of SOST and
DMP1
expression depending on their age/maturation. We performed our
experiments in this heterogeneous population. In an effort to establish a more
homogeneous osteocytic population, we also performed FACS on Ocy454 to
isolate single cell subclones. Ocy454 and several single cell clones 4 , have the
same osteocyte marker expression and response to stimuli.
51
For two-dimensional cell culture, cells (Ocy454, IDG-SW3', and primary long
bone osteoblasts isolated from 4-week old SV40TAg mice) were plated at 10 5
cells/ml, allowed to reach confluence at the permissive temperature (33 OC) for 3
days.
Subsequently,
cells
were
either differentiated
at
the
permissive
temperature or switched to the semi-permissive temperature (37 'C) for the
indicated time points. MLO-Y4 cells were plated at 10 5 cells/ml and RNA
extracted at 4 days. For primary osteocytes, cells were isolated from 4-week old
DMP1-GFP long bones. In brief, long bones were flushed of bone marrow with
PBS, subjected to sequential collagenase digestions, minced, and bone chips
placed in tissue culture plates. Subsequently, at the two-week time point FACS
was performed. GFP- and GFP+ positive populations were directly collected into
RNA extraction buffer (Qiagen).
The routine culturing conditions to maintain the Ocy454 osteocytic phenotype
was twice weekly sub-passages (1:5) for up to 4 months from a frozen stock.
For 3D cell culture, 1.6 x 105 Ocy454 cells were plated on 200 pm polystyrene
Alvetex (Reinnervate) well insert scaffolds. Scaffolds were collagen coated per
manufacture protocols for indicated experiments. All other chemicals were from
Sigma-Aldrich (St. Louis, MO) or Fisher Scientific.
4.3.2
Quantitative real time pcr
Total RNA was isolated (RNAEasy, Qiagen, Valencia, CA) per manufacturer's
recommendations and RNA quantified (NanoDrop, Thermo Scientific, Rockford,
IL). cDNA synthesis was preformed (Qiagen, Valencia, CA or Clontech, Mountain
View, CA ) on 0.5-1 pg total RNA, followed by SYBR qPCR (StepOnePlus, Life
Technologies, Grand Island, NY). Primer sequences are available upon request.
Beta-actin (ACTB) was used for normalization of gene expression and ACT
computed within each sample to the housekeeping reference and AACT across
experimental conditions. Experiments were run in triplicates, unless otherwise
indicated.
52
4.3.3
Western blot
Whole cell lysates (MPER, Thermo Scientific, Rockford, IL) from 2D-cell culture
conditions were prepared according to the manufacturer's recommendations.
Protein concentrations were quantified
(Bio-Rad Protein Assay, Bio-Rad,
Hercules, CA), 10 pg was separated on a 4-20% Tris-Glycine denaturing gel (Life
Technologies, Grand Island, NY), and transferred to a PVDF membrane using
the Trans-blot Turbo (Bio-Rad) system per manufacturer's recommendations.
The membrane was blocked with 3% BSA and 5% non-fat milk in Tris buffered
saline containing 0.05% Tween-20 (TBST) for 1 hour and then incubated with
goat polyclonal mouse sclerostin antibody (1:200, R&D Systems, Minneapolis,
MN) overnight at 4C [30].
After washing, secondary antibody (1:5000) was
incubated for 1 hour at room temperature and then developed using enhanced
chemiluminescence
(Thermo
Scientific, Rockford,
IL)
[30].
For
Gsa
immunoblotting, similar procedures were followed using an anti-Gsa antibody
(Millipore, catalogue number 06-237).
4.3.4
Sclerostin immunohistochemistry
Three-dimensional scaffolds were washed once with phosphate-buffered saline
(Life Technologies), frozen embedded (OCT, Tissue Tek), and 10 pM sections
cut onto standard microscope slides. In brief, proteinase K was used for antigen
retrieval for 15 minutes, followed by a quench in 3% H 20 2/Methanol for 10
minutes, washed in H 2 0 and rinsed in 1X TBS. Next, biotinylated anti-sclerostin
antibody (R&D Systems, BAF1589) diluted 1:50 in TNB was incubated for 1 hour,
washed three times with 1X TNT, SA-HRP diluted 1:100 in TNB was then added
to slides and incubated for 30 minutes, washed three times with 1X TNT, and
incubated with DAB HRP substrate (Vector Labs) for 5 minutes, and coverslipped.
53
4.3.5
Sclerostin elisa
Four ml of cell culture supernatants from slow-turning rotating wall bioreactor
experiments at indicated time points were spun at 850 rpm for four minutes and
volume reduced to 250 ml with a 10kDa centrifugal filter unit (Millipore, Billerica,
MA) per manufacturer recommendations.
Supernatants were assayed for
sclerostin using a commercially available assay (ALPCO, Salem, NH) per
manufacture recommendations. For additional sclerostin ELISA experiments, an
antibody matched pair ELISA assay was used46' . In brief, for the matched pair
sclerostin ELISA, conditioned medium (36-48 hours) was harvested from Ocy454
cells as indicated in the figure legends and stored at -80 C until further use. High
binding 96 well plates (Fisher, 21-377-203) were coated with Scl-Ab VI capture
antibody (3 pg/ml) in PBS for one hour at room temperature. Plates were washed
(PBS plus 0.5% Tween-20) and blocked with wash buffer supplemented with 1%
BSA and 1 % normal goat serum for one hour at room temperature. Samples (60
pl/well) were then added along with a standard curve of murine recombinant
Sclerostin (ALPCO) and plates were incubated overnight at 4C. Plates were
washed three times, incubated with HRP-coupled ScI-Ab VII detection antibody
(0.5 pg/ml) for one hour at room temperature. After washing, signal detection was
performed using Ultra TMB-ELISA (Pierce, 34028), stopped by 2N sulfuric acid,
and read at 450 nm. Prior to harvesting supernatant, cell number per well was
determined
using
PrestoBlue
assay (Life Technology)
according to the
manufacturer's instructions.
For shRNA experiments, shRNA (Broad Institute, Cambridge, MA) lentiviral
particles in puromycin resistant vector, targeted against luciferase (control,
shLuciferase) or shGsa were used to infect cells plated one day prior at 0.5 x 10 5
cells/ml. Subsequently, infected cells were puromycin selected (2 pg/mL) at the
permissive temperature (33 'C) for seven days, and subsequently allowed to
differentiate for 14-16 days at the semi-permissive temperature. Table 4-1
provides the shRNA target sequences.
54
Table 4-1: shRNA Target Sequence
shRNA
LacZ
Luciferase
GNAS E3
GNAS B2
GNAS G2
GNAS G9
GNAS C2
Target Sequence
CCAACGTGACCTATCCCATTA
AGAATCGTCGTATGCAGTGAA
CGCAGATAAGAAACGCAGCAA
GCCAAGTACTTCATTCGGGAT
TCGGGATGAGTTTCTGAGAAT
CCTGCATGTTAATGGGTTTAA
CCTGAAGAATCTGTGCCATTT
4.4
Results
4.4.1
Osteocytic cell line basal and hormonal characterization
Our method for osteocyte cell line development coupled fluorescent sorting for an
osteocytic marker (DMP1) with functional hormonal screening to accurately
ensure the cell line possessed the key functional responses of mature osteocytes
in vivo. Out of several preparations one population of sorted DMP-GFP-SV40TAg
(Ocy454) cells was selected for further characterization on the basis of its high
expression of SOST at early time points at the semi-permissive temperature.
Ocy454 osteocytic cells displayed a dendritic morphology (Figure 4-1A) similar to
other osteocytic cell lines' 2 and at two weeks at the semi-permissive temperature
(37'C) expressed the DMP1 -GFP transgene (Figure 4-1 B, C).
55
C
37 C
33C
12d
Figure 4-1: (A) Representative dendritic morphology of osteocytic cell line (Ocy454), (B)
DMP1-GFP expression at 3-days for permissive temperature (33'C), (C) DMP1-GFP
expression time course at 5d and 12d for both permissive (33'C) and semi-permissive
temperature (37'C).
After two weeks at 370C, Ocy454 cells expressed significantly higher levels of
SOST and DMP1 compared to long bone primary osteocytes as well as the only
other available osteocytic cell lines, MLO-Y4 and IDG-SW3 (Figure 4-2A,B).
Upon further study, we also observed Ocy454 differentiated upon contact
inhibition at the permissive temperature (Figure 4-1C, 2B).
However, Ocy454
differentiated at a slower pace at the permissive temperature. For example, at
the one-week time point there was lower levels of SOST at the permissive
temperature compared to the semi-permissive temperature (Figure 4-2B).
In
addition, Ocy454 expressed levels of SOST that were significantly higher than
those expressed by long bone osteoblasts (Figure 4-2B) as early as one week at
37C in the absence of differentiation factors. Sclerostin was detected in the cell
culture supernatant at day 11 and continued to increase with time in culture
(Figure 4-2B). Furthermore, after two weeks at the semi-permissive temperature
56
(37C) Ocy454 cells expressed high levels of other characteristic osteocytic
genes such as DMP1 (Figure 4-2B). In contrast, these cells had low levels of
expression of genes characteristic of immature osteocytes and late osteoblasts,
such as keratocan (Kera) (Figure 4-2B) 78, . After one week in culture at 370C,
Ocy454 cells expressed levels of DMP1 (Figure 4-2B) that were significantly
higher than those expressed by long bone osteoblasts (LB-OBs). In addition,
RANKL was highly expressed at the permissive temperature and then expression
dropped to levels comparable to wild-type osteoblasts and IDG-SW3 cell line with
differentiation at the semi-permissive temperature (Figure 4-21B). Interestingly the
expression of FGF23 in Ocy454 followed a biphasic pattern of expression with
significantly more mRNA at one week in semi-permissive culture than at later
time points (Figure 4-2B).
57
A
SOST
800
L
7 300Con.
200100-
z
0-
B
-5
*
M
60
4000.52000.0-
DMP1
Sclerostin
100-
-I
5
80-
i
*#
***
5I
Ocy454
C3 MLO-Y4
SOST
3
250
1
RANKL
DMP1
*
400-
*# ' 60-
I
Ocy454-33C
Ocy454-37C
LB-OBs
IDG-SW3
LB: GFP-
40-
Ocy: GFP+
M1
1520
20-
RANKL
FGF23
15-
15-
10-
10-
10
KERA
4- NDNDN
IIo-
5-
5-
II
NV
C
0
0
CP
#
Figure 4-2: (A) Ocy454 at 2 weeks (37'C), black bars, express characteristic osteocytic
markers vs. MLO-Y4 in the absence of differentiation media, (D) Ocy454 at I weeks and 2
weeks (37 'C), black bars, express characteristic osteocytic markers SOST, Sclerostin,
DMP1, FGF23, RANKL, and lack Kera expression in the absence of differentiation media
compared to long bone osteoblasts, IDG-SW3 (2 weeks), long bone DMPI-GFP- and long
bone DMP1-GFP+ osteocytes. * p<0.001 1wk, 2wk at semi-permissive growth temperature
(37'C) vs. permissive growth temperature (33'C, 3 days), ** p<0.001 1wk, 2wk at semipermissive temperature vs. permissive growth temperature at indicated time points,
p<0.001 Ocy454 vs. long bone osteoblasts (LB-OB), W p<0.001 Ocy454 vs. long bone
DMP1-GFP+ osteocytes at indicated time points. ND: Not detected. Error bars represent
one SD.
58
We next assessed Ocy454 cell responsiveness to known osteocyte regulators.
Short-term (4 hour) treatment with human (h)PTH(1-34), forskolin (FSK), or (16
hour) prostaglandin E2 (PGE 2) induced a statistically significant down-regulation
of SOST (Figure 4-3A, p < 0.001 for all), and sclerostin both in whole cell lysate
and condition media (Figure 4-3B, C). These results are consistent with the
known inhibitory effects of these agents on SOST expression. In contrast, a
PGE 2 inhibitor, indomethacin caused an increase in SOST (Figure 4-3B) showing
Ocy454 cells have an intact hormonal axis that increases SOST expression.
In addition, hPTH(1-34) dose- and time-response experiments showed Ocy454
to be sensitive to down regulation of SOST in as short as 2 hours (1OOnM, Figure
4-3D) and a 50% suppression at doses as low as 0.1 nM hPTH(1 -34) (Figure 43D). Similarly, hPTH(1-34) (4-hr), FSK, and PGE 2 caused concurrent increases
in RANKL mRNA (Figure 4-3E). hPTH(1-34) suppressed Mef2C mRNA (Figure
4-3F), consistent with previous reports [44,45] and DMP1 mRNA (Figure 4-3G).
There was no regulation of FGF23 mRNA by 4-hour PTH treatment in Ocy454 at
1-week or 2-weeks in two-dimensional non-collagen and collagen coated 6-well
plate culture conditions (data not shown).
59
A
B
Sclerostin
SOST
VEH
PTH
FSK
PGE2
S 1.5-
I0
Sclerostin
M
T
m
VEH
PTH
300-
m
3
i
200-
VEH
Indometh 1 uM
Indometh 10 uM
PTH 10 nM
100-d
0.0
14d
C
I
2
3
4
16d
5
Sclerostin
GAPDH
PTH-4HR Dose Response
PTH: SOST Timecourse
2.0-
1.5-
z~*1.5-
z
Eo
*
1 0.5-
0
g'*0.5-
*
*
-
~if
.0-
E
*
D
V
*
0.I
G
F
E
RANKL
m
20
m
U*
40
DMPI
Mef2C
VEH
PTH
FSK
PGE2
m VEH
m
1.0
T
PTH
2.01.5-
m VEH
m
PTH
1.0-
E .j
0.54K
15
-r
Z*
0.5-
O.Oj
0.0-
Figure 4-3: Ocy454 (A) decreases SOST with 4-hour (h)PTH(1-34) (100 nM), Forskolin
(10 nM) and 16-hour (PGE 2, 5 nM) treatment to known negative regulators at 2 weeks at
semi-permissive temperature, (B) 16-hour hPTH(1-34) (100 nM) decreases secreted
sclerostin as measured by ELISA (ALPCO) and 48-hr indomethacin treatment (1 IM
and 10 pM) increases secreted sclerostin by ELISA (Amgen), (C) 4-hour hPTH(1-34)
(100 nM) treatment of Ocy454 at 2-weeks suppresses total cell lysate sclerostin: Lane
1-2: VEH, Lane 3-4: hPTH(1-34), Lane 5: Sclerostin standard (APLCO), (D) hPTH(1-34)
time course and dose response for SOST suppression, (E) increases RANKL with 4hour treatment to hPTH(1-34), forskolin, and 16-hour PGE 2 at 2 weeks, (F) 4hr hPTH(134) treatment suppresses Mef2C, (G) and DMP1. * p<0.001 for all SOST time course
and hormone/cytokine treatment vs. VEH. Error bars represent one SD.
60
We and others have previously reported that mice lacking 39 Gsa have increased
levels of SOST/Sclerostin. To confirm these in-vivo results in Ocy454 cells, we
used shRNA to knock-down Gsa in Ocy454, as was done previously for
HDAC5 4. The range of sclerostin secretion (normalized to cell number) was
determined in each experiment using 10 separate control lentiviruses expressing
shRNAs against non-expressed genes (LacZ, luciferase, GFP, RFP). Dotted
lines indicate two standard deviations above the mean value of sclerostin
secretion (normalized to cell number) in the presence of the control hairpins. As
shown in Figure 4-4A, two out of five hairpins tested to achieve LV-mediated
shRNA knockdown of GNAS (but not related heterotrimeric G proteins GNAQ or
GNA1 1) consistently increased sclerostin secretion (individual hairpins labeled
next to corresponding data points). The individual hairpins that reduced GNAS
mRNA levels accordingly increased SOST expression (Figure 4-4B), thereby
confirming the expected knockdown/phenotype relationship for this known SOST
regulator. GNAS hairpins "G2" and "G9" both effectively reduced Gsa protein
levels (Figure 4-4C), and hairpin G2 was selected for further study. Sclerostin
secretion in control (shLuciferase) and GNAS G2 shRNA-expressing cells was
determined over time. As shown in Figure 4-4D, GNAS shRNA causes an
increase in sclerostin secretion at all-time points, with the most dramatic results
at early times after switching cells from 330C to 37C. Finally, GNAS shRNA cells
were tested for PTH responsiveness.
Figure 4-4E shows that GNAS shRNA
increases basal SOST expression (after 14 days at 37TC); furthermore, while
control cells respond to PTH at this time point with suppression of SOST levels,
this is not the case when Gsa levels are reduced. Taken together, these data
confirm a cell-intrinsic role for Gsa in osteocytes, and further support the use of
Ocy454 cells for studying SOST gene regulation.
61
B
A
400.
8
G2
300G9
E
W0
200 1309
B2
m10
4-
A A
-- 2
V
........M..C2...
.....
~
-E-40C2
A
A.A .............
6e*
G9
-20
Nv
0
20
B2
E3
40
60
80
% GNAS knockdown
shLacZ
D
C
shGNAS
100008000(
shRNA
6000
(
IBGs,
.2
m
mn
4000
200
IBTubulin
SOST
E
X10-
E
mVEH
8.
PTH
4
E.
0z
shLuciferase shGNAS C2 shGNAS G2
Figure 4-4: (A) Ocy454 cells were infected with control shRNA-expressing lentiviruses
(shGFP, shLuciferase, shRFP, shLacZ) and 5 separate hairpins targeting the indicated
gene. Each data point represents sclerostin/cell number values obtained for an individual
hairpin. Dotted lines indicate values two standard deviations above and below those of the
controls. For GNAS, individual hairpins are labeled on the data plot. (B) Ocy454 cells were
infected with shGFP and the indicated GNAS shRNA lentiviruses and then switched to
37'C. 14 days later, RNA was isolated and RT-qPCR performed for beta actin, GNAS, and
SOST. (C) As in (B), except lysates were generated for immunoblotting. (D) As in (B),
except conditioned medium was collected at the indicated times for sclerostin ELISA. (E)
As in (B), expect cells were treated with vehicle or hPTH(1 -34) (50 nM) for 4 hours followed
by RT-qPCR for SOST and beta actin. *, p<0.01 hPTH(1-34) vs VEH. ** p < 0.001 shGNAS G2
vs. shLuciferase and shGNAS C2. *** p <0.001 shGNAS vs. shLacZ for all time points.
62
4.4.2
Three-dimensional culture enhances osteocytic phenotype
To evaluate the effects of a three-dimensional culture environment on the
expression of osteocyte-specific genes and to provide a scaffold for cell
attachment in the rotating wall bioreactor system used to simulate microgravity,
Ocy454 cells were seeded onto scaffolds and cultured for an additional 7 to 14
days. Consistent with our two-dimensional culture results, we observed also a
significant down regulation of SOST (Figure 4-5A), increases in RANKL (Figure
4-5B), and decreases in DMP1 (Figure 4-4D) in three-dimensional cultures (p <
0.001 for all) upon PTH treatment. Previous reports have demonstrated that
TGFp1 increases SOST/sclerostin levels during mechanical loading [47,48]. In
contrast to prior reports, treatment of Ocy454 cells with TGFP1 (10 ng/ml, 24
hours) resulted in a down regulation of SOST (Figure 4-4A), increases in RANKL
(Figure 4-5B), and a known TGFP1 responsive gene Serpinel was increased
2.3
0.1 fold (p< 0.007). Interestingly, in contrast to two-dimensional cultures,
culture in three dimensions with 4-hr PTH treatment resulted in a five-fold (p <
0.001) increase in FGF23 expression (Figure 4-5C). In a direct comparison
between three-dimensional and two-dimensional culture at an early time point (3
days at 370C), Ocy454 had significantly higher amounts of SOST and RANKL in
the three-dimensional culture conditions (Figure 4-5G) than in the twodimensional setting. Furthermore, Ocy454 displayed dendritic morphology in
three-dimensional culture conditions (Figure 4-5E) and we observed decreases
in sclerostin protein expression with hPTH(1-34) treatment in three-dimensional
culture (Figure 4-5F).
63
EE
~B
A
A
RANKL
SOST
U
t
15-
1.5-
VEH
PTH
TGFP
I
_r
10-
z
Of
E
5-
0.50
*
z
0.0-
C
0-
D
FGF23
6-
F
DMPI
Sclerostin
*
1.5-
zc
4-
1.0AN
E
0.5-
2-
Z~i
0-
G
C
0
60-
*t
1.5-
t
10-
PTH 4HR
DMP1
15-
100-
N
RANKL
SOST
s0-
VEH
- 0.0-
-r
f2D
mScaffold
-I-
z .1
(U 40E8
I
5-
0.5-
200- MI.
3d
0-
-
0.0-
3d
3d
Figure 4-5: Ocy454 on 3D scaffold (collagen coated, A-D for hPTH(1-34) experiments). (A)
4-hour hPTH(1-34) (100 nM) and 24-hour TGFp 1 (10 ng/ml) treatment at 12-14 days
decreases SOST, (B) increases RANKL, 4-hPTH(1-34) (C) increases FGF23, (D) decreases
DMPI expression, (E) representative H&E stain of Ocy454 cell within the 3D scaffold. (F) 4hour hPTH(1-34) (100 nM) treatment decreases sclerostin expression of Ocy454 on 3D
scaffold. (G) Ocy454 gene expression for SOST, DMP1, and RANKL on three-dimensional
scaffolds vs. two-dimensional culture at semi-permissive growth temperature for 3-days
(37*C). * p<0.001 for hPTH(1-34) or * p< 0.007 TGFp 1 vs. VEH. Error bars represent one SD.
64
4.5
Conclusion
We have successfully generated a novel osteocytic cell line Ocy454 that
recapitulates known in-vivo osteocytic functions without the requirement for long
term high-density cultures and in the absence of differentiation media conditions.
Thus, for the first time, we have established osteocytes cell lines that can be
routinely
cultured
in
short
time
periods
with
high-level
expression of
SOST/Sclerostin that is responsive to hormonal (PTH), cytokine stimuli, and
matches the known response to SOST/Sclerostin of several in-vivo knockout
animal models.
65
1
2
3
4
5
6
7
8
9
Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I. & Bonewald, L. F. Cell
line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro
and accelerates bone formation in vivo. J Bone Miner Res 26, 2634-2646,
doi:10.1002/jbmr.465 (2011).
Bonewald, L. F. Establishment and characterization of an osteocyte-like
cell line, MLO-Y4. J Bone Miner Metab 17, 61-65 (1999).
Fulzele, K. et al. Myelopoiesis is regulated by osteocytes through
Gsalpha-dependent signaling. Blood 121, 930-939, doi:10.1182/blood2012-06-437160; 10.11 82/blood-2012-06-437160 (2013).
Wein, M. N. et al. HDAC5 controls MEF2C-driven sclerostin expression in
osteocytes. Journal of bone and mineral research : the official journal of
the
American
Society
for
Bone
and Mineral
Research,
doi:10.1002/jbmr.2381 [doi] (2014).
Kalajzic, I. et al. Dentin matrix protein 1 expression during osteoblastic
differentiation, generation of an osteocyte GFP-transgene. Bone 35, 7482, doi:10.1016/j.bone.2004.03.006 S8756328204001097 [pii] (2004).
Yu, L. et al. Sclerostin expression is induced by BMPs in human Saos-2
osteosarcoma cells but not via direct effects on the sclerostin gene
promoter
or
ECR5
element.
Bone
49,
1131-1140,
doi:10.1016/j.bone.2011.08.016; 10.1016/j.bone.2011.08.016 (2011).
Igwe, J. C., Gao, Q., Kizivat, T., Kao, W. W. & Kalajzic, I. Keratocan is
expressed by osteoblasts and can modulate osteogenic differentiation.
Connective
tissue
research
52,
401-407,
doi:10.3109/03008207.2010.546536;
10.3109/03008207.2010.546536
(2011).
Paic, F. et al. Identification of differentially expressed genes between
osteoblasts
and
osteocytes.
Bone
45,
682-692,
doi:b875b3282(09)01634-2 [pii] 10.1016/j.bone.2009.06.010 (2009).
Wu, J. Y. et a/. Gsalpha enhances commitment of mesenchymal
progenitors to the osteoblast lineage but restrains osteoblast
differentiation in mice. The Journal of clinical investigation 121, 34923504, doi:10.1 172/JC146406; 10.1172/JC146406 (2011).
66
Chapter 5
5
Osteocytes as bone's gravity sensor: cell autonomous
increases in sclerostin in mechanical unloading
This thesis chapter, in part, previously published as the manuscript: Spatz, et al.,
The Wnt-inhibitor Sclerostin
is Up-regulated by Mechanical Unloading in
Osteocytes in-vitro, JBC, 2015.
67
5.1
Rationale
In bed rest and immobilization, an increase in circulating sclerostin (Chapter 9) is
associated with a decrease in parathyroid hormone (PTH)'
2
. The increase in
SOST/sclerostin could be ascribed to direct osteocyte responses to changes in
mechanical cues or due to endocrine regulators, such as PTH. To investigate
whether
up-regulation
SOST/sclerostin
in
mechanical
unloading
is
an
endogenous, osteocyte intrinsic response (e.g. Wolff's Law) or a hormonal
response to decreased PTH levels, to maintain nominal blood calcium levels, we
have subjected osteocytes to the simulated microgravity environment of the
NASA/Synthecon Rotating Wall Vessel (RWV) bioreactor system.
5.2
The
Introduction
precise
mechanisms
whereby
osteocytes
respond
to
and convert
mechanical stimuli to biochemical signals remain elusive because of a lack of
appropriate in vitro models (Chapter 4). At the molecular level, osteocytes are
thought to regulate the bone's response to mechanical loading by at least two
key molecules, sclerostin and receptor activator of nuclear factor kappa-B ligand
(Rank) 3 ,4. Mature osteocytes are one of the few cells that postnatally produce
sclerostin, encoded by the SOST gene. Sclerostin inhibits bone formation, both in
vitro and in vivo, by directly reducing proliferation and differentiation of
osteoblasts via the canonical Wnt signaling pathway. Sclerostin is thought to act
by binding the low-density lipoprotein receptor 5 and 6 (LRP5 and 6) to inhibit
Wnt-pcatenin-signaling 5-7 . Moreover, sclerostin appears central to the bone's
response to mechanical loading. SOST/sclerostin expression increases with
mechanical unloading 28,
9
and decreases with loading 0 . Furthermore, serum
sclerostin is significantly increased during prolonged, 90 day, bed rest in healthy
volunteers (Chapter 9), in obese patients undergoing weight loss", and acutely
in postmenopausal stroke patients. In addition to the effects of sclerostin, it was
recently shown that soluble Rankl also secreted by osteocytes 3' 4 contributes to
the control of bone remodeling. However, Rankl has also been found to be
68
expressed in a variety of other cell types including osteoblasts, bone lining cells,
keratinocytes, T and B lymphocytes, mammary epithelial cells, and undefined cell
types within the brain1 3 . Thus, it is currently unknown whether osteocytes can
increase Rankl in a cell autonomous manner, potentially serving as an initiator of
the cascade of bone resorption seen in mechanical unloading and microgravity.
Regardless of the initiation mechanisms, the hallmark of immobilization and
microgravity in humans is an increase in bone resorption1 1 4 , resulting in
subsequent transient hypercalcemia with persistently increased urinary and fecal
calcium loss'. The endocrine counter regulatory mechanisms to maintain normal
serum calcium are a reduction in serum parathyroid hormone (PTH) and
consequently lower 1,25 -dihydroxyvitamin D concentrations'. However, PTH is
also a known potent regulator of SOST/sclerostin in osteocytes, both in humans
and
in
animal
models 1 5 1 7 , raising the
possibility that the increase
in
SOST/Sclerostin during unloading or bed rest might be a consequence of
decreased serum PTH rather than direct mechanical sensing by osteocytes.
Indeed, there is an inverse correlation between PTH and sclerostin in male
hypoparathyroid subjects and PTH infusion in healthy men induces a decline in
circulating sclerostin1 7 . Both in vivo and in vitro, PTH decreases sclerostin
expression via activation of the PTH receptor expressed on osteocytes1 5 and
mice lacking the PTH receptor specifically in osteocytes have elevated
expression of sclerostin 9 . Thus, in-vivo studies cannot determine whether
suppression of PTH, or other changes in cytokines, such as PGE 22 0, are driving
the increases in serum sclerostin following unloading. More broadly, there is no
evidence to assess whether the increase in SOST/sclerostin is a direct osteocyte
response to mechanical unloading as postulated by the mechanostat theory
.
postulated by Harold Frost21
The primary hypothesis and objective of this study was to determine whether
mechanical unloading is sensed in an osteocyte endogenous manner and
investigate
the
cellular
mechanism(s)
69
osteocytes
employ
to
regulate
SOST/sclerostin. We hypothesized that simulated unloading (microgravity), as
achieved in the NASA rotating wall bioreactors would increase SOST/sclerostin
in a cell autonomous fashion and that this increase would be suppressible by
negative regulators (PTH, PGE 2) of SOST/sclerostin. As reported herein,
osteocytic cells are indeed capable of responding to reduced mechanical forces
with a time-dependent increases in SOST/sclerostin expression. In addition, the
gene expression profile in simulated microgravity (e.g. SOST, Osteocalcin, Phex,
MEPE) is distinct from that seen with mechanical loading, as achieved by fluid
shear stress.
Moreover,
the increase
in
SOST/sclerostin
expression
is
suppressed by PTH and PGE 2, suggesting upstream mechanistic overlap
between mechanical sensing and G-protein-coupled receptor signaling and the
potential to use targeted therapies in these signaling pathways as treatments for
disuse induced bone loss.
5.3
Materials and methods
5.3.1
Simulated microgravity
Ocy454 cells were plated on three-dimensional scaffolds as described (Chapter
4) and allowed to grow at the permissive temperature (33 'C) for three days.
Subsequently, scaffolds were moved to the semi-permissive temperature (37 OC)
for an additional culturing time before being loaded into the NASA/Synthecon
RWV bioreactors. Scaffolds were cut into 3 mm discs using disposable biopsy
punches (Integra Miltex, Plainsboro, NJ) and placed into non-rotating (static) or
rotating (simulated microgravity) 110 ml slow-turning-lateral-vessels (STLV;
Synthecon, Houston, TX) for three days. For the rotating vessels, rotation speed
was set to 18.6 rpm for the first 24 hours and increased to 20.9 rpm to maintain
.
solid body rotation kinetics throughout the experiment 22
70
5.3.2
Two dimensional laminar fluid shear stress
cells were plated
Ocy454
on glass
microscope culture slides (Flexcell
International Corp., NC) at 2 x 10 5 cells/ml and allowed to grow at the permissive
temperature (33 OC) for three days. Subsequently, slides were moved to the
semi-permissive temperature (37 OC) for an additional culturing time (11-14
days). Media was changed to static slide or slides were loaded into the laminar
fluid flow shear stress device (Flexcell Streamer, Flexcell International Corp, NC)
connected to an electronically controlled peristaltic pump with pulse dampers
integrated into the flow circuit to allow for continuous unidirectional shear stress.
Cells were exposed to 0.5 or 2 dynes/cm 2 for a period of either 2 hours or 3.
days 23 -25
5.3.3
Three dimensional laminar fluid shear stress
Alvetex scaffolds were seeded with 1.6 million cells and allowed to grow at the
permissive temperature (33 'C) for 2 days prior to transferring to (37 OC) for
differentiation. Cells were differentiated for 14 days prior to transferring to the
Reinnervate Perfusion Plate. The perfusion plates were attached to a Masterflex
Peristaltic Pump (#7520-57) with a Masterflex Standard Pump Head (#7014-20)
and exposed to either 0.5 or 2 dynes/cm 2 for a period of either 1-day or 3-days.
5.3.4
Statistical Analysis
All values are reported as the mean
SD, unless otherwise noted. Group mean
differences were evaluated with Student t-test and considered significant at
p <0.05.
71
5.4
Results
5.4.1
Fluid shear stress regulation of Ocy454 in two-dimensional culture
Ocy454 were subjected to continuous unidirectional fluid shear stress in twodimensional culture conditions. Consistent with previous reports using UMR
106.01 osteoblast-like cells
23,
short-term (2 hour) fluid shear stress, significantly
suppressed SOST mRNA levels at low and high shear stresses (Figure 5-1).
Whereas Rankl was reduced at low shear stress (0.5 - 2 dyne/cm 2), and Rankl
and DMP1 were increased at higher shear stress (8 dyne/cm 2 ), as shown in
Figure 5-1. These results demonstrate that Ocy454 cells are exquisitely
responsive to mechanical forces with an intact SOST, DMP1 and RANKL
regulation to overloading stimuli. Our results also suggest differential regulation
of SOST and DMP1 to fluid shear stress, but not to simulated microgravity;
whereas the response to hPTH(1-34) is same (Figure 5-2).
C
B
A
SOST
DMPI
RANKL
2.0
1
2.
mStatic
0.5 dynes/cm 2
8
Static
-W C=
< 146
I .
2 dynes/cm 2
4.Static
8 dynes/cm 2
'
.
,;
H R.2
2
0.
R
0.02H
Figure 5-1: Short-term (2 hour) fluid shear stress in two-dimensional-culture reduces (A)
SOST, (B) increased DMP1 at high shear stress (8 dyne/ cm2), (C) reduces RANKL at lowshear stress (0.5 - 2 dyne/cm2) and increases RANKL at high shear stress (8 dyne/cm2).
* p<0.001, ** p< 0.05 static vs. fluid shear stress. Error bars represent one SD.
5.4.2
Simulated microgravity increases SOST/sclerostin and Rankl
We utilized the NASA-developed rotating wall bioreactor system to mimic
microgravity to assess whether osteocytes can directly sense mechanical
72
unloading and regulate the expression of sclerostin and Rankl, known be
involved in the bone's response to unloading.
Indeed, under simulated
microgravity conditions (3 days), there was a statistically significant increase of
3.5
1.9 fold (p < 0.001) in SOST expression compared to static controls (Figure
5-2A). Secreted sclerostin, as assessed by ELISA, was also increased as early
as 1 day by 1.4
0.001
0.1, by 2.7
0.4 at 2-days, and by 4.7
0.1 at 3-days (p<
for all) (Figure 5-2B). There were no significant changes in other
osteoblastic genes (osteocalcin, alkaline phosphatase, osterix mRNA) between
the loaded and unloaded bioreactors demonstrating that the increase in
SOST/sclerostin expression was not a consequence of an altered cell state as
we observed in our prolonged two-dimensional fluid shear stress experiments. In
an effort to identify upstream regulator of SOST/Sclerostin expression, we
assessed changes in reported and potential regulators of SOST in the Mef2
pathway (Mef2A-D), PGE 2 pathway (mPTGES-1, 15-HGPD, EP 2 , EP 4 ), SIRT1,
Osterix, PTHrP, PTH receptor, and periostin. We observed no changes in mRNA
levels for any of these known regulators of SOST (Table 5-1) following simulated
microgravity.
73
B
A
SOST
Sclerostin
.1
z
Static
C Simulated uG
I
0
412
150
m
-
6
m
'"
#
100
Static
Simulated uG
Lu2-
z
S
i
20f*
.
~..
PTH
VEH
C
NDND
nImI
PGE2
DMP1
OPG
RANKL
4-
1.5-
2X a9
*
3-
Static
Simulated uG
,
2-
2-
0.5-
0ma
0-
10-
0.0-1
gp38
MEPE
Phex
2.0-
""
3-
1.0-
*111-
E
m
**
Vp
z
RANKL/OPG
4-
4-
CU
3-
i
2.5-
Osteocalcin
1.5-
1.5,
**
o
1.5-
<0
,I
2.01.5-
1.
1.0-
0.5-
0.5-
0.
0.0-
1.0-
1.0-
0.5-
0z
0.0-
0.5-
0.0-
UT
#
Figure 5-2: 3-day simulated microgravity (white bars) increases (A) SOST compared to static
controls (black bars). 4-hour hPTH(1-34) (50 nM) and 16-hour PGE 2 (5 nM) decrease SOST in
both simulated microgravity and static controls. (B) Sclerostin increases as early as 1-day
exposure to simulated microgravity and remains elevated through 3-days, overnight (16hour) hPTH(1-34) (50 nM) treatment on days 2-3 suppresses secreted sclerostin as
measured by ELISA (APLCO). 3-day simulated microgravity increases (C) DMP1, Rankl,
Rankl/OPG ratio, gp38, and MEPE, decreases OPG, has no effect on Phex or Osteocalcin
p<0.001, ** p<0.05 for simulated microgravity vs. static controls, * p< 0.001 for all
hormone/cytokine treatments vs. VEH. ND: Not detected. Error bars represent one SD.
74
Consistent with previous reports of osteoblasts increasing Rankl expression in
simulated microgravity conditions [49], we observed increased Rankl mRNA
(Figure 5-2C) and a concurrent modest reduction in OPG mRNA (Figure 5-2C)
resulting in a statistically significant increase in the Rankl/Opg ratio in unloaded
vs. static conditions (Figure 5-2C). We also detected a modest increase mRNA
encoding DMP1, MEPE, and gp38 with no change in Phex or osteocalcin mRNA
under simulated microgravity conditions (Figure 5-2C). Thus, we report these
regulatory changes to osteocytic genes as a signature of osteocytes exposed to
simulated microgravity.
Table 5-1: Evaluated Regulators of SOST/Sclerostin in Simulated Microgravity
ECR5 Enhancers
Mef2A, C, D, Mef2B (not expressed)
SOST Promoter Transcription Factors
TFGB1
Osterix
Runx2
SIRT1
Pax6
Periostin
MyoD: Not expressed in Ocy454
Gsa
PGE2 Pathway
EP2, EP4
Cox-2
mPTGES-1
15-HGPD
Cell Membrane Receptors
PTH1R, PTHrP: Not expressed in Ocy454
P2XR1 -7
5.4.3
GPCR responsiveness: SOST/Sclerostin in simulated microgravity
To determine whether activation of PTH receptors (or other G-protein coupled
receptors) could still suppress SOST/Sclerostin in microgravity, we tested the
effects of PTH and PGE 2 treatment in simulated microgravity. PTH (Figure 5-2A)
suppressed SOST and sclerostin levels of expression (Figure 5-2B) to the same
75
extent in static and unloaded conditions (p < 0.001). Similarly, PGE 2 caused the
same magnitude of suppression of SOST expression in both static and simulated
microgravity. These results demonstrate that, while the increase in SOST
expression is not dependent on reductions in GPCR expression (PTHR, EP2/4)
or Gsc. activity (Table 5-1), modulating GPCR signaling can still regulate
SOST/Sclerostin expression in the setting of microgravity or unloading, such as
disuse-induced bone loss.
5.4.4
Long Term fluid shear stress regulation of Ocy454
One limitation of the NASA rotating wall bioreactor system is the possible
generation of minimal fluid shear stress demonstrated to be on the order of 0.5-2
dynes/cm
2
22,26
. To investigate whether the changes in gene expression observed
in the NASA bioreactor were indeed due to simulated microgravity and not
minimal shear stress, we subjected Ocy454 cells to long-term exposure (24 hour
or 72 hour) to low laminar fluid shear stresses (0.5 - 2 dyne/cm 2) in threedimensional (Alvetex) culture conditions. At 2-dyne/cm 2 we observed a significant
reduction in SOST mRNA and no change in DMP1 mRNA at 24-hr (Figure 5-3).
AL
U.5
Uyne/ucr2 we observed significant suppression of S ST mRNA, a
significant increase in DMP1 mRNA, decreases in Opg, MEPE, gp38 (24 hour),
and osteocalcin mRNAs with no effect on Rankl or Phex mRNAs (Figure 5-3).
Similar results for 2-dyne/cM2 were observed at 72-hr (Figure 5-3), with the
exception of a lack of regulation of DMP1 mRNA. These data clearly indicated
that the up-regulation of SOST/sclerostin present in the NASA rotating wall
bioreactor system was indeed due to simulated microgravity and not minimal
shear stress.
76
3
OPG
RANKL
DMPI
SOST
15-
15-
10-
10.*
2.0-
0 C,-
Statc
2
0.5 dynes/cm
2
2 dynes/cm
1.5-
2-
1.0
5-
;5.
E
*
5-
0.5
5
oz
0
-*-
*
3d
ld
0.0
0
0- A4
3d
Id
Phex
Id
3d
Osteocalcin
gp38
3-
5-
4-
4-
3-
2
3d
Id
MEPE
3-
*
1z
E0
2-
*
3-
C.
22E
2-
*
0ld
3d
0
0-
0
Id
3d
Id
3d
Id
3d
Figure 5-3: Fluid shear stress of Ocy454 in three-dimensional culture at 1-day and 3-day
2
fluid at low shear stress of 0.5 dyne/cm2 and 2 dyne/cm (A) reduces SOST, (B) increases
DMP1, (C) decreases OPG, (D) Phex, (E) MEPE, (F) increases gp38, (G) and decreases
osteocalcin at the shear stresses and time points. * p<0.001, ** p<0.05 static vs. fluid shear
stress. Error bars represent one SD.
In addition, as shown in Figure 5-4, we subjected Ocy454 to 2D long-term low
fluid flow. These low flow conditions induced changes in the differentiation state
of Ocy454, as illustrated by significantly elevated levels of expression of SOST,
DMP1, Rankl, Opg (24 hours), Phex, MEPE, and gp38 with a reduction of
osteocalcin expression (Figure 5-4).
77
Overall, these two-dimensional and three-dimensional long-term mechanical
over-loading results demonstrated that our simulated microgravity experiments
reflect a unique osteocyte cellular response to mechanical under-loading stimuli.
OPG
DMPI
SOST
30-
150-
20-
100.
4-
Static
2
0.5 dynes/cm
Static
2
2 dynes/cm
3d
.2
.2U
2-
E
.r0
10
z E'U 10
azE
50-
0*
0
03d
3d
3d
Phex
MEPE
RANKL
25-
15-
.2
20Ce 4
0
2:
15-
.- 10-
E
tS
z
-I
50-
p
I
3d
=
<0
E
2-
I
04U
3d
3d
gp38
Osteocalcin
3
3-
4
22
.$
.z E
1.
0
1L
3d
3d
Figure 5-4: Long-term (3-day) fluid shear stress in two-dimensional-culture increases
SOST, DMP1, RANKL, OPG (24 hour), Phex, MEPE, gp38, and decreases osteocalcin at the
indicated shear stresses and time points. * p<0.001, ** p<0.05 static vs. fluid shear stress.
Error bars represent one SD.
78
5.5
Discussion
The primary objective of this study was to determine whether increases in
SOST/sclerostin and Ranki seen in the context of disuse induced bone loss are
an intrinsic osteocytic response to mechanical unloading. While it has been
established that osteocytes are key players in the bone's response to mechanical
stimuli 10,2 3,24 ,2 7,2 8, it is still unclear whether their response to unloading is a direct
response to reduction in load, as theorized by Wolf's Law, or a consequence of
changes
in
systemic
endocrine
or
paracrine
factors.
Furthermore,
the
biochemical response of the osteocytic network to overloading 10 ,2 3 ,2 4 does not in
it of itself provide evidence for a direct response to unloading stimuli. Here we
present new data showing that osteocytes elicit an intrinsic response to
mechanical loading which is independent of the known external hormonal
influence of PTH and other factors.
Prior studies in rodents have reported increases in SOST/sclerostin in bone
tissue9 '1 0 and in circulating sclerostin 9 during unloading. In addition, increased
circulating serum sclerostin levels with a concurrent reduction of PTH levels have
been reported in the context of disuse-induced bone loss in rodents 29 [55] and
humans (Chapter 9). However, as PTH is a strong negative regulator of
SOST/sclerostin, these in-vivo studies cannot address the question of whether
osteocytes can directly sense mechanical unloading or respond to hormonal
changes.
Importantly, our results suggest that the increase in bone resorption in
mechanical unloading and microgravity with associated transient hypercalcemia
and reduced parathyroid hormone levels is not the driving force for increases in
SOST/sclerostin and Rankl expression. Thus, for the first time, we have
observed isolated osteocytes sensing mechanical unloading and responding with
increases in SOST/sclerostin and the Rankl/Opg ratio.
79
The transcriptional regulators of SOST/Sclerostin in mechanical unloading are
currently unknown. However, Mef2 transcription factors have been shown in
several contexts to bind a distal enhancer (ECR5) in the SOST locus resulting in
the increased expression of SOST/sclerostin 30,31. However, we observed no
transcriptional changes in the potential regulators of SOST in the Mef2 pathway
(Mef 2A, C, D) (Table 5-1). Furthermore, since PGE 2 is a known negative
regulator of SOST/Sclerostin in a Mef2 independent mechanism
32
and reductions
in PGE 2 production genes (Cox-2) have been observed in osteoblasts exposed
to microgravity 33, we assessed changes in the PGE 2 production or degradation
pathways (mPTGES-1,
15-HGPD) and receptor expression (EP2, EP4), as
shown Table 5-1. Notably, no changes in mRNA of transcripts responsible for
PGE2 production, PGE 2 degradation, or PGE 2 receptors were observed between
static and unloaded cultures implying that the increases of SOST/sclerostin in
mechanical unloading are presumably not arising from changes in the PGE 2
pathway. Several transcription factors have also been reported to suppress the
SOST promoter (like SIRT1, Osterix) 34 '3 5 or act at the distal enhancer (ECR5)
(like TGFp 1 .3) 3 0. However, in the context of mechanical unloading we observed
no change in the SIRT1 Osterix, nr T(FR1
3 mRNA
(Table 5-1). It has also been
proposed the periostin (Postn) matricellular protein suppresses SOST in a
Mef2C-dependent mechanism that is regulated by PTH 36 . However, in Ocy454
we observed no correlation between sclerostin, PTH, and Postn mRNA or protein
expression in two-dimensional cultures or in the context of mechanical unloading
(data not shown). Thus, future studies, investigating the novel transcriptional or
post-transcriptional regulation of SOST/sclerostin in the context of mechanical
unloading and microgravity are warranted.
G-protein coupled hormonal (PTH) and cytokine regulators (PGE 2) were capable
of suppressing the increases of SOST/Sclerostin seen in mechanical unloading.
Thus, while our results show osteocytes can directly sense mechanical
unloading, they also suggest the overall level of sclerostin in-vivo appears as an
integral response of the osteocyte network to mechanical loading, hormonal, and
80
cytokine cues. Of particular note, we have shown mice lacking PTHR in
osteocytes lose bone in the hind limb unloading model consistent with our in-vitro
findings that GPCR signaling may play a minimal role in disuse induced bone
loss. One study has recently reported that SOST regulation in mechanical
unloading in rodents could be site-specific with modest (-1.5%) down regulation
in cancellous metaphyseal and cortical bone while up regulation was seen in
diaphyseal cortical
37
regions. Our results are consistent with these findings as
our cell lines were isolated from the diaphysis of long bones. However, as the
majority of osteocytes in the load-bearing skeleton are located in the diaphysis of
long bones and circulating levels of sclerostin are elevated in the setting of
disuse-induced bone loss, the clinical significance of the heterogeneity nature of
the osteocytic network remains to be further explored. Furthermore, while the
NASA rotating wall bioreactor provides a solid-body rotation with a minimal fluid
shear stress in the range of 0.5 - 2 dynes/cm 2
22,26,
no currently existing in-vitro
ground-based model of microgravity can fully eliminate the low level of shear
stress inherent in our model.
However, short mechanical loading10
38
and fluid shear stress 2 3 are known to
cause decreased, not increased, levels of SOST/sclerostin and Rankl as we
have observed (Figure 5-2). To further investigate this confounding variable of
minimal fluid shear stress in the NASA bioreactor, we subjected Ocy454 cells in
two-dimensional and three-dimensional culture conditions to low unidirectional
fluid shear stress. Importantly, neither two-dimensional nor three-dimensional
fluid shear stress matched the pattern of osteocytic gene expression seen in
simulated microgravity. In addition, cells on the surfaces of the scaffolds are
likely exposed to shear stresses higher in range than cells within the scaffold.
However, the same seeding technique was used in all scaffold experiments so
non uniformity in cell distribution could in it of itself not account for the significant
down-regulation of SOST in three-dimensional fluid flow (Figure 5-3) compared
to the increased in SOST (Figure 5-2) we observed in the simulated microgravity
experiments. Finally, additional variables such as nutrient availability could also
81
be playing confounding factors to our observed results. However, the simulated
microgravity experiments utilized a 110-ml bioreactor. Daily changes of 10%
volume of media were also performed to facilitate elimination of bubbles. Thus,
these culture conditions for both static and microgravity conditions are nutrient
rich. Our interpretation notwithstanding, we acknowledge such confounding
variables specific to osteocytic cell cultures in simulated microgravity will need to
be addressed in future experiments under conditions of true-microgravity
(Chapter 6).
5.6
Conclusion
In conclusion, isolated osteocytes can directly sense a mechanical unloading
stimulus resulting in the increases in expression of both inhibitors of bone
formation, SOST/sclerostin, and stimulators of bone resorption, notably Rankl
and the Rankl/Opg ratio. Future therapies, aimed at modulating the osteocyte's
gravity-sensing pathways could lead to improved therapies for a range of bone
disorders.
82
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85
86
Chapter 6
6
Preparation for an osteocyte cell line experiment to the
International Space Station
This thesis chapter, in part, previously published as the manuscript: Pajevic,
Spatz, et al., Osteocyte biology and space flight, Current Biotechnology 2013
2013; 2(3): 179-183.
87
6.1
Rationale
The NASA rotating wall bioreactor operates on the principle of subjecting cells to
continuous rotating fluid environment that randomizes the gravity vector over one
revolution 2 . However as an analog model of microgravity, it has a significant
limitation of also exposing cells to fluid shear stress'. To determine the molecular
mechanisms, in-vitro, of osteocytes exposed to true microgravity, we integrated
Ocy454
into flight proven cell culture hardware
(Osteo-4) onboard the
International Space Station. This chapter describes the validation of osteocytes
(Ocy454) into the Osteo-4 payload in preparation for an ISS mission.
6.2
Key Findings
The osteocytic cell line (Ocy454) grown in spaceflight bioreactors (Osteo-4)
maintained cell functions, such as responsiveness of SOST and Rankl (Figure 66) to the treatment of human parathyroid hPTH(1-34) (100 nM) hormone. This
demonstrated osteocytes can be grown in the conditions necessary for an ISS
experiment.
Simulated
launch random vibrations had minimal
statistically
significant effects on known mechanically responsive genes, SOST and DMP1
(Figure 6-7), highlighting the importance of accounting for the confounding
effects of the launch in the overall experimental design. Experiments were also
conducted to demonstrate that post experiment, bioreactor cooling at 8 'C for up
to 24 hours had no significant effect on RNA recovery and gene expression
(Figure 6-8). These RNA recovery experiments highlighted the importance of
ensuring astronaut resources were allocated to collect science within 24 hours of
bioreactor fixation.
During the ISS Osteo-4 mission, launched April
14 th
2015, media samples were
collected at 6 hours and 24 hours post-launch to examine the effects of launch
vibrations. In addition, these samples serve to provide early time points in
microgravity to examine secreted proteins and cytokines of osteocytes. Two
bioreactors were fixed in RNA preservation buffer (RNAProtect cell reagent,
88
Qiagen)
and
one
was
fixed
for
histology
and
protein
recovery
(1%
paraformaldehyde) at 2-, 4-, and 6- days post launch to study both early and late
responses of osteocytes to microgravity.
6.3
Flight Hardware for Microgravity Bone Biology Studies
At present, few long-term cell culture payloads have flown successfully in space,
and limited capability exists on the International Space Station (ISS) for
mammalian cell culture experiments. One of the payloads developed and used
successfully for several previous missions to study bone cell cultures in
microgravity is the Osteo (Figure 6-1, ISS-configuration) payload. The Osteo
system was originally developed for the Canadian Space Agency (CSA) as a cell
culture rack operated by astronauts for Space Shuttle missions. It flew on several
missions, including STS-95 and STS-107 (no data was collected from STS-107).
During the Columbia investigation and "return to flight" timeframe, Osteo was
upgraded by CALM Technologies, Kingston, Canada, to a fully automated
payload, renamed eOSTEO (also referred to as Osteo-3). A Russian FOTON
biosatellite in 2007 flew with two eOSTEO systems aboard; one for CSA
experiments
and
one for
European
Space
Agency (ESA)
experiments.
Investigations for Canadian scientists included effects of parathyroid hormone
(PTH)-related peptide (PTHrP) on osteoblasts, the CD200 ligand pathway in
osteoblasts and osteoclasts, and the cytoskeleton of bone cells in microgravity.
European
investigators
looked at fibrogenesis of osteoblasts, osteoclast
differentiation, and the effects of microgravity on the cross-influence of
osteoclasts and osteoblasts 3 Upgrades to eOSTEO were begun in 2012, to
further improve the scientific yield, and allow the hardware to be flown on ISS.
This upgraded hardware was renamed Osteo-4, and the system included
automated hardware specifically designed for bone cell cultures.
Osteo-4 is
currently the system of choice for culturing bone and bone-derived cells in
microgravity with significant flight heritage.
89
Another fully automated payload recently used to investigate the effect of
microgravity on bone marrow derived mesenchymal stem cells (BMSC), is a
bioreactor from Kayser-Italia 3 . The spaceflight bioreactor is comprised of five
cylindrical plunger compartments in which cells are cultured on porous ceramic
disks (Skelite) and maintained in a culture chamber 3. The latest NASA payload
for cell culture is the Cell Bio Tech Demo (CBTD), a precursor to new hardware
currently developed by the agency and scheduled for delivery to the ISS in
middle of this decade.
Osteo payloads operate as a close system that, in contrast to a standard in vitro
culture conditions, is not exposed to a controlled carbon dioxide (C02)
concentration (i.e., 5% CO2 for routine culturing of Ocy454). This closed system
does not allow for free exchange of oxygen and C02, requiring either the use of
specialized culture medium or the addition of buffers to maintain the pH of the
cell cultures within the physiological range. For Osteo-4, we chose to supplement
our media with 25 mM HEPES and perform daily media changes to maintain pH
and nutrient availability to the osteocytic cell line (Ocy454). Under these
conditions, Ocy454 were successfully qrown in the Osteo-4 hardware for up to 14
days in pre-mission ground-based testing experiments.
6.4
Osteo-4 upgrades for ISS compatibility
The Osteo-4 hardware modifications were upgrades to the Osteo-3, to meet
requirements of flying the hardware on the International Space Station. ISS
interface requirements were met through the use of a modified standard ISS
locker, which allows installation and operation of the payload in specialized
payload racks, called "expedite the processing of Experiments for Space Station
(EXPRESS)
racks". In addition to the interface upgrades, science related
activities were facilitated through the on-orbit removal of bioreactors at
experimental time points to allow astronauts to store the fixed bioreactors in cold
(-95
0C)
stowage. Design modifications to the existing Osteo-3 flight trays and
bioreactors were avoided to minimize cost and risk by reusing hardware that had
90
previously and successfully completed cell culture experiments in microgravity.
Historically, the Osteo systems have flown in a stacked configuration, comprised
of four trays with a base unit containing communication avionics. In these prior
Osteo configurations, the adjacent top tray provided the function of a lid and
there was no provision for on-orbit removal of bioreactors. As the ISS Osteo-4
configuration allows for the removal of individual trays and bioreactors at
scientifically relevant time points, the Osteo-3 stacking approach to the structural
layout of the payload was no longer suitable for the Osteo-4 mission.
Thus, the Osteo-4 approach (Figure 6-1), consists of three removable trays
(Figure 6-2, 6-3) that are integrated into an EXPRESS locker (Figure 6-3). The
locker system provides a common power, data, communications, and thermal
interface (avionics air for within tray cooling to 8-to-16 0C) for the trays, and a
means of securing the trays for launch and storage. This approach required that
each tray provide qualified levels of containment to meet NASA on-orbit safety
standards. In order to achieve this, clear polycarbonate tray lids were added to
each tray (Figure 6-3). In addition, quick disconnects on each bioreactor (Figure
6-5) and access panels were included in the lid design to allow on-orbit recovery
of the bioreactors by the astronauts for subsequent cold stowage of the
bioreactor after each of the desired experimental time points.
Figure 6-1:Osteo-4
ISS payload within EXPRESS rack locker (gray)
91
Waste Media Sample Chamber
Waste Bag
Valve Actuators
Syringes
Bioreactor
Bioreactor Cooling
Tray Electronics
Valve Actuators
Figure 6-2: 3D Solid Model of Single Osteo-4 Tray (Interior)
Figure 6-3: Tray Removal from Express Rack (top) Osteo-4 tray with lid (bottom, left), and
Osteo-4 lid showing access ports for bioreactor removal (bottom, right)
92
6.4.1
Osteo-4 Fluid Pathway
The Osteo-4 fluid pathway (Figure 6-4) is used as the first level of containment
and contains all the fluid reagents (syringes) and cell cultures (bioreactors) for
the science.
6OmL Syringes
Sample
MEDIA
14
MEDIA
One-way
Valves
I
Waste Bags
Cannula/Septum
Disconnect
-n
0
MEDIA
MEDIA
Main Waste Bag
)
-11E
I
0
Disconnec
P--nt
Cell Cuture
Bioreactor
Modules
Automated
Valves
Figure 6-4: Osteo-4 Fluid Pathway Configuration Diagram
93
Figure 6-5: Osteo-4 bioreactor with 2 ml sample bag (top), Ocy454 on Alvetex Scaffolds
integrated into Osteo-4 assembly (bottom, left), Osteo-4 bioreactor integrated into fluid
pathway (bottom, right)
In operation, the Osteo-4 tray performs fluid delivery in the closed fluid pathway
by opening one of four valves (3 Bioreactor, 1 flush, Figure 6-4) allowing fluids
from one of the four syringe- based fluid reservoirs to be delivered to the selected
bioreactor or flush line. When a bioreactor fluid sample is desired, one of 3
sample waste valves is opened during a feed to collect a small 2 mL sample. As
this is a flow-through system, waste fluid is collected in the main waste bag.
6.5
Material and Methods
6.5.1
Cell Culture
The Ocy454 cell line was cultured at permissive growth temperature (33
0C)
maintained on rat type I collagen coated flasks grown in Minimum Essential
Media Alpha Media aMEM supplemented with 10% fetal bovine serum (Gibco,
Grand Island, NY) and 1% Antibiotic-Antimycotic (Gibco, Grand Island, NY).
94
Upon
confluence, Ocy454
were
plated at
1.6 million
on Alvetex
cells
(Reinnervate, UK) 6-well insert scaffolds and grown for 3 days at 33 'C, media
changed, and grown for 3 days to 2 weeks at 37 0C prior to integrating five
alvetex scaffolds (9
mm)
into
Osteo-4 bioreactors.
Media
for Osteo-4
experiments was additionally supplemented with 25 mM Hepes buffer to maintain
physiological pH in the non-CO 2 closed environment bioreactors for up to 48
hours. To preserve RNA for later analysis, selected Osteo-4 bioreactors were
fixed with an RNA stabilization buffer (RNAprotect Cell Reagent, Qiagen). To
preserve bioreactors for histology and protein analysis, selected Osteo-4
bioreactors were fixed with 1% paraformaldehyde in phosphate buffered saline,
pH 7.4.
6.5.1
Quantitative Real Time PCR
Two 9 mm scaffolds were pooled and total RNA was isolated (RNAEasy, Qiagen,
Valencia,
CA)
per
manufacture's
recommendations
followed
by
RNA
quantification (NanoDrop, Thermo Scientific, Rockford, IL). cDNA synthesis was
performed on 500 to 1000 ng total RNA, followed by SYBR qPCR (StepOnePlus,
Life Technologies, Grand Island, NY).
6.6
Results
6.6.1
Osteo-4 spaceflight bioreactors
We performed experiments in Osteo-4 bioreactors (Figure 6-4, 6-5) to test the
scaffold insert holder biocompatibility and the no-CO 2 environment of the Osteo-4
spaceflight-qualified bioreactors. To this end several commercially available noCO 2 media
(Life Technologies) were tested.
However, the phosphate buffers
used in commercially available no-CO 2 media adversely effected basal gene
expression of Ocy454 (data not shown).
aMEM supplemented with 10% fetal
bovine serum (FBS) and 25 mM Hepes was chosen because it preserved the
osteocytic gene expression pattern and maintained the pH of the medium within
physiological range for a minimum of 48 hours between media changes. In some
experiments, at the end of the culture period bioreactors were fed with medium
95
containing vehicle or 1OOnM human PTH(1-34) and the experiment was
terminated 4 hours later with the injection of fixatives to preserve samples for
RNA
(RNAProtect
isolation
cell
reagent,
Qiagen)
or
histology
(1%
paraformaldehyde) as performed during the ISS flight mission. As shown in
Figure 6-6, Ocy454 cells maintained their PTH responsiveness and readily
suppressed SOST by 60% and increased RANKL by 35% upon hormonal
treatment. These results match the in-vivo and in-vitro response of osteocytes
(see Chapter 3-5) and reflect the cells maintain their osteocytic phenotype in the
Osteo-4 bioreactors.
RANKL
Sclerostin mRNA
VEH
80
4
*
3-
60
Ir '0 2
E .4
40
ePTH
:E1.20
0z*
0
0
2wk
2wk
Figure 6-6: Ocy454 responsiveness to PTH. Ocy454 were seeded into 9mm Alvetex scaffolds and
cultured for 3 days at 33C. Cells were then loaded into single loop bioreactor and cultured for 14 days.
Medium was changed manually every other day. Cells were treated with PTH for 4 hour prior to RNA
isolation and gene analysis. Data normalized by beta actin. * p<0.05.
To assess sclerostin expression in these Ocy454 within the Osteo-4 bioreactors,
we performed immunohistochemistry (IHC) for sclerostin in Ocy454 cells grown
for 7 days in an Osteo-4 bioreactor and treated with PTH or vehicle for 4 hours at
the end of the culturing period. Sclerostin was readily detected in Ocy454 cells
(Figure 6-7A), and as expected, its expression was significantly suppressed by
PTH treatment, as revealed by reduced immunostaining, (Figure 6-7B). Tunel
staining (Figure 6-7C,D) of the same scaffolds revealed minimal cell death under
these conditions demonstrating high cell viability in the Osteo-4 bioreactor
configuration.
96
..........
V,
C
il
J
D
Figure 6-7: Sclerostin and Tunel staining in Ocy454 cells. Ocy454 cells were seeded into 9 mm
scaffold and cultured for 3-4 days at 33 C before being switched to non-permissive temperature
(37C) for an additional 14 days. Scaffold were then loaded into a Osteo-4 bioreactor and cultured for
7 days with a daily medium change. At the end of the 7 days experiments , bioreactor was treated
with PTH of vehicle for 4 additional hr. Scaffold were fixed and processed for IHC and Tunel assay.
Sclerostin IHC in vehicle (A) and hPTH (1-34), 100 nM (B)
Importantly, these result demonstrate Ocy454 have intact osteocytic signaling
pathways in the Osteo-4 bioreactors and will enable us to test the response of
OCY454 to microgravity stimuli during the ISS mission.
6.6.2
Osteocytic response to random launch vibration
As osteocytes are sensitive to mechanical loading, we performed experiments in
Osteo-4 to investigate the effects of launch vibrations on Ocy454. In brief,
Ocy454 were grown for 1-week prior to integration into the Osteo-4 bioreactors
and two identical payloads. Next, one Osteo-4 payload was subjected to single
axis random launch vehicle (SpaceX Falcon 9, Dragon Capsule) vibrations on a
shaker table (Draper Laboratories, Cambridge, MA) for a duration of 180
seconds. The other Osteo-4 payload served as the non-vibrated control.
Subsequently, RNA was isolated at 2- and 24-hours following the random
vibration test completion. The number of bioreactors at each time point was: two
static and one random vibration bioreactor at 2-hour time point, and one static
and three bioreactors at 24-hour time point.
97
DMP1
Osteocalcin
2.0
1.&
Sclerostin mRNA
3-
M Static
.111
.0,
z "
1m Random Vibration
1.0-
2
0.&
1
0 1.0-
.
E
ill.
0z
0.i
0.0
2 HR
24 HR
24 HR
2HR
24HR
Figure 6-8: Ocy454 gene expression changes following single axis random vibration test
of the Osteo-4 payload simulating SpaceX Falcon 9 Dragon launch
For two known mechanically responsive genes (DMP1
and SOST), non-
statistically significant changes were observed at both time points, except for a
small statistically significant up-regulation of SOST and down-regulation of
osteocalcin at the 24-hour time point (Figure 6-8). However, as SOST is known
be down regulated with mechanical loading and fluid shear stress (Chapter 5),
these findings given the limited number of biological and technical replicates,
should be interpreted accordingly. To help account for this potential effect of
launch loading as a confounding variable for the Osteo-4 ISS flight experiment,
2-ml of medium was collected from both ground and flight bioreactors at 6- and
24- hours post launch.
6.6.3
Preservation of Osteo-4 bioreactors in space flight environment
Given the unique crew constraints of conducting a cell culture experiment on the
ISS, coupled with our ability to provide temporary within-tray cooling of
bioreactors to 8-to-12
stowage at -95
DC),
0C
(pending crew recovery of bioreactors for long term
we sought to investigate the effects of the key osteocyte
genes under these thermal conditions. Importantly, we discovered SOST, a key
osteocytic gene under investigation for the Osteo-4 mission, was significantly
down regulated after extended cooling (48 hours) when Ocy454 was grown
under normal growth conditions, integrated into Osteo-4 bioreactors (Figure 6-9),
and fixed with a buffer (RNAProtect cell reagent, Qiagen) to preserve RNA for
98
subsequent isolation and analysis upon sample returned to earth. This data
highlighted the importance of astronauts moving Osteo-4 bioreactors to deep
cold stowage (-95
0C)
post fixation with the RNA preservation buffer (RNAProtect
cell reagent, Qiagen) within 24-hours, to obtain optimum science return. This
data set, related to the performance of RNA cell protection buffers to minimize
changes in gene expression if science is recovered within 24-hours is widely
applicable
to
science
other
conducting
investigations
ISS
cell
culture
experiments.
B
A
Sclerostin mRNA
SOST
2.0
1.-
M 24HR
48HR
01.
24HR
48HR
0
01.0
E
0.5
0
0
*
Z
0.
0.0
8C
12C
16C
Figure 6-9: (A) Ocy454 SOST regulation on three-dimensional scaffolds (Alvetex) grown
under normal growth conditions for 4-days (37 *C, 5% CO 2 ), fixed with Cell Protect RNA
preservation buffer, and RNA recovered at indicated time points. (B) Ocy454 SOST
regulation on three-dimensional scaffolds (Alvetex) grown under normal growth
conditions for 4-days (37 "C, 5% CO 2), integrated into Osteo-4 bioreactors for 4-days, fixed
with Cell Protect RNA preservation buffer, and RNA recovered at indicated time points.
99
6.7
Conclusions
We have shown Ocy454 grew well within Osteo-4 bioreactors with no significant
cell apoptosis. In addition, Ocy454 maintained osteocytic responses in the
Osteo-4 spaceflight qualified bioreactors by reducing SOST and increasing Rankl
upon PTH treatment, showing that important osteocytic biological pathways
remain intact in the spaceflight qualified bioreactors and flight conditions. In
addition, we demonstrated the importance of astronauts recovering Osteo-4
science for long term cold-stowage within 24-hours of bioreactor fixation. Lastly,
the Osteo-4 hardware
successfully completed
all technical and
science
operations for the ISS mission and bioreactors have been returned to earth for
processing and downstream analysis.
1
2
3
Gutierrez, R. A. & Crumpler, E. T. Potential effect of geometry on wall
shear stress distribution across scaffold surfaces.
Martinez, E. M., Yoshida, M. C., Candelario, T. L. & Hughes-Fulford, M.
Spaceflight and simulated microgravity cause a significant reduction of key
gene expression in early T-cell activation. doi:D - NLM: PMC4360066
[Available on 03/15/16] OTO - NOTNLM.
Pajevic, P. D., Spatz, J. M., Garr, J., Adamson, C. & Misener, L.
Osteocyte biology and space flight.
100
Chapter 7
7
Sclerostin antibody inhibits skeletal deterioration due to
reduced mechanical loading
This thesis chapter, in part, previously published as the manuscript: Spatz, et al.,
Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical
loading, J Bone Miner Res. 2013 Apr;28(4):865-74. doi: 10.1002/jbmr.1807.
101
7.1
Introduction and Rationale
Despite intensive exercise countermeasures, muscle and bone loss remain
among the top medical challenges to extended space missions. While recent
advances in exercise countermeasures (Advanced Resistive Exercise Device,
ARED) demonstrated on the International Space Station have reduced both
muscle and bone loss for extended missions in microgravity, bone loss at weight
bearing skeletal sites is still -0.5%
per month and highly variable between
individuals'. In addition, serum markers of bone resorption and urinary calcium
were significantly elevated in astronauts using the intensive exercise regimes
provided by the ARED 1 . Thus, significant bone turnover and bone loss is
occurring in long duration astronauts and remains an area of concern for deepspaceflight human missions.
7.2
Pharmacologic prevention of bone loss
Having been shown to reduce fracture risk in large-scale randomized clinical
trials, several drugs are currently approved for prevention of bone loss and
treatment of osteoporosis. Broadly, there are two classes of clinically approved
therapies: 1) anti-resorptive, including the bisphosphonates, calcitonin, selective
estrogen receptor modulators (SERMs), estrogen, and the newest biologic antiRankl antibodies; and 2) anabolic, including teriparatide (PTH (1-34)).
Among the anti-resorptive therapies, the oral bisphosphonates are the most
widely used. Whereas they potently inhibit bone resorption, and therefore afford
protection from bone loss associated with disuse, they are retained in bone for
years, raising some concerns about their use in younger individuals, such as the
astronaut population. Moreover, due to the coupling of bone resorption and bone
formation,
bisphosphonates
also
suppress
bone
formation.
Whereas
bisphosphonates are highly efficacious for reducing vertebral, hip and nonvertebral fracture risk in postmenopausal women, a number of rare, but serious
side effects may be associated with bisphosphonate use, including gastro-
102
intestinal disorders, osteonecrosis of the jaw, atypical femoral shaft fractures,
delayed fracture healing, and bone pain. These concerns notwithstanding, a
study recently completed
tested the effect of the oral
bisphosphonate
alendronate plus exercise in preventing the declines in bone mass and strength
and elevated levels of urinary calcium and bone resorption in astronauts during
5.5 months of spaceflight 2. The combination of the ARED and bisphosphonate
use attenuated the declines in altered bone physiology during spaceflight
including, bone mineral density of the spine, hip, and pelvis assessed by dualenergy x-ray absorptiometry (DXA), compartmental losses in trabecular and
cortical bone mass in the hip assessed by quantitative computed tomography,
.
elevated levels of bone resorption markers, and urinary excretion of calcium 2
However, several individual astronauts were unable to participate or complete
the study due to the aforementioned side effects, particularly gastro-intestinal
concerns during pre-flight phase-in dosing of the oral bisphosphonate.
Taken
together,
these
observations
reduce
enthusiasm
for
use
of
bisphosphonates in astronauts, and provide rationale for developing new
targeted biologic therapies to inhibit spaceflight induced bone loss.
7.3
Key findings
We tested the ability of a murine sclerostin antibody (SclAbll) to prevent bone
loss in adult mice subjected to hind limb unloading (HLU) via tail suspension for
21 days. Mice (n = 11-17/group) were assigned to control (CON, normal weight
bearing) or HLU and injected with either SclAblI (subcutaneously, 25 mg/kg) or
vehicle (VEH) twice weekly. SclAbll completely inhibited the bone deterioration
due to disuse, and induced bone formation such that bone properties in HLUSclAbll were at or above values of CON-VEH mice. For example, hind limb bone
mineral density (BMD)
increased 4.2%
decreased -9.2%
0.7%, 13.1%
1.0%
1.0%, and 30.6%
in HLU-VEH,
whereas it
3.0% in CON-VEH, HLU-
SclAbll, and CON-SclAbll, respectively (p<0.0001). Trabecular bone volume,
assessed by micro-computed tomography (pCT) imaging of the distal femur, was
103
lower in HLU-VEH versus CON-VEH (p < 0.05), and was 2- to 3-fold higher in
SclAblI groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by
three-point bending, and distal femoral strength, assessed by micro-finite
element analysis (pFEA), were significantly higher in SclAblI versus VEH-groups
in
both
(134
loading
5pg/mL)
conditions.
compared
Serum
to
sclerostin
CON-VEH
(116
was
higher
6pg/mL,
in
HLU-VEH
p'<0.05).
Serum
osteocalcin was decreased by hind limb suspension and increased by SclAblI
treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone
outcomes appeared to be enhanced by normal mechanical loading. Altogether,
these results confirm the ability of SclAbil to abrogate disuse-induced bone loss
and demonstrate that sclerostin antibody treatment increases bone mass by
increasing
bone
formation
in
both
normally
loaded
and
under
loaded
environments.
7.4
Background
Pharmacologic inhibition of sclerostin induces bone formation in normal and
ovariectomized animals that are fully weight-bearing3-7 and also following
unilateral limb immobilization in rats". In addition, there is only a short-term (7
days) study that has examined sclerostin antibody treatment in the wellcharacterized hind limb unloading (HLU) model 9 . However, because of the limited
duration of unloading, this study did not demonstrate bone microarchitectural
changes due to HLU, nor report effects on bone mechanical properties of
unloading or sclerostin antibody treatment. Further, there are conflicting reports
in the literature as to whether the optimal anabolic effect of sclerostin antibody
treatment requires normal mechanical loading 38, ,9. Finally, although there is
evidence that SOST is increased by mechanical unloading 0'",
there is limited
data on serum levels of sclerostin following reduced mechanical loading in
animal models.
Thus, in this study we sought to demonstrate the anabolic effects of
pharmacologic inhibition of sclerostin in the HLU model. We hypothesized that
104
sclerostin antibody treatment would
not only inhibit bone loss and the
deterioration of mechanical properties associated with disuse-induced bone loss,
but would also induce bone formation. We also determined whether the skeletal
effects of sclerostin antibody treatment depend on mechanical loading by
comparing the response to pharmacologic inhibition in normally loaded animals
to those exposed to HLU, and by comparing the responses in the forelimbs and
hind limbs of HLU mice. Finally, we determined whether serum sclerostin
increased following HLU to elucidate whether in addition to SOST, the sclerostin
protein is mechanically regulated by disuse.
7.5
Material and methods
7.5.1
Overview of study design
Female adult mice (C57BI/6J, 12 weeks of age; Jackson Laboratory, Bar Harbor,
ME, USA) were subjected to either HLU via tail suspension
, or normal
loading (CON) and injected twice weekly with sclerostin antibody (SclAbll, 25
mg/kg, subcutaneously; Amgen, Thousand Oaks, CA, USA) or vehicle (VEH) for
the 21-day experiment. Thus, mice were assigned to one of four groups: HLUVEH (n = 13), HLU-SclAbll (n = 11), CON-VEH (n = 17), or CON-SclAbll (n =
11). Animals were assigned to groups by total body bone mineral density (BMD)
and body mass in a manner to minimize differences between groups at baseline.
All mice were weighed daily for the first 5 days and biweekly thereafter, with
adjustments made to ensure the hind limb paws could not touch the ground. The
average weight-bearing on the forelimbs of HLU groups was 43% 1.4% of total
body mass. Mice were maintained on a 12/12 hour light/dark cycle and had ad
libitum access to standard laboratory rodent chow and water. Control animals
were singly housed to mimic the increased stress environment of singly housed
HLU animals. Mice were euthanized by CO 2 inhalation at the end of the
experiment.
All animal procedures were
approved by and performed
accordance with the guidelines of the Institutional Animal
Care and Use
Committee (IACUC) at the Beth Israel Deaconess Medical Center.
105
in
7.5.2
Bone mineral density and body composition
In vivo assessment of total body (exclusive of the head region), hind limb, and
forelimb BMD (g/cm 2) was performed at baseline and end of the study using
peripheral dual-energy X-ray absorptiometry (pDXA PiXImusIl; GE Lunar Corp.,
.
Madison, WI, USA), as described 4
7.5.3
Specimen harvesting and preparation
Femurs, tibias, and humeri were harvested and cleaned of soft tissue. The right
femurs and humeri and were prepared for imaging and biomechanical testing by
wrapping in saline-soaked gauze and freezing at -20*C.
The left femur was
prepared for histology in 10% neutral buffered formalin at 40C for 48 to 72 hours,
and then transferred to 70% ethanol at 4*C. Wet weight of the gastrocnemius
and soleus muscles were obtained at the end of the study, and normalized to
body weight.
7.5.4
Bone turnover markers
Mice were fasted for 2 hours before blood was collected at the time of
euthanasia. Serum was used to measure sclerostin (in VEH-treated mice only)
and bone turnover markers. Osteocalcin and sclerostin (in VEH-treated mice
only) were assessed using the species-specific single-plex Luminex assays
(Millipore,
Billerica,
MA,
USA).
Serum
concentrations
of amino-terminal
propeptide of type I procollagen (P1 NP), tartrate-resistant acid phosphatase 5b
(TRACP5b), and type I collagen C-telopeptide (sCTX) were measured by using
mouse ELISA kits (IDS, Fountain Hills, AZ, USA). All assays were run according
to the manufacturers' protocols.
7.5.5
Histology and quantitative histomorphometry
Qualitative
histologic
analysis
and
quantitative
static
and
dynamic
histomorphometry were performed as described15 . To examine bone formation
106
rates, calcein (15 mg/kg) was injected intraperitoneally at 8 days and alizarin red
or demeclocycline 2 days prior to euthanasia. Histomorphometric measurements
were performed on the secondary spongiosa of the distal femoral metaphysis
using an OsteoMeasure morphometry system (Osteometrics, Atlanta, GA, USA).
For dynamic histomorphometry, mineralizing surface per bone surface (MS/BS,
%)
and mineral apposition rate (MAR, pm/d) were measured in unstained
sections under ultraviolet light, and used to calculate bone formation rate with a
surface referent (BFR, pm 3/pm 2/d). Eroded surface per bone surface (ES/BS, %),
number of osteoblasts, osteoclasts per bone surface, number of osteocytes per
bone area (identified as filled lacunae), and number of adipocytes per marrow
area were also measured, as described1 5 . Terminology and units follow the
recommendations of the Histomorphometry Nomenclature Committee of the
.
American Society for Bone and Mineral Research 6
7.5.6
Mechanical testing
Femurs were mechanically tested at a constant displacement rate of 0.03 mm/s
to failure in three-point bending (Bose ElectroForce 3200 with 150 N load cell;
Bose Corporation, Eden Prairie, MN, USA). Fresh-frozen femurs were thawed to
room temperature then centered longitudinally, with the anterior surface on the
two lower support points spaced 10 mm apart17 . Force-displacement data were
acquired at 30 Hz and used to determine maximum force (N) and stiffness
(N/mm). Assessment of bone morphology and microarchitecture was performed
with high-resolution micro-computed tomography (pCT40; Scanco Medical,
Bruttisellen, Switzerland). In brief, the distal femoral and humeral metaphysis
were scanned using 70 KvP, 50 mAs, and 12-pm isotropic voxel size. The
femoral metaphysis region began 240 pm distal to the growth plate and extended
1.8 mm distally. Similarly, the humeri region began 240 pm distal to the growth
plate and extended 1.2 mm distally. Cancellous bone was separated from cortical
bone with a semi-automated contouring program. For the cancellous bone region
we assessed bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th,
mm), trabecular separation (Tb.Sp, mm), trabecular number (Tb.N, 1/mm),
107
connectivity density (ConnD,
1/mm 3),
and
structure
model
index (SMI).
Transverse CT slices were also acquired at the femoral midshaft to assess total
cross-sectional area, cortical bone area, and medullary area (TA, BA, and MA,
respectively, all mm 2); bone area fraction (Ct.BA/TA, %), cortical thickness
(Ct.Th, mm), porosity (Ct.Po, %) and minimum (Imin, mm 4 ), maximum (Imax,
mm ), and polar (pMOI, mm ) moments of inertia. Cortical bone was analyzed
from the metaphysis (surrounding the trabecular volume of interest) and from a
0.6-mm-long mid-diaphyseal region. Bone was segmented from soft tissue using
the same threshold, 247 mg HA/cm 3 for trabecular and 672 mg HA/cm 3 for
cortical bone. Scanning and analyses adhered to published guidelines 8
To assess the effect of unloading and sclerostin antibody treatment
on
mechanical properties of metaphyseal bone, pCT-derived data was used to
perform linear micro-finite element analysis (pFEA) of the distal femur using the
manufacturer's software (Scanco Medical AG, Bassersdorf, Switzerland), which
implements a voxel-based pFEA method 19. The pFE model of the metaphyseal
region, including both cortical and trabecular bone, was subjected to applied
uniaxial compression, with an elastic modulus of 10 GPa and Poisson's ratio of
0.3 for each element. Outcomes included axial stiffness (N/mm) as well as the
percentage of load carried by the cortical and trabecular compartments.
7.5.7
Statistical analysis
All data were checked for normality, and standard descriptive statistics
computed. Treatment effects were evaluated using analysis of variance (ANOVA)
or repeated measures ANOVA for all continuous variables. We used two-factor
ANOVA to determine whether the effect of sclerostin antibody treatment
depended on the loading condition. Main ANOVA effects and post hoc testing
were considered significant at p < 0.05, whereas the interaction between
treatment and loading was considered significant at p < 0.1. Data are reported as
mean
sem, unless noted.
108
7.6
Results
7.6.1
Body mass
Body mass increased slightly in the CON-VEH and CON-SclAbil groups and
declined transiently in the HLU groups in the first 3 days by -8% to -9% but then
below baseline for remainder of study (p < 0.05 versus
stabilized at -5%
baseline). As a result, the HLU-SclAbll and HLU-VEH weighed less than their
respective CON groups at the end of the study (-9.1% and -11.5%, respectively,
p < 0.05).
7.6.2
Muscle mass
Soleus wet weight was 51% and 38% lower than CON in HLU-SclAbll and HLUVEH, respectively (p < 0.0001, Figure 7-1A). Gastrocnemius wet weight was 27%
and 19% lower than CON in HLU-SclAbll and HLU-VEH, respectively (p <
0.0001, Figure 7-1 B). There were no differences in muscle mass between VEHtreated and SclAblI-treated groups in either loading condition.
-
A
.
0.3
B
Soleus
t
*VEH
l SclAbIl
Gastrocnemius
6-
40.2
3
2
$.0
0
CON
~
A
0
HLU
CON
HLU
Figure 7-1: Effects of unloading and sclerostin antibody treatment on normalized wet weight
of (A) soleus and (B) gastrocnemius muscles. t p < 0.01 for CON versus HLU within treatment
group. Error bars represent 1 SEM.
7.6.3
Bone mineral density
BMD increased slightly in CON-VEH at all sites, whereas HLU caused significant
bone loss at the hind limb and total body, but not the forelimb (Figure 7-2).
Treatment with sclerostin antibody not only prevented the bone loss due to HLU,
109
but also led to marked increases in BMD in CON and HLU groups, both versus
baseline and versus VEH-treated groups. For example, hind limb BMD declined
-9.3%
+
1.1% in HLU-VEH, whereas it increased 4.3%
and 30.6%
0.7%, 13.2%
1.0%,
3.0% versus baseline in CON-VEH, HLU-SclAbll, and CON-SclAblIl,
respectively (p < 0.001 for all). The pattern was similar for total body BMD
(Figure 7-2A). Forelimb BMD was unchanged in HLU-VEH (-1.1%
tended to increase in CON-VEH (4.1%
2.6%) and
3.0%, p = 0.2 versus baseline).
Forelimb BMD increased in SclAbll-treated HLU (15.1%
versus baseline) and CON groups (28.6%
2.9%, p < 0.001
2.4%, p < 0.001, Figure 2C); and
these increases were significantly greater than the BMD changes in VEH-treated
animals (p < 0.0001 for both).
A
B
C
Forelimb
Hindlimb
Total Body
35-
EVEH
[ SclAbIl
30
S25E20-
15
10
00-
-
6Z -10
-15
CON
HLU
CON
HLU
CON
HLU
Figure 7-2: Effect of unloading and sclerostin antibody treatment on (A) total body BMD,
(B) hind limb BMD, and (C) forelimb BMD. *Significantly different from baseline (p < 0.001).
Horizontal bars denote significant differences (p < 0.01) between VEH and SclAblI within
loading groups; tp < 0.05 for CON versus HLU within a treatment group. Error bars
represent 1 SEM. (One CON-ScIAblI and three CON-VEH and animals were excluded from
forelimb BMD measurements due to poor positioning on either baseline or follow-up scan.
7.6.4
Bone microarchitecture
Consistent with hind limb BMD measurements, HLU resulted in significant bone
deterioration, particularly in the trabecular compartment (Figure 7-3, Table 7-1).
Compared to CON-VEH, HLU-VEH mice had lower Tb.BV/TV, Tb.N, and Tb.Th
in the distal femur. Cortical bone was also negatively affected by unloading,
because HLU-VEH had lower cortical bone area, cortical bone area fraction,
110
cortical thickness, and polar moment inertia than fully loaded animals at both the
distal femoral and mid-diaphyseal sites (Table 7-1). In addition, HLU-VEH had
higher cortical porosity than CON-VEH at the distal femoral cortex. At the
humerus, trabecular bone parameters were unaffected by HLU; however, cortical
bone area, bone area fraction, thickness, and polar moment of inertia were
slightly lower in HLU-VEH versus CON-VEH (Table 7-2).
111
Table 7-1: Effect of HLU and SclAbil-Il treatment on femoral trabecular and cortical bone microarchitecture,
assessed by pCT (mean
ANOVA Results
HLU
Controls
SciAbll
Vehicle
Site
SEM).
SciAbll
Vehicle
Pload
Ptreatment
Pinteraction
Distal Trabecular
BV/TV (%)
10.3 0.4b
0.04b
32.0
1.5ab
4.32 0.05a, b
8.0
3.72
0.3
21.7
1.2 a
<0.0001
<0.0001
<0.0001
0.05
4.05
0.05a
0.0006
<0.0001
0.1
Tb.N (mm-1)
3.86
Tb.Th (mm)
0.054
0.001 b
0.097
0.002a, b
0.048
0.001
0.075
0.003a
<0.0001
<0.0001
<0.0001
0.252
0.003b
0.198
0.003ab
0.261
0.003
0.217
0.005a
0.008
<0.0001
0.2
3a
0.3
<0.0001
0.06
Tb.Sp (mm)
)
ConnD (mm-
3
)
SMI
Distal Cortical
Tt.CSA (mm 2
2
74
2.99
3b
0.06
2.49 0.03
107
3a
108
55
0.12ab
3.05
0.05
1.98
0.08a
0.01
<0.0001
0.002
2.68 0.05ab
2.48
0.05
2.54
0.05
0.3
0.008
0.9
1.4
0.71
0.01 b
0.96 0.01 a,b
0.57
0.01
0.80
0.02a
<0.0001
<0.0001
0.7
Ct.BA/TA (%)
28.5
0.4b
35.5
0.7a b
22.8
0.4
31.5
0.2a
<0.0001
<0.0001
0.06
Ct.Por (%)
5.9
0.2b
5.3
7.9
0.4
5.3
0.4 a
0.0006
<0.0001
0.003
0.2
0.12 0.002b
0.15 0.002ab
0.10
0.001
0.13
0.001a
<0.0001
<0.0001
0.08
0.50
0.01 b
0.71
0.39
0.01
0.57
0.02a
<0.0001
<0.0001
0.3
1.56
0.018
0.010
0.03
<0.0001
0.05
0.010b
1.58 0.026
0.70 0.012a
0.001
0.66
1.69 0.024ab
0.86 0.015ab
1.54 0.030
)
)
Ct.BA (mm
<0.0001
0.02
Ct.MA (mm 2
0.90
0.014b
0.83
0.013a
0.99 0.023
0.88 0.016a
0.0004
<0.0001
0.2
<0.0001
<0.0001
0.04
<0.0001
<0.0001
0.03
7
0.09
0.1
0.7
0.01 a
<0.0001
<0.0001
0.04
Ct.Th (mm)
4
)
pMOI (mm
)
Midshaft Cortical
Tt.CSA (mm 2
2
)
Ct.BA (mm
Ct.BA/TA (%)
Ct.Th (mm)
TMD (mgHAlccm)
)
pMOI (mm
4
42.1 0.5b
0.16
1148
0.002b
5
0.28 0.01 b
0.02a,b
50.8
0.21
0.4a,b
0.003ab
1161
8
0.37 0.01ab
0.55
35.8
0.13
1135
0.24
44.2
0.5
0.3a
0.17 0.002a
0.002
4
1147
0.01
0.29
a: p<0.05 SclAbll vs. VEH within loading condition; b: p<0.05 CON vs. HLU within treatment condition
112
Table 7-2: Effect of HLU and sclerostin antibody treatment on bone microarchitecture at the humerus (mean
Site
Vehicle
ANOVA Results
HLU
Controls
SciAbli
Vehicle
SEM)
SciAbll
Pload
Ptreatment
Pinteraction
0.01
Proximal Trabecular
BV/TV (%)
Tb.N (mm-1)
Tb.Th (mm)
Tb.Sp (mm)
ConnD (mm- 3)
SMI
Midshaft Cortical
Tt.CSA (mm 2)
2
Ct.BA (mm )
Ct.MA(mm 2)
Ct.BA/TA (%)
9.2 0.4
23.0
3.85 0.07
0.049 0.001
0.26 0.005
41 4
2.8 0.05
4.55
0.076
0.20
87
2.0
0.73
0.41
0.32
0.01
0.80
0.006b
0.006b
0.54
0.26
TMD (mgHA/ccm)
56.3 0.3b
0.24 0.02
0.16 0.001b
1174 6
pMOl (mm 4)
0.072
Ct.Por (%)
Ct.Th (mm)
0.002b
1.3ab
0.1Oa
0.001a, b
0.006a
4a
0. 1ab
0.01 a,b
0.005ab
0.006a
67.7 0.5ab
0.20 0.02
0.21 0.002ab
1167 6
0.093
0.002ab
8.5 0.4
18.0
1.0 a
0.004
0.0001
0.7
<0.0001
0.03
<0.0001
<0.0001
0.008
<0.0001
<0.0001
<0.0001
0.04
1
0.2
3.99
0.047
0.25
37
3.0
0.07
0.001
0.005
5
0.06
4.30
0.066
0.22
83
2.2
0.112
0.001a
0.007a
0.08a
1
0.5
0.009
0.71
0.01
0.009
0.75
0.01a
0.03
0.0001
0.2
0.48 0.007a
0.27 0.006a
<0.0001
0.009
<0.0001
<0.0001
0.2
0.3
64.0 0.3a
0.20 0.01
0.19 0.001a
1171 8
<0.0001
0.3
<0.0001
0.18
<0.0001
0.1
<0.0001
0.6
0.2
0.6
0.2
0.1
0.003a
0.0008
<0.0001
0.3
0.37
0.35
0.006
51.5 0.5
0.21 0.005
0.14 0.002
1153
0.065
9
0.003
0.081
7a
a: p < 0.05 SclAbll vs VEH within loading condition; b: p<0.05 HLU vs CON within treatment condition
Abbreviations: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity
density (ConnD), structure model index (SMI), total cross-sectional area (Tt.CSA), cortical bone area (Ct.BA), medullary area (Ct.MA), cortical
bone area fraction (Ct.BA/TA), cortical porosity (Ct.Por), cortical thickness (Ct.Th), polar moment of inertia (pMOl), tissue mineral density (TMD)
113
B
A
35-
*VEH
30 SclAbII
302520
to 15
1051
0CON
HLU
Figure 7-3: Effect of unloading and sclerostin antibody treatment at the distal femur. (A)
Trabecular BV/TV (%); (B) 3D rendering of pCT image from representative animals from
each group. Horizontal bars designate significant differences between VEH and SclAblI
within loading group (p < 0.001); tp < 0.01 for CON versus HLU within a treatment group;
and #significantly greater effect of SclAblI in CON versus HLU. Error bars represent 1
SEM.
Treatment with SclAbli improved bone properties in normally loaded animals and
fully inhibited disuse-induced bone loss, improving cortical and trabecular bone
parameters to levels at or above the fully-loaded VEH-treated group. Specifically,
SclAbll-treated animals, both loaded and unloaded, had significantly higher
Tb.BV/TV, Tb.Th, and Tb.N, along with lower Tb.Sp, better connectivity density,
and more plate-like architecture (SMI) than VEH-treated animals at both the
femur and humerus (Tables 7-1 and 7-2).
Treatment with SclAblI also improved cortical bone properties in both loaded and
unloaded animals, increasing cortical bone area, thickness, and bone area
fraction at both the femur and humerus, and prevented the increase in cortical
114
porosity seen in the HLU group. SclAbli treatment led to lower midshaft
medullary area in both HLU and CON, consistent with endocortical bone
apposition. Mid-femoral cross-sectional area was increased in CON-SclAbll, but
not
HLU-SclAbll, suggesting that
normal
loading may
augment
SclAbll
treatment's ability to induce periosteal bone apposition. Consistent with this,
SclAbli treatment led to increased mid-diaphyseal cross-sectional area in the
humeri of both the loaded and unloaded animals (Table 7-2). The positive effect
of SclAblI treatment was significantly greater in loaded than unloaded animals for
femur Tb.BV/ TV, Tb.Th, and SMI, and midshaft cortical bone area (Figure 7-3,
Table 7-1, Pinteraction < 0.001), and for Tb.BV/TV and Tb.Th in the proximal
humerus.
7.6.5
Mid-femoral biomechanics and pFEA of the distal femur metaphysis
Femoral bending stiffness and maximum load were 19% lower in HLU-VEH
compared to CON-VEH (p < 0.05, Figure 7-4). Mice treated with SclAbll had
better mechanical properties compared to VEH-treated groups in both loading
conditions, with maximum load and bending stiffness 40% to 50% higher than
VEH (Figure 7-4).
B
A
Bending Stiffness
Maximum Force
20
90
80
15
--
t
VEH
ElScIAbII
70
f
60
E
60-550
Z40
30-
5
-
2010-
0
0
CON
CON
HLU
HLU
Figure 7-4: Effect of unloading and sclerostin treatment on femoral strength as assessed
by three-point bending, (A) maximum force, and (B) bending stiffness. Horizontal bars
designate significant differences between VEH and ScIAblI within loading group (p < 0.01);
tp < 0.01 CON versus HLU within a treatment group.
115
pFEA showed that compressive stiffness of the combined cortical and trabecular
regions were 18% lower in HLU-VEH than CON-VEH (p < 0.05) and 50% higher
in SclAbli versus VEH-treated mice in both loading conditions (Figure 5A).
Interestingly these changes in stiffness were nearly twofold greater than the
respective changes in bone volume (-10% for HLU-VEH and +25% for SclAblltreated animals), suggesting that changes in bone mass underestimate changes
in mechanical properties.
C
A
pFEA - Distal Femur Stiffness
CON-VEH
HLU-VEH
CON-SciAbIl
HLU-SclAbI
* VEH
10A
CON
H-LU
E 6B
~
szFEA - Distal Femur Cortex
2
100
98-
EScAbI
CON
0.0
HLU
10.0
Von Mises Stress (MPa)
Figure 7-5: Effect of unloading and scierostin antibody treatment on bone strength, as
%
assessed by micro-finite element analysis (pFEA) of the distal femur: (A) stiffness, (B)
cortical load, (C) representative pFEA Von Mises Stress color map images. Horizontal bars
designate significant differences between VEH and ScIAblI within loading group (p < 0.05);
fp < 0.01 for CON versus HLU within a treatment group; #significantly greater effect of
ScIAblI in CON versus HLU (p < 0.02); Error bars represent 1 SEM.
HLU did not change the distribution of load sharing between the trabecular and
cortical compartments. In contrast, the proportion of load carried by trabecular
and cortical bone compartments were increased and decreased, respectively, in
ScIAbil-treated mice compared to VEH groups, consistent with a shift toward
more uniform load sharing following ScIAbl treatment in both loading conditions
(Figure 5). Notably, differences in pFEA-estimated stiffness (-18% in HLU-VEH
116
versus CON-VEH, and +50% in SclAbll-treated mice) were larger than the
differences in total bone volume (-10% in HLU-VEH versus CON-VEH, and
+25% in SclAblI-treated mice), suggesting that changes in bone mass alone
underestimate the effects of unloading and sclerostin antibody treatment on bone
biomechanical properties.
7.6.6
Serum sclerostin and bone turnover markers
5 versus 116 6 pg/mL, p <
HLU-VEH mice had higher serum sclerostin (134
0.05), lower osteocalcin, and lower CTX1 than CON-VEH, but similar TRACP5b
(Figure 6). Compared to VEH-treated mice, those treated with SclAbll had higher
osteocalcin and lower TRACP5b (in HLU only), but had similar CTX1 levels
(Figure 6).
1:
2
A
40-
C
t
20 -
100
EVE
ScIAbII
E
15-
0
I..
C
40
2020
EH
S5-
0
CON-VEH
HLU-VE
B
2500
0
0
0
0
6
t
0-
H
60-
E E
El T ScIAbli
CON
D
1-
.0
HLU
*VEH
EIScAbI
50-
200150-
-E 40-
t
0
100-
Q20
50-
10
0CON
HLU
CON
HL.
Figure 7-6: Effect of unloading and sclerostin antibody treatment on serum sclerostin and
markers of bone turnover. (A) Sclerostin (vehicle groups only); (B) Osteocalcin; (C)
TRACP5b, and (D) CTXI. Horizontal bars designate significant differences between VEH
and ScIAblI within loading group (p < 0.05). tp < 0.01 CON versus HLU within a treatment
group. Error bars represent 1 SEM.
117
7.6.7
Histomorphometry
Static and dynamic histomorphometry results are summarized in Table 7-3. Due
to technical issues with fluorescent label incorporation in some animals, sample
sizes in some groups were limited to 3 animals for dynamic outcomes. In VEHtreated animals, HLU led to reduced MAR and greater marrow adiposity. SclAblI
treatment led to significantly higher bone formation indices (MAR, MS/BS, and
BFR/BS) compared to VEH-treated mice in both loading conditions, but had no
effect on marrow adiposity.
Table 7-3: Effect of unloading and SclAblI treatment on static and dynamic quantitative
histomorphometry of the distal femur (mean SEM).
Controls (CON)
Vehicle
SciAbli
Hind limb Unloaded (HLU)
Vehicle
SclAbll
Static indicest
N.Ob/BS (#/mm 2)
N.Oc/BS(#/mm 2)
24 4
3.9 1.1
23 2
4.4 1.0
26 4
6.5 1.3
32 2
5.1 1.3
N.Ot/BA (#/mm 2 )
740
44
591
34
653
93
647
38
N.Ad/MA(#/mm )
16 4P
17
4b
34
7
35
6
Ad.Diam (pm)
OS/BS(%)
ES/BS (%)
Dynamic indicesti
44 2.5c
24.0 2
6.3 1.5
MS/BS (%)
13.6
3.3
24.9
2.2a
11.6
5.5
21.1
5.0a
1.2
0.2b
1.6
0.2a
0.5
0.2
2.0
1.1a
0.2
0.1
0.4
0.1a
0.09
0.1
0.9
0.6a
2
MAR (pm/day)
3
2
BFR/BS (pm /pm /d)
36 1.1b
25.7 2
6.3 0.7
51 4.0
25.2 5
8.7 1.9
50.5 2.4
33.7 2
10.1 2.4
a p<
0.05 SclAblI vs VEH treatment within the CON or HLU groups;
p<0.05 HLU vs CON within the SclAbll and VEH treatment groups.
C p=0.08 HLU vs CON within VEH-treated group
b
t Sample size for static indices: n=6 / group
t Sample size for dynamic indices: CON-VEH=6, CON-SclAbll=5, HLU-VEH=3,
HLU-SclAbll=3.
Abbreviations: osteoblast number per bone surface (N.Ob/BS), osteoclast number per bone
surface (N.Oc/BS), osteocyte number per bone area (N.Ot/BA), adipocyte number per
marrow area (N.Ad/MA), adipocyte diameter (Ad.Diam), osteoid surface per bone surface
(OS/BS), eroded surface per bone surface (ES/BS), mineralizing surface (MS/BS), mineral
apposition rate (MAR), bone formation rate per bone surface (BFR/BS).
118
7.7
Discussion
The primary objective of this study was to determine the musculoskeletal effects
of pharmacologic inhibition of sclerostin in mice exposed to hind limb unloading.
We hypothesized that sclerostin antibody treatment would prevent bone loss and
the deterioration of mechanical properties associated with disuse by promoting
bone formation. Treatment with sclerostin antibody led to skeletal anabolic
activity in the setting of unloading, as evidenced by increases in BMD, trabecular
and cortical microarchitecture, and femoral strength values in the HLU-SclAblI
group that were at or above values in the CON-VEH group. Furthermore,
treatment with sclerostin antibody resulted in an increase in serum bone
formation
markers and histologic evidence of enhanced trabecular bone
formation. These observations of skeletal anabolic activity following treatment
with sclerostin antibody in disuse are consistent with other studies of sclerostin
antibody treatment during immobilization in rodents 2 0 2 1 . Further, the increases in
total body and hind limb BMD observed in our normally loaded control animals
induced by sclerostin antibody treatment were similar to prior observations in
normally loaded animals 4
8 22
. In addition, serum bone turnover markers and
dynamic histomorphometry outcomes were consistent with sclerostin antibody
treatment
increasing bone
mass in rodents mainly by enhancing
bone
formation 8,9
Sclerostin antibody treatment had inconsistent effects on indices of bone
resorption, with decreased serum TRACP5b levels, but no differences in serum
CTX. Serum was collected only at a single time point (e.g., end of study) and
thus the serum measures cannot reflect the changes over the entire experiment
in osteoclast number versus their net activity that are theoretically reflected in the
TRACP5b and CTX measures, respectively. Other studies in rodents have also
reported declines in TRACP5b following sclerostin antibody treatment9.
The current study also explored whether the skeletal effects of sclerostin
antibody treatment are sensitive to mechanical loading by examining effects in
119
hindlimb-unloaded versus fully-loaded controls, and effects in the unloaded hind
limb versus loaded forelimb. In the femur, the skeletal response to sclerostin
inhibition tended to be enhanced in the normally loaded mice compared to those
exposed to hind limb suspension, with significantly greater response in trabecular
bone volume and microarchitecture, cortical bone area and thickness, and distal
femur pFEA-estimated stiffness, as well as a trend for greater gain in leg BMD (p
= 0.20 for load-treatment interaction). Furthermore, femoral midshaft crosssectional area was greater than VEH-treated mice only in the fully loaded
SclAbll-treated animals, suggesting that periosteal apposition
induced by
sclerostin inhibition requires mechanical loading. At the humerus, whereas the
effects of sclerostin antibody on BMD and cortical bone morphology were similar
in HLU and fully-loaded groups, the increases in trabecular bone volume,
number, and thickness were greater in CON than HLU. Although speculative, the
finding that the response to sclerostin inhibition is altered in the "loaded" humerus
of the HLU group suggests that systemic effects of HLU (i.e., stress) that are
unrelated to mechanical loading influence the response to sclerostin inhibition. In
a study of rats exposed to unilateral hind limb immobilization via bandages, Tian
and colleagues8 also found that the trabecular bone response to sclerostin
inhibition tended to be enhanced in the loaded versus unloaded limbs, whereas
responses in cortical bone were similar in both groups. In contrast, in rats
injected unilaterally with botulinum toxin A (botox) to induce hind limb paralysis,
proximal tibia primary spongiosa
.
the response to sclerostin inhibition was similar in the loaded and unloaded
Although far from conclusive, taken together these observations suggest that the
anabolic effects of sclerostin inhibition are enhanced with normal mechanical
loading. As proposed by Tian and colleagues8 , the relative excess of sclerostin in
unloaded bone could reduce the anabolic effects of sclerostin inhibition relative to
those seen in fully loaded bone. Alternatively, although sclerostin appears to be a
central mediator of the bone's response to mechanical loading
3,
it may not be
the only mechanism by which the osteocytic network responds to mechanical
120
unloading.
For
example, another
mechanism
by which osteocytes
may
orchestrate a response to altered mechanical loading is suggested by the
observation that osteocytes are a major source of the osteoclastogenic cytokine
receptor activator of NFkB ligand (RANKL) 24 , and further, that mice lacking
RANKL in osteocytes are protected from bone loss induced by hind limb
unloading 25 . Thus, sclerostin-independent effects, notably RANKL-mediated
effects or, for example, the detrimental effects of increased marrow adiposity on
osteoblasts, could also be responsible for a differential response to sclerostin
antibody treatment in unloaded versus loaded bone. Clearly, further studies are
needed to further investigate the interaction between mechanical loading and the
anabolic effects of sclerostin antibody treatment.
Hind limb unloading in rodents and bed rest studies in humans have reported an
increase in marrow adiposity 2 ,2 7. Interestingly, sclerostin antibody treatment did
not prevent the increased marrow adiposity with HLU and had no effect on
marrow adiposity in normally loaded animals. Canonical Wnt signaling inhibits
adipogenesis and promotes survival of committed preadipocytes 2 8 3- 0 . Patients
with activating mutations in low-density lipoprotein receptor-related protein 5
(LRP5) leading to increased Wnt signaling are associated with increased
trabecular bone volume and reduced bone marrow fat in iliac crest biopsies, as
well as increased osteogenesis and reduced adipogenesis of mesenchymal stem
cells3 1 . However, specific Wnt targets and the role of noncanonical Wnt signaling
in adipogenesis remain incompletely understood3 2 . Because sclerostin binds to
the LRP4/5/6 receptor to inhibit Wnt signaling, our observation that sclerostin
antibody treatment had no influence on bone marrow adiposity suggests that
other mechanisms besides sclerostin-mediated Wnt signaling must be involved in
the increased marrow adiposity seen with unloading.
Several limitations of this study merit mention. We studied only female mice at
one time point, with a single-dosing regimen for sclerostin inhibition. Thus it is not
clear whether the anabolic effects of sclerostin antibody would continue with
121
longer treatment, or whether a higher dose or more frequent dosing would
promote even greater anabolic effects in the skeleton or eliminate the loadtreatment interactions we observed.
These limitations notwithstanding, this study provides novel information about the
ability of sclerostin antibody to induce bone formation in the situation of reduced
mechanical loading. We showed that changes in bone volume underestimate
both the loss of bone strength with disuse and the gain of bone strength with
sclerostin inhibition, and we also explored the question whether mechanical
loading influences the anabolic actions of sclerostin inhibition by comparing the
skeletal responses of HLU and CON mice at both the forelimbs and hind limbs.
7.8
Conclusion
In summary, treatment with sclerostin antibody induces an anabolic skeletal
response in an established rodent model of disuse-induced bone loss, such that
unloaded animals treated with sclerostin antibody had BMD, microarchitecture,
and mechanical strength values at or above the normally loaded control mice.
These results provide strong rationale for testing the ability of sclerostin antibody
treatment to improve skeletal fragility in patients with spinal cord injuries, stroke,
muscular dystrophy, cerebral
palsy, and other diseases and conditions
associated with short-term or chronic disuse.
122
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125
126
Chapter 8
8
Sclerostin antibody inhibits skeletal deterioration in mice
exposed to partial weight-bearing in mice
8.1
Rationale
The biological mechanism of osteocytes' mechanosensing remains elusive,
particularly in the spectrum of clinically relevant reduced, but not full reduction in
weight-bearing activities. Furthermore, future long duration spaceflights to
terrestrial destinations, such as the Moon and Mars, will have astronauts
spending considerable amounts of time in partial gravity environments. Currently,
both the U.S. and Russian bioastronautics medical community consider the
unknown risks of partial weigh bearing effects on the human body a top medical
concern.
As we have shown, mice can be protected in the setting of full unloading with the
sclerostin antibody treatment, but several bone parameters had an enhanced
anabolic effects with SclAbli in normal mechanical loading (Chapter 7). To
further investigate the response to sclerostin inhibition in the presence of partial
mechanical unloading, we tested the ability of sclerostin antibody to inhibit
skeletal deterioration during exposure to prolonged (21 -day) partial weightbearing at 20%, 40%, and 70% of normal loading. We hypothesized that
sclerostin antibody treatment would prevent bone loss across the spectrum of
partial unloading and we would observe a load dependent effect of sclerostin
antibody treatment. Consistent with our findings in hind limb unloading, we
observed sclerostin antibody led to anabolic activity across the spectrum of
partial unloading. In this chapter, we also compare the musculoskeletal
responses of full loading to partial weight-bearing environments, and assessed
whether the efficacy of anti-sclerostin antibody depends on the loading condition.
127
8.2
Key findings
While all partial weight-bearing groups had bone parameters well above the
CON-VEH group, we observed a modest load dependent effect in our sclerostin
antibody partial weight-bearing groups. At the dose used in our studies, these
results suggest even small amounts of loading (i.e., PWB20, PWB40) provide
enough mechanical loading stimuli to provide optimum benefit of sclerostin
antibody inhibition.
8.3
Introduction
The profound effects of mechanical unloading on muscle atrophy and skeletal
fragility are well established. However, there has been little investigation into the
physiological effects of partial reduction of weight-bearing. To address this gap in
knowledge, our group previously developed the partial weight-bearing (PWB)
model'
2
that enables long-term exposure of mice to partial loading while
maintaining quadrupedal locomotion. We have used this model to show that
bone and muscle loss is linearly proportional to the reduction of mechanical
loads 2 . In addition, we previously reported that in mice subjected to mechanical
disuse via high limb unloading (HLU), treatment with sclerostin antibody not only
inhibits bone loss, but leads to improved bone mass via increased bone
formation (Chapter
7)3.
Notably, for a few bone outcomes, we observed greater
anabolic effects of sclerostin inhibition in the normally loaded mice (Chapter 7)3.
To further investigate the skeletal responses to sclerostin inhibition in a
mechanical unloading environment, we tested the ability of sclerostin antibody to
inhibit skeletal deterioration during exposure to partial weight-bearing at 20%,
40%, and 70% of normal loading. We hypothesize that treatment with sclerostin
antibody would improve bone mass, microarchitecture and strength in all loading
conditions, but that there would be a greater skeletal response in the normally
loaded mice than in unloaded mice.
128
8.4
Material and methods
8.4.1
Overview of study design
We tested the ability of sclerostin antibody (SclAbIl) to prevent bone loss in adult
female mice (C57BI/6J, 11 wks. of age) subjected to mechanical unloading for 21
days. Mice were assigned to one of four loading groups (n=8 to 17 / group):
partial weight-bearing at 20% (PWB20), 40% (PWB40), 70% (PWB70) of normal
weight-bearing, or control (CON, normal weight-bearing). Animals were assigned
to groups by total body bone mineral density (BMD) and body mass in a manner
to minimize differences between groups at baseline. Mice in each group were
injected with either SclAbIl at 25 mg/kg or vehicle (VEH) twice weekly. All mice
had access to standard rodent chow and water ad libitum. The protocol was
approved by the Institutional Animal Care and Use Committee at Beth Israel
Deaconess Medical Center.
8.4.2
Partial weight-bearing (PWB) model
For partial weight-bearing, we followed the methods described previously by
Ellman et al.2 . In brief, two to three days prior to unloading, mice assigned to
PWB groups were placed in the PWB jacket and singly housed in standard
vivarium cages for acclimation. On day 0, mice were placed in a two-point full
suspension rig, as described previously 2 3 . A clasp on the jacket and a tail wrap
were connected by a chain and spaced by a hollow metal rod to distribute
loading. This apparatus was then joined to a spring and hung from a wheel with
linear freedom along a rail across the top of a cage. Adjustments to actual
weight-bearing, or effective mass, were made by threading the spring through its
support thereby changing the length of spring engaged by the harness, and
providing differential vertical force to support mouse's body weight. Effective
mass was measured daily by quiet standing on a scale and the spring tension
adjusted to maintain the desired amount of unloading.
129
8.4.3
Bone mineral density and muscle mass
In vivo assessment of total body (exclusive of the head region) and leg (femur
)
and tibia exclusive of femoral neck and foot) bone mineral density (BMD, g/cm 2
was performed at baseline and end of the study using peripheral dual-energy Xray
absorptiometry
(pDXA,
PIXImusll,
GE
Lunar
Corp.),
as
previously
described2 . Muscle atrophy was assessed by wet weight of the soleus and
gastrocnemius muscles at necropsy.
Muscle mass was normalized to animal
body weight.
8.4.4
Specimen harvesting and preparation
At the end of the study, mice were euthanized via C02 overdose. Femurs were
harvested and cleaned of soft tissue. The right femur was prepared for imaging
-
and biomechanical testing by wrapping in saline-soaked gauze and freezing at
200C.
8.4.5
Serum markers of bone metabolism
Mice were fasted for 2 hours before blood was collected at the time of euthanasia
and used to measure serum sclerostin (in vehicle treated mice only) and bone
turnover markers. Osteocalcin was assessed using the species-specific singleplex Luminex assays (Millipore,
Billerica, MA).
Serum concentrations of
TRACP5b, and type I collagen C-telopeptide (sCTX) were measured by using
mouse ELISA kits (IDS, Fountain Hills, AZ). All assays were run according to the
manufacturers' protocols.
8.4.6
Bone microarchitecture
Assessment of bone microarchitecture was performed with microcomputed
tomography (pCT40, Scanco Medical, BrOttisellen, Switzerland) 2 -4 . In brief, the
distal femoral metaphysis were scanned using 70 KvP, 50 mAs, and 12-pm
isotropic voxel size. The femoral metaphysis region began 240 pm distal to the
growth plate and extended 1.8 mm distally. Cancellous bone was separated from
130
cortical bone with a semi-automated contouring program. For the cancellous
bone region we assessed bone volume fraction (Tb.BV/TV, %), trabecular
thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), trabecular number
(Tb.N, 1/mm), connectivity density (ConnD 1/mm 3), and structure model index
(SMI).
Transverse CT slices were also acquired at the femoral midshaft to
assess total cross-sectional area, cortical bone area and medullary area (Tt.Ar,
Ct.Ar and Me.Ar, mm 2 ); bone area fraction (Ct.BA/Tt.TA, %), cortical thickness
(Ct.Th, mm), and polar (pMOI, mm 4 ) moment of inertia. Bone was segmented
from soft tissue using the same threshold for all groups: 247 mg HA/cm 3 for
trabecular and 672 mg HA/cm 3 for cortical bone. MicroCT scanning and analyses
adhered to published guidelines4.
8.4.7
Mechanical testing
Femurs were mechanically tested at a constant displacement rate of 0.03
mm/sec to failure in three-point bending (Bose ElectroForce 3200 with a 150 N
load cell, Bose Corporation, Eden Prairie, MN). Fresh-frozen femurs were
thawed to room temperature then centered longitudinally, with anterior surface on
the two lower support points spaced 10 mm apart2 3 . Force-displacement data
were acquired at 30 Hz and used to determine maximum force (N) and stiffness
(N/mm).
8.4.8
Statistical analysis
All data were checked for normality, and standard
descriptive statistics
computed. Overall treatment and loading effects were evaluated using analysis
of variance (ANOVA) for all continuous variables. Fisher exact post-hoc testing
were considered significant at p<0.05. Data are reported as mean
noted.
131
SEM, unless
8.5
Results
8.5.1
Body mass and muscle mass
PWB20-VEH, PWB40-VEH, PWB40-VEH, and PWB70-Sclabll groups had lower
body mass (<5% declines) through the first two weeks of unloading and
recovered to baseline at end of study. PWB70 and CON groups had minimal
changes with unloading and both gained (<5% body mass) at the end of the
study. Soleus wet weight (normalized to body weight) was 20%, 17%, and 6%
lower in PWB20-VEH, PWB40-VEH, and PWB70-VEH, respectively, than CONVEH (p<0.05) consistent with our prior observations in the PWB model that
muscle loss is proportional to the degree of unloading 2. Gastrocnemius wet
weight (normalized to body weight) was 12%, 11 %, and 6% lower in than lower in
PW1B20-VEH, PWB40-VEH, and PWB70-VEH, respectively, than CON-VEH
(p<0.05). There were no differences in muscle mass between VEH and SclAblI
treated mice at any unloading level, demonstrating that the activity of sclerostin
antibody is specific to bone (data not shown).
8.5.2
Bone mineral density
Partial weight-bearing caused significant bone loss, whereas sclerostin antibody
treatment increased total body and leg BMD in all groups (Figure 8-1).
Specifically, leg BMD declined -9.8
0.7%, -7.0 in
0.7%, and -4.9%
1.1% in
PWB20-VEH, PWB40-VEH, and PWB70-VEH, respectively, whereas it increased
3.9
0.4% in CON-VEH (p<0.0001 vs baseline for all, and p<0.001 for all PWB
groups vs CON). Leg BMD increased by 14 to 18% in all PWB groups treated
with SclAblI (p<0.001 vs VEH for all). In comparison, leg BMD increased 30
in CON-SclAblI
(p<0.001
3%
vs VEH-treated animals). The effect of SclAblI
treatment on BMD did not vary with the level of PWB and there was no difference
between the response of any PWB group and fully-loaded controls to SclAbll (p =
0.22). The pattern was similar for total body BMD (Figure 8-1).
132
A
B
Total Body
Hindlimb
n
....flil
40-
40*
30-
20-
20.u~n.,0
2
C.)
C
10-
10-
0-
0-
-10-
-10-
-20-J PWB20
PWB40
PWB70
CON
M VEH
M SciAbli
*
*
30-
-20" PWB20
PWB40
PWB70
CON
Figure 8-1: Effect of partial weight-bearing and sclerostin antibody treatment on (A) hind
limb BMD, (B) total body BMD. Changes in leg BMD were significantly different from
baseline in all groups (p<0.001). * p < 0.05 for VEH vs SclAbIl within loading group; **
p<0.01 for CON-VEH vs all other VEH-treated groups. Error bars represent one SEM.
8.5.3
Bone volume and microarchitecture
Consistent with in vivo BMD measurements, partial weight-bearing led to
significant bone deterioration, particularly in the trabecular compartment (Table
8-1). Treatment with SclAblI led to increased Tb.BV/TV, regardless of loading
condition, such that trabecular bone volume of SclAbll-treated groups was 2.5 to
3-fold higher than that of VEH animals. SclAbll treated animals, both normally
loaded and partially unloaded, had significantly higher trabecular bone volume
(Figure 8-2A), thickness and number, along with lower trabecular separation and
A
M
B
VEH
C3 SciAbilI
40
*
VEH
*
SciAbil
0
40-
30
20
I-
100.
00
0
+,
20
40
70
100
% Loaded
Figure 8-2: Effect of partial weight-bearing and sclerostin antibody treatment on (A)
trabecular bone volume at the distal femur and (B) linear regression (solid line) shown
with 95% confidence bands (dashed lines) across the loading groups for trabecular bone
volume at the distal femur. * p<0.0001 for VEH vs. ScIAblI within a loading group, **
p<0.0001 for CON-VEH vs all other VEH-treated groups. Error bars represent one (A) SEM.
133
more plate-like architecture than vehicle-treated animals (Table 8-1). The
improvement in trabecular microarchitecture with sclerostin antibody treatment
was dose dependent with loading across the PWB groups (Figure 8-2B, r 2
0.76, p = 0.1).
Cortical bone was also negatively affected by partial unloading, as PWB mice
had lower cortical bone area, cortical bone area fraction, cortical thickness, and
polar moment inertia than CON animals at the mid-femoral diaphysis (Table 8-1).
Treatment with SclAblI led to improved cortical bone properties in all groups,
such that cortical properties in unloaded animals were equal to or better than the
normally loaded, vehicle-treated group.
134
Table 8-1: : Effect of PWB and SciAbII treatment on femoral trabecular and cortical bone microarchitecture,
SEM
Vehicle
N=10
Distal Femur Trabecular
BV/TV (%)
Tb.N (mm-1)
Tb.Th (mm)
Tb.Sp (mm)
7.3 0.5b
3.59 0 .1b
0.048 0.001b
0.27
3
46
0.006b
7b
0.1b
ConnD(mm- )
SMI
Femur Midshaft Cortical
3.2
Tt.CSA (mm 2)
1.54 0.02
2
TMD (mgHA/ccm)
0.54 0.01b
1.0 0.02b
35.0 0.4b
1101 7b
pMOl (mm 4)
0.23
Ct.BA (mm )
Ct.MA (mm 2)
Ct.BA/TA (%)
0.01b
SclAbli
N =9
1.62 0.01 b
0.73 0.01ab
0.89 0.01ab
45.1 0.5ab
1116 3b
0.31
0.01ab
SclAbll
N=8
Vehicle
N 10
SclAbll
N 8
Vehicle
N = 17
24.7 +1 .0 ab
4.08 + 0.06ab
0.082 + 0.001ab
0.21 + 0.005a,b
106 4a
1.9 0.07ab
0.6
3.78 0.1b
0.049 0.001b
0.26 0.007b
71 8
2.9 0.05
24.8 0.4 a,b
4.1 0.03ab
0.078 + 0.001ab
0.21 0.002ab
113 1
1.8 0.03ab
10.3 0.4
3.85 0.04
0.054 0.001
0.25 0.003
69 3
3.0 0.06
1.62 0.01 b
0.73 0.01 ab
0.89 + 0.01ab
45.0 0.2ab
1113 lb
Vehicle
N 14
8.8 0.3
22.2 0.8ab
3.79 0.06b
3.95 0.05a,b
0.049 0.001 b
0.077 0.001a b
0.26 0.004 (p=0 06)
0.22 0.004a,b
56 4
104 5a
3.0 0.02
2.0 0.06a,b
Fully Loaded
PWB70
PWB40
PWB20
Site
.
assessed by tCT (mean
9.6
1.50
0.56
0.95
36.8
1133
0.03
0.09b
0.02b
0.5b
8b
0.03b
0.75+ 0.01ab
0.84 0.02a
47.1 0.3a,b
1167 5a
1.59 0.01
0.57 0.01b
1.0 0.01b
35.8 0.6b
1105 9b
0.23
0.01b
0.31
0.25
1.58
0.01 a,b
0.01b
0.31
0.01 ab
SclAbIl
N = 11
32.0
4.3
0.097
0.20
107
1.4
1.5a
0.05a
0.003a
3
0.12a
1.56
0.66
0.9
42.7
1148
0.02
0.01
0.01
0.5
5
1.69
0.86
0.83
50.8
1175
0.02a
0.02a
0.01a
0.4a
3
0.27
0.007
0.37
0.012
a: p<0.05 SclAbll vs. VEH within loading condition; : p<0.05 CON vs. PWB within treatment condition. Abbreviations: bone volume fraction (BV/TV),
trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (ConnD), structure model index (SMI), total
cross-sectional area (Tt.CSA), cortical bone area (Ct.BA), medullary area (Ct.MA), cortical bone area fraction (Ct.BA/TA), cortical thickness (Ct.Th),
polar moment of inertia (pMOI), tissue mineral density (TMD).
135
0.002a
8.5.4
Femoral strength
VEH-treated PWB groups had on average 23% lower maximum force and 18%
lower bending stiffness compared to normally loaded VEH-treated animals (Figure 83). Mice treated with SclAbll had improved mechanical properties compared to VEHtreated groups in all loading conditions. Specifically, maximum bending force was
50% higher, bending stiffness was 40% higher, and work to failure was 30-50%
higher (Figure 8-3C) in SclAbll treated mice than VEH in all-loading conditions.
Estimated bending modulus (data not shown) and post-yield displacement (Figure 83D) did not vary with unloading or sclerostin-antibody treatment.
A
B
20-
z
100
*
VEH
3 SciAbIl
*
10-
*
22
C
E2
E
M
*
2
DO
E
20
z
E
00
Figure 8-3: Effect of partial weight-bearing and sclerostin antibody treatment on femoral
strength as assessed by three-point bending, (A) maximum force and (B) bending stiffness,
(C) work to failure, (D) post-yield displacement. * p<0.05 for VEH vs. SclAbll within a loading
group, ** p<0.05 for CON-VEH vs all other VEH-treated groups. Error bars represent one SEM.
136
8.5.5
Bone turnover markers
Compared to normally loaded controls, PWB led to reduced sCTX, unchanged levels
of TRACP5b, and decreased osteocalcin (Figure 8-4). Independent of loading, mice
treated with SclAbll had increased bone formation, as evidenced by approximately
70% higher osteocalcin levels compared to VEH controls. SclAbll treated mice also
exhibited unchanged or decreased bone resorption, with variable effects on sCTX
and generally lower TRACP5b compared to VEH-treated groups.
A
B
601
20-
T
C
300E
C3 SclAbil
1
0-Il10-
Figure 8-4: Effect of unloading and sclerostin antibody treatment on serum markers of bone
turnover: A) CTX1; B) TRACP5b; and C) Osteocalcin,. * p < 0.05 SclAbli vs VEH control within
a loading condition. ** p<0.01 for CON-VEH vs all other VEH-treated groups. Error bars
represent one SEM.
8.6
Discussion
The primary objective of this study was to determine the skeletal effects of
pharmacologic inhibition of sclerostin in mice exposed to different levels of unloading
via the partial weight-bearing model. We hypothesized that sclerostin antibody
treatment would improve bone mass, microarchitecture and strength across the
spectrum of partial unloading and that the anabolic effects of sclerostin antibody
would vary with the degree of unloading. Consistent with our previous finding that
sclerostin antibody improves bone outcomes in the hind limb unloading model 3 , we
found here that sclerostin antibody significantly increased trabecular and cortical
bone mass and improved bone microarchitecture and strength across a range of
partial unloading. Although improvements in trabecular bone following sclerostin
antibody treatment were greater with higher levels of weight-bearing, it should be
noted that the current results, along with our prior study in hind limb unloaded mice
137
indicate a robust response to sclerostin antibody treatment, even in the absence of
normal loading. Moreover, femoral bending strength and cortical bone responses to
sclerostin antibody treatment did not vary with unloading condition.
However, our
finding that trabecular, but not cortical, bone improvements following sclerostin
antibody treatment were enhanced with greater levels of partial weight-bearing
should be considered in the context of the relatively high, and frequent dosing
regimen (25 mg/kg, twice weekly) that was used in this study. Previous rodent
studies have shown that both 5 and 25 mg/kg doses of sclerostin antibody produce
similar increases in bone mass and strength at the femur5'6 , suggesting that maximal
responses are already seen at the lower dose. Thus, future studies could further
explore if lower doses of sclerostin antibody (< 5 mg/kg) and/or less frequent dosing
regimens would enhance evaluation of possible load-dependent skeletal responses
to sclerostin antibody. Ultimately, studies investigating sclerostin antibody in clinical
situations of reduced mechanical loading, such as bed rest or spinal cord injury
(SCI), will be needed to investigate the clinical utility of sclerostin antibody for
preventing disuse-induced bone loss.
For example, a recently completed rodent
complete SCI model showed sclerostin antibody inhibition, at the same dosing
reqimen we used, resulted only in maintenance of trabecular bone comparable to
sham operated controls and no effect on cortical bone microarchitecture . In
contrast, our partial weight-bearing results showed all partial weight groups to had
both trabecular and cortical bone equal to or greater than those of CON, VEHtreated mice suggesting a synergistic response to SclAblI and partial loading.
Serum indices of bone turnover measured at the end of the study showed that partial
weight-bearing was associated with a reduction in bone formation and resorption. In
rodent models of disuse, bone formation markers have been previously reported as
being suppressed8 . However bone resorption markers have a more complicated
time-course, showing transient increases that preceded suppression of bone
formation markers in rodents 8 . It may be useful to assess bone turnover indices at
an earlier time point measurement to capture the initial changes in bone metabolism
following unloading and sclerostin inhibition. Similar to prior studies 4' 59' , serum
138
markers of bone turnover indicated that treatment with sclerostin antibody increased
bone formation and decreased or maintained bone resorption across all partial
weight-bearing groups.
This study was limited only studying female mice at one time point, with a single
dosing regimen for sclerostin inhibition. Furthermore, we did not conduct dynamic
histomorphometry studies, which would further aid in defining the skeletal response
to partial weight-bearing coupled with sclerostin antibody treatment. Nonetheless,
this study provides novel information about the ability of sclerostin antibody to induce
bone formation in the situation of reduced, but not full mechanical unloading.
Consistent with our prior work demonstrating a robust skeletal response to sclerostin
antibody in the hind limb unloading model 3 , here we showed that while even small
amounts of loading (i.e., 20% of normal weight-bearing) enable the skeleton to
achieve a profound improvement in bone structure in response to sclerostin
inhibition, greater weight-bearing may lead to even greater benefits, particularly in
the trabecular compartment.
This observation suggests that patients with limited
mobility and/or weight-bearing would have marked benefits from treatment with
sclerostin antibody, even if their responses were attenuated relative to persons with
normal skeletal loading.
8.7
Conclusion
Treatment with sclerostin antibody induces an anabolic skeletal response in a rodent
model of partial weight-bearing, such that partially unloaded animals treated with
sclerostin antibody had bone mineral density, microarchitecture, and mechanical
strength values at or above the normally loaded control mice. These results provide
strong rationale for testing the ability of sclerostin antibody treatment, in conjunction
with even limited weight-bearing exercise, to improve skeletal fragility in patients with
spinal cord injuries, stroke, muscular dystrophy, cerebral palsy, and other diseases
and conditions associated with short-term or chronic musculoskeletal disuse.
139
1
2
3
4
5
6
7
8
9
Wagner, E. B. et al. Partial weight suspension: a novel murine model for
investigating adaptation to reduced musculoskeletal loading. J Appl Physiol
(1985) 109, 350-357, doi: 10.11 52/japplphysiol.0001 4.2009 (2010).
Ellman, R. et al. Partial reductions in mechanical loading yield proportional
changes in bone density, bone architecture, and muscle mass. J Bone Miner
Res 28, 875-885, doi:10.1 002/jbmr.1814 (2013).
Spatz, J. M. et al. Sclerostin antibody inhibits skeletal deterioration due to
reduced mechanical loading. J Bone Miner Res 28, 865-874,
doi:10.1002/jbmr.1807 (2013).
Bouxsein, M. L. et al. Guidelines for assessment of bone microstructure in
rodents using micro-computed tomography. J Bone Miner Res 25, 14681486, doi:10.1002/jbmr.141 (2010).
Ominsky, M. S. et al. Two doses of sclerostin antibody in cynomolgus
monkeys increases bone formation, bone mineral density, and bone strength.
J Bone Miner Res 25, 948-959, doi:10.1002/jbmr.14 (2010).
Tian, X., Jee, W. S., Li, X., Paszty, C. & Ke, H. Z. Sclerostin antibody
increases bone mass by stimulating bone formation and inhibiting bone
resorption in a hindlimb-immobilization rat model. Bone 48, 197-201,
doi:10.1016/j.bone.2010.09.009 (2011).
Beggs, L. A. et al. Sclerostin Inhibition Prevents Spinal Cord Injury-Induced
Cancellous
Bone
Loss.
J Bone
Miner
Res
30,
681-689,
doi:10.1002/jbmr.2396 (2015).
Kurokouchi, K. et al. Changes in the markers of bone metabolism following
skeletal unloading. Environmental medicine : annual report of the Research
Institute of Environmental Medicine, Nagoya University 39, 21-24 (1995).
Li, X. et al. Inhibition of sclerostin by monoclonal antibody increases bone
IIrm~atIUIn, bUIne masd, d bI
tent in aged male ra ts. J Bone tviiner
Res 25, 2647-2656, doi:10.1 002/jbmr. 182 (2010).
140
Chapter 9
9
Serum sclerostin increases in healthy adult men in bed
rest
This thesis chapter, in part, previously published as the manuscript: Spatz, et al.,
Serum sclerostin increases in healthy adult men in bed rest, J Clinical
Endocrinology Metab. 2012 Sep;97(9):E1736-40. doi: 10.1210/jc.2012-1579.
Epub 2012 Jul 5.
141
9.1
Rationale
Animal
models and human studies suggest that osteocytes regulate the
skeleton's response to mechanical unloading in part by an increase in sclerostin.
However, few studies have reported changes in serum sclerostin in humans
exposed to reduced mechanical loading. We determined changes in serum
sclerostin and bone turnover markers in healthy adult men undergoing controlled
bed rest.
9.2
Introduction
Reduced mechanical loading of the skeleton is invariably associated with muscle
atrophy and bone loss. Bone loss is observed clinically after prolonged bed rest,
immobilization, stroke, and spinal cord injury- 6 . In addition, microgravity causes
profound muscle atrophy and bone loss in astronauts 7. However, the precise
mechanisms underlying disuse-induced and microgravity bone loss in humans
are incompletely understood. Previous bed rest studies have reported that bone
loss at weight-bearing skeletal sites is accompanied by decreased serum PTH,
along with increased urinary calcium excretion and bone resorption markers with
the mechanisms underlying these changes unknown.
Osteocytes play a key mechanosensing role, modulating bone modeling and
by orchestrating the activity of osteoblasts and osteoclasts8
.
remodeling
Consistent with the negative effect of sclerostin on bone formation, transgenic
mice overexpressing SOST are osteopenic9 , whereas SOST-null animals have
high bone mass 0 , similar to the human conditions of van Buchem disease and
sclerosteosis. Importantly, the SOST/sclerostin pathway has been implicated in
the response of bone to mechanical loading in murine models because increased
skeletal loading reduces SOST expression, whereas decreased mechanical
loading increases it". Furthermore, SOST-null animals are resistant to disuseinduced bone loss 0 . An alternate mechanism by which osteocytes respond to
altered mechanical loading is suggested by the observation that osteocytes are a
142
major source of the osteoclastogenic cytokine RANKL 8 , and further, that mice
lacking RANKL in osteocytes are protected from bone loss induced by hind limb
unloading.
Despite evidence of several molecular mechanisms by which osteocytes may
regulate the response to mechanical loading in animal models, little is known
about how osteocytes orchestrate skeletal adaptation to mechanical unloading in
humans. In a cross-sectional study, older women who had suffered a stroke 10
months beforehand had 3-fold higher serum sclerostin levels than age matched,
fully ambulating controls (4), consistent with the notion that sclerostin levels
increase in response to mechanical unloading. Yet, men with unloading due to
chronic spinal cord injury (mean
SD, 22.4
+
11.2 yr.) have lower sclerostin
levels than ambulating control subjects 6 . To address a gap in the literature with
regard to the acute response to unloading, we evaluated the longitudinal
changes in serum sclerostin levels in healthy men that participated in a 90-d,
controlled bed rest study, with the hypothesis that acute unloading would lead to
an increase in serum sclerostin.
9.3
Subjects and materials
9.3.1
Subjects
Seven healthy men were recruited by the National Aeronautics and Space
Administration (NASA) Johnson Space Center (JSC) to participate in a 90-d bed
rest experiment. Eligibility included physically fit men between the ages of 25 and
55 yr. who were not taking prescribed medication that would interfere with
physiological measurements. Mean ( SD) age of the participants was 31
3 yr.
(range, 28-36 yr.). Mean ( SD) height, weight, and body mass index of subjects
were 183
6 cm, 88
12 kg, and 26
2.8 kg/m2, respectively. Mean dietary
calcium and vitamin D intake were fixed at 1674
251 mg/d and 384
72 IU/d,
respectively. Subjects were continuously monitored via remote controlled
cameras and remained in 60 head down tilt for 90 d with controlled nutrition,
143
water intake, day-night cycles, ambient room temperature, and in-bed hygiene.
Daily vital sign measurements were collected. Additional details regarding the
University of Texas Medical Branch (UTMB)-NASA bed rest protocol can be
found in prior publications1
35
- ,
. The institutional review boards of both NASA JSC
and UTMB approved the study protocol, and all subjects gave written informed
consent.
9.3.2
Serum sclerostin and bone turnover markers
Serum (fasting, 0630 h collection) and urine samples were collected at two prebed rest time points (10 and 3 d before bed rest) and at bed rest d 28 (BR-28),
bed rest d 60 (BR-60), and bed rest d 90 (BR-90). For all serum and urine
markers, the results from the two pre-bed rest measurements were averaged to
provide a baseline (BL) value for each subject. In addition, urinary collections
were normalized to a 24-h time period at each time point. Serum sclerostin levels
were assayed in duplicate using an ELISA kit (ALPCO/Biomedica, Salem, NH).
All sclerostin samples were assayed with a single assay. The intra-assay
variability as reported by the manufacturer is 5%. We also measured markers of
bone metabolism, including serum PTH, 25 (OH) vitamin D, and 1,25 (OH) 2
vitamin D; serum markers of bone formation (bone-specific alkaline phosphatase
(BSAP) and osteocalcin); serum markers of osteoclast activity, soluble RANKL,
osteoprotegerin (OPG); urinary calcium and phosphorus excretion; and urinary
markers of bone resorption, N-terminal telopeptide (NTX),
deoxypridinoline
.
(DPD), pyridinium crosslinks (PYD)], using previously reported methods 2
9.3.3
Bone mineral density
Areal BMD (grams per square centimeter) was measured by dual-energy x-ray
absorptiometry (DXA Hologic Discovery; Hologic Inc., Bedford, MA). BMD of the
whole body, lumbar spine, averaged left and right hips, heel, and forearm was
assessed at BL and repeated at BR-60 and 5 d after the end of the bed rest
period (BR + 5). One subject was lost to follow-up at BR + 5. BMD
144
measurements for each subject reported are the mean of triplicate scans.
9.3.4
Statistical analysis
All data are summarized by mean
SD unless otherwise specified. All data were
analyzed with repeated-measures ANOVA. Analyses were performed with SAS
9.2 (SAS Institute Inc., Cary, NC) using the proc mixed procedure with an
autoregressive covariance structure. Given the small sample size, a two-sided a
of 0.10 for the overall ANOVA model was accepted as significant to proceed to
pre-specified pairwise comparisons of specific time points vs. BL (BL vs. BR-28,
BR-60, and BR-90), for which two-sided of a = 0.05 was considered significant.
9.4
Results
9.4.1
Serum sclerostin and PTH
Serum sclerostin increased after bed rest in all subjects (Fig. 9-1A). Specifically,
serum sclerostin levels increased above BL at BR-28 (+29
20%; p = 0.003) and
appeared to plateau in most subjects at BR-60 (+42
31%; p
< 0.001).
Sclerostin levels remained mildly elevated at BR-90, although this result did not
reach statistical significance (+22
levels declined at BR-28 (-17
21%; p = 0.07). In contrast, serum PTH
16%; p = 0.02) and BR-60 (-24
remained reduced at BR-90 (-21
14%; p = 0.03),
21%; p = 0.14), but did not reach statistical
significance.
145
B
A
,o- Sclerostin
-0-. PT H
02
7r
2
M
BR-60
3
BR-90
O
0.
-2
CHC:I
BL
BR-28
BR-60
BR-90
,
*4*
.
T
_20
Figure 9-1: A, Effect of bed rest on serum sclerostin and PTH. B, BMD (mean SD).
* p < 0.05; **, p < 0.005; ***, p < 0.0001, indicating significant change from BL.
9.4.2
Bone mineral density
Subjects had normal BMD because BL Z-scores at the distal radius, lumbar
spine, total hip, and femoral neck were 0.2
0.7, -0.6
1.0, 0.2
1.0, and 0.0
1.3, respectively. BMD declined significantly at BR-60 and BR+5 at all weightbearing skeletal sites, including the lumbar spine, hip, femoral neck, and
calcaneus (Fig. 9-1 B; p < 0.05 for all). There was no change in forearm BMD.
9.4.3
Serum and urinary markers of bone turnover
As summarized in Table 9-1, urinary levels of bone resorption markers (NTX,
DPD, PYD) increased significantly compared with BL at all-time points. Urinary
calcium excretion was also significantly increased throughout the study, whereas
urinary phosphorus levels were elevated at BR-28 and BR-60 (p < 0.005 for
both), with a return to BL at BR-90. Serum bone formation markers (serum
BSAP, osteocalcin), serum RANKL, OPG, and the RANKL/OPG ratio did not
change. 25 (OH) vitamin D was above BL at all-time points (p < 0.005), whereas
1,25 (OH) 2 vitamin D was significantly lower than BL at BR-28 (-13%; p < 0.05)
and tended also to be lower at BR-60 (p = 0.06) and BR-90 (p = 0.07).
146
Table 9-1: Serum and urinary measurements of bone turnover makers, urinary calcium and
phosphorous at baseline (BL), BR-28, BR-60, and BR-90 (mean SD).
BL
BR-28
BR-60
BR-90
35.4 7
31.5 12
13 3
45.3 9.4**
25.5 10.5*
17 3**
48.8 4.8**
24 10.9*
19 2***
42.1 4.7
25.6 13.6
18 3***
Serum
Sclerostin (pmol/l)
PTH (pg/ml)
25 (OH) Vitamin D (ng/ml)
1,25 (OH) 2 Vitamin D
(pg/ml)
BSAP (U/L)
Osteocalcin (ng/ml)
Soluble RANKL (pmol/1)
OPG (pmol/1)
RANKL/OPG (pmol/I)
24-Hour Pooled Urine
NTX (nmol)
38
27
5
6
34
29
7*
5
34
30
9
5
14
0.3
3
0.2
13
0.3
2.5
0.12
3
0.2
0.9
0.12
13 2
0.3 0.3
2.4 1
0.13 0.11
2.2 1
0.14 0.13
482
127
795
182***
735
153**
DPD (nmol)
PYD (nmol)
67
221
16
63
108
367
22***
119 **
112
380
Calcium (mmol/day)
Phosphorus (mg/day)
5.9
849
1.3
117
7.9 0.6***
1039 200 **
7.4
1074
1.1*
160**
33
32
13
0.3
7
3
4
0.3
2.2 1
0.18 0.18
796
165*
25***
116
26***
145**
409
171**
7.5
946
1.2*
136
*p < 0.05, ** p < 0.005, *** p<0.0001 compared to BL. Assay manufacturers were: sclerostin,
soluble RANKL, and OPG-ALPCO/Biomedica, Salem, NH; PTH-Scantibodies, Santee, CA; 25
(OH) vitamin D and 1,25 (OH)2 vitamin D-DiaSorin, Stillwater, MN; BSAP, DPD, and PYDQuidel, San Diego, CA; osteocalcin-Biomedical Technologies, Stoughton, MA; NTX and
phosphorus-Alere North American, Waltham, MA; and calcium-Atomic Absorption, Perkins
Elmer Flame; PerkinElmer Inc., Waltham, MA.
9.5
Discussion
We found that in healthy men exposed to bed rest, serum sclerostin levels
increased significantly by as early as 1 month and remained elevated for another
month. Consistent with prior bed rest studies 3, serum PTH declined, urinary
calcium and bone resorption markers increased, and BMD decreased at weightbearing sites. There were no detectable changes in serum markers of bone
formation.
Our observation of increased serum sclerostin after bed rest is consistent with
previous reports of elevated sclerostin levels in animal and human models of
disuse4
11
. A prior cross-sectional study reported that 10 months after suffering a
147
stroke, postmenopausal women (mean age = 80 yr.) had 3-fold higher serum
sclerostin levels than age-matched healthy controls 4 . In comparison, in the
current longitudinal study (subject mean age = 36), the maximum increase in
serum sclerostin levels was +42% vs. BL at BR-60. Several differences between
these two studies may have contributed to the different magnitudes of sclerostin
increases after disuse, including: 1) we assessed the longitudinal response to
acute mechanical unloading, whereas the study of stroke patients was a crosssectional study of long-term disuse; 2) we enrolled healthy young men, whereas
the stroke study examined elderly postmenopausal women; and 3) we studied
strictly controlled bed rest, whereas activity levels of the stroke patients were
more variable. Our data also differ from a previous cross-sectional study in
middle-aged men with chronic spinal cord injury, in whom sclerostin levels were
lower than normally ambulating age-matched controls (6). However, the increase
in serum sclerostin that we observed supports a conceptual model where serum
sclerostin rises acutely and then is suppressed in the chronic bone wasting
phase. Clearly, additional human studies are needed to better define the time
course of changes in serum sclerostin in response to disuse, to address whether
there is a neuroloqical component to its regulation, and to test the efficacy of
sclerostin antibody treatment in the setting of acute-onset, disuse-induced bone
loss.
Decreased levels of serum PTH accompanied the increases in serum sclerostin
after bed rest, presumably driven by a transient increase of serum calcium levels
by bone resorption and a measured increase in urinary calcium. In animal
models, PTH decreases sclerostin expression via activation of the PTH receptor
expressed on osteocytes1 3 . Furthermore, there is an inverse correlation between
PTH and sclerostin in male hypothyroid subjects1 4 , and PTH infusion in healthy
men induces a decline in serum sclerostin levels 15. However, in these studies, we
cannot determine whether the reduction in PTH levels is driving the observed
increase in sclerostin or whether sclerostin increases in disuse due to non-PTHmediated mechanisms. To answer this question, future bed rest studies could
148
employ more frequent measures of serum calcium, sclerostin, and PTH to more
precisely define the time course of changes in each. Also, blocking the increase
in serum calcium, perhaps by administration of an antiresorptive agent, might
prevent the decrease in serum PTH and allow one to determine whether the
increased sclerostin in bed rest is independent of serum PTH. Finally, we have
conducted in-vitro unloading experiments in isolated osteocytes (Chapter 5) to
determine if osteocytes inherently can sense simulated microgravity in the
absence of PTH to give rise to an increase in SOST and Sclerostin to directly decouple the confounding effects of PTH and mechanical unloading in in-vivo
experiments.
9.6
Conclusion
In conclusion, serum sclerostin levels increased significantly in healthy young
men exposed to 90 d of head down tilt bed rest16 . Bed rest was also associated
with a decrease in serum PTH, an increase in bone resorption markers, and a
decrease in BMD at weight-bearing sites. These are the first data to show the
acute, longitudinal changes in serum sclerostin in response to bed rest. Given
the key role that sclerostin plays in mediating bone metabolism and formation,
additional studies exploring the regulation of sclerostin in disuse are warranted,
particularly given the emergence of anti-sclerostin pharmacological therapies.
149
1
2
3
4
5
6
7
8
9
Zwart, S. R. et al. Effects of 21 days of bed rest, with or without artificial
gravity, on nutritional status of humans. J Appi Physiol 107, 54-62,
doi:91136.2008 [pii] 10.11 52/japplphysiol.91136.2008 (2009).
Zwart, S. R. et al. Nutritional status assessment before, during, and after
long-duration head-down bed rest. Aviat Space Environ Med 80, Al 5-22
(2009).
Inniss, A. M., Rice, B. L. & Smith, S. M. Dietary support of long-duration
head-down bed rest. Aviat Space Environ Med 80, A9-14 (2009).
Gaudio, A. et al. Increased sclerostin serum levels associated with bone
formation and resorption markers in patients with immobilization-induced
bone loss. J Clin Endocrinol Metab 95, 2248-2253, doi:jc.2010-0067 [pii]
10.1210/jc.2010-0067 (2010).
Spector, E. R., Smith, S. M. & Sibonga, J. D. Skeletal effects of longduration head-down bed rest. Aviat Space Environ Med 80, A23-28
(2009).
Morse, L. R. et al. Association between sclerostin and bone density in
chronic spinal cord injury. Journal of bone and mineral research : the
official journal of the American Society for Bone and Mineral Research 27,
352-359, doi:10.1 002/jbmr.546 (2012).
LeBlanc, A. D., Spector, E. R., Evans, H. J. & Sibonga, J. D. Skeletal
responses to space flight and the bed rest analog: a review. J
Musculoskelet Neuronal Interact 7, 33-47 (2007).
Nakashima, T. et al. Evidence for osteocyte regulation of bone
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doi:10.1038/nm.2452; 10.1038/nm.2452 (2011).
Winkler, D. G. et al. Osteocyte control of bone formation via sclerostin, a
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novel BMP~~aIantagonist.
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via Antagonizing Wnt/beta-Catenin Signaling. J Bone Miner Res 24, 16511661 (2009).
Robling, A. G., Bellido, T. & Turner, C. H. Mechanical stimulation in vivo
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Xiong, J. et al. Matrix-embedded cells control osteoclast formation. Nat
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receptor signaling in osteocytes. PLoS One 3, e2942,
doi: 10.1371/journal.pone.0002942 (2008).
Costa, A. G. et al. Circulating sclerostin in disorders of parathyroid gland
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Yu, E. W., Kumbhani, R., Siwila-Sackman, E. & Leder, B. Z. Acute Decline
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M~
10
11
12
13
14
15
11 A-AA
0 I ^^ ^r 17 ^-
150
16
Spatz, J. M. et al. Serum sclerostin increases in healthy adult men during
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151
152
Chapter 10
10
Summary and conclusions
10.1
Summary of hypotheses
As outlined in section 1.2, these investigations were designed to explore four
primary hypotheses.
Hypothesis 1: Conditionallyimmortalized osteocytic cell lines, derived from
long bones, can be establishedthat express markers of mature osteocytes
and follow the hormonal responses of in-vivo osteocytes.
As described in Chapter 4, a novel osteocytic cell line (Ocy454) was developed
and validated as a new in-vitro tool to study osteocyte biology. The significant
advancement for the field of bone biology is that Ocy454 can be cultured without
differentiation factors and that Ocy454 exhibits robust expression of markers of
mature osteocytes within two-weeks.
Hypothesis 2: Osteocytic cells directly sense mechanical unloading to
increase SOST and sclerostinin simulatedmicrogravity.
We showed that exposure of an osteocytic cell line (Ocy454) to simulated
microgravity results in increased expression of both inhibitors of bone formation
(e.g., SOST/sclerostin), and stimulators of bone resorption, notably RANKL and
the RANKL/OPG ratio. In addition, the response to a mechanical loading
stimulus, achieved by subjecting Ocy454 to fluid shear stress, does not in itself
account for the observed gene regulation seen in simulated microgravity.
Hypothesis 3: Pharmacologic inhibition of sclerostin prevents bone loss
and induces bone formation in adult mice subjected to hind limb
unloading, and partial weight bearing.
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This hypothesis was confirmed by showing that adult mice exposed to both full
unloading (Chapter 7) and partial weight bearing (Chapter 9) and treated with
sclerostin antibody had BMD, microarchitecture, and mechanical strength values
at or above the normally loaded control mice. Moreover, in the hind limb
unloading study we confirmed that inhibition increased bone formation by
quantitative histomorphometry. Interestingly, we observed that although robust in
all conditions, the skeletal response to sclerostin antibody tended to be better in
normally-loaded compared to unloaded mice suggesting that limited weightbearing exercise may improve the skeletal response to sclerostin inhibition in
patients with spinal cord injuries, stroke, muscular dystrophy, cerebral palsy, and
other
diseases
and
conditions
associated
with
short-term
or
chronic
musculoskeletal disuse.
Hypothesis 4: The osteocyte secreted protein sclerostin, is elevated in
healthy adult men subjected to 90 days of controlledbed rest.
The hypothesis that sclerostin may play an important role in the human response
to disuse-induced bone loss is supported by our observation that serum
sclerostin levels increased significantly in healthy young men exposed to 90 days
of head down tilt bed rest.
10.2
Future work
The development of a novel osteocyte cell line (Ocy454) that is easy-to-use and
re-capitalizes key aspects of in-vivo osteocytes provide a useful tool for studies
of bone biology and endocrine research community. Already, collaborators have
utilized the latest advances in biotechnology, such as Crispr/Cas9 and shRNA, in
Ocy454 to modify the genome of osteocytes, knockdown,
knockout, or
overexpress genes of interest thereby facilitating diverse studies of the roles of
osteocytes that would have otherwise been difficult prior to the invention of the
Ocy454 cell line. Furthermore, the Ocy454 cell line is heterogeneous cell line
representative of different stages of osteocyte maturation enabling future
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researchers to single cell clone Ocy454 to meet particular experimental needs as
the understanding of osteocyte biology evolves.
With the successful completion of the ISS OSTEO-4 mission, the Ocy454 cell
line was the first osteocyte culture to be flown in microgravity. Future work will
compare whole genome expression changes observed in the ISS flight study to
those occurring in the ground-based simulated microgravity models. The long
term goal is identify novel targets of the osteocyte proteome that are regulated by
mechanical unloading to determine how osteocytes integrate the mechanical
unloading stimuli in hopes of providing additional therapeutic targets for disuseand microgravity-induced bone loss and other conditions of skeletal fragility, most
notably osteoporosis.
Sclerostin antibody remains a promising anabolic therapy for a wide variety of
bone disorders. While we have clearly demonstrated its potential in pre-clinical
models of disuse- and microgravity-induced bone loss, there remains an
important next step and need to demonstrate its effectiveness in well-controlled
human studies of disuse-induced bone loss (e.g. bed rest) and, eventually, in
long duration spaceflight experiments.
10.3
Conclusions
In summary, we have shown both with in-vitro and in-vivo experiments that
osteocytes play an important role in bone's remarkable adaption to mechanical
unloading. Most notably, the development of a novel, easy to use, mature
osteocytic cell line is a particularly unique contribution of this thesis. In addition,
the confirmation of elevated serum sclerostin levels in human bed rest studies
and the protection of sclerostin antibody treatment in disuse-induced bone loss
highlight emerging opportunities to modulate osteocytes and their secretome to
cure a multitude of human bone diseases.
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