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The Pennsylvania State University
The Graduate School
Department of Materials Science and Engineering
ON THE PERMANENT LIFE OF TISSUE OUTSIDE THE ORGANISM
A Thesis in
Materials Science and Engineering
by
Dhurjati Ravi
© 2007 Dhurjati Ravi
Submitted in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
December 2007
The thesis of Dhurjati Ravi was reviewed and approved* by the following:
Erwin A. Vogler Professor of Materials Science and Engineering and Bioengineering Thesis Advisor Chair of Committee
Andrea M. Mastro Professor of Microbiology and Cell Biology Paul W. Brown Professor of Ceramics Ronald Hedden Assistant Professor of Materials Science and Engineering Sandeep K. Prabhu Assistant Professor of Veterinary and Biomedical Science
Gary L. Messing Distinguished Professor of Ceramic Science and Engineering Head of the Department of Materials Science and Engineering
*Signatures are on file in the Graduate School
iii
ABSTRACT
Growth and maintenance of cells/tissue outside the body is central to the practice of
biomedical sciences and technology. In vitro culture techniques, developed almost a century
ago, are fundamental enabling tools for the study of life processes and are routinely
employed in many applications that impact human health care. Emergence of the field of
tissue engineering, focused on developing tissue surrogates for transplantation, has renewed
the emphasis on critically examining the conditions under which we culture cells/tissue
outside the body. It has been well recognized that isolation of cells from the complexity of
their native physiological environment results in adaptive responses that often limit cell
viability and function in vitro. Bridging the gap between physiological complexity and in
vitro culture environments is critical to the successful implementation of the tissue
engineering strategy. We show herein that a compartmentalized bioreactor based on the
principle of continuous-growth–and-dialysis provides stable culture conditions that better
simulate the physiological environment. The bioreactor was used to grow mineralizing,
collagenous bone tissue up to 150 mμ thick from an inoculum of isolated murine (mouse
calvaria MC3T3-E1, ATCC CRL-2593) or human (hFOB 1.19 ATCC CRL-11372)
osteoblasts over uninterrupted culture periods up to a year. Proliferation and phenotypic
progression of an osteogenic-cell monolayer into a tissue comprised of cell layers of
mature osteoblasts in the bioreactor was compared to cell performance in conventional
tissue-culture polystyrene (TCPS) controls. Cells in the bioreactor basically matched results
obtained in TCPS over a 15 d culture interval, but loss of insoluble ECM (iECM) and ~2X
increase in apoptosis rates in TCPS after 30 d indicated progressive instability of cultures
maintained in TCPS with periodic refeeding but without subculture. By contrast, stable
cultures were maintained in the bioreactor for up to a year, suggesting that extended-term
6≥
iv
tissue maintenance is feasible with little-or-no special technique. Month’s long culture
interval in the bioreactor lead to progression of pre-osteoblasts to osteocyte-like cells
embedded in mineralized matrix observed in normal bone and production of visually-
apparent (macroscopic) bone. Challenging bioreactor-derived bone tissue at different stages
of development with metastatic breast cancer cells (MDA-MB-231) known to invade the
skeleton created a system-in-crisis that captured early stages breast cancer colonization of
bone. In situ confocal microscopy revealed sequential steps of breast cancer cell adhesion,
penetration, and degradation of the 3D bone-like osteoblast tissue over co-culture intervals
ranging from 3 to 10 days. Bioreactor enabled direct observation of interactions of breast
cancer cells with engineered 3D bone like tissue and simulated an important step in the
progression of the disease that may be a target for therapeutic intervention. Using bone as a
model tissue, this study demonstrates that complex physiological and pathological processes
of great importance to human health can be simulated using engineered cellular
environments for potential applications in drug discovery and toxicology.
v
TABLE OF CONTENTS
LIST OF TABLES ........................................................................................................vii
LIST OF FIGURES..................................................................................................... viii
Acknowledgements ........................................................................................................xii
Chapter 1 Introduction 1
In vivo and in vitro 1
The gap between in vivo and in vitro 3
Bridging the culture gap 4
References 7
Chapter 2 Literature Survey 13
Osteogenesis In Vivo 13
Bone Development 13
Cellular Origins Of Bone Cells 15
Bone Formation 15
The Osteoblast Milieu 18
Osteogenesis In Vitro 19
Organ Cultures 19
vi
Culture Of Bone Slices And Explants 20
Culture Of Isolated Bone Cells 22
In Vitro Models Of Bone Formation: An Assessment 23
Homogeneity Versus Heterogeneity 23
Length Of Culture Interval 25
Effect Of The Microenvironment 25
3D Models of Bone Formation 29
References 33
Chapter 3 Extended-Term Culture Of Bone Cells in a
Compartmentalized Bioreactor
61
Introduction 63
Methods And Materials 66
Results And Discussion 75
Conclusions 80
References 83
Chapter 4 Metastatic Colonization Of Bone Tissue By Breast
Cancer In Vitro
101
Introduction 102
Materials and Methods 105
Results and Discussion 108
Conclusions and Future Work 111
References 113
vii
LIST OF TABLES
Chapter 1 Introduction 1
Table 1 Comparison of in vivo and in vitro techniques 10
Chapter 2 Literature Survey 13
Table 1 Principal extracellular matrix proteins secreted by the
osteoblasts and their sequence of expression
56
Table 2 Local factors in the bone microenvironment that
condition the growth and differentiation of the
osteoblasts
57
Table 3 Historical time line of the development of in vitro
models of bone formation
58
Table 4 Representative bioreactors for bone tissue engineering
applications
59
Table 5 Unique design features of the compartmentalized bioreactor 60
Chapter 3 Extended-Term Culture of Bone Cells in a
Compartmentalized Bioreactor
61
Table 1 hFOB 1.19 cells in conventional culture compared to
compartmentalized bioreactor
88
Table 2 MC3T3-E1 Cells in conventional culture compared
to compartmentalized bioreactor
89
viii
LIST OF FIGURES
Chapter 1 Introduction 1
Figure 1 Cells and/or tissues are cultured in an artificial environment within
a defined control volume. Physical, biological and biochemical
factors that condition the cellular response within the control
volume are listed
12
Chapter 2 Literature Survey 13
Chapter 3 Extended-Term Culture of Bone Cells in a
Compartmentalized Bioreactor
61
Figure 1 Compartmentalized bioreactor design. Panel (a) is a cross-sectional
diagram through the device showing separation of the cell-growth
space (A) from the basal-medium reservoir (B) by a dialysis
membrane (C). Cells are grown on gas-permeable but liquid-
impermeable film (E). The device is ventilated through film (D),
which is the same material as (E) as described herein but can be
different. The whole device is brought together in a liquid-tight
fashion using screws shown in the laboratory implementation Panel
(b) and Panel (c) which is an exploded-view identifying separate
components. Liquid-access is through leur-taper ports (J, K) which
mate to standard pipettes. See Methods and Materials for theory of
operation.
93
Figure 2 Growth dynamics of hFOB 1.19 on tissue-culture-grade polystyrene
controls (TCPS, filled triangles) compared to the
compartmentalized bioreactor (open squares; note logarithmic
ordinate). Inset expands short-term attachment rates using a linear
ordinate. Note that the initial two-fold cell-attachment preference
for TCPS persists through the exponential-growth (3 7t 2< < hr)
and post-confluence expansion ( 72 720t< < hr) phases
94
ix
Figure 3 Comparison of hFOB 1.19 on TCPS (Panels A, B) and in the
compartmentalized bioreactor (Panels C, D) by cross-sectional
TEM. Notice that cell layers are thicker in the bioreactor than on
TCPS (compare Panels A, B to C, D). and formation of apoptotic
bodies (arrows) after 30 d culture in TCPS
95
Figure 4 Comparison of insoluble extracellular matrix (iECM) production by
hFOB 1.19 on TCPS (dark bar) and in the bioreactor (light bar).
Notice that iECM significantly decreased after 30 d on TCPS but
remained constant in the bioreactor. Bar values are mean of
duplicate samples measured in triplicate with standard deviation
represented by error bars
96
Figure 5 Representative confocal image of apoptotic bodies (green stain) in
TCPS and in the bioreactor selected from 8-10 similar in-depth
image planes probing hFOB 1.19-derived tissue. All cells were
stained red (Sytox Orange, scale bar = 50 μm). Percent apoptotic
cells quoted in each panel are the range of values observed within
the image planes
97
Figure 6 Ultramorphology of hFOB 1.19–derived tissue (15 d) recovered
from the bioreactor by cross-sectional TEM. Arrows point to cell
protrusions that occasionally connect two cells, as shown in Panel
C. Annotations: jNC = gap junction; MV = matrix vesicles; N =
nucleus; rER = rough endoplasmic reticulum
98
Figure 7 Analysis of mineral nodules taken from MC3T3-E1 cultured in a
bioreactor. Panel A is an SEM of a large nodule taken from a 70 d
bioreactor. Panel B is a high-magnification, cross-sectional TEM of
a similar nodule. Panel C is an x-ray spectrum obtained by
SEM/EDAX of a nodule taken from a 30 d bioreactor confirming
Ca and P as major constituents
99
Figure 8 Microscopic examination of matrix derived from MC3T3-E1
cultured in a bioreactor for 70 d. Panel A is an SEM of the outer
layer showing a highly fibrous network with bone nodules. Panel B
is an optical micrograph of a histologic workup showing layers of
100
x
collagen with imbedded osteoblasts. Panel C is a cross-sectional
TEM showing mineralized fibers running in (IP annotation) and
out (OP annotation) of the plane
Chapter 4 Metastatic Colonization of Bone Tissue by Breast
Cancer In Vitro
101
Figure 1 Schematic showing the sequential steps of cancer cell attachment,
penetration, replication and possible host tissue destruction
observed in the bioreactor as a result of interactions between GFP-
labeled breast cancer cells and multiple layer 3D osteoblast tissue.
118
Figure 2 (A) Light micrographs of a Hemotoxylin and Eosin stained 70-day
tissue (40X) reveal osteoblasts (pale red with dark-red nuclei) lining
a collagenous matrix (pale pink). (B)Transmission electron
micrographs (TEM) of 22-day tissue cross sections show up to 5 cell
layers (1500X). (C) Scanning electron micrographs of the surface of
a 70 day culture are studded with mineral nodules (1000X) that
prove positive for calcium and phosphorous by energy-dispersive
analysis of x-rays (not shown). (D) Bone chip recovered from a 5
month MC3T3-E1 bioreactor. X-ray diffraction (inset) indicates
similarity with a bovine bone reference spectrum (lines).
119
Figure 3 Transmission electron micrograph (TEM) of a cross-section of an
isolated MDA-MB-231 breast cancer cell crawling on the base film
of the bioreactor used in this work (cultured for 3 days in
osteoblast-conditioned medium) reveals an amoeboid morphology
with protrusive structures at the leading edge, consistent with a
migratory phenotype (Panel A, 1500X). TEM of breast cancer cells
(BC in Panel B, 5000X) co-cultured for 10-days with a 60-day
MC3T3-E1 tissue shows that cancer cells displaced osteoblasts
originally adhering to the base film (see Figure 2) and exhibit
protrusive structures that may be involved in penetration. (arrows;
compare to Panel A). Fluorescence microscopy oft breast cancer
cells (green) in co-culture with osteoid tissue (red) organize in
120
xi
single-file order (Panel C, 40X), captured in cross-section by TEM
(inset, 2000X).
Figure 4 Serial optical sections through an MC3T3-E1 derived osteoid tissue
(5-month culture interval stained with Cell Tracker Orange ™) co-
cultured with GFP-labeled MDA-MB-231 breast cancer cells
(green) for 3 days. Optical sections are from bottom [0μm] to top
[16μm]) of the tissue. (40X, scale bar = 50 μm)
121
Figure 5 3D reconstruction of serial optical sections taken through the
thickness of the 5 month osteoblast tissue cultured in the bioreactor
and subject to MDA-MB-231 breast cancer challenge over 3 day co-
culture interval.
122
xii
ACKNOWLEDGEMENTS
I would like to acknowledge my gratitude to Dr. Vogler for sharing his tremendous
enthusiasm and passion for science. It has been a pleasure to learn from a gifted teacher,
generous with his time and energies for all his students. I appreciate the continuous,
unwavering support through the years of graduate school and will always treasure the
conversations I have had with him about science and the philosophy of science.
I would also like to extend my gratitude to my thesis committee members- Dr. Mastro,
Dr. Brown, Dr. Prabhu and Dr. Hedden for helpful suggestions, critiques and constant
encouragement. I have benefited from my interactions with every one of my committee
members representing a diverse group of faculty from engineering and life sciences. I am
particularly indebted to Dr. Mastro for giving me the opportunity to work in the area of
cancer biology and for generously sharing her expertise and her valuable insight from the
many years of research on breast cancer.
I have had the opportunity to work with a wonderful group of people as part of this project.
My colleagues deserve special thanks for sharing the many joys and the minor tribulations of
the life in research.
Chapter 1
Introduction
1.1 In Vivo and In Vitro
Fundamental biological processes can be studied within the living
organism (in vivo) or in a controlled artificial environment outside the body (in
vitro). Investigations of biological phenomena in vivo using animal models has
the distinct advantage that the responses to particular dose are integrated across
the length scales of cells, tissues and organs, and are, therefore, representative of
physiological complexity. However, from an experimental design standpoint, it
is not very easy to separate cause and effect because the response to a dose
cannot be limited to a particular physiological compartment. In vivo studies offer
very limited control over the experimental conditions and it is difficult to tease
out the relationships between the numerous variables involved. Therefore, the
very complexity that confers physiological relevance to in vivo studies often
makes them difficult to interpret (Table 1).
Isolation of cells, tissues or even organs into a controlled artificial
environment has several advantages. In vitro techniques provide greater control
over the experimental conditions and the ability to limit the number of variables.
In other words they allow us to isolate a control volume defined by the biological
1
structure under investigation and its environment. Techniques that make it
possible to isolate, grow and sustain cells and tissues in controlled environments
outside the body were developed in the early part of the 20th century by pioneers
such as Harrison and Carrell.1-3 The incredible power of this approach to
provide fundamental insights into biological phenomenon was illustrated by the
very first experiment utilizing the technique. In 1907, Harrison2 isolated
fragments of the nerve tube of larval frog into a medium of clotted frog lymph in
a hollowed out glass slide and observed the outgrowth of nerve fibers from the
explants. Harrison’s seminal experiment2,4 provided, for the first time, the means
to observe living processes outside the body under controlled conditions. The
experimental setup allowed Harrison to separate the growth of the embryonic
nerve tissue from the overall complexity of embryonic development. Harrison’s
method was perfectly suited for testing his hypothesis that the nerve fibers were
outgrowths from the neurons and it helped him furnish experimental evidence to
resolve definitively one the major scientific controversies of his time on the
development of the nervous system.2,4
Early in vitro culture techniques including those used by Harrison and
Carrell were primarily culture of explants or fragments of tissue. The shift from
culture of tissues to the culture of isolated cells became possible only with the
development of enzymatic methods of cell separation in the 1950s.5 Rapid
2
adoption of these methods has resulted in culture of isolated cells being the norm
today. These developments have been consolidated into the standard practice of
monolayer cell culture where cells are cultured on a 2D substrate surrounded by
a medium that sustains the metabolic requirements of the cell.5 In almost a
century since their development, in vitro culture techniques have become integral
to the practice of biomedical sciences and technology. These enabling techniques
remain to this day, fundamental tools for the study of life processes and are
routinely employed in many applications that impact the delivery of human
health care.5
1.2 The Gap between In Vivo and In Vitro
In spite of the widespread success in the use of in vitro techniques, it is
important to recognize that there are profound differences between in vivo and in
vitro cellular environments. In vivo, cells held together by cell-cell contacts and
the extracellular matrix in a 3D structure, interact with neighboring cells of the
same type and/or different type with their responses coordinated by a variety of
biochemical and mechanical factors.6 In other words, cells function within the
context of a highly specialized microenvironment that is specific to the cell type
and the anatomical location. Growth, differentiation and ultimate fate of cells is
determined by complex interactions between the cell and its
microenvironment.7-9
3
Culture of cells outside the body using standard 2D monolayer techniques
deprives cells of their physiological context and brings into sharp focus the
impact of culture conditions on cellular function. Physical, biological and
biochemical factors affect cellular function within this controlled environment
(Fig. 1-1). To illustrate the nature of these environmental effects, consider the
affect of changes in osmolarity of the medium on cell volume. Decreasing the
osmolarity of the medium surrounding a cell leads to a compensatory response
of cell swelling; increase in osmolarity leads to cell shrinkage.10 In addition to
these physical factors, biological factors like cell-cell11 and cell-matrix
interactions7,8 condition cellular responses. Absence of proper environmental
cues often results in a less differentiated phenotype in vitro.7,8,12 Thus, there is
strong experimental evidence that cells undergo several adaptive responses
when transferred from their physiological environment to a radically different
exogenous environment.13 In aggregate, these adaptive responses have been
characterized as “culture shock” and they are often thought to limit cell viability
and function in vitro.13
1.3 Bridging the culture gap
The basic principles underlying in vitro culture techniques have not
changed significantly over the many years of their development. It is only with
4
the emergence of fields like tissue engineering14 that the environment in which
we routinely grow cells is being critically appraised. The gap between
conventional cell culture and the physiological environment is driving efforts to
develop engineered 3D cellular environments that can better simulate
physiological complexity.6,15 These approaches have the potential to integrate
physiological responses across the length scales of cells and tissues under
controlled conditions enabling us to model complex biological processes in vitro.
Working on the overall theme of the influence of the microenvironment
on cellular responses, this study showed that improved simulation of
physiological complexity can be achieved through suitable engineering of the
cellular environment. Particular focus was on the stability of culture
environment- an issue that has not received much attention in the literature.
Stable culture environments were engineered by separating cell growth and cell
feeding functions and through retention of cell-secreted growth factors and
cytokines in the cellular microenvironment.16 Using bone forming cells as a
model system, it was shown that stable culture environments promote assembly
of isolated cells into complex biological structures that can develop/mature over
extended periods.16 These in vitro grown structures permit a high level of
control over important experimental variables yet provide sufficient biological
complexity (e.g. three-dimensional, tissue-like cell density) that outcomes are a
5
reasonable predictor of the whole-organism physiological response to the
experimental variables. These tissue surrogates will be used as model systems to
study biological phenomenon of importance to human health care such as bone
formation and breast cancer colonization of bone.
6
References
1. Carrel A, Burrows MT. Cultivation of Adult Tissues and Organs Outside
the Body. J. Amer. Med. Assn. 1910;55:1379-1381.
2. Harrison RG. Experiments in transplanting limbs and their bearing upon
the problems of the development of nerves. Volume 4; 1907. p 239-281.
3. Oppenheimer JM. Taking Things Apart and Putting them Together Again.
Bull. Hist. Med. 1978;52(2):149-161.
4. Harrison RG. The outgrowth of the nerve fiber as a mode of protoplasmic
movement. Volume 9; 1910. p 787-846.
5. Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. New
York: Wiley-Liss Inc; 2000.
6. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro.
Nat Rev Mol Cell Biol 2006;7(3):211-224.
7. Roskelley CD, Desprez PY, Bissell MJ. Extracellular matrix-dependent
tissue-specific gene expression in mammary epithelial cells requires both
physical and biochemical signal transduction. Proc. Natl Acad. Sci. USA
1994;91:12378-12382.
8. Bissell MJ, Rizki A, Mian IS. Tissue architecture: the ultimate regulator of
breast epithelial function. Curr. Opin. Cell Biol. 2003;15:753-762.
7
9. Gurdon J, Lemaire P, Kato K. Community effects and related phenomena
in development. Cell 1993;75(5):831-4.
10. Sorkin AM, Dee KC, Knothe Tate ML. "Culture shock" from the bone cell's
perspective: emulating physiological conditions for mechanobiological
investigations. Volume 287; 2004. p C1527-1536.
11. Lauffenburger DA, Griffith LG. Who's Got Pull around Here? Cell
Organization in Development and Tissue Engineering. Proceedings of the
National Academy of Sciences of the United States of America
2001;98(8):4282-4284.
12. Abbot J, Holtzer H. The loss of phenotypic traits by differentiated cells. J.
Cell Biol. 1966;28:473-487.
13. Sherr C, DePinho R. Cellular senescence: mitotic clock or culture shock?
Cell 2000;102(4):407-10.
14. Langer R, Vacanti JP. Tissue Engineering. Science 1993;260(5110):920-926.
15. Griffith LG, Naughton G. Tissue engineering current challenges and
expanding opportunities. Science 2002;295:1009-1014.
16. Dhurjati R, Liu X, Gay CV, Mastro AM, Vogler EA. Extended-term culture
of bone cells in a compartmentalized bioreactor. Tissue Engineering
2006;12(11):3045-3054.
8
List of Table Legends
Table 1: Comparision of in vivo versus in vitro techniques.
9
Table 1: Comparison of in vivo and in vitro techniques
In Vivo In Vitro 1. Difficult to separate cause and effect because of the complexity of physiological environment and the number of independent variables involved.
1. Easier to separate cause and effect under controlled conditions and isolate the effect of individual variables.
2. Responses to dose are integrated across the length scales of cells, tissue and organs and are therefore representative of physiological reality.
2. Results obtained at the different length scales have to be integrated to create informed hypotheses about function at the organism level.
3. Tissue structure and organization and intercellular relationships are largely preserved.
3. Tissue structure and organization is often disrupted. Depending on the purpose of the investigation cellular physiology is sometimes studied in the absence of cell-cell and cell-ECM relationships found in vivo.
4. Does not easily permit reductionist approach of studying complex problems by breaking them down to their individual components.
4. Permits reductionist approach.
5. Difficult to study molecular level interactions including signaling and regulatory mechanisms.
5. Easier to study molecular level interactions.
6. Expensive, time-consuming and requires the maintenance of animals under the required test conditions for extended periods.
6. Simpler and cheaper to execute and permit greater control over experimental conditions
7. Studies using animals have to consider the inherent heterogeneity of subjects and require careful statistical analysis.
7. Permits greater control over test conditions and therefore repeatability.
10
List of Figure Legends
Figure 1-1: Cells and/or tissues are cultured in an artificial environment within a
defined control volume. Physical, biological and biochemical factors that
condition the cellular response within the control volume are listed.
11
Cont
rol V
olum
e
Tem
pera
ture
(typ
ical
ly 3
7oC)
Gas
com
posi
tion
(5 %
CO
2)M
ediu
m c
ompo
sitio
n, p
H, a
nd O
smol
arity
Oxy
gen
tens
ion
Mec
hani
cal s
tres
s
Pres
ence
of c
ytok
ines
, gro
wth
fact
ors,
ser
um-d
eriv
ed
prot
eins
, or
othe
r m
acro
mol
ecul
es in
the
med
ium
eith
er
by d
esig
n or
thro
ugh
secr
etio
n by
the
cells
as
part
of t
heir
norm
al fu
nctio
n.
Cell-
Cell
, Cel
l-EC
M a
nd C
ell-
Subs
trat
e In
tera
ctio
ns.
Phys
ical
Fac
tors
Biol
ogic
al F
acto
rs
Cellu
lar
Envi
ronm
ent a
nd C
ellu
lar
Func
tion
Fig
ure
1
12
Chapter 2
Literature Survey
This chapter starts with a short tutorial on bone growth and development and
introduces critical aspects of the physiology of bone-forming cells. Historical review of in
vitro models used to study these cells revealed specific design considerations for meeting the
need for improved bone cell culture models.
2.1 Osteogenesis In Vivo
2.1.1 Bone Development
Bone is a highly specialized organ that performs multiple functions within the body.1
In addition to providing structural support, bone acts as an storehouse of essential minerals
and is the site for hematopoiesis.2 Bone consists of a network of interconnected cells,
mineralized extracellular matrix and spaces that include bone marrow cavity, vascular
supply, canaliculi and lacunae.2,3 Growth, development and maintenance of the structural
integrity of bone require the coordinated actions of a complex cellular network.2,4,5
The primary modes of bone formation are endochondral and intramembranous.1,2
The process of endochondral bone formation that gives rise to the long bones and the
vertebrae requires the presence of a cartilaginous template that is replaced by or remodeled
into bone.2 Structurally, endochondral bone is further organized as either a dense outer
13
shell called the cortical bone or as a relatively thin inner network of connecting rods and
plates called trabecular bone.2,3,5 Intramembranous bones like the flat bones of the skull,
scapula and the ilieum form directly through differentiation of the mesenchymal cells into
bone forming osteoblasts without the need for an intermediate cartilage model.6
The principal functional activities involved in bone development and maintenance
are the formation of bone by osteoblasts7 and resorption of bone by osteoclasts.8 Removal of
bone from one site and resorption of bone at a different site occurs through a process called
“modeling” and is responsible for the shape and structure of bone during development.9 In
adults, resorption and new bone formation occur at the same site resulting in regeneration of
bone. This process called “remodeling” continues throughout life and becomes the
dominant process by the time bone reaches its peak mass in the 20s.3 Remodeling results in
complete regeneration of the adult skeleton every 10 years.3
Bone remodeling occurs through a tightly regulated sequence of osteoclastic bone
resorption followed by osteoblastic bone formation.10 The specific sequence of events
involved are osteoclast resorption and apoptosis followed by migration of osteoblast
precursors to the resorption site, proliferation and differentiation of osteoblasts, formation of
the extracellular matrix and its mineralization and finally cessation of osteoblast activity.11
Remodeling occurs through focal and discrete packets through the agency of a temporary
cellular unit called the “basic multicellular unit” (BMU) comprising of osteoblasts and
osteoclasts, central vascular capillary, nerve supply and related connective tissue.11 It is
estimated that in healthy human adults, 1 million BMUs operating at any given time and
over a course of a year 3-4 million BMUs are initiated to support the highly dynamic
function of bone tissue.11 The resorption stage of the bone remodeling sequence takes place
14
over the time frame of 10-14 days whereas formation of new bone at the site of resorption
takes up to 6 months.11 Delicate balance between resorption and bone formation that is the
hallmark of the remodeling process becomes disrupted in several pathological conditions12
including osteoporosis and breast cancer metastasis13 to bone resulting in loss of the
structural integrity of bone.
2.1.2 Cellular Origins of Bone Cells.
Osteoblasts and osteoclasts are derived from precursor cells within the bone
marrow.14 The bone marrow consists of cellular elements belonging to either the stromal
lineage or the hematopoietic lineage. The hematopoietic group is responsible for giving rise
to blood cells such as lymphocytes, erythrocytes, granulocytes, megakaryocytes and
monocytes.15 Osteoclasts are derived from hematopoietic cells of the
monocyte/macrophage lineage.15 Osteoblasts belong to the stromal group and are derived
from multipotent mesenchymal stem cells, which also give rise to bone marrow stromal cells,
chondrocytes, muscle cells and adipocytes.16 The complex sequence of steps involved in the
transformation of these early progenitor cells into functional osteoblasts and the various
factors that regulate the lineage development are only beginning to be understood.16 The
focus of this study was the functional expression of the osteoblast phenotype through the
sequence of proliferation, extracellular matrix formation, mineralization and finally
osteocytic transformation.17
2.1.3 Bone Formation
Bone consists of a mineral phase (70-90%) dispersed within an organic matrix
(osteoid) consisting primarily of Type I collagen. The rest of the organic matrix is composed
15
of non-collagenous proteins such as osteocalcin, osteonectin, bone sialoprotein, fibronectin,
vitronectin , thrombospondin as well as several proteoglycans, glycosoaminoglycans and
lipids (Table 1).18,19 The organic matrix of bone also sequesters several cytokines and growth
factors derived from bone cells and other cells that play important role in modulating growth
and differentiation of bone cells.20 These include growth factors such as Insulin-like growth
factor (IGF-I), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF),
Transforming Growth Factor (TGF-β), Bone Morphogenetic Proteins (BMP) and cytokines
such as IL-1 (Table 2).
Synthesis, deposition and mineralization of the organic matrix of bone is carried out
by osteoblasts.7 Bone formation involves proliferation of preosteoblasts and their
differentiation into functional osteoblasts capable of deposition, organization and progressive
mineralization of the organic matrix of bone.21 Stages of pre-osteoblast proliferation,
differentiation, extracellular matrix formation, mineralization and osteocytic transformation
advance through a tightly regulated spatial and temporal sequence.22
In the final stage of development osteoblasts can either undergo programmed cell
death (apoptosis) or transform into bone lining cells or become trapped within the bone
matrix as osteocytes.6 The proportion of osteoblasts that follow a particular fate is highly
variable and depends on several factors including age, species and anatomical location.6 In
the human cancellous bone it has been estimated that about 65% of the osteoblasts undergo
apoptosis23 with 30% transforming into osteocytes.24 In addition to pre-osteoblasts, active
osteoblasts, bone lining cells and osteocytes, a number of transitional stages have been
identified in the developmental program of the osteoblasts.25 These transitional stages
include preosteoblastic osteoblast, osteoblastic osteocyte (Type I pre-osteocyte), osteoid-
16
osteocyte (Type II pre-osteocyte), Type III pre-osteocyte, young osteocyte and old osteocyte.
Characteristic feature of this developmental program is that osteoblasts at different stages of
differentiation are part of a functional network of cells that communicate with each other
through gap junctions.26,27,28 As a result, bone tissue has been described as a functional
syncytium with cells ranging from osteoprogenitors to mature osteocytes connected in a vast
network,28 and coordinating responses to various mechanical and biochemical stimuli
through dynamic cell-cell contacts.
Various stages in the osteoblast differentiation are identified on the basis of
morphology, function, responsiveness to hormones such as parathyroid hormone and the
expression of a suite of molecular markers.29 Pre-osteoblasts are actively dividing cells that
express bone specific markers such as alkaline phosphatase and osteonectin.4 Osteoblasts are
characterized as non-dividing, cuboidal shaped cells, with large eccentric nucleus and one to
three nucleoli. Osteoblasts have extensive rough endoplasmic reticulum and golgi areas
consistent with their primarily secretory function.4 Osteoblasts can be distinguished from
pre-osteoblasts by the upregulation of number of bone markers including osteocalcin, bone
sialoprotein and type I collagen, alkaline phosphatase, vitamin D3 receptor and others.29 As
osteoblasts lay down the matrix, they become trapped within matrix and undergo
transformation to osteocytes.30,31 Nascent osteocytes radiate several cell processes32,33
towards the mineralizing matrix and have organelles very similar to those of osteoblasts. As
the osteocytes become embedded deeper in the matrix, changes in morphology and cell
organelle distribution occur.34 Mature osteocytes have a characteristic stellate shape, are
embedded within spaces called lacunae and extend long thin cellular processes that contact
other osteocytes and osteoblasts.32 Osteocytic transformation results in up to 70% decrease
in cell volume compared with original osteoblasts, and simplication of organelles with
17
decrease in golgi apparatus, decrease in the size of mitochondria and decrease in
endoplasmic reticulum.25,34 Osteocytes with an estimated half life of 25 years in humans
have a very long life span compared to the 3-4 month life span of the osteoblasts.33
Osteocytes die as a result of senescence, degeneration, and/or entrapment by osteoclasts.30
Several aspects of osteocyte function and lifecycle are yet to be defined because it is very
difficult to study osteocytes outside the body.30 Osteocytes are non-dividing, and they do not
maintain their differentiated state and function when isolated from the matrix that surrounds
them in their native state. As a result of this, several fundamental questions regarding
osteocytic transformation and the function of osteocytes are not yet fully understood.3,29,30 In
summary, the complex developmental program of osteoblasts associated functionally with
the process of bone formation ranges from pre-osteoblasts to osteocytes with a number of
transitional stages between these two stages and is modulated by a number of cellular and
molecular level interactions.33,35 In spite of significant progress in our understanding of the
developmental program of osteoblast and the various transitional stages there are still
considerable gaps in our knowledge. Filling these gaps requires experimental models that
can define the various stages not only in terms of morphology but also by the levels of
expression of specific molecular markers like alkaline phosphatase, osteocalcin, osteonectin,
parathyroid hormone related protein, as well as markers considered to be to specific to
osteocytes such as E11 and DMP1.
2.1.4 The Osteoblast Mileu
Growth, development, and function of osteoblasts is modulated by number of local
factors through both autocrine and paracrine mechanisms.20 The vast majority of these
factors are secreted by the osteoblasts themselves (Table 2).5 Mediators of osteoblast
response are also derived from other cells in the marrow environment and from the bone
18
matrix which has a high affinity for systemic and local factors.5 The complexity of the
osteoblast milieu is defined by cell-cell, cell-matrix interactions as well as the presence of a
number of regulators in the local environment of the osteoblasts that include growth factors
such as insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth
factor (FGF), transforming growth factor (TGF-β), bone morphogenetic proteins (BMP)
and cytokines such as IL-1. For extensive discussion of these regulatory factors see
References 17 and 20.
2.2 Osteogenesis In Vitro
Tissue culture techniques have contributed tremendously to our understanding of the
mechanisms of bone formation. Bone has been studied in vitro as an organ, tissue or as
isolated cells. This section traces the historical development of these techniques outlining
the advantages and disadvantages associated with each of these approaches.
2. 2. 1 Organ Cultures
Strangeways and Fell pioneered the use of organ cultures36 to study the embryonic
development of skeletal tissue. Early organ cultures involved isolation of explants from the
chick embryo at different stages of development into a semi-solid medium of clotted plasma
and embryo extract. Cultures were incubated in test tubes36, watch glasses37 or in hollowed
out glass slides.38 Watch glass cultures were further enclosed in a petridish with a layer of
moist cotton at the bottom to provide a humidified culture environment.37 In the first
successful adoption of the technique, Strangeways and Fell36 demonstrated the growth and
development of cartilage from the leg buds of a 3-day chick embryo over a 14 day culture
interval. These early experiments revealed the potential of the technique to study the
developmental processes of bone under controlled conditions. Organ culture technique was
19
further refined and developed by Fell to study the development of avian embryonic long
bones. In 1929, Fell published the first paper37 dealing with calcification of bone in vitro
using organ cultures of isolated femora and tibiae from 5 1/2 to 6 day chick embryo. In the
1930s Fell used this technique to establish the importance of alkaline phosphatase in
mineralization,37,39,40 to demonstrate the osteogenic capacity of the periosteum and
endosteum in vitro38 and to investigate the in vitro development of the avian knee joint.41
These early studies and Fell’s contributions in particular are highlighted here because they
represent a significant progress from static histological studies to the dynamic study of the
development of bone. Fell’s pioneering studies form the basis for our understanding of
different stages of cartilage and bone cell differentiation during the process of embryonic
bone development. Organ cultures have been used since then to describe the embryonic and
fetal development of the chick long bone42 and to study bone development in mammalian
models like fetal and rat long bones.43 Organ cultures have also been found to be particularly
suitable to evaluate the action of vitamins and hormones on bone44 and to carry out
resorption studies.43,45
2.2.2 Culture of Bone Slices and Explants
Culture of slices of bone derived from the long bone, calvaria or metatarsal tissue
provide another in vitro alternative to study bone.46-48 The most successful study employing
slices of bone tissue49 in vitro was carried out by Rose in 1960 using tissue culture chambers
made of cellophane.50-52 Rose was among the first to recognize the importance of retaining
cell-secreted factors in the cellular environment for maintaining the differentiated state of the
cells. Culture chambers employed by Rose separated cell growth and cell feeding functions
through use of a dialysis membrane and represented a significant innovation in technique
20
(see chapter 3 for extended discussion). Rose reported outgrowth of osteoblasts and
fibroblasts from embryonic chick bone slices in culture.49 Although calcification was not
demonstrated, explants were maintained for extended periods up to weeks.49,52 Utility of
these early studies in understanding the cellular basis of bone formation was limited by the
heterogeneity of the cell populations involved. In the absence of modern genomic and
proteomic tools it was difficult to identify and separate the responses of bone cells from other
cell types involved. Therefore, the primary contribution of these early studies using slices of
bone tissue in vitro is limited to short-term studies on the metabolism of bone and in
determining the effect of hormones on bone tissue.46-48
Explant cultures of the periosteum and endosteum have also been used to study bone
formation.38 Culture of periosteum was initiated by Fell who showed that explants of
periosteum from 6-10 day chick embryos could form osteoblast-like cells that produced an
osteoid. Culture of folded periostea, developed by Nijweide, is a particularily useful form of
the explant technique. 53 Nijweide and Vanderplas showed that in cultures of folded
periostea from 16-18 day old embryonic chick calvaria, osteoprogenitor cells at the center of
the fold differentiate into matrix-secreting osteoblasts.53 Folded periostea preserve critical
cell-cell contacts and the overall 3D structural organization of the tissue and serve as a
functional model for the study of osteogenesis in vitro. Culture of folded periostea from
embryonic chick calvariae has been used to study hormonal regulation,54,55 metabolic
effects55, the regulation of calcium transport53,55, to demonstrate osteoid formation53,56 and
mineralization in the presence of β-glycerophosphate57, and to study the effect of
dexamethasone on bone formation in vitro.58
21
2.2.3 Culture of Isolated Bone Cells
The real breakthrough in our understanding of the physiology of bone forming cells
came through the development of enzymatic methods of isolation of bone cells by Peck and
his colleagues59-61 in the 1960s. Peck61 employed buffered collagenase to isolate rat calvarial
osteoblasts and cultured them using monolayer cell culture techniques. Isolated cells were
shown to be viable in culture for days although matrix deposition or bone formation was not
noted.61 In subsequent studies, it was shown that osteoblastic cells synthesize collagen,
respond to ascorbic acid with enhanced collagen synthesis59 and react to hormones such as
parathyroid hormone.62 Isolation of osteoblasts in culture under these controlled conditions
made it possible for the first time to undertake a wide range of biochemical and metabolic
studies. It was possible, for example, to study calcium transport,63,64 oxygen consumption
and lactic acid production65 over short-term incubation periods.
Methods of enzymatic isolation developed by Peck and his co-workers and the
refinements of these methods66 provided the basis for much of subsequent work on
understanding the function of bone forming cells. Sequential extraction of bone cells using
proteases such as trypsin and collagenase67-71 resulted in isolation of subpopulations of
osteogenic mouse calvarial cells. A further advance in developing homogenous cell
populations was the dissociation and cloning of fetal rat and mouse calvarial cells72-79 in the
early 1980’s. Cloning allowed isolation of cell populations based on specific characteristics
such as growth patterns, responsiveness to parathyroid hormone or alkaline phosphatase
activity. Formation of discrete mineralized bone nodules in vitro was reported in cultures of
fetal rat calvarial cells in the presence of ascorbic acid and β-glycerophosphate.80 Ability of
cultured osteoblasts to form mineral deposits was found to be dependent on several factors
22
including initial cell density, length of culture interval and the presence of ascorbic acid and
organic phosphates like β-glycerophosphate.72-74,80-89 Isolated osteoblasts in culture were
shown to synthesize several proteins involved in bone formation including osteocalcin, type I
collagen, osteonectin and osteopontin.22,90 Identification of these proteins and enzymes and
their temporal expression during the bone development process was actively investigated.22,91
Studies using cultures fetal rat and chick calvarial osteoblasts identified the developmental
sequence of osteoblasts consisting of proliferation, extracellular matrix formation and
mineralization and co-related this sequence with the gene expression of specific proteins
involved in bone formation.22
2.3 In Vitro Models of Bone Formation: An Assessment
In summary, bone cells/tissue in culture isolated from different
sites (long bones, calvaria, mandibles or the iliac crest) and from different species (rat,
mouse, avian, human) using a variety of isolation techniques (enzymatic dissociation,
outgrowths from explants) have contributed to our understanding of bone formation (Table
3). These isolation and culture techniques continue to be in use today.92 Careful analysis of
these disparate studies reveal important issues that are critical to understanding past efforts at
culturing bone cells and devising better culture models for the study of bone formation.
Some of these issues are discussed in the next few sections.
2.3.1 Homogeneity versus heterogeneity
A convincing argument can be made that the overwhelming trend in the
historical development of bone culture models is a reductionist shift from the heterogeneity
and complexity of tissue/organ/explant cultures to the simplicity and homogeneity of
cultures of isolated bone cells. Shift to the study of homogenous cell populations was driven
23
by a need to isolate the cellular level responses of specific population of bone cells from
various other influences (other cell types, biochemical factors) in the bone
microenvironment. However a purely reductionist approach brings into question the
physiological relevance of culturing isolated bone cells deprived of critical cell-cell and cell-
ECM interactions. Choice between organ cultures and cultures of isolated bone cells is
ultimately an experimental design issue and has to be resolved by the needs of the problem
being investigated. Traditionally organ cultures have been used for studying the systemic
effects of various hormones, drugs or other agents on bone and while cultures of isolated
bone cells have been used investigate cellular and molecular level interactions involved in the
formation of bone. Culture of tissue explants have been the bridge between the length scales
of cell and organ.
Another way of resolving the issue of length scale is to define a functional unit of structure
with the necessary complexity to simulate the physiological process of bone formation.
Osteoblasts have been known to lose their differentiated state when cultured as a monolayer
and reassume their differentiated state when cultured as multiple layers. There is also
extensive evidence in the literature that multiple-layered osteoblast tissue is required to
demonstrate mineralization in vitro.93-95 Tissue surrogates grown from isolated cells can
integrate the requirements of 3D structure,93,94 increased cell-cell contact and cell-ECM
interactions in the developmental processes of the bone cell and in the formation of bone in
vitro. Developing 3D tissue from isolated cells in a controlled manner has the potential to
realize functional biological structures that can bridge the gap between culture of isolated
bone cells and organ cultures. Further, tissue engineering methods differ fundamentally
from cultures of explant tissues because they allow us to build complexity in a controlled
manner using relatively homogenous cells as the starting materials. Aspects of cell
24
proliferation and stages of differentiation can be controlled by providing suitable
environmental cues through use of scaffolds, specially designed culture vessels called
bioreactors96 and by controlling the presence of various biochemical factors in the cellular
environment. Numerous studies in recent years have adopted this approach95,97-104 to develop
3D bone tissue from isolated bone cells seeded on to scaffolds and cultured in vitro in dishes,
spinner flasks, perfused catridges or rotating vessels.95,100,105-108
2.3.2 Length of Culture Interval
The length of culture interval is a critical variable in studies of bone
formation in vitro.109,110 Demonstration of osteogenesis in vitro requires continuous culture
intervals of weeks to months because the underlying physiological processes of bone cell
proliferation, extracellular matrix formation and mineralization takes place over extended
periods up to months. Quick survey of the historical development of in vitro bone cultures
shows that the most successful studies of osteogenesis involve extended culture intervals.
The classic example is the study by Binderman and his colleagues80 where they
demonstrated for the first time mineralization in cultures of isolated bone cells after 8 weeks
of continuous culture. In vitro cultures of bone cells must not only be viable over these
extended culture intervals109 but they must simulate the different stages in the development of
bone forming cells in a physiologically relevant manner. Recent studies have also shown
that the formation of bone in vitro is dependent on the length of culture interval.109
2.3.3 Effect of the Microenvironment.
Biochemical Factors: Growth and function of bone cells is regulated by the presence of a
number of local factors in their microenvironment.20,111-113 Osteoblasts secrete a number of
regulatory factors that in turn control the growth and differentiation of these cells through
25
autocrine and paracrine mechanisms.111,114 Importance of retaining these biochemical factors
in the cellular microenvironment was first articulated by Rose in the 1960s49,52 and has been
elegantly demonstrated by Tenenbaum and Heersche using cultures of folded chick
periosteaum.53,57 Success of periosteal osteogenesis model has to do largely with the fact
that folding the tissue not only increases cell-cell contact but also provides a
microenvironment in which the cell-secreted products are sequestered. To demonstrate this
effect, periostea were cultured without folding under two different configurations. In the first
configuration cell-secreted products were allowed to accumulate in the immediate
environment of the tissue. In the alternate configuration cell-secreted factors could diffuse
away from the tissue leading to reduced local concentrations. Under these experimental
conditions it was found that osteo-differentiation took place only in those situations where
the cell-secreted products were trapped within the local cellular environment.53,57
Refinements of the experimental setup employing Diaflo membranes with different
molecular weight cut-offs showed that allowing large molecules (100-300kD) to accumulate
in the local cellular environment was critical for inducing differentiation of the
osteoprogenitor cells. Recognition of these ideas has to the led to the discovery of several
factors in the bone microenvironment that play a critical role in the development of the bone
cell.113 Soluble factors that influence bone formation include bone morphogenetic proteins
(BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet-derived
growth factor (PDGF) and Interleukin-1 (IL-1). Complete description of the complex role
these factors play in the cellular physiology and development of bone is beyond the scope of
this work. However, it is critical to incorporate the effect of local regulators of bone
formation in the development of in vitro models of osteogenesis. Recent studies using 3D cell
culture models have confirmed the importance of the mass transfer of biochemical factors in
the development of bone tissue.115
26
Influence of Medium/Substrate: Early in vitro studies of bone tissue were carried out in the
semi-solid medium of plasma clot/embryo extracts and with air as the gas phase.
Refinement of explant culture techniques have resulted in culture of explants on a floating
raft/grid/gel at the interface between liquid medium and air to ensure sufficient gas
exchange.116,117 Development of techniques for the culture of isolated bone cells resulted in
widespread adoption of fluid medium consisting primarily of buffered salt solution with
addition of various nutrients and vitamins.117-119 The conventional practice is to culture
monolayer of cells in polystyrene dishes/flasks completely surrounded by a fluid medium118
containing nutrients and to incubate these dishes in 5% CO2 in air.
Higher CO2 tensions used in conventional culture have been shown to stimulate
bone cell proliferation and bone formation and calcification in organ cultures.120 In studies
examining the role of oxygen tension, lower O2 tension has been shown to stimulate bone
matrix formation and calcification in some culture systems.117,120-122 Studies of oxygen
consumption are diverse in terms of cells/tissues used and therefore it is difficult to
extrapolate the results of these studies to arrive at a specific oxygen requirement for bone
cells. Ensuring optimal delivery of this critical nutrient is critical to the development of
in vitro bone cell culture models. Oxygen tensions have to be tailored to the thickness of the
cell/tissue layer and the specific metabolic requirements of the cells being cultured.
Conventional polystyrene flasks are relatively impermeable to CO2 and O2 and therefore
oxygen supply is mostly through the dissolved oxygen supplied through the medium.
Oxygen diffusivity in aqueous solutions is rather low.123,124 Depending on the rate of oxygen
consumption, cells in conventional culture may be placed in anoxic conditions or hyperoxic
conditions. Therefore, the level of medium in the culture dishes (between 2-5mm) has to be
27
carefully controlled to provide sufficient oxygen tensions for the metabolic requirements of
the cells. Local oxygen concentrations regulate cell behavior in many different ways and
therefore it is important to consider not just the bulk oxygen concentration but also the
effects of oxygen gradients on cellular function.125 In summary, supply of oxygen in optimal
pericellular concentrations is a critical design consideration for developing better in vitro
models of bone formation.
Mechanical Stress: Growth and development of bone is conditioned by mechanical loads
imposed on the skeleton. Number of model systems have been developed to study the effect
of stretching and pressure forces on bone and organ cultures.126-132 More recently fluid shear
stresses133 have been shown to have an important role in regulating the development of bone
tissue in 3D cell culture models. Although, mechanical conditioning of in vitro bone
cells/tissue is an important area of research, it is not being addressed directly through this
study.
Review of the literature on bone cell physiology and historical development of the
in vitro methods used to study the function and development of bone cells, revealed
fundamental requirements for the design of culture models that can bridge the gap between
multiple length scales and demonstrate osteogenesis in vitro. These requirements include 3D
structural organization, optimal mass transport of nutrients and oxygen tension, stability of
the culture environment achieved through retention of cell-secreted biochemical factors and
the ability to grow/maintain tissue over extended time periods. Design and implementation
of a compartmentalized bioreactor that meets these requirements is described in the next
chapter.
28
2.4 3D models of Bone Formation
Identification of improved methods of hard tissue repair, augmentation or
replacements represents a major clinical and socioeconomic need.12,134-136 The field of bone
tissue engineering has emerged in recent years to meet this need through the development of
engineered bone tissue from a combination of cells, scaffolds and growth factors. Rapid
growth in the field has led to the development of several 3D models of bone formation
employing the basic strategy of culturing cell-scaffold constructs under controlled conditions
in devices called bioreactors.95,100,137 Bone tissue engineering approach has received extensive
attention in recent literature. Examples of primary studies,95,100,138,139 reviews,97,98,101,104 and
even books devoted to the subject 140,141 indicate the extent of interest in the field.
Central to the development of engineered bone tissue are the design and
implementation of bioreactors that provide a controlled physical environment and suitable
microenvironmental cues to direct the assembly of isolated bone cells into 3D tissue.142
Numerous studies in recent years have investigated the use of bioreactors to engineer bone
tissue to repair or replace bone lost due to disease or injury.142 Many different bioreactor
designs such as spinner flasks105,143, rotating wall vessels144, perfusion reactors106,145 and
rotating vessels139,146 have evolved for this purpose in recent times (Refer to Table 4 for a few
examples drawn from many refs. 147-150) , each with unique attributes and drawbacks.
Common design considerations underlying these different approaches to bioreactor
development are discussed below -
2.4.1 Cell-scaffold Interactions: The surface of the scaffold is designed to promote cell-
attachment through careful choice of scaffold material and biomaterial surface engineering.
The internal structure of the 3D scaffold is designed to promote efficient cell seeding, to
29
provide a suitable microenvironment for the cells, to allow for transfer of fluids and to
provide a template for the development of 3D tissue. Scaffolds made of biodegradable
polymers are designed to degrade and to be ultimately replaced by the developing tissue.100
2.4.2 Mass Transport of Nutrients and Oxygen: Critical limiting factors in the growth and
maintenance of 3D tissue are the delivery of soluble nutrients and oxygen to the cells while
simultaneously removing metabolic waste products. Design of bioreactors has to take into
account concentration gradients in nutrients and oxygen concentrations that may develop
through the thickness of the tissue and the tissue/scaffold constructs. Without adequate
nutrient supply, the scaffolds may either develop a necrotic core or diminished cell survival
and function at the center of the scaffold compared to cell/tissue layer at the surface.
Bioreactor designs take different approaches to address this fundamental issue. Spinner
flasks provide increased mass transport through continuous mixing of the medium.
However, the turbulence generated in spinner flasks can be detrimental to tissue
development. Mass transport through the tissue construct could be increased by continuous
rotation of the culture vessel itself or by continuous direct perfusion of medium through the
cell/scaffold constructs. Rotating wall vessels employ dynamic laminar flow under
conditions of low shear stress for efficient mass transport.
2.4.3 Mechanical conditioning: Bioreactors have also been designed with specific
emphasis on mechanical conditioning to the 3D bone tissue constructs.151 Mechanical
conditioning can be provided through direct application of stress or through application of
fluid shear stress.133,152-154
30
2.4.4 Mass transport of biochemical factors: A design feature that has received far less
attention in the development of bioreactors is the mass transport of biochemical factors.
Source of biochemical factors could be either endogenous (cells) or exogenous (added to the
bioreactor environment). It is well recognized that cell-secreted growth factors and cytokines
play important roles in modulating cellular function through both autocrine and paracrine
mechanisms. Retaining these factors within the cellular microenvironment could be critical
for the growth/differentiation and maturation of the 3D bone tissue. (For complete
discussion see section 2.3.3 in this chapter). Continuous or scheduled perfusion of medium
typically employed in bioreactors can result in removal of cell-secreted regulatory factors,
perturbation of the cellular environment and instability in cultures. There is experimental
evidence to suggest that increased flow rates through 3D bone constructs lead to diminished
cell survival and function that could be a result of the loss of cell-secreted regulatory
molecules.155
As a consequence of the above factors, we have focused our attention on the
design and implementation of a compartmentalized bioreactor based on the principle of
continuous-growth and dialysis. The compartmentalized bioreactor was a significant
departure from existing designs because of its emphasis on stability of the pericellular
environment (Table 5). Extraordinarily stable culture environments were achieved in the
bioreactor through the separation of cell-growth and cell- feeding functions by a dialysis
membrane. Carefully selected molecular weight cut-off resulted in continuous supply of
nutrients and removal of low molecular weight waste products while retaining cell-secreted
biochemical factors. Whole device was enclosed by gas permeable films that provide
sufficient oxygen tension to meet the metabolic requirements of the cells. Continuous
dialysis resulted in controlled delivery of nutrients and removal of waste products. As a
31
result of the compartmentalized design, it was not necessary to disturb the medium in the
cell chamber through the course of the month’s long experiment. At the same time, nutrients
from the medium reservoir gradually dialyzed into the cell chamber while low-molecular
weight nutrient were continuously removed from the cell chamber. Hence a decided
advantage to the simultaneous-growth-and-dialysis critical to this study is that the
microenvironment remained substantially constant. Design features that further distinguish
the bioreactor include scaffold-free design that permitted direct observation of the growth of
3D bone- like tissue and its interaction with metastatic cancer cells through in situ confocal
microscopy. Scaffold free design also permitted routine examination of tissue through
phase-contrast microscopy and relatively easy isolation of tissue (avoiding the complexity of
separating tissue from a 3D scaffold) at different stages of development for various analyses.
The surface area of the films was designed to be equivalent to standard T-25 tissue culture
dishes (25 cm2) to enable direct comparison culture conditions in the bioreactor and standard
2D cell culture. The bioreactor was designed, in principle, to accommodate application of
mechanical stress on the cells. This design feature, however, was not evaluated in this
particular study.
The implementation of the compartmentalized bioreactor for extended term culture of bone
cells is discussed in the next chapter.
32
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List of Table Legends
Table 1: Principal extracellular matrix proteins secreted by the osteoblasts and their
sequence of expression.
Table 2: Factors in the bone microenvironment secreted that condition the growth and
differentiation of the osteoblasts.
Table 3: Historical time line of the development of in vitro models of bone formation.
Table 4: Representative bioreactors for bone tissue engineering applications.
Table 5: Unique design features of the compartmentalized bioreactor
55
Principal Extracellular Matrix Proteins
MW(kDa)
Characteristics Sequence of Expression
Collagen type I ~320 Principal structural component of bone extracellular matrix.
Expressed early, during the proliferation period. Gene expression gradually downregulated with mRNA maintained at low ostoecalcin basal level at subsequent stages of differentiation.
Osteopontin 60 Acidic glycoprotein.Arg-Gly-Asp containing sequence mediates cell attachment. Binds to Hydroxyapatite.
Expressed early and late in the osteoblastdevelopment sequence.
Osteocalcin 5.7 Binds to Hydroxyapatite.Vitamin K dependentCharacterized by three γ-carboxyglutamic acid residues(Gla). Marker for mature osteoblasts. Possible role in coupling of bone formation to resorption.
Expressed post-proliferatively with onset of nodule formation.
Maximal expression with mineralizaton.
Expression highly correlated to calcification of the extracellular matrix.
Phosphorylatedglycoprotein, binds calcium, cell attachment.
Osteonectin 33 Associated with mineralization.
Table1: Principal extracellular matrix proteins secreted by the osteoblasts and their sequence of expression. Adapted from Reference (22)
56
Table 2: Factors in the bone microenvironment secreted that condition the growth and differentiation of the osteoblasts.
Protein MW(kDa) IGF-I 7.5 IGF-II 7.5 aFGF ~16 bFGF ~16
IL-1
Growth factors and cytokines secreted by osteoblasts
TGF-β 25 BMP 18.5 OIF 22-28
PDGF 30 EGF ~6.4
BDGF 11
Factors derived from the Bone and other sources
Prostaglandins <1 IGF- Insulin Derived Growth Factor aFGF- Acidic Fibroblast Derived Growth Factor bFGF-Basic Fibroblast Derived Growth Factor BMP- Bone Morphogenic Protein OIF-Osteoinductive Factor PDGF- Platelet Derived Growth Factor IL-Interleukin EGF-Epidermal Growth Factor BDGF-Blood Derived Growth Factor * Cytokines are polypeptides (proteins) that regulate many cell functions. Cytokines that act on the same cell that produced them are called autocrine factors; those that act on other cells are called paracrine factors; those that act systemically (through the vascular system) are referred to as endocrine factors. Molecules that regulate cell division are referred to as growth factors.
57
Table 3: Historical time line of the development of in vitro models of bone formation.
Organ Cultures Fell and Robison (1929)
Cultivation of isolated femora and tibiae from 51/2 to 6 day chick embryos using watch glass technique.
First successful demonstration of calcification of bone in vitro
[39]
Explant Cultures Fell (1932)
Culture of periosteum and endosteum from embryonic chick calvaria using hanging drop method.
Demonstration of the osteogenic capacity of the periosteum and endosteum in vitro.
[40]
Rose (1960)
Culture of bone explants from the long bone of embryonic chick in culture chambers made of cellophane
Weeks long culture of explants in specially designed culture chambers
[51]
Nijweide (1975) Culture of folded periostea from embryonic chick calvaria
Study of osteoid formation , calcium transport and effects of hormones
[55]
Tenenbaum and Heersche (1982)
Culture of folded periostea from embryonic chick calvaria
Osteoid deposition and mineralization in the presence of β-glycerophosphate
[58]
Culture of Isolated Osteoblasts Peck et al. (1964) Isolation of osteoblasts in
culture from rat calvariae using buffered collagenase.
Landmark study that pioneered isolation of osteoblasts using enzymatic techniques.
[63]
Smith et al. (1973) Isolation of four different types of cells by mechanical separation from rat calvaria
Studies on the short term (hours) metabolism of isolated bone cells.
[67]
Binderman et al (1974)
Isolation of osteoblasts in culture from rat calvariae using trypsin and EDTA
Weeks long culture of osteoblasts showing deposition and mineralization of extracellular matrix.
[82]
Wong and Cohn (1974) Rao et al. (1977) Leuben et al . (1977)
Isolation of osteoblasts using sequential enzymatic treatment
Isolation of sub-populations of osteogenic cells
[69] [71] [73]
Kadis et al. (1980) Aubin et al. (1982) Ecarot-Charier et al. (1983) Sudo et al (1983) Bellows et al (1986)
Development of clonal populations from fetal rat and mouse calvarial cells
Increased homogeneity in cell populations
[78] [81] [84] [75] [74]
Robey and Termine (1985)
Establishment of human bone cells in vitro
[88]
Nefussi et al (1985) Bellows et al(1986)
Formation of discrete mineralized nodules in the presence of ascorbic acid and organic phosphate.
Mineralized nodule formation in culture
[87] [74]
Lian and Stein (1990)
Sequential progression of osteoblast differentiation using fetal rat and chick calvarial oteoblasts
Osteoblast development sequence
[22]
58
Table 4: Representative bioreactors for bone tissue engineering applications.
Types of Bioreactors
Principle of Operation Scaffolds Cells Ref.
Rotating-wall vessels (RWV)
RWV consists of two concentric cylinders- a stationary gas-permeable inner cylinder and a rotating outer cylinder. Scaffolds are placed in the annual space immersed in culture medium. Carefully selected rotational rates balance gravitational and centrifugal forces acting on the scaffolds resulting in microgravity-like conditions.
Microcarrier beads made of dextran based polymers.
Rat osteosarcoma cell line ROS 17/2.8 cells
[144]
Perfusion Culture Reactors
Continuous perfusion of medium through the scaffolds using a peristaltic pump.
Porous ceramic materials composed of β-tricalcium phosphate
Osteoblasts derived from the bone marrow of 7 week-old Fischer 344 male rats
[106]
Rotating Vessels
50 ml polypropylene centrifuge tube with a 30x40mm silicone window for gas exchange placed on roller culture apparatus rotating at 6 RPM
3D poly(DL-lactide-co-glycolide) PLGA foams
Osteoblastic cells from ther marrow of 3-7 days old neonatal Lewis rats
[146]
Spinner Flasks
Scaffolds seeded with cells are attached to needles hanging from the cover of the flask. Magnetic stir bar at the bottom of the flask is used to effect mixing of the medium
3D poly(DL-lactide-co-glycolide)PLGA foams
Rat marrow stromal cells
[143]
59
Table 5: Unique Design Features of the Compartmentalized Bioreactor Requirement Design Implementations Mass Transport of Nutrients Continuous- growth and dialysis Mass Transport of Biochemical Factors Limited to the cell growth compartment by the
dialysis membrane Flow of Medium Static Delivery of Oxygen Gas-permeable films made of a co-polymer of
ethylene methacrylate designed to provide optimal oxygen tensions.
Cell-Scaffold Interactions Scaffold free design. Cell-substrate function is performed by the dialysis membrane.
Microscopy Optically clear gas-permeable films enable observation of tissue through phase contrast or confocal microscopy.
60
Chapter 3
Extended Term Culture of Bone Cells in a Compartmentalized Bioreactor
Abstract
A specialized bioreactor is used to grow mineralizing, collagenous tissue up to
150 mμ thick from an inoculum of isolated murine (mouse calvaria MC3T3-E1,
ATCC CRL-2593) or human (hFOB 1.19 ATCC CRL-11372) osteoblasts over
uninterrupted culture periods longer than 120 d (4 months ). Proliferation and
phenotypic progression of an osteogenic-cell monolayer into a tissue comprised
of 6≥ cell layers of mature osteoblasts in the bioreactor was compared to cell
performance in conventional tissue-culture polystyrene (TCPS) controls. Cells in
the bioreactor basically matched results obtained in TCPS over a 15 d culture
interval, but loss of insoluble ECM (iECM) and ~2X increase in apoptosis rates in
TCPS after 30 d indicated progressive instability of cultures maintained in TCPS
with periodic refeeding but without subculture. By contrast, stable cultures were
maintained in the bioreactor for more that 120 d, suggesting that extended-term
tissue maintenance is feasible with little-or-no special technique. TEM
ultramorphology of tissue derived from hFOB 1.19 cells recovered from the
bioreactor after only 15 d of culture showed evidence of osteocytic-like processes
61
and gap junctions between cells like that observed in vivo, over-and-above
elaboration of the usual osteoblastic markers such as alkaline phosphatase
activity and mineralization (alizarin-red). Thus, the bioreactor design based on
the principle of simultaneous-growth-and-dialysis was shown to create an
extraordinarily-stable pericellular environment that better simulates the in vivo
condition than conventional tissue-culture. The bioreactor shows promise as a
tool for the in vitro study of osteogenesis and osteopathology.
62
1. Introduction
Cells extracted from the native, in vivo physiological state and placed into
a culture system undergo adaptive responses often referred to as “culture shock”
(1). The impact and duration of culture shock strongly depends on the stability
of the pericellular (micro) environment, the extent to which this
microenvironment simulates the in vivo condition, and the ability of cells to
actively interact/transform the pericellular milieu by secreting a variety of
macromolecules found in extracellular matrix (ECM) (2, 3). Indeed, culture
shock and the accumulated damage cells sustain in culture has long been
thought to limit cell viability in vitro (4, 5). Thus, one of the bioengineering
objectives for any in vitro culture device is to create a stable pericellular
environment simulating the in vivo condition in a manner that mitigates, rather
than amplifies, the impact of culture shock. The conventional tissue-culture
approach is to surround cells held in dishes or flasks with a buffered medium
containing various nutrients (amino acids, glucose, serum proteins, vitamins,
etc.). As cells grow, nutrients are depleted, waste products accumulate
(especially lactic acid), and pericellular pH decreases to unacceptable levels. The
typical solution to this problem is to exchange spent growth medium with fresh,
either on a continuous basis (perfusion) or as a matter of scheduled maintenance.
A decided disadvantage with either of these approaches is that the
63
aforementioned secreted macromolecules are removed along with waste
products. In the case when periodic media exchanges are employed, the
resulting oscillation in both pH and nutrient concentrations imparts an instability
in cultures that can lead to excessive cell vacuolization, ruffled cell margins, and
increasing rates of cell-surface detachment. Thus, conventional tissue-culture
strategies, especially the flask-and-dish type serviced with periodic media
exchanges, fail to maintain a stable pericellular environment critical to recovery
from culture shock. As applied specifically to the culture of bone cells in vitro,
continuous removal or periodic exchange of growth medium also means that
cell/protein-mediated dissolution-precipitation reactions involved in
mineralization-resorption are likewise perturbed. Thus it is difficult to simulate
the natural developmental sequence of bone cells that includes proliferation,
post-mitotic expression of a differentiated phenotype, extracellular matrix
formation, and mineralization. As an example relevant to this work,
transformation of mature osteoblasts into an osteocytic phenotype in vitro has not
been reported to our knowledge.
More than a decade before widespread use of conventional tissue culture
in biotechnology, G. G. Rose pioneered the “simultaneous-growth-and-dialysis”
method (6) by culturing cells beneath a cellulosic membrane (commercial
cellulose wrapping film in Rose’s implementation). The core idea was to
64
continuously feed cells with low-molecular-weight metabolites by dialysis
through a cellulose membrane that also retained secreted high-molecular-weight
macromolecules within the growth space created by the bounding membrane.
Metabolic waste products such as lactic acid dialyzed out of the pericellular
space and into the basal medium where waste could be removed without
perturbing cells by wholesale growth-medium removal. Rose’s idea languished
in the literature until its rediscovery more than twenty years later when
simultaneous-growth-and-dialysis was bioengineered into a routinely-usable, so-
called compartmentalized culture device (7, 8), one version of which is shown
Fig. 1. This bioreactor was shown to facilitate a number of advantages in the
culture/maintenance of soft-tissue and hybridoma cells, perhaps most notably of
which was the ability to sustain cells for more than 30 d without user
intervention and the accumulation of biosynthesized macromolecules (such as
IgG) within the growth space for periodic harvest (8). Unpublished work
(Vogler) suggests advantages in the culture of recalcitrant primary cells and in
vitro immunization as well.
Our interest in the development of improved orthopedic biomaterials and
scaffolds for orthopedic tissue engineering (9-11) has led the need for in vitro test
vehicles that permit extended-term (>30 d) contact of osteoblasts, osteoclasts, and
co-cultures thereof with candidate biomaterials. Finding many limitations with
65
conventional culture, not the least of which was loss of culture integrity over
long culture intervals, we were motivated to implement the compartmentalized
bioreactor for bone-cell culture. Herein we report phenotypic development of
two continuous osteoblast cell lines from an osteogenic-cell monolayer to a
mineralizing, collagenous tissue; mouse calvaria MC3T3-E1 (ATCC CRL-2593)
and human fetal osteoblasts (hFOB 1.19, ATCC CRL-11372). TEM
ultramorphology of hFOB 1.19-derived tissue recovered from the bioreactor after
15 d shows evidence of osteocytic-like processes and regions of intercellular
contacts between cells, suggesting that osteoblasts undergo complex phenotypic
development within the bioreactor like that observed in vivo when osteoblasts
are engulfed in a mineralizing tissue. Thus, the compartmentalized bioreactor
shows promise as a tool for the in vitro study of osteogenesis and osteopathology.
2. Methods and Materials
Bioreactor Design and Implementation: The compartmentalized
bioreactor (Fig.1, Panel a) separates a cell-growth space (A, 5 mL) from a larger-
volume medium reservoir (B, 30 mL) with a dialysis membrane (C). Cells were
cultured on a transparent, liquid-impermeable film (E) selected for
cytocompatibility (12-14) and gas (O2 and CO2) permeability (see further below).
During culture, cells were continuously bathed in pH-equilibrated and
oxygenated medium dialyzing from the reservoir (B). At the same time,
66
metabolic waste products such as lactic acid dialyzed out of the growth
compartment (A), maintaining low-pericellular concentrations. Serum
constituents or macromolecules synthesized by cells with molecular weights in
excess of the dialysis-membrane cutoff (6-8 kDa in our implementation) were
retained and concentrated within the growth compartment. The entire vessel was
ventilated through transparent gas-permeable films bounding cell growth and
reservoir compartments (D, E). The assembled bioreactor had a 25 cm2 cell–
growth area. The medium reservoir volume was designed for medium-
replenishment intervals ranging from 15-45 d, depending on the metabolic
activity of the cells. Access to the cell-growth space and medium reservoir was
through luer taper ports (J, K; Panel c Fig. 1) using standard pipettes. The
bioreactor was designed to work with most standard inverted phase-contrast
microscopes with minor stage modification and allowed adequate optical
microscopy throughout the culture interval; although resolution at the cellular
level naturally was compromised by the development of a thick, collagenous
multi-cellular tissue. The bioreactor was machined from 316L stainless-steel
stock (8). The body of the compartmentalized device consisted of four main rings
(F, G H, I; Panel c). Two chambers (A, B) were created with three films (C, D, E)
sandwiched between inner rings (F, G). The whole device was held together in a
liquid tight fashion using 6 stainless steel screws, as can be seen in the laboratory
implementation of Panel b. Access to, and venting of, growth and reservoir
67
chambers was through Luer-taper ports (J, K). The gas permeable films (G, H)
forming the outer barriers for chambers (A, B) were ~3 mil thick and made by
hot pressing Surlyn 1702 resin (DuPont, Wilmington, DE) by simultaneous
application of heat (220 oC) and pressure (245 Pa) in a laboratory hot press
(Model 2699, Fred S. Carver Inc.). The internal film barrier was cellulosic-
dialysis membrane (Spectrapor-13266, Spectrum Medical Industries) and was
hydrated in deionized water for at least 1 hr before assembly of the bioreactor.
Fabricated bioreactors were filled with 0.1% sodium azide prepared in phosphate
buffer saline, packaged in plastic bags, and sterilized using 10 Mrad γ-ray
irradiation at the Breazeale Nuclear Reactor on the campus of the Pennsylvania
State University. Sterile-packed bioreactors were opened within the confines of a
laminar-flow biosafety cabinet, drained of azide storage solution, and 3X rinsed
with basal medium using conventional aseptic technique just before cell
inoculation as described below.
Cells and Cell Culture: Murine calvaria pre-osteoblasts (MC3T3-E1,
ATCC CRL-2593) and human fetal osteoblastic cells (hFOB 1.19, ATCC CRL-
11372) were obtained form American Type Culture Collection (ATCC). MC3T3-
E1 were cultured in alpha minimum-essential medium (α-MEM) supplemented
with 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin.
Post-confluent MC3T3-E1 were cultured in a differentiation medium additionally
68
containing 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate. hFOB 1.19
were cultured using Dulbecco’s modified Eagle medium-Ham’s F-12 (1:1) basal
media supplemented with 10% charcoal-stripped fetal bovine serum, and 1%
penicillin-streptomycin. Post-confluent hFOB 1.19 were cultured in a more
complex differentiation medium additionally containing 50 µg/mL ascorbic
acid, 10-8M 1,25-dihydroxy vitamin D3 and 10-8M menadione. The hFOB 1.19 cell
line is conditionally immortalized by transfection with a gene encoding for the
temperature-sensitive mutant (tsA58) of the SV40 large T antigen. Transfection
confers a 33.5 oC continuous proliferation “permissive” temperature and 39 oC
quiescent temperature at which cells stop proliferating without undergoing
apoptosis (15). We cultured hFOB 1.19 at 37 oC and obtained a doubling time
similar to that observed at the permissive temperature (16).
Culture experiments were initiated in rinsed bioreactors (see above) by
filling the growth compartment with approximately 5 mL of serum-containing
medium containing a suspension of approximately 2X104 cells/mL (sub-
confluent cell density). The reservoir was filled with serum-free basal medium
containing no proteins. Cells from the same inoculum were plated in 25 cm2
standard tissue-culture-grade polystyrene (TCPS) dishes (Corning Life Science)
which served as the controls. Cultures were maintained in a water-jacketed 5%
CO2 incubator (Model 3110, ThermoForma) held at 37 oC. After cells had
69
reached confluence (3-5 d), growth medium was replaced with differentiation
medium and cells were permitted to grow in control plates and in the bioreactor
without subculture. Medium was replaced in TCPS controls every 3-5 d as
dictated by color of the methyl red indicator, with a hint of yellow (acid
indicator) leading to full exchange. Basal media within the bioreactor reservoir
(but not growth space) was refreshed every 15-30 d, again depending on the
perceived color of the pH indicator, with a hint of yellow leading to full
exchange of only the reservoir contents. For example, 120 d MC3T3-E1 cultures
in the bioreactor were sustained with four replacements of basal media within
the reservoir every 30 d.
Cell Attachment and Proliferation Rates: Short-term ( 0 3t< < hr) cell
attachment assays were performed as described previously (16, 17). Briefly, cells
were plated onto 15 identically-prepared plates at 2X104 cells/cm2 in whole
medium. TCPS culture dishes were the comparison controls. Dishes with the
substratum used in the bioreactor were prepared by adhering Surlyn film into
the bottom of TCPS culture dishes using double-sided adhesive tape. Cells were
allowed to adhere from the sessile medium while incubated at 37 oC in a CO2
incubator. At various time intervals, a single plate was selected for destructive
analysis (every 5 min from 0 30t< < min, every 10 min from 30 60t< < min,
every 15 min for 60 120t< < min, and every 30 min for 120 180t< < min) by
70
discarding suspension medium and rinsing substrata 3X with PBS. Attached
cells were released with trypsin and counted by hemacytometry. Surfaces were
analyzed in triplicate. Proliferation assays were performed in the same basic
way, except that after 3h attachment time, substrata were rinsed with PBS to
remove non-adherent cells and refreshed with growth medium. Remaining cells
were allowed to proliferate for 6, 12, 24, 48, and 72 h before destructive analysis.
Long term ( 15≥ d) proliferation was monitored by SYBR green nuclear-staining.
Duplicate TCPS and substrata removed from the bioreactor were fixed with 4%
paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, stained
with 0.02% SYBR Green in PBS (Molecular Probes), and visualized using an
Olympus BX-60 epi-fluorescent microscope. Average nuclei number was
determined by counting 15 representative spots on each substrate using image
analysis (ImagePro Software, MediaCybernetics Inc).
Alkaline phosphatase activity: Alkaline phosphatase (ALP) activity was
quantified at the end of each culture period using a chromogenic assay involving
conversion of p-nitrophenyl phosphate to p-nitrophenol as described in (18).
Briefly, cells washed in PBS were lysed by rinsing with PBS and 0.1% (w/v)
Triton X-100 (Sigma) in PBS. Lysates were subjected to two freeze-thaw cycles,
after which ALP reaction buffer (1:1 mixture of 0.75 M 2-amino-2-methyl-1-
propanol and 2 mg/mL p-nitrophenol phosphate) was added and incubated at
71
37°C for 15 min. This reaction mixture was stopped with 0.05 N NaOH, and the
absorption was measured at 410 nm with a Beckman Spectrophotometer (DU-70
series). A calibration curve was prepared by serial dilution of p-nitrophenol
standard solution. A portion of the reaction mixture was used to measure total
protein concentration using Bio-Rad protein assay kit. ALP activity was
normalized to total cell protein. Experiments were conducted in triplicate for
duplicate cultures. Results are reported as mean±standard deviation (1σ ).
Apoptosis: Cell apoptosis was analyzed using the enzyme terminal
deoxynucleotidyl transferase assay (TUNEL assay, Promega) to catalytically
incorporate fluorescein-12-dUTP at 3’-OH DNA ends, so that apoptotic cells
fluoresce green. All cells were stained red with SYTOX® Orange (Molecular
Probes) so that apoptotic cells could differentiated from normal by fluorescent
microscopy. At the end of each growth period, duplicate cultures were rinsed
twice with PBS, fixed with 4% paraformaldehyde, and permeablized with 0.2%
Triton X-100. Cells were then equilibrated in buffer for 5-10 min at room
temperature, followed by incubation with TdT buffer (that included equilibration
buffer, nucleotide mix, and TdT enzyme) in a humidified chamber for 60 min at
37oC. The reaction was terminated using 2X “SSC washes” for 15 min at room
temperature according to the TUNEL assay protocol. A positive and negative
control was prepared in parallel. After removing unincorporated fluorescein-12-
72
dUTP, cells were stained with SYTOX® Orange for 10 min at room temperature.
After staining, substrata were mounted with ProLong® Gold and photographed
with a Laser Scanning Confocal microscope (Olympus Fluoview300). Images
were analyzed with ImagePro software (MediaCybernetics Inc.). The percentage
of apoptotic cells in the whole cell population was calculated from 10-20 random
spots on each substrate.
Insoluble Extracellular Matrix (iECM) Protein assay: Cells were
incubated in a bioreactor or in TCPS for 15 and 30 d. After medium was
removed, cell layers were washed twice with PBS and extracted in 4 M guanidine
hydrochloride, 50mM Tris-HCl, 100mM 6-aminocaproic acid, 5 mM benzamidine
hydrochloride, and 1mM phenylmethylsulphonyl fluoride, pH 7.4, at 4oC with
constant rocking. Cells were separated from the extract by centrifugation (2000g
X 5 min; the extract was routinely checked for cell debris by microscopy with
trypan-blue staining to enhance visualization of cell debris). Protein
concentration in the supernatant was quantified using Bio-Rad protein assay kit,
and normalized with cell number for each growth condition.
Hematoxylin and Eosin Staining: Tissue excised from the bioreactor was
fixed in 2.5% glutaraldehyde. Dehydration and paraffin embedding were carried
using an automated tissue processor (Citadel 2000, Thermo Electron). 5μm
73
sections were cut using a microtome (Model 2040, Leica Micro System) and
mounted on glass coverslips. Hematoxylin and Eosin staining was carried
according to a standard protocol using automated staining equipment (Shandon
Gemini, Thermo Electron) and the stained sections were observed under an
optical light microscope (Olympus BX50, HiTech).
Scanning and Transmission Electron Microscopy (SEM and TEM):
Cultures were washed in buffer (0.1M, pH 7.2 sodium cacodylate) and fixed
overnight with 2.5% glutaraldehyde in cacodylate buffer at 4oC followed by
staining with 1% osmium tetroxide in cacodylate buffer for an hour at room
temperature. Fixed cells were dehydrated using graded series of ethanol
concentrations, dried in a critical point dryer (BALTEC SCD030, Techno Trade
Inc.) using dry CO2 , mounted on to an aluminum stub, and sputter coated with
10 nm of gold/palladium in an automated sputter coater (BALTEC SCD030,
Techno Trade Inc.). Cells were examined under an SEM (JSM 5400, JEOL,
Peabody, MA) at an accelerating voltage of 20 kV. Energy dispersive X-Ray
analysis was carried out using image analysis software (IMIX-PC v.10, Princeton
Gamma Tech Inc.). For TEM, primary fixation with 2.5% glutaraldehyde and
secondary fixation with 1% osmium tetroxide were followed by en bloc staining
with 2% aqueous uranyl acetate for an hour. Dehydration was carried out using a
graded series of ethanol concentrations followed by impregnation and
74
embedding in Spurr’s resin. Ultra-thin sections were cut with a diamond knife
(Diatome Ultra 45) on a microtome (Ultracut UCT, Leica), placed on uncoated
copper grids and stained with both 0. 2% aqueous uranyl acetate and 0.2% lead
citrate. The cross-sections were examined using TEM (JEM 1200 EXII, JEOL) and
images were collected using an attached high-resolution camera (Tietz F224,
Gauting).
3. Results and Discussion
Tables 1 and 2 compile semi-quantitative cell-growth data over 30 d for
hFOB 1.19 and 120 d for MC3T3-E1 cells, respectively, in the bioreactor
compared to that obtained in conventional tissue-culture grade polystyrene
(TCPS). Neither cells grown on TCPS nor those grown in the bioreactor were
subcultured over the entire culture interval (see Methods and Materials). For
both hFOB 1.19 and MC3T3-E1, between 1-2 cell layers formed in TCPS
compared to 4-6 layers in the bioreactor over 30 d culture interval (the exact
number of layers varied with position in the bioreactor and how a cell layer was
defined). Evidence of a mature osteoblastic phenotype was indicated by alkaline
phosphatase activity which was positive for both cell lines in both culture
devices. Mineralization was especially evident by von Kossa and formation of
75
nodules in the MC3T3-E1 case, but hFOB 1.19 did not mineralize as well in either
culture device. Based on the data summarized in Tables 1 and 2, it is evident that
performance of hFOB 1.19 and MC3T3-E1 in the bioreactor was similar that of
cells grown on TCPS in the first 15 d of culture. However, cell performance in
the bioreactor clearly exceeded that for TCPS over a 15-30 d culture interval, with
double the number of cell layers and increased alkaline phosphatase activity and
alizarin-red staining. TCPS controls were not carried for more than 30 d because
it was both visually and quantitatively evident that these cultures were failing
(see further below).
Fig. 2 compares attachment and growth dynamics of hFOB 1.19 in the
bioreactor and TCPS over 0 720t< < hr culture period. In the short-term
( 0 3t< < hr), there was ~ 2X attachment preference for TCPS over the polymer-
film substrata used in the bioreactor (see inset expanding time axis and
expressing % inoculum on a linear axis; see also ref. (17) for more discussion of
polymer-film cytocompatibility). However, proliferation into confluent
monolayer (on TCPS) from this sub-confluent cell density (3 72t< < hr) and post-
monolayer growth into multilayers (72 720t< < hr) was nearly the same in TCPS
and the bioreactor. In fact, actual growth rates k within 3 72t< < hr were
statistically identical at 95% confidence (TCPS = 0.23 ± 0.01 10-2 hr-1; bioreactor =
0.29 ± 0.03 10-2 hr-1). Data within the 72 720t< < hr interval were too sparse for
76
statistical comparison, but it is interesting that the total-cell number (at constant
surface area = 25 cm2) continued to significantly increase within both the
bioreactor and TCPS at approximately the same rate, achieving ~5X increase in
total cell number over this time span. The data show that the initial two-fold
cell-attachment preference for TCPS persisted through the exponential-growth
(3 72t< < hr) and post-confluence expansion ( 72 720t< < hr) phases. Further
interpretation in terms of the data in Table 1, this persistent-attachment
preference implies that more cells are packed into fewer cell layers on TCPS than
within the bioreactor, consistent with the general observation that cells on TCPS
appear thinner than in the bioreactor (compare, for example, Panel A, C Fig. 3);
although a focused study would be required to be definitive in this regard.
Despite of the initial advantage on TCPS, hFOB 1.19 began to appear less
robust than in the bioreactor within 720 hr (30 d), as evidenced by TEM images
showing numerous apoptotic bodies, cytoplasmic vesiculation, and chromatin
margination (Fig. 3) and a distinct loss of insoluble iECM (Fig. 4). TUNEL assay
results (summarized in Fig. 5) confirmed that at 30 d, apoptosis was indeed ~2X
greater in TCPS than in the bioreactor (37-47% in TCPS compared to 16-23% in
the bioreactor), although 30 d apoptosis levels were roughly 3X 15 d levels in
both devices. Examination of hFOB 1.19 tissue recovered from the bioreactor
after 15 d culture by cross-sectional TEM (Fig. 6) revealed 3-4 layers of cells
77
(Panels A and B) with many cytoplasmic protrusions (arrowheads) and healthy-
appearing nuclei showing no evidence of apoptosis. The general impression
derived from an examination of many such TEM cross-sections is that cell layers
closest to the substratum (presumably older cells) were flatter and more dish-
shaped compared to cells within upper layers (presumably younger cells) that
had a more cuboidal shape. No effort was made to quantify this visual
interpretation. Arrow annotations on Fig. 6C point to what appear to be
osteocytic-like processes between two cells and Fig. 6D shows a close contact
between cells. Exocytosis of vesicles is evident in Fig. 6D.
Results obtained with MC3T3-E1 osteoblasts in TCPS and the bioreactor
were similar to those seen for hFOB 1.19 with the notable exception that
mineralization was much more striking with MC3T3-E1 cells. One of many large
nodules (Panel A, SEM) that were found on and within MC3T3-E1 tissue after 70
d culture (see also Fig. 8 Panel A) was analyzed (Fig. 7). Panel B is a high-
resolution cross-sectional view through a nodule similar to that shown in Panel
A. Nodules such as these proved positive for calcium and phosphorous by
energy-dispersive x-ray analysis (Panel C, SEM/EDAX of a nodule taken from a
30 d bioreactor). These nodules were enmeshed in a fibrous, apparently
collagenous, network that was very evident in SEM preparations such as that
shown in Fig. 8A. Histological preparations of MC3T3-E1 tissue recovered from
78
70 d bioreactors (Panel B) confirm substantial matrix development with
osteoblasts oriented along sheets of matrix. TEM of von Kossa-stained 70 d
tissue shown in Panel C is consistent with collagen fibers running in (IP
annotation) and along (OP annotation) the plane with interspersed mineral
nodules (arrows). We estimated that tissue taken from a 120 d bioreactor
was 150 mμ thick. After 120 d in culture, there was no indication that MC3T3-E1
tissue grown in the bioreactor was unstable and the absolute term-of-culture
within the bioreactor is regarded as indefinite until experimentation finds an
endpoint at which significant instability can be detected.
79
4. Conclusions
Evidence summarized in Fig. 2 and supporting micrographs (Figs. 3-8)
show that long-term maintenance of hFOB 1.19 (ATCC CRL-11372) and MC3T3-
E1 (ATCC CRL-2593) cells without subculturing leads to formation of cell
multilayers within a thick, mineralized matrix that cannot be realized when cells
are regularly passaged at confluence. Achievement and maintenance of such a
3D ‘tissue’ that promotes cell-cell contact has been shown to be critical to the
development of osteogenic cells into a fully-differentiated osteoblast phenotype
(19-22). We demonstrated that such tissue can be grown and maintained long-
term (> 120 d) in a bioreactor based on the principle of simultaneous-growth-and
dialysis with improved efficiency over conventional tissue-culture labware
(TCPS). In fact, tissue maintained 30 d in TCPS with medium replacement every
3 d became unstable, with 2X apoptosis rates and loss of insoluble extracellular
matrix (iECM) compared to cells grown in the bioreactor, even though results
obtained in TCPS and the bioreactor were similar in the first 15 d of culture.
Furthermore, morphology of cells grown in the bioreactor suggested
development of an osteoid structure with older cells taking on a flattened
osteocytic-like phenotype with inter-cellular connections. The matrix
surrounding up to 6 layers of cells was collagenous and mineralized. This
comparison of the bioreactor to TCPS suggests that the eventual instability
observed in TCPS was primarily due to the accumulated damage arising from
80
periodic medium replacement that removes not only waste products but also
‘luxury macromolecules’ secreted by the cells. Hence, the standard cell-culture
method tends to propagate, rather than mitigate, the effects of culture shock. By
contrast, the simultaneous-growth-and-dialysis culture method maintained an
extraordinarily-stable pericellular environment. With neither subculturing steps
to push cells closer-and-closer to the Hayflick limit (4) nor the environmental
stress of an ever-changing pericellular milieu, tissue grown with the bioreactor is
apparently stable for very long periods of time measured at least in months.
Thus the compartmentalized bioreactor shows promise as a tool for the in vitro
study of osteogenesis and osteopathology.
81
Acknowledgements
This work was supported, in part, by a grant from the Pennsylvania Department of
Health. Pennsylvania Department of Health specifically disclaims responsibility for any
analyses, interpretations or conclusions. This work was also supported by The Pennsylvania
State Tobacco Settlement Formula Fund and NIH AG13087-10. This work was also
supported, in part, by grants from the US Army Medical and Materiel Command Breast
Cancer Program (W81XWH-06-1-0432), and the Susan G. Komen Breast Cancer
Foundation (BCTR 0601044. Authors appreciate additional support from the Huck
Institutes of Life Sciences, Materials Research Institute, and Departments of Bioengineering
and Materials Science and Engineering of the Pennsylvania State University.
82
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5. Rubin, H. Telomerase and Cellular Lifespan: Ending the Debate?
Nature Biotechnology 16, 396, 1998.
6. Rose, G.G. Cytopathophysiology of Tissue Cultures Growing
Under Cellophane Membranes. In: Richter, G.W., and Epstein,
M.A., eds. Int. Rev. Exp. Pathology. New York: Academic Press,
1966, pp. 111-178.
7. Vogler, E.A. Compartmentalized Cell-Culture Device and Method.
USA: DuPont de Nemours Inc., 1988.
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8. Vogler, E.A. A Compartmentalized Device for the Culture of
Animal Cells. J. Biomaterials, Artificial Cells, and Artificial Organs
17, 597, 1989.
9. Donahue, H.J., Siedlecki, C.A., and Vogler, E. Osteoblastic and
Osteocytic Biology and Bone Tissue Engineering. In: Hollinger, J.O.,
Einhorn, T.A., Doll, B., and Sfeir, C., eds. Bone Tissue Engineering.
Boca Raton: CRC Press, 2004, pp. 44-54.
10. Lim, J.Y., Liu, X., Vogler, E.A., and Donahue, H.J. Systematic
Variation in Osteoblast Adhesion and Phenotype with Substratum
Surface Characteristics. J. Biomed. Mater. Res. 68A, 504, 2004.
11. Lim, J.Y., Taylor, A.F., Li, Z., Vogler, E.A., and Donahue, H.J.
Integrin Expression and Osteopontin Regulation in Human Fetal
Osteoblastic Cells Mediated by Substratum Surface Characteristics.
Tissue Engineering 11, 19, 2005.
12. Vogler, E.A., and Bussian, R.W. Short-Term Cell-Attachment Rates:
A Surface Sensitive Test of Cell-Substrate Compatibility. J. Biomed.
Mat. Res. 21, 1197, 1987.
13. Vogler, E.A. Thermodynamics of Short-Term Cell Adhesion in vitro.
Biophysical J. 53, 759, 1988.
14. Vogler, E.A. A Thermodynamic Model of Short-Term Cell
Adhesion In vitro. Colloids and Surfaces 42, 233, 1989.
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15. Harris, S.A., Enger, R.J., Riggs, B.L., and spelsberg, T.C.
Development and Characterization of a Conditionally
Immortalized Human Fetal Osteoblastic Cell Line. J. Bone and
Mineral Res. 10, 178, 1995.
16. Lim, J.Y., Liu, X., Vogler, E.A., and Donahue, H.J. Systematic
variation in osteoblast adhesion and phenotype with substratum
surface characteristics. J. Biomed. Mat. Res. 68A, 504, 2004.
17. Vogler, E.A., and Bussian, R.W. Short-term cell-attachment rates: a
surface-sensitive test of cell-substrate compatibility. J. Biomed. Mat.
Res. 21, 1197, 1987.
18. Lim, J.Y., Taylor, A.F., Li, Z., Vogler, E.A., and Donahue, H.J.
Integrin expression and osteopontin regulation in human fetal
osteoblastic cells mediated by substratum surface characteristics.
Tissue Engineering 11, 19, 2005.
19. Freed, L.E., and Vunjak-Novakovic, G. Culture of organized cell
communities. Advanced Drug Delivery Reviews 33, 15, 1998.
20. Gurdon, J., Lemaire, P., and Kato, K. Community effects and
related phenomena in development. Cell 75, 831, 1993.
21. Ferrera, D., Poggi, S., Biassoni, C., Dickson, G.R., Astigiano, S.,
Barbieri, O., Favre, A., Franzi, A.T., Strangio, A., Federici, A., and
Manduca, P. Three-dimensional cultures of normal human
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osteoblasts: proliferation and differentiation potential in vitro and
upon ectopic implantation in nude mice. Bone 30, 718, 2002.
22. Gerber, I., and Gwynn, I. Differentiation of rat osteoblast-like cells
in monolayer and micromass cultures. Eur Cell Mater 3, 19, 2002.
86
List of Table Legends:
Table 1. hFOB 1.19 Cells in Conventional Culture Compared to
Compartmentalized Bioreactor
Table 2. MC3T3-E1 Cells in Conventional Culture Compared to
Compartmentalized Bioreactor
87
Table 1. hFOB 1.19 Cells in Conventional Culture Compared to Compartmentalized Bioreactor
Conventional Culture Compartmentalized
Bioreactor
Days in Culture 15 30 15 30
Number of cell layers 1-2 1-2 3-4 4-6
Alkaline Phosphatasea, b 4.39 ± 0.26 4.47 ± 0.38 28.89 ± 1.99 5.79 ± 0.40
Alizarin redc (μmol) 2.89 ± 0.04 3.03 ± 0.02 2.97 ± 0.18 5.17 ± 0.14
von Kossac - - - -
Mineralized nodules - - - -
+ = observed; - = not observed; ameasures of osteoblast maturity; b(nmol/mg
protein/min); cextent of mineralization.
88
Table 2. MC3T3-E1 Cells in Conventional Culture Compared to Compartmentalized Bioreactor
Conventional Culture Compartmentalized Bioreactor
Days in Culture 15 30 15 30 60 90 120
Number of cell layers 1-2 1-2 1-2 4-6 4-6 4-6 4-6
Alkaline Phosphatasea, b 1.14±0.01 1.27±0.17 2.94±0.48 3.36±0.46 ND ND ND
Alizarin redc (μmol) 4.59±0.12 5.07±0.09 5.28±0.05 6.88±0.55 ND ND ND
von Kossac + + + ++ ++ ++ ++
Mineralized nodules - +/- + ++ ++ ++ ++
+ = observed; - = not observed; ameasures of osteoblast maturity; b(nmol/mg protein/min); cextent of
mineralization; ND = not determined
89
List of Figure Legends:
Figure 1: Compartmentalized bioreactor design. Panel (a) is a cross-sectional
diagram through the device showing separation of the cell-growth space (A)
from the basal-medium reservoir (B) by a dialysis membrane (C). Cells are
grown on gas-permeable but liquid-impermeable film (E). The device is
ventilated through film (D), which is the same material as (E) as described herein
but can be different. The whole device is brought together in a liquid-tight
fashion using screws shown in the laboratory implementation Panel (b) and
Panel (c) which is an exploded-view identifying separate components. Liquid-
access is through leur-taper ports (J, K) which mate to standard pipettes. See
Methods and Materials for theory of operation.
Figure 2: Growth dynamics of hFOB 1.19 on tissue-culture-grade polystyrene
controls (TCPS, filled triangles) compared to the compartmentalized bioreactor
(open squares; note logarithmic ordinate). Inset expands short-term attachment
rates using a linear ordinate. Note that the initial two-fold cell-attachment
preference for TCPS persists through the exponential-growth (3 72t< < hr) and
post-confluence expansion ( 72 720t< < hr) phases.
90
Figure 3: Comparison of hFOB 1.19 on TCPS (Panels A, B) and in the
compartmentalized bioreactor (Panels C, D) by cross-sectional TEM. Notice that
cell layers are thicker in the bioreactor than on TCPS (compare Panels A, B to C,
D). and formation of apoptotic bodies (arrows) after 30 d culture in TCPS.
Figure 4: Comparison of insoluble extracellular matrix (iECM) production by
hFOB 1.19 on TCPS (dark bar) and in the bioreactor (light bar). Notice that iECM
significantly decreased after 30 d on TCPS but remained constant in the
bioreactor. Bar values are mean of duplicate samples measured in triplicate with
standard deviation represented by error bars.
Figure 5: Representative confocoal image of apoptotic bodies (green stain) in
TCPS and in the bioreactor selected from 8-10 similar in-depth image planes
probing hFOB 1.19-derived tissue. All cells were stained red (Sytox Orange,
scale bar = 50 μm). Percent apoptotic cells quoted in each panel are the range of
values observed within the image planes.
Figure 6: Ultramorphology of hFOB 1.19–derived tissue (15 d) recovered from
the bioreactor by cross-sectional TEM. Arrows point to cell protrusions that
91
occasionally connect two cells, as shown in Panel C. Annotations: jNC = gap
junction; MV = matrix vesicles; N = nucleus; rER = rough endoplasmic
reticulum.
Figure 7: Analysis of mineral nodules taken from MC3T3-E1 cultured in a
bioreactor. Panel A is an SEM of a large nodule taken from a 70 d bioreactor.
Panel B is a high-magnification, cross-sectional TEM of a similar nodule. Panel C
is an x-ray spectrum obtained by SEM/EDAX of a nodule taken from a 30 d
bioreactor confirming Ca and P as major constituents.
Figure 8: Microscopic examination of matrix derived from MC3T3-E1 cultured
in a bioreactor for 70 d. Panel A is an SEM of the outer layer showing a highly
fibrous network with bone nodules. Panel B is an optical micrograph of a
histologic workup showing layers of collagen with imbedded osteoblasts. Panel
C is a cross-sectional TEM showing mineralized fibers running in (IP annotation)
and out (OP annotation) of the plane.
92
50mm
(E)(E)
(C)(C)
(D)(D)
(I)
(H)(H)
(F)(F)
(G)(J)(J)
(K)(K)
a c
b
Figure 1
93
Time (hr)
Log
(% In
ocul
um)
0
1
2
3
4
0 1 3 360 720
Confluent monolayer
72
Time (min)0 50 100 150 200
% In
ocul
um0
20
40
60
80
Figure 2
94
A B
DC
substrate
substrate
substrate
substrate
Figure 3
95
iEC
M p
rodu
ctio
n (n
g/ce
ll)
0.0
0.1
0.2
0.3
0.4
0.5
0.6TCPSBioR
15 days 30 days
Growth period
* * * *
Figure 4
96
Figure 5
97
Substrate A
n
n
NN
ASubstrateSubstrate A
rER
BSubstrate
N
N
MV
N
C
rER
rER
MV
DMV
jNC
N
NN
ECM
ECM
ECM
Figure 6
98
A 5μm
500nm
B
C
Figure 7
99
5μm
cfl
cfccfl
50μm
Collagen
OB
C
Bone Nodule
Bone Nodule
A B
C
Figure 8
100
Chapter 4
System in Crisis- Colonization of Bone Tissue by Metastatic Breast Cancer In Vitro
Abstract
Metastasis is the ultimate cause of mortality in over 90% of cancer patients. Metastasis is a
multi-step process that starts with the shedding of neoplastic cells from the primary tumor,
entry into circulation, exit into a distant organ and ends with colonization of the target
organ. The final step in the metastatic cascade, colonization of distant tissue, is a critical
step that determines the eventual disease outcome and is therefore a valuable target for
therapeutics. Dynamic interaction between tumor cells and the host microenvironment
determines the survival, growth and the eventual development of clinically relevant tumors
in the target organ. Lack of model systems with sufficient biological complexity to serve as
relevant surrogates for the host microenvironment is a significant impediment to
understanding the cellular and molecular basis for colonization and discovery of therapeutic
interventions. We show herein that a three-dimensional, mineralizing, bone-like tissue
grown from isolated mouse calvarial pre-osteoblasts (MC3T3-E1) over months-long culture
in a specialized bioreactor is an effective surrogate for studying bone-tissue colonization by
metastatic breast cancer. Bioreactor derived bone-like tissue challenged with metastatic
breast cancer cells (MDA-MB-231) known to invade the skeleton captured important
hallmarks of metastatic colonization. In situ confocal microscopy revealed sequential steps
of breast cancer cell adhesion, penetration, and apparent degradation of the 3D bone-like
tissue over co-culture intervals ranging from 3 to 10 days. Bioreactor enabled direct
observation of interactions of breast cancer cells with engineered bone-like tissue simulating
101
an important step in the progression of the disease that may be a target for therapeutic
intervention.
Introduction Tissue engineering has focused considerable interest in the growth of three-dimensional
(3D), multiple-cell-layer tissues from isolated cells of purposely-selected type(s). It has been
well recognized that these engineered tissues can be used to great advantage in drug
discovery, development of therapeutic strategies, and toxicology1 by bridging the gap
between conventional monolayer (2D) cell culture and whole animals. These in vitro tissue
models confer considerable control over important experimental variables yet provide
sufficient biological complexity that outcomes are a reasonable predictor of the whole-
organism physiological response to experimental variables.2 A further advance in the use of
engineered tissue for drug discovery is to create an in vitro “system in crisis” that simulates an
in vivo disease state. Various candidate drugs and/or therapeutic strategies could then be
screened to find those that most effectively resolve the purposely-imposed crisis. Tissue
surrogates are useful in this pursuit because systemic aspects of disease can be broken down
into physiological subsystems to be studied separately in vitro. Efficiency of drug discovery
can be greatly improved with a clear understanding of the cellular and molecular basis of
disease because opportune targets of therapeutic intervention can be clearly identified.
We have focused on metastatic breast cancer colonization of bone as a proof-of-principle
problem with both stringent analytical requirements and great importance in human
healthcare. Colonization is the final step in the multi-step process of metastasis that starts
with the shedding of neoplastic cells from the primary tumor, entry of these cells into
circulation, arrest in a distant organ and finally growth and tumor formation in the target
organ.3 Cancers that colonize bone are particularly pernicious because, once bone
102
colonization occurs, the cure rate is almost zero.4-6 Cancers in bone progress with significant
morbidity related to massive bone loss, bone pain, hypercalcemia, pathological fractures,
and spinal cord compression.7 Of the major cancers that colonize bone (breast, prostate, and
multiple myeloma of hematopoietic origin), breast cancer has perhaps attracted the most
attention because of its devastating effects on women - approximately 25% of breast cancers
metastasize and the target organ is bone in 46% of the primary cases. Cancer can reoccur
after successful treatment of primary tumor and years of remission – in patients with first
relapse the frequency of metastatic bone disease is much higher at 70%.8 Critical rate
limiting steps in cancer colonization of bone include survival of tumor cells in the bone
microenvironment, proliferation and organization into overt tumors and angiogenesis or
recruitment of blood vessels necessary to sustain the tumor mass.9 Cancer cells can also
remain dormant and persist as isolated cells for years or even decades ready to be activated
into fully developed tumors under the right set of conditions.9 Cancer dormancy is a poorly
understood stage in disease progression characterized by the presence of cancer cells in bone
that have not progressed to form clinically detectable tumors. Difficulty in accessing the
bone marrow cavity probably accounts for the fact that bone tumors are usually detected
after cancer colonization has reached a very advanced stage, further complicating effective
treatment strategies.
Bone degradation in metastatic breast cancer is thought to be primarily mediated by bone-
resorbing cells (osteoclasts)10-13 that are activated by cancer cells. Excessive resorption results
in the formation of osteolytic lesions that reduce the strength and integrity of bone.
Consequently, drugs of the bisphosphonate family are currently used to inhibit osteoclasts.
Bisphosphonate treatment slows lesion formation but does not result in the restoration of
bone. Furthermore, it is not a cure in that it does not eliminate the cancer cells.14
Osteoblasts, responsible for bone formation, also play a critical role in cancer colonization.
103
Osteoblasts not only regulate osteoclast activity but also interact with cancer cells.11,15,16 As a
consequence, we have focused our attention on understanding the cellular and molecular
basis of cancer-cell colonization of bone and the related interactions with osteoblasts.17-22
These interactions include suppression of osteoblastic differentiation, increased osteoblast
apoptosis, an immune-like osteoblastic stress response, and the up-regulated secretion of
cytokines observed in conventional 2D cell culture. These findings imply a significant
osteoblastic pathology associated with cancers in bone, in addition to (and perhaps coupled
with) the known activation of osteoclasts. Understanding the effect of breast cancer on
osteoblast function can lead to therapeutic strategies that not only inhibit development of
osteolytic lesions but also restore or renew the normal functional activity of bone formation.
In spite of its clinical importance, the biology of cancer colonization is not completely
understood. Lack of experimental models that simulate cellular interactions involved in
breast cancer colonization of bone is a significant impediment to progress in the field.
Whole animal models often obscure details of the colonization process, especially when the
target is a difficult-to-access tissue such as bone. Models based on excised bone are not only
technically problematic but also difficult to interface with modern microscopic methods of
investigation. Further, most experimental models access only the end stage of the disease
defined by presence of clinically relevant tumors – critical early stages that determine the
eventual disease outcome remain largely “hidden” and inaccessible. In principle, a sub-set
of the metastatic process can be studied in vitro if the model system under consideration
retains sufficient biological complexity to be a reasonable surrogate for host tissue. Three-
dimensional (3D) tissue models have become a focus of recent investigation for this reason.
We have shown recently that a compartmentalized bioreactor based on the principle of
simultaneous–growth-and dialysis can promote the development of isolated osteoblasts into
mature 3D tissue over extended culture intervals up to a year.23 Resulting bone like
104
osteoblast tissue recapitulated progression of pre-osteoblasts to osteocyte-like cells observed
in normal bone, including production of visually- apparent (macroscopic) bone. Challenging
the bioreactor derived osteoblast tissue at various stages of development with metastatic
breast cancer cells known to invade the skeleton revealed the sequential steps of cancer cell-
tissue adhesion, penetration, and ultimate degradation of the host tissue, modeling a critical
stage in the progression of the disease that may be a target for therapeutic intervention.
Materials and Methods
Cells and Cell Culture: Murine calvaria pre-osteoblasts (MC3T3-E1, ATCC CRL-2593)
were a gift from Dr. Norman Karen at the University of Delaware. Human metastatic breast
cancer cell line (MDA-MB-231, ATCC-HTB 26) labeled with green fluorescent protein
(GFP) were a gift from Dr. Danny Welch from the University of Alabama at Birmingham.
MC3T3-E1 were inoculated into the bioreactors at a sub-confluent density (104 cells/cm2)
and cultured in alpha minimum-essential medium (α-MEM) (10-022-CV, Mediatech Inc,
Manassas VA) supplemented with 10% fetal bovine serum (FBS) and 100U/ml penicillin
and 100μg/ml streptomycin. Once the cells reached confluence, the medium was replaced
with differentiation medium containing the additional ingredients 50μg/mL ascorbic acid
and 10mM β-glycerophosphate. MC3T3-E1 cultures were maintained in the bioreactor
without sub-culture for extended intervals up to10 months using culture conditions and
protocols described in chapter 3. MDA-MB-231 cells were cultured in standard tissue
culture dishes in Dulbecco’s modified Eagle medium (DMEM) containing 5% fetal bovine
serum and 100U/ml penicillin and 100μg/ml streptomycin.
105
Bioreactor Co-Cultures: MC3T3-E1 osteoblast tissue in the bioreactor was stained with
Cell Tracker Orange ™ (Invitrogen Corp., Carlsbad, CA; prepared according to vendor
instructions) by replacing original culture medium with fresh medium containing 2.5μM cell
tracker orange for 45 minutes at 37oC, followed by 30 minutes incubation in dye-free culture
medium. Co-cultures were initiated by inoculating a suspension of GFP-labeled MDA-MB-
231 in osteoblast differentiation medium into the bioreactor on top on the MC3T3-E1 tissue
at an estimated cancer cell/ osteoblast ratio of 1/10. All operations were performed under
sterile conditions in a level II biosafety cabinet. Bioreactor co-cultures were maintained for
periods ranging from 3-10 days under standard culture conditions (5% CO2 and 95% air) in a
humidified incubator at 37oC. In situ confocal microscopy was used to follow the interaction
of breast cancer cells with 3D osteoblast tissue. At the end of the culture interval, the
bioreactor was dismantled and substratum film with adherent tissue was cut into pieces for
various assays. Bioreactors cultured with MC3T3-E1 for equivalent periods of time under
exactly the same conditions with similar medium changes but without the addition of cancer
cells were used as controls.
Confocal Microscopy: In situ laser-scanning confocal microscopy of co-cultures in the
bioreactor was performed using Olympus FV-300 laser scanning microscope (Olympus
America Inc, Melville, NY). Sections were observed with a 40X Olympus UPlanF1
objective with a numerical aperture of 0.85. Cell Tracker Orange ™ was excited using a 543
nm line from a helium-neon laser and collected through a 565 nm long-pass filter. GFP was
excited using a 488 nm argon laser and collected through 510 nm long-pass and 530nm
short-pass filters. A 570nm dichroic long-pass filter was used to split the emission. Serial
optical sections were taken at 1μm intervals throughout the tissue. Confocal images were
processed using Fluoview 300, Version 4.3b, Olympus America Inc. 3D optical
106
reconstructions of 2-D serial sections were obtained using AutoQuant AutoDeblur and
AutoVisualize software (Version 9.3, Media Cybernetics, Inc., Silver Spring, MD).
Transmission Electron Microscopy: Tissue excised from the bioreactor was fixed overnight
with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at 4oC. Secondary fixation with
1% osmium tetroxide in 0.1M sodium cacodylate was carried out for an hour was followed
by enbloc staining with 2% aqueus uranyl acetate. Buffer washes in 0.1M sodium cacodylate
(3 washes, each 5 minute duration) were carried out after each fixation step and after the
enbloc staining step. Tissue was then dehydrated using a graded series of ethanol
concentrations ( 50 %, 70%, 85%, 95%, 100%) and finally immersed in 100% acetone.
Infiltration with Spurrs resin was carried out gradually over 3 infiltration steps with each step
carried out overnight at room temperature. Samples were embedded in Spurr’s resin an
cured overnight at 55oC. The cured block of embedded tissue was thin-sectioned with a
diamond knife (Ultra 45, Diatome, Hatfield PA) on a microtome (Ultracut UCT, Leica,
Bannockburn, IL). Thin sections of tissue were placed on uncoated copper grids and stained
with both 0.2% aqueous uranyl acetate and 0.2% lead citrate. Cross-sections of tissue were
examined using TEM (JEM 1200 EXII, JEOL, Peabody MA) and images were collected
using an attached high-resolution camera (Tietz F224, Tietz Video and Image Processing
Systems GmbH, Gauting Germany).
Scanning Electron Microscopy: Cultures were washed in buffer (0.1M, pH 7.2 sodium
cacodylate) and fixed overnight with 2.5% glutaraldehyde in cacodylate buffer at 4oC
followed by staining with 1% osmium tetroxide in cacodylate buffer for an hour at room
temperature. Fixed samples were dehydrated using graded series of ethanol concentrations,
107
dried in a critical point dryer (BALTEC SCD030, Techno Trade Inc.) using dry CO2,
mounted on to an aluminum stub, and sputter coated with 10 nm of gold/palladium in an
automated sputter coater (BALTEC SCD030, Techno Trade Inc.). Samples were examined
under an SEM (JSM 5400, JEOL, Peabody, MA) at an accelerating voltage of 20 kV.
Hematoxylin and Eosin Staining: Tissue excised from the bioreactor was fixed in 2.5%
glutaraldehyde. Dehydration and paraffin embedding were carried using an automated
tissue processor (Citadel 2000, Thermo Electron, Waltham MA). 5μm sections were cut
using a microtome (Model 2040, Leica Micro System, Peabody, MA) and mounted on glass
coverslips. Hematoxylin and Eosin staining was carried according to a standard protocol
using automated staining equipment (Shandon Gemini, Thermo Electron, Waltham MA)
and the stained sections were observed under an optical light microscope (BX50, Olympus
USA, Center Valley, PA).
Results and Discussion:
Figure 1 represents schematically results of interaction between GFP-labeled human breast
cancer cells (MDA-MB-231) and the multiple layer, mineralizing osteoblast tissue in the
bioreactor. We observed (Figure 2) that murine MC3T3E-1 osteoblasts maintained in the
bioreactor for periods up to 10 months develop a tissue-like network (Panel A and B) of 6-8
layers of differentiated cells within a highly collagenous matrix that stain for alkaline
phosphatase activity and mineralization by Von Kossa (Panel B), as well as form
mineralized nodules (Panel C) that prove positive for calcium and phosphorous by scanning
electron microscopy/energy-dispersive analysis of x-rays. 5 months of MC3T3-E1 culture
resulted in formation of visually apparent macroscopic bone especially in the form of
108
deposits on the dialysis membrane (Fig. 2D). Injection of MDA-MB-231 human breast
cancer cells (Figure 3, Panel A) directly onto the osteoblast tissue leads to cancer cell,
adhesion and tissue penetration within 24 hours (Panel C) probably accomplished through
the extension of long cellular processes (Panel B). In figures 4 and 5 are results of in situ
confocal microscopy study of breast cancer cell penetration of, and replication within, an
MC3T3E-1 osteoblast tissue developed in the bioreactor over 5 months of continuous
culture. Figure 4, a collection of optical sections through the thickness of the tissue
corresponding to 1-3 days of cancer cell/bone tissue co-culture indicates that solitary cancer
cells (day 1), replicate (day 2 ) and co-localize with the osteoblasts and penetrate the entire
thickness of the tissue (day 3). Figure 5 shows 3D reconstructions of optical sections like
those shown in Figure 4 corresponding to days 1-3 of co-culture, respectively. We interpret
these images to be multi-layers of MC3T3E-1 incorporated in a thick collagenous matrix
(black, compare to Figure 2) into which columns of cancer cells penetrate in a few locations
during the first day of co-culture (Panel B). Within 2 days of co-culture, breast cancer cells
replicate (Panel B) and begin to organize into linear files especially evident in Panel C of
Figure 4. Close inspection of both 2D optical sections and 3D reconstructions (Figure 5)
suggests concomitant remodeling of the osteoid tissue. Before cancer-cell challenge,
osteoblasts exhibit a rounded, cuboidal morphology. Over 3 days of cancer cell co-culture,
osteoblasts take on a definitively elongated appearance and align with cancer cells. Similar
morphological changes have been observed in osteoblasts exposed to breast-cancer-cell-
conditioned medium in conventional culture. It was also observed that tissue from co-
culture experiments was much more fragile than originating tissue not exposed to cancer
cells, requiring very careful processing to prevent wholesale cell sloughing during the wash
steps involved in preparation of specimens for histology and electron microscopy. It was
plainly evident that the tissue matrix was eroded and easily separated from the base film of
the bioreactors used in this work. We speculate that osteoblast tissue destruction occurs
109
through increased apoptosis and suppression of osteoblast differentiation that has been
observed in conventional 2D tissue culture24, as well as wholesale degradation of the thick
extracellular matrix (ECM) in which osteoblasts are embedded (Figure 1) through the action
of matrix metalloproteinases and cathepsin K synthesized by cancer cells.25-27 Further studies
using genomic and proteomic tools can delineate the specific mechanisms responsible for
degradation of osteoblast tissue by breast cancer cells observed in the bioreactor.
Cancer-related bone loss appears to occur through multiple pathways, including the
osteoclast-mediated resorption.10,11,28 In particular, destruction of devitalized bone directly
by cancer cells has been reported10,11,28 and it has been found that, late in metastasis when
bone-degradation rate is highest, osteoclast cell numbers are actually in precipitous decline.29
These lines of evidence support the idea that osteoclasts are not solely responsible for
excessive bone degradation and that cancer cells directly contribute to bone loss.
Degradation of the osteoblast-derived osteoid tissue by co-culture with breast cancer cells
observed in the model system presented here (that purposely excludes osteoclasts) strongly
suggests that yet another mechanism of bone loss is related to disruption of the bone-
accretion process by destruction of osteoblastic tissue. There is clinical and experimental
literature to support this concept. For example, quantitative histomorphometric analyses of
bone biopsies from patients with hypercalcemia due to bone metastasis indicated a dramatic
decrease in osteoblast activity.30 Histomorphometric analysis of rodents inoculated with lytic
human breast cancer cells (MDA-MB-231) indicated that, even though administration of
risedronate (a bisphosphonate) reduced the number of osteoclasts, slowed bone lysis, and
significantly reduced tumor burden, there was no evidence of new bone deposition or repair.
Similarly, administration of bisphosphonates to humans with osteolytic metastasis slowed
lesion progression but did not bring about healing.31 All taken together, these observations
strongly suggest that normal osteoblast function (i.e. deposition of matrix) is not only
110
impaired in the presence of breast cancer cells but, in fact, osteoblastic tissue is destroyed by
an orchestrated attack by cancer cells, possibly by enlisting a cooperative response by
osteoblasts themselves.
Conclusions and Future Work:
We have shown that metastatic breast cancer challenge of 3D osteoblast tissue grown in a
specialized bioreactor over 5 months of continuous culture permitted direct observation of
interaction of breast cancer cells with the osteoblast tissue. Pathological outcomes such as
tissue penetration and cancer cell filing which are not well modeled in conventional culture
were clearly observed in the bioreactor. The bioreactor allowed us to control and select
stages in osteoblast development for challenge with cancer cells. By varying the breast
cancer cells/ osteoblast ratio the model allows us to probe the conditions under which cancer
cells remain as solitary cells or associate to form tumors simulating early stages of cancer
colonization of bone. A decided advantage of the bioreactor cultures is that medium in the
cell culture chamber is not disturbed throughout the course of the experiment and various
cell-secreted factors are allowed to accumulate within the cell chamber. Supernatant from
the culture medium can be collected at periodic co-culture intervals to assay for specific
factors that may be secreted by cancer cells over the course of their interaction with 3D
tissue. Isolating the RNA from the osteoblast cultures at different co-culture intervals can
elucidate the mechanisms that lead to changes in osteoblast physiology in the presence of
breast cancer. The bioreactor model also permits study of the effect of specific drugs that
may inhibit cancer cell penetration, tumor formation and osteoblast tissue destruction under
controlled conditions over extended culture intervals. Further understanding of the cellular
and molecular basis for breast-cancer interactions with osteoblasts and discovery of
111
therapeutic interventions will be greatly expedited by the use of three-dimensional bone
tissue models such as the one reported herein.
112
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List of Figure Legends
Figure 1: Schematic showing the sequential steps of cancer cell attachment, penetration,
replication and possible host tissue destruction observed in the bioreactor as a result of
interactions between green fluorescent protein (GFP) labeled breast cancer cells and multiple
layer 3D osteoblast tissue.
Figure 2: (A) Light micrographs of a Hemotoxylin and Eosin stained 70-day tissue (40X)
reveal osteoblasts (pale red with dark-red nuclei) lining a collagenous matrix (pale pink).
(B)Transmission electron micrographs (TEM) of 22-day tissue cross sections show up to 5
cell layers (1500X). (C) Scanning electron micrographs of the surface of a 70 day culture are
studded with mineral nodules (1000X) that prove positive for calcium and phosphorous by
energy-dispersive analysis of x-rays (not shown). (D) Bone chip recovered from a 5 month
MC3T3-E1 bioreactor. X-ray diffraction (inset) indicates similarity with a bovine bone
reference spectrum (lines).
Figure 3: Transmission electron micrograph (TEM) of a cross-section of an isolated MDA-
MB-231 breast cancer cell crawling on the base film of the bioreactor used in this work
(cultured for 3 days in osteoblast-conditioned medium) reveals an amoeboid morphology
with protrusive structures at the leading edge, consistent with a migratory phenotype (Panel
A, 1500X). TEM of breast cancer cells (BC in Panel B, 5000X) co-cultured for 10-days with
a 60-day MC3T3-E1 tissue shows that cancer cells displaced osteoblasts originally adhering
to the base film (see Figure 2) and exhibit protrusive structures that may be involved in
penetration. (arrows; compare to Panel A). Fluorescence microscopy oft breast cancer cells
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(green) in co-culture with osteoid tissue (red) organize in single-file order (Panel C, 40X),
captured in cross-section by TEM (inset, 2000X).
Figure 4: Serial optical sections through an MC3T3-E1 derived osteoid tissue (5-month
culture interval stained with Cell Tracker Orange ™) co-cultured with GFP-labeled MDA-
MB-231 breast cancer cells (green) for 3 days. Optical sections are from bottom [0μm] to top
[16μm]) of the tissue. (40X, scale bar = 50 μm)
Figure 5: 3D reconstruction of serial optical sections taken through the thickness of the 5
month osteoblast tissue cultured in the bioreactor and subject to MDA-MB-231 breast cancer
challenge over 3 day co-culture interval.
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Figure 1
118
BC
A B
Figure 2
D
119
BC
BC
B
5µm
A
C
Figure 3
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Day 2
Day 3
Day 1
0µm 4µm 8µm 12µm 16µm
Figure 4
121
A
B
C
Figure 5
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VITA
Dhurjati Ravi
Dhurjati Ravi was born in Kakinada in India on December 08, 1976. Dhurjati graduated from
Kerala University with a degree in Mechanical Engineering. Dhurjati came to the United States
to pursue higher education and received his Masters Degree in Materials from Penn State
University in 2003. Dhurjati has been pursuing doctoral degree in Materials Science and
Engineering since Jan 2004. Dhurjati’s research interests are in the broad area of tissue
engineering and with specific interest in simulating complex physiological and pathological
processes using engineered tissue.
Research Accomplishments
Osteogenesis and Osteocytic Transformation In Vitro
Continuous culture of bone forming cells for extended periods up to a
year resulted in a specialized bioreactor resulted in growth/maturation
of 3D multilayered tissue, osteocytic transformation and macroscopic
bone formation.
System in Crisis: Breast Cancer Colonization of Bone In Vitro
In vitro bone tissue surrogate challenged with metastatic breast cancer
cells, effectively captured fundamental aspects of breast cancer
colonization of bone.
Publications Ravi D, Liu X, Mastro AM, Gay CV and Vogler EA Extended-Term Culture of Bone Cells in a Compartmentalized Bioreactor Tissue Engineering, [12], 3045-3054, Nov 2006.
Liu X, Lim JY, Donahue JH, Ravi D, Mastro AM and Vogler EA Influence of Substratum Surface Chemistry/Energy and Topography on the Human Fetal Osteoblast Cell Line HFOB 1.19: Phenotypic and Genotypic Responses Observed In Vitro Biomaterials, [28], 4535-4550 Nov 2007
Ravi D, Green DJ. Sintering Stresses and Distortion Produced By Density Differences inBi-layer Structures. J. Eur. Ceram. Soc, [26], 17-25, Jan 2006