November 2016 | Volume 4 | Article 871
REVIEW
published: 15 November 2016
doi: 10.3389/fbioe.2016.00087
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org
Edited by:
Alessandro Tognetti,
University of Pisa, Italy
Reviewed by:
Giovann Vozzi,
University of Pisa, Italy
Jonathan Gooi,
University of Melbourne, Australia
*Correspondence:
Cecile M. Perrault
c.perrault@shefeld.ac.uk
Specialty section:
This article was submitted to
Bionics and Biomimetics,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
Received: 04September2016
Accepted: 25October2016
Published: 15November2016
Citation:
WittkowskeC, ReillyGC, LacroixD
and PerraultCM (2016) In Vitro Bone
Cell Models: Impact of Fluid Shear
Stress on Bone Formation.
Front. Bioeng. Biotechnol. 4:87.
doi: 10.3389/fbioe.2016.00087
In Vitro Bone Cell Models: Impact
of Fluid Shear Stress on Bone
Formation
Claudia Wittkowske
1,2
, Gwendolen C. Reilly
2,3
, Damien Lacroix
1,2
and Cecile M. Perrault
1,2
*
1
Department of Mechanical Engineering, University of Shefeld, Shefeld, UK,
2
INSIGNEO Institute for insilico Medicine,
University of Shefeld, Shefeld, UK,
3
Department of Material Science, University of Shefeld, Shefeld, UK
This review describes the role of bone cells and their surrounding matrix in maintain-
ing bone strength through the process of bone remodeling. Subsequently, this work
focusses on how bone formation is guided by mechanical forces and uid shear stress
in particular. It has been demonstrated that mechanical stimulation is an important
regulator of bone metabolism. Shear stress generated by interstitial uid ow in the lacu-
nar-canalicular network inuences maintenance and healing of bone tissue. Fluid ow is
primarily caused by compressive loading of bone as a result of physical activity. Changes
in loading, e.g., due to extended periods of bed rest or microgravity in space are asso-
ciated with altered bone remodeling and formation invivo. In vitro, it has been reported
that bone cells respond to uid shear stress by releasing osteogenic signaling factors,
such as nitric oxide, and prostaglandins. This work focusses on the application of invitro
models to study the effects of uid ow on bone cell signaling, collagen deposition,
and matrix mineralization. Particular attention is given to invitro set-ups, which allow
long-term cell culture and the application of low uid shear stress. In addition, this review
explores what mechanisms inuence the orientation of collagen bers, which determine
the anisotropic properties of bone. A better understanding of these mechanisms could
facilitate the design of improved tissue-engineered bone implants or more effective bone
disease models.
Keywords: bone remodeling, collagen orientation, osteoblast, osteocyte, uid shear stress
1.BONE PHYSIOLOGY
Bone is a highly specialized, rigid tissue which provides structural support for the body, allows
movement through muscle attachment sites, protects organs, and serves as calcium and growth
factor storage (
Clarke, 2008). Bone has the power to regenerate and repair constantly throughout
the entire life. is process, referred to as bone remodeling, involves dierent cell types and can be
Abbreviations: ALP, alkaline phosphatase; ATP, adenosine triphosphate; BMP-2, bone morphogenetic protein 2; BSP, bone
sialoprotein; COX-2, cyclooxygenase-2; Cx43, connexin 43; DMP-1, dentin matrix protein 1; DNA, deoxyribonucleic acid;
ECM, extracellular matrix; ER, endoplasmic reticulum; FGF-23, broblast growth factor 23; FSS, uid shear stress; hFOB,
human fetal osteoblast-like cells; hOB, human osteoblast-like cell; ISF, interstitial uid; LCS, lacunar-canalicular system;
M-CSF, macrophage colony-stimulating factor; MSC, mesenchymal stem cell; NO, nitric oxide; OCN, osteocalcin; OPG,
osteoprotegerin; OPN, osteopontin; PIV, particle image velocimetry; PPFC, parallel-plate ow chamber; PGE
2
, prostaglandin
E
2
; RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; RNA,
ribonucleic acid; Runx2, Runt-related transcription factor 2.
FIGURE 1 | Bone remodeling cycle. Bone remodeling is initiated by microcracks or changes in mechanical loading and consists of four consecutive steps:
activation, resorption, reversal, and formation. Activation of osteoclasts is controlled through the RANK/RANKL/OPG pathway. Following bone deposition,
osteoblasts can differentiate to osteocytes (osteocytogenesis), turn to bone-lining cells, or enter apoptosis.
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initiated in response to changes in biomechanical loading or to
replace old, microdamaged bone with new, mechanically stronger
bone (
Kini and Nandeesh, 2012).
1.1.Bone Remodeling
Bone remodeling is an essential process in maintaining bone
strength and mineral homeostasis. Remodeling allows the repair
of old and damaged bone and adjustment of the bone’s archi-
tecture to changes in external loading. Specialized cells, namely
osteoclasts which remove mineralized matrix and osteoblasts
which deposit new bone matrix, work together during this pro-
cess. eir collaboration is tightly controlled through biochemi-
cal pathways (
Hadjidakis and Androulakis, 2006). For example,
the release receptor activator of nuclear factor kappa-B ligand
(RANKL) by osteoblasts induces osteoclast activation through
binding to RANK receptors on the surface of osteoclast precur-
sors. is process can be inhibited by osteoprotegerin (OPG)
which competitively binds to RANKL (
Boyce and Xing, 2008).
e remodeling cycle (Figure1) is composed of four consecutive
phases (Clarke, 2008):
• Activation: hormonal or physical stimuli recruit mononuclear
pre-osteoclasts from the circulation to the bone remodeling
site. Following attachment to the bone surface, cells fuse to
multinucleated osteoclasts.
• Resorption: osteoclasts initiate resorption of organic and
mineral bone components which takes between 2 and 4weeks.
Osteoclasts form characteristic Howship’s lacunae in trabec-
ular bone and a cutting cone in cortical bone. Aer these
cavities reach a certain size, apoptosis of osteoclasts terminates
bone resorption (
Sikavitsas etal., 2001).
• Reversal: the resorbed surface is smoothed by mononuclear
macrophage-like cells and prepared for matrix deposition.
• Formation: osteoblasts lay down new bone by secreting a colla-
gen matrix and controlling its mineralization. roughout this
process, some osteoblasts become buried within the matrix
and dierentiate to osteocytes which reside in the fully min-
eralized lacunar-canalicular system (LCS). Aer 4–6months,
this phase is completed and osteoblasts either turn into
bone-lining cells or enter apoptosis.
In cortical bone, a remodeling rate of 2–3% per year is sucient
to maintain bone strength. Trabecular bone presents a higher
turnover rate, indicating the importance of bone remodeling for
calcium and phosphorus metabolism (
Clarke, 2008).
1.2.Bone Cells
Bone cells work together in a coordinated way during bone
remodeling by maintaining a balance between osteoblasts depos-
iting new bone tissue, osteoclasts breaking down bone matrix,
and osteocytes orchestrating the activity of osteoblasts and
osteoclasts as a response to mechanical loading (
Hadjidakis and
Androulakis, 2006
; Bonewald and Johnson, 2008).
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1.2.1.Osteoblasts
Osteoblasts are bone-forming cells which are derived from mes-
enchymal stem cells (MSC) (
Caplan, 1991). MSCs dierentiate
into osteoblasts under the appropriate stimuli, but they can also
turn into cartilage, muscle, tendon, and fat cells (
Caplan and
Bruder, 2001
). e osteoblast dierentiation and maturation
process is governed by both mechanical and biochemical path-
ways. For example, Runt-related transcription factor 2 (Runx2)
is essential in preosteoblast development where it activates
osteoblast-specic genes, including osteopontin, type I collagen,
osteocalcin, and alkaline phosphatase (ALP) (
Ducy etal., 1997;
Xu etal., 2015). Mature osteoblast dierentiation is controlled by
the Wnt signaling pathway, which is activated either by hormones
or mechanically (Westendorf etal., 2004).
e morphology of preosteoblasts is very similar to bro-
blasts; however, the latter are not able to produce a mineralized
matrix. Mature osteoblasts are typically cuboidal in shape (
Franz-
Odendaal etal., 2006
). Osteoblasts directly regulate bone matrix
synthesis and mineralization by their own secretion mechanism.
Bone resorption is indirectly controlled by osteoblasts through
paracrine factors acting on osteoclasts. For example, the release
of receptor activator of RANKL initiates bone resorption through
binding to RANK receptors on the surface of osteoclast precur-
sors (
Boyce and Xing, 2008).
e average life-span of osteoblasts ranges from a few days
to about 100days (Rosenberg etal., 2012). At the end of their
life, osteoblasts can either (1) become embedded in newly formed
bone matrix and dierentiate to osteocytes, (2) transform into
inactive bone-lining cells which protect inactive bone surfaces,
or (3) initiate apoptosis (
Manolagas, 2000).
1.2.2.Osteocytes
Osteocytes are terminally dierentiated osteoblasts which became
trapped within newly deposited bone matrix (
Franz-Odendaal
etal., 2006
). Although osteoblast and osteocytes have the same
origin, they signicantly dier in morphology and function.
During osteocytogenesis, i.e., dierentiation from osteoblasts to
osteocytes, the cell body size decreases and cell processes start to
radiate toward the mineralizing matrix which may be controlled
by E11/gp38, a marker for early osteocytes (
Schulze etal., 1999).
Aer the transition, gene expression of ALP, type I collagen,
and bone morphogenetic protein 2 (BMP-2) are reduced. Other
proteins, including osteocalcin, E11/gp38, sclerostin (Sost), and
dentin matrix protein 1 (DMP-1) are upregulated or introduced
(
Mullen etal., 2013).
ere is little knowledge about the cues which regulate osteo-
cytogenesis (Dallas et al., 2013). e mechanical properties of
the deposited osteoid, which is soer compared to mineralized
bone tissue, might guide dierentiation (Mullen etal., 2013). In
addition, mineralization of the osteoid and hypoxic conditions
might also be a driver for osteocyte formation (Irie etal., 2008;
Prideaux etal., 2012).
Research in osteocytes has gained interest in recent years, since
they are no longer seen as the “passive place holder in bone,” but as
cells with very dierent functions (
Bonewald, 2011). Osteocytes,
which are the most abundant cell type in bone (90–95% of total
bone cells), are thought to respond to mechanical loading by
releasing signal factors. rough these factors, they coordinate
bone remodeling by regulating osteoclast and osteoblast activity
(
Knothe Tate etal., 2004).
1.2.3.Osteoclasts
Osteoclasts are specialized cells which can resorb mineralized
bone matrix by secreting acid and lytic enzymes. ey are multi-
nucleated cells derived from mononuclear precursor cells which
are located in the bone marrow (
Boyle et al., 2003). eir dif-
ferentiation (osteoclastogenesis) is controlled by cytokines, such
as RANKL and macrophage colony-stimulating factor (M-CSF),
which are produced by neighboring stromal cells and osteoblasts.
Dierentiation of osteoclasts can be inhibited by OPG which
binds RANKL with high anity and prevents its attachment to
the RANK receptor (
Suda etal., 1999).
1.3.Bone Extracellular Matrix
e extracellular matrix (ECM) of bone is a composite mate-
rial consisting of 50–70% inorganic mineral, 20–40% organic
materials, less than 3% lipids, and water (
Clarke, 2008). e exact
composition depends on factors such as age, bone site, gender, or
medical conditions including osteoporosis (Boskey, 2013).
e mineral part of bone closely resembles hydroxyapatite
and provides the bone with mechanical rigidity and load-bearing
strength (
Boskey, 2007). is phase can be best described as
a crystalline complex of calcium and phosphate which also
contains impurities, such as sodium, magnesium, citrate, and
uoride (
Khan etal., 2013). Elasticity and exibility of the bone
is provided by the organic components which include structural
proteins, such as collagen and bronectin (Nair etal., 2013). e
organic phase is also composed of other non-collageneous matrix
proteins which serve important functions controlling matrix
organization and mineral deposition (
Young, 2003; Boskey,
2013
). For example, mineralization is likely to be controlled by the
small Ca
2+
-binding protein osteocalcin. Mechanotransduction is
facilitated by glycoproteins, such as osteopontin and osteonectin,
which can attach to integrins on cell surfaces. Osteopontin also
enables the attachment of osteoclasts to bone surfaces (
Gundberg,
2003
). e small amount of lipids is crucial for cell signaling and
ion ow (
Clarke, 2008).
1.3.1.Collagen Assembly
e assembly of collagen brils is a complex process involving
intracellular and extracellular steps. Collagen is rst synthesized
as precursor molecules (procollagen) in the intracellular space
before these molecules are assembled to long bers outside the
cell (Figure2).
Collagen formation is initiated in the nucleus of collagen-
producing cells, such as osteoblasts and also broblasts. In the
nucleus, a particular segment of deoxyribonucleic acid (DNA) is
transcribed into messenger ribonucleic acid (mRNA). Aer the
mRNA has moved out of the nucleus into the cytoplasm, it is
translated into polypeptide chains, known as pre-pro-collagen.
Each chain is about 300nm in length and 1.5nm in diameter.
ey are characterized by a strict pattern consisting of multiple
triplet sequences of Gly–Y–Z. Glycine residues (Gly) have to be
present in every third position to allow proper folding of these
FIGURE 2 | Formation of extracellular matrix: collagen assembly and mineralization.
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chains later on. Although Y and Z can be any amino acid, they
are commonly proline and hydroxyproline (
van der Rest and
Garrone, 1991
). Each chain is terminated by a few characteristic
amino propeptides and carboxy propeptides on either side. ese
terminal propeptides are essential in preventing self-assembly of
long collagen bers within the cell.
Proline and lysine residues are then hydroxylated in the endo-
plasmatic reticulum (ER) which will aid cross-linking of peptide
chains later. is enzymatic step requires ascorbic acid (vitamin
C) as a cofactor. A lack of ascorbic acid would either result in
the formation of looser collagen triple helices or prevent collagen
synthesis altogether, resulting in diseases such as scurvy (
Canty
and Kadler, 2005
). ree modied peptide chains will form a
triple helix which is further stabilized by disulde bonds. In the
case of type I collagen, two α1 chains and one α2 chain assemble
to form a triple helix, referred to as procollagen.
Aer the proteins have achieved their helical conformation,
they move from the ER to the Golgi apparatus where they are
packed into secretory vesicles. ese carriers vary in size and
morphology and have been either described as vacuoles around
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500nm in length (Leblond, 1989), or as larger tubular-saccular
structures (
Polishchuk etal., 2000). e vesicles move along the
microtubules toward the plasma membrane where they release
the procollagen in the extracellular space.
Once procollagen has been released from the cell, collagen
bers start to form directly on the cell membrane. is proxim-
ity potentially allows the cell to directly control brogenesis
and possibly even the formation of long-range assemblies, e.g.,
parallel bundles in tendon and ligament or interlocking weaves
in bone (
Kadler etal., 2008). Fibronectin and specic cell-surface
integrins, such as the collagen-binding α2β1 integrin, have been
found to be essential in the organization and deposition of bril-
lar collagen (
McDonald et al., 1982; Li et al., 2003). Wenstrup
etal. (2004)
also found that small amounts of type V collagens are
necessary for the induction of brillogenesis of collagen I bers
invivo.
Collagen bers can only form aer specic enzymes remove
the terminal propeptides from the procollagen which are then
called tropocollagen (
Prockop etal., 1998). Tropocollagen units
assemble spontaneously into collagen bers. Several hundred
tropocollagen molecules line up in a characteristic “quarter
staggered” array, so that the composite ber appears as a striated
pattern by electron microscopy. e striated pattern results from
the longitudinal staggering of the molecules which leaves a “hole”
roughly the size of one quarter of the length of tropocollagen
(67nm) between the end of one molecule and the beginning of the
next (
Scott, 1995). e bers are further supported through the
formation of covalent bonds. e enzyme lysyl oxidase catalyzes
the formation of bonds by converting hydroxyl groups on lysines
and hydroxyl lysines into aldehyde groups (
Kagan and Li, 2003).
Consequently, the bers increase up to 10-fold in diameter and
dramatically in length following lateral and end-to-end fusion
(
Birk etal., 1995).
1.3.2.Collagen Orientation
Two types of bone can be distinguished based on the orientation
of collagen bers within the bone matrix: (1) woven bone which
consists of randomly oriented collagen brils and (2) lamellar
bone which is characterized by highly orientated collagen bers
(
Kini and Nandeesh, 2012). Collagen bers in lamellar bone
are arranged in arrays of parallel bers, which successively
change orientation to form a “twisted plywood-like” structure
(
Weiner et al., 1997). e alternating orientation of collagen
arrays results in signicantly higher strength of lamellar bone
compared with woven bone (Clarke, 2008). is plywood
structure can be found in the cylindrical osteons which are the
primary building blocks of compact bone. Osteons are made
up of several concentric lamellae which are arranged in dier-
ent orientations around the Haversian channel containing the
blood and nerve supply (Figure3). e long axis of an osteon is
usually parallel to the long axis of the bone, and the dominant
collagen ber orientation commonly follows the direction of
load (
Martin and Boardman, 1993; Hert etal., 1994; Seto etal.,
2008
). Furthermore, longitudinal collagen bers are primarily
present in regions supporting tensile loads, while regions under
compressive loading are composed of transverse bers (
Riggs
etal., 1993; Martin etal., 1996).
Besides the spatial orientation of collagen bers, mechanical
properties of bone are also determined by several other factors
which include total bone mass and material properties of bone
mineral (
Viguet-Carrin et al., 2006). While the latter is more
important with respect to bone stiness and yield strength, the
collagen network appears to have a greater impact on post-yield
properties, including ultimate strength and toughness of bone
(
Garnero, 2015). Even though the dependence of ber orienta-
tion on bone strength is well accepted, it remains dicult to
estimate exactly the eect collagen orientation has on mechanical
properties of bone (
Viguet-Carrin etal., 2006).
e mechanisms which guide the spatial arrangement of
collagen bers have been a matter of research for more than
40years. In 1975,
Jones etal. (1975) observed that collagen bers
were oriented in the same direction as osteoblasts producing
the collagen, but they could not determine whether the cells
controlled the orientation of the collagen bers or viceversa. e
central question since then has been revolving around whether
the 3D organization of collagen brils is a result of self-assembly
(
Giraud-Guille et al., 2008) or cell activity (Matsugaki et al.,
2015b
). It is generally accepted that collagen ber formation on
the nanometer length scale is driven by self-assembly (
Kadler
etal., 2008
), while, on a macro scale, there is clear evidence of
collagen ber orientation following external loading patterns as
described above (Martin etal., 1996; Puustjärvi etal., 1999). As a
result, research focusses on determining the mechanisms which
regulate collagen bril assembly on intermediate length scales and
whether cells inuence this process (
Kerschnitzki etal., 2011).
Mechanically weaker woven bone is produced in situations
where no bone matrix is present, i.e., during bone formation in
the fetus and newborn or in early phases of bone repair following
fractures and osteotomies. Under such conditions, mesenchymal
osteoblasts secret collagen bers rapidly and randomly in all
directions (
Shapiro, 1988, 2008). However, most bone in a healthy
adult which is formed as a result of the remodeling process
described above is composed of highly orientated, lamellar bone
(Figure3). It is thought that the production of parallel collagen
bers requires collective, organized action of bone-producing
cells (
Kerschnitzki etal., 2011).
Weakly organized woven bone or pre-existing old bone
matrix is believed to act as trigger for cells enabling lamellar
bone formation. In particular, the nanobrillar topography of
bone appears to be a powerful substrate-specic cue for cell and
collagen alignment. In vitro studies have shown that osteoblasts
align uniformly along the direction of grooves in the micrometer
(
Wang etal., 2000, 2003) and nanometer range (Zhu etal., 2005;
Yang etal., 2009; Lamers etal., 2010). Highly oriented natural and
articial collagen ber matrices can also act as scaold induc-
ing alignment of osteoblasts (
Delaine-Smith, 2013; Matsugaki
et al., 2015b
). ese studies further show that newly secreted
collagen bers and apatite crystals follow the cell direction. In
contrast, Matsugaki et al. (2015a) recently cultured osteoblasts
on nanogrooved biomedical alloys. However, they observed a
mismatch between cell orientation following the direction of
the grooves and collagen matrix and apatite crystals orientation
which aligned perpendicular to the cell direction. e authors
explained this “abnormal” orientation with a yet-to-dene impact