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Mechanotransduction in Osteoblats Biomechanical Stimulation and its Clinical Prospects 53
Figure 2: Partial schematic summary of cellular events following mechanical stimulation in
osteoblast.
associated also with the increased expression of Runx2. Downstream to the PGE2
activity the anabolic TGF- mRNA was found to be elevated. TGF- triggers
mitosis and matrix synthesis induction [9]. In osteoblasts the TGF- receptor 1
(TGFR1) was identified as a cardinal target gene for Runx2.
Runx
MECHANICAL STIMULATION
Membrane receptors
-IGF
-INTEGRINS
-TGF-b/BMP
-Wnt
-b-catenin
-Raf
-ERK
-SMADs
Cytoplasmic signal
transmitters
Nucleal signal transmitters
and transcription factors
-Runx2
-Dlx5
-AP-1
-Osterix
TGF-b
Wnt
INTEGRIN
IGF
Ras
b-catenin
SMADs
Raf
ERK
AP-1
Dlx5
Osterix
54 Biomechanical Stimulation and its Clinical Prospects Berkovich et al.
Animal studies demonstrated the increased nitric oxide (NO) secretion in response
to flow-stimulated or cyclically stretched primary osteoblasts. NO binds to a
regulatory site on Ras and potentially triggers cellular proliferation and ECM
production through the Ras-Raf-mitogen-activated protein kinase (ERK) cascade.
Downstream to the NO activity, cyclo-oxygenase (Cox) 1 and 2 are activated and
stimulate the anabolic activity in osteoblasts.
Mechanical stimulation also triggers the up regulation of IGF-I, independently from
PGE2. Growth factors, such as VEGF, BMP-2 and BMP-4, are essential for this
mechanism [10]. The activated growth factors trigger phosphoinositide 3-kinase
(PI3-K)-protein kinase B (Akt), MAPKs and SMAD signal transduction pathways.
Following mechanical stimulation the BMPs induce anabolic effect in an
autocrine and paracrine mode of action through the activation of tyrosine kinases
and serine/threonine kinases. BMP-2 induces expression of three osteogenic
transcription factors: Runx2, Osx and Dlx5 [11]. Mechanical strain also triggers
up regulation of growth related genes c -fos, early growth response factor 1 (egr-
1) and autocrine basic fibroblast growth factor (bFGF).
The example of overall effect of physiological mechanical loading on osteoblasts
is revealed in the study when the fifteen seconds long pulses of gravity stress on
osteoblasts, similar to the in vivo physiological strain generated during walking or
running, stimulate osteoblast proliferation. Different mechanical strains can
trigger bone versus cartilage formation. Low frequency strain and high frequency
strain stimulate individual bone cells to deposit bone ECM in vitro as well as
in vivo. Surprisingly the high magnitude compression forces on osteoblasts trigger
the extracellular antagonists of BMPs to inhibit osteoblastogenesis [12].
In vitro the constant compression load on osteoblasts triggers production of
inflammatory cytokines and their receptors. Interleukin (IL)-1β synthesis
increases in an autocrine fashion. IL-1 is induced by mechanical stress and
probably has a role in the RANKL - RANK signalling pathway. This pathway
stimulates osteoclastogenesis.
RANKL, a cell surface molecule, belongs to the tumour necrosis factor (TNF)
super family. RANKL is expressed under steady state conditions in osteoblasts in
response to osteogenic inducers, such as vitamin D, PTH and prostaglandins. The
RANKL-RANK interaction is modulated by osteoprotegrin (OPG), a soluble
glycoprotein that strongly suppresses osteoclast differentiation in vitro and in
vivo. OPG binds by the TNF receptor (TNFR) to the TNF domains on RANKL.
This activity suppresses the resorption and causes bone mass gain [13].
Mechanotransduction in Osteoblats Biomechanical Stimulation and its Clinical Prospects 55
Short intervals of fluid flow or cyclic substrate loads at physiological levels
stimulate the proliferation and survival of the osteoblasts. These physiological
strains trigger the expression of survival cues in the osteoblasts. Therefore the
adequate physical activity increases the bone density, which is reduced after the
cessation of exercise.
Gravitation promotes pro-survival behaviour of osteoblasts. Lack of gravitation
induces cytokine-mediated decrease of survival-promoting transcription factors to
DNA and damages mitochondrial function by altering Bcl to Bax ratio, leading to
apoptosis of osteoblasts. Therefore a lack of mechanical stimulation in vivo leads
to osteoblasts’ apoptosis and osteoporosis. On the other hand, supra-physiological
strain leads to cells detachment, following rupture of cell adhesions, thereby
eliminating the ability to withstand high physiological strain. The detachment of
cells from their supporting extracellular matrix also leads to cell death by the
apoptosis [14].
Overall, osteoblasts (Fig. 3) govern the osteogenesis through interactions with
osteocytes, endothelial cells etc. It is clear that these cells are highly active
metabolically (Fig. 4) and due to their key role in osteogenesis these cells are
extensively targeted in the research of osteoinductive mechanotransduction.
Figure 3: Human osteoblasts in culture (scale - 50µ). Multipolar shape of the cells is evident.
56 Biomechanical Stimulation and its Clinical Prospects Berkovich et al.
Figure 4: Microscopic image of osteoblasts stained by mitotracker green (MTG). Strings of green
stained mitochondria are apparent indicating on high metabolic activity. Confocal microscopy,
scale - 20 µ.
CONFLICT OF INTEREST
The author confirms that author has no conflict of interest to declare for this
publication.
ACKNOWLEDGEMENTS
Declared None.
REFERENCES
[1] Tami, A.E. The role of interstitial fluid flow in the remodeling response to fatigue loading. J Bone
Miner Res2002; 17: 2030-2037.
[2] Rosenberg N, Rosenberg O, Soudry M. Osteoblasts. In Bone Physiology - Mini Review Rambam
Maimonides Med Jl 2012; 3(2): 1-7.
[3] Frost, H.M. Bone’s mechanostat: a 2003 update. Anat Rec A Discov Mol Cell Evol Biol 2003; 275:
1081-110.
Mechanotransduction in Osteoblats Biomechanical Stimulation and its Clinical Prospects 57
[4] Xiao, G. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1
stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem 2005; 280: 30689-30696.
[5] Santos A, Bakker AD, Zandieh-Doulabi B, de Blieck-Hogervorst J.M. Klein-Nulend J Early
activation of the beta-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-
3 kinase/Akt, and focal adhesion kinase. Biochem Biophys Res Commun 2009.
[6] Liu, W. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia
with multiple fractures. J Cell Biol 2001; 155: 157-166.
[7] Marie, P.J. Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys 2008; 473:
98-105.
[8] Weinbaum S, Cowin S.C., Zeng Y A model for the excitation of ostocytes by mechanical loading-
induced bone fluid shear stresses. J Biomech1994; 27: 339-360.
[9] Celil, A.B. and Campbell, P.G. BMP-2 and insulin-like growth factor-Imediate Osterix (Osx)
expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling
pathways. J Biol Chem 2005; 280: 31353-31359.
[10] Gu, M. Nitric oxide increases p21 (Waf1/Cip1) expression by a cGMP-dependent pathway that
includes activation of extracellular signal-regulated kinase and p70 (S6k). J Biol Chem 2000; 275:
11389-11396.
[11] Lee, M.H. BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2.
Biochem Biophys Res Commun 2003; 309: 689-694.
[12] Duncan, R.L. Transduction of mechanical strain in bone. ASGSB Bull 1995; 8: 49-62.
[13] Herman, S. Molecular mechanisms of inflammatory bone damage: emerging targets for therapy.
Trends Mol Med 2008; 14: 245-253.
[14] Zhan, M. Signalling mechanisms of anoikis. Histol Histopathol 2004; 19: 973-983.
Mesenchymal Cell Activation by Biomechanical Stimulation and its Clinical Prospects, 2016, 59-64 59
Nahum Rosenberg (Ed)
All rights reserved-© 2016 Bentham Science Publishers
CHAPTER 6
Mechanotransduction in Chondrocytes
Marwan Haddad, Jacob Shapira and Nahum Rosenberg
*
Department of Orthopaedic Surgery, Rambam Health Care Campus, POB 9602, Haifa 31096,
Israel
Abstract: Mechanical load is the cardinal factor in regulating the metabolic activity of
chondrocytes. Chondrocytes are sensitive to load pattern and can adjust their
metabolism according to the magnitude and frequency of mechanical stress. Quantity
and quality characterization of the chondrocyte response to stress is yet to be revealed
and may lead to novel therapeutic methods in preventing and treatment of cartilage
degeneration diseases.
Keywords: Cartilage, chondroblat, chondrocyte, ECM, GAG.
BACKGROUND
Articular cartilage serves as load-bearing surface and therefore is subjected to
significant pressure. During normal activity, pressure may reach as high as 100 -
200 atmospheres (10-20MPa) within milliseconds. Cartilage compression of 15%-
45% is in the physiological range and be resulted from long-term or static
compression [1]. Occasionally physiological short loads occur with high-
frequency stress. The ability of cartilage to withstand the physiological
compressive, tensile, and shear forces depends on the composition and structural
integrity of its extracellular matrix (ECM).
Chondrocytes, which comprise 3-5% of the tissue volume, are responsible for
maintaining ECM integrity and proper adaptation of cartilage to stress (Fig. 1).
ECM consists mostly of type II collagen and aggregating proteoglycans. These
molecules give cartilage its characteristic rigidity and tensile strength, thus
enabling it to withstand stress.
The composition of cartilage reflects the chondrocytes adaptation to mechanical
stress. Cartilage proteoglycans concentration is the highest in high-loaded regions.
Removal of compression leads to decrease in proteoglycans concentration with
subsequential cartilage degeneration [2].
*Corresponding author Rosenberg Nahum: Department of Orthopaedic Surgery, Rambam Health Care
Campus, POB 9602, Haifa 31096, Israel; E-mail: nahumrosenberg@hotmail.com
60 Biomechanical Stimulation and its Clinical Prospects Haddad et al.
Chondrocytes maintain normal tissue homeostasis by synthesizing and replacing
ECM macromolecules. Cartilage cells respond to mechanical forces, and, within
the normal limits of these forces, remodel cartilage in order to adapt to the
mechanical stress. In load bearing zone of a healthy joint cartilage is thicker with
higher proteoglycans concentration and higher mechanical strength in comparison
to non-load bearing zone. Microscopically chondrocytes in the load-bearing areas
are larger with greater volume of intracellular organelles [3].
Figure 1: Micrograph of human hyaline articular cartilage in tissue culture. Scale - 50µ.
Chondrocyte (arrow) are imbedded in extracellular matrix.
Abnormal turn-over of the ECM macromolecules may lead, with time, to
alteration in the mechanical properties of the tissue involved. Cartilage may
undergo "wasting" in response to immobilization, or more accurately, to load
removal, by the IL-1 and proteoglycan synthesis suppression. After load-bearing
is restored, proteoglycans concentration is also gradually restored [4].
Mechanosensors of Chondrocytes
Mechanotransduction in chondrocytes is a process of chondrocytes’ sensing the
mechanical cues from the environment and respond by a synthetic cascade,
eventually leading to structural changes in cartilage matrix. Mechanical changes
in the ECM-cell surface interface are sensed by mechanoreceptors such as
integrins and ion channels. These receptors in turn trigger cell proliferation and
Mechanotransduction in Chondrocytes Biomechanical Stimulation and its Clinical Prospects 61
matrix deposition [5]. Integrins are heterodimeric transmembrane receptors that
interact with cytoskeletal proteins such as fibronectin, osteopontin and vitronectin.
Additional receptors that transfer mechanical stimulus in cartilage cells exist, such
as the non-selective transient receptor potential vanilloid V4, which is extensively
expressed stretch receptor in chondrocytes. Additionally immunohistochemical
studies demonstrated the expression of aquaporin water channels, ENaC, NMDA,
calcium, chloride, sodium and potassium ion channels that are involved in
mechanotransduction in chondrocytes [6]. The role of ion channels in
chondrocytes is intriguing and not obvious because chondrocytes are non-
excitable cells and do not conduct action potential. However it was demonstrated
that potassium channels play a role in apoptosis, volume homeostasis, maturation
and chondrogenesis and might be targeted in the future in treating cartilage
degenerating disease such as osteoarthritis.
Biomechanical Signaling
Fluid flow, electrical current across chondrocytes’ membranse, mechanical
compression, shear stress, hydrostatic pressure, ionic and osmotic pressures are all
responsible for alteration in chondrocytes gene transcription, cell signaling and
protein synthesis [7].
In general chondrocytes respond to at least two sources of stress. First mechanism
is outflow of water outwards during joint loading. This in turn increases
osmolarity. As pressure is decreased, water rush back in and osmolarity decreases.
Intracellular osmolarity must be equalized by compatible ion channels to maintain
homeostasis of ions and water.
Mechanical signals regulate different stages in signaling cascade including
transcription, translation and post-translation modification. In addition to
signaling cascade, multiple organelles and cytoskeletal structures are involved in
ECM modification. Peptides are translated in a coordinated manner as response to
mechanical stress and transported to the Golgi apparatus. Then the aggrecans are
modified post-translationally in the Golgi apparatus. Glycosaminoglycans (GAG)
chains are extended on core proteins by GAG primers in the Golgi/rough
endoplasmatic reticulum complex. It is becoming well accepted that dynamic
compression and shear stress in frequencies between 0.01-1.0 Hz increase matrix
synthesis. Static forces, on the other hand, decrease production of ECM by
chondrocytes [8]. It has been demonstrated that static compression leads to
decrease in the number of GAG per core protein and an increase in GAG chain
length by a process of post-translational modification [9]. Additionally static
62 Biomechanical Stimulation and its Clinical Prospects Haddad et al.
hydrostatic pressure leads to increasingly longer GAG chain length relative to the
non-stimulated group [10]. Mechanical stress is also responsible for increase in an
aggrecans expression and decrease in MMP-3 gene expression. In this process
there is a release of IL-4 and α5β1 integrin. Interestingly in osteoarthritis (OA)
this signaling pathway is absent, suggesting that abnormal signaling pathway
contribute to osteoarthritis (OA) disease exacerbation.
NF-B transcription factors are involved in immune and inflammatory processes.
These factors regulate expression of genes involved in inflammation, apoptosis,
matrix breakdown and cell cycle. IL-1β, TNFα, lipopolysaccharides and other
stimuli trigger activation of NF-B, which in turn enters the nucleus to initiate
transcription of its target genes. In OA, induced in animal model, low progression
of joint articular cartilage damage was demonstrated when joint movement was
initiated early. This phenomenon was accompanied by a suppression of NF-B
expression and subsequential suppression of joint inflammation [11]. More
studies have shown that the degenerative process of cartilage in articular joints
might be suppressed by cycled mechanical joint loading.
Research Equipment
In order to explore the mechanical effects on chondrocytes the in vitro
experimental models are constantly being developed and improved, because of the
obvious difficulties to study these phenomena in vivo. Cartilage explants are often
utilized when cells are isolated from a tissue specimen. Three dimensional
cultures, when chondrocytes are grown on gel matrices, are popular because of
their ability to simulate the in vivo conditions. These systems allow quantifying
the relationship between mechanical and biological factors. The interactions
between the cells and matrix components, as well as chondrocytes gene
expression, can be monitored using explants and chondrocytes-gel cultures [12].
Explants are able to achieve equilibrium of matrix synthesis and turnover and thus
can be used for studying changes caused by application of mechanical stimuli.
Three dimensional agarose gel and alginate gel culture systems are used for
studying chondrocytes phenotypic expression, proliferation and accumulation of
proteoglycans-rich ECM during prolonged culturing. These systems were created
in order to study and understand the role of the ECM and the chondrocytes-ECM
interaction in the response to mechanical stress [13]. Phenomena such as
mechanical compression, hydrostatic pressure, physiochemical stimuli (PH and
osmolarity) and electrical current are now can be studied using these tissue and
cellular systems.
Mechanotransduction in Chondrocytes Biomechanical Stimulation and its Clinical Prospects 63
A versatile shear and compression apparatus is commonly used for studying the
mechanical forces’ application on cartilage tissue and cell samples. This type of
device is usually housed in a culture incubator in order to function during a long-
term incubation. Such device is capable to provide closed loop feedback control
of displacement, load, shear, angle and torque. In these studies explants or gel
culture disks are examined within sterile closed chambers, which are subjected to
tuneable compression forces (Fig. 2)[14].
Figure 2: Schematic representation of the commonly used experimental device for mechanical
stimulation of cartilage in tissue culture. A motor generates force, through a piston movement, on
cartilage sample in the culture media. The setup is embedded into CO
2
humidified incubator.
CONFLICT OF INTEREST
The author confirms that author has no conflict of interest to declare for this
publication.
ACKNOWLEDGEMENTS
Declared None.
incubator
vertical movement
cartilage
sample
media
level
motor
force (N)
signal generator
Hz
time
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