Mechanobiology of Bone - Engineered Bone: Tissue Engineering Intelligence Unit - page 33

the induction of prostaglandin G/H synthase-2 (or cyclo-oxygenase 2, COX-2) expression. 19
Again osteocytes were much more responsive than osteoblasts and osteoprogenitor cells, as
only 15 min treatment with PFF increased COX-2 mRNA expression by 3-fold in osteocytes,
but not in the other two cell populations. 19 Upregulation of COX-2, but not COX-1 by PFF
had been shown earlier in a mixed population of mouse calvarial cells 60 and was also demon-
strated in primary bone cells from elderly women, 61 while the expression of COX-1 and 2 in
osteocytes and osteoblasts in intact rat bone has been documented. 62 The in vitro experiments
on immuno-separated cells suggest that as bone cells mature, they increase their capacity to
produce prostaglandins in response to fluid flow. 10 First, their immediate production of PGE 2 ,
PGI 2 and probably PGF 2 α 60 in response to flow increases as they develop from osteoprogenitor
cell via the osteoblastic stage into osteocytes. Second their capacity to increase expression of
COX-2 in response to flow, and thereby to continue to produce PGE 2 even after the shear
stress has stopped, 19 increases as they reach terminal differentiation. As induction of COX-2 is
a crucial step in the induction of bone formation by mechanical loading in vivo 63 these results
provide direct experimental support for the concept that osteocytes, the long-living terminal
differentiation stage of osteoblasts, function as the “professional” mechanosensors in bone
tissue.
Pulsating fluid flow also rapidly induced the release of NO in osteocytes, but not
osteoprogenitor cells. 17 Rapid release of NO was also found when whole rat bone rudiments
were mechanically strained in organ culture 18 and in human bone cells submitted to fluid
flow. 64 In line with these in vitro observations, inhibition of NO production inhibited me-
chanically induced bone formation in animal studies. 65,66 NO is a ubiquitous messenger mol-
ecule for intercellular communication, involved in many tissue reactions where cells must col-
laborate and communicate with each other. 67 An interesting example is the adaptation of blood
vessels to changes in blood flow. In blood vessels, enhanced blood flow as for instance during
exercise, leads to widening of the vessel, to ensure a constant blood pressure. This response
depends on the endothelial cells, which sense the increased blood flow, and produce intercellu-
lar messengers such as NO and prostaglandins. In response to these messengers, the smooth
muscle cells around the vessel relax, to allow the vessel to increase in diameter. 51 The capacity of
endothelial cells to produce NO in response to fluid flow is related to a specific enzyme,
endothelial NO synthase or ecNOS. Interestingly, this enzyme was found in rat bone lining
cells and osteocytes 68,69 and in cultured bone cells derived from human bone. 70 Treatment with
pulsatile fluid flow increased the level of ecNOS RNA transcripts in the bone cell cultures, 70 a
response also described in endothelial cells. 71,72 Enhanced production of prostaglandins is also
a well described response of endothelial cells to fluid flow. 51,71 It seems therefore that endothe-
lial cells and osteocytes posses a similar sensor system for fluid flow, and that both cell types are
“professional” sensors of fluid flow. Mutatis mutandis , this is an indication that osteocytes sense
bone strains via the (canalicular) fluid flow resulting from bone strains.
Mechanotransduction starts by the conversion of physical loading-derived stimuli into cel-
lular signals. Several studies suggest that the attachment complex between intracellular actin
cytoskeleton and extracellular matrix macromolecules, via integrins and CD44 receptors in the
cell membrane, provides the site of mechanotransduction. 15,73-76 An important early response
is influx of calcium ions, through mechanosensitive ion channels in the plasma membrane and
release of calcium from internal stores. 54,55,75,77,78 The signal transduction pathway then in-
volves protein kinase C and phospholipase A 2 to activate arachidonic acid production and
PGE 2 release. 75 However, many other steps in the mechanosignaling cascade are still unknown,
in osteocytes as well as other mechanosensory cells.
Cell Stretching: Technique, Cell Responses
To mimic the effect of physiological bone loading in monolayer cell cultures, several au-
thors have used cell stretching, via deformation of the cell culture substratum 18,42,69,79-82 (see
Burger and Veldhuijzen 83 for a review of the older literature). Stretch-loading by hypo-osmotic
cell swelling was also used. 84 The results are generally in agreement with the studies using fluid