Mechanobiology of Bone - Engineered Bone: Tissue Engineering Intelligence Unit - page 32

In fracture healing and DOG, cells within the callus tissue are loaded quite differently
compared to bone cells in normal healthy bone. The strain values during distraction are much
higher than in adult bone. These higher strains are found to stimulate cell proliferation, matrix
production and cell and matrix alignment in vitro. 41-45
Mechanical Stimulation of Bone Cells in Vitro
Fluid Shear Stress: Technique, Cell Responses
In healthy, adequately adapted bone, small matrix strains produce flow of extracellular tis-
sue fluid through the lacuno-canalicular network. 46,47 This strain-derived extracellular fluid
flow may help to keep osteocytes healthy, particularly the deeper ones, by facilitating exchange
of nutrients and waste products between the Haversian channel and the osteocyte network of
an osteon. 48 However a second function of this strain-derived interstitial fluid flow is thought
to be the transmission of “mechanical information”. The magnitude of interstitial fluid flow
through the lacuno-canalicular network is directly related to the amount of strain of the bone
organ. 49 Because of the narrow diameter of the canaliculi, bulk bone strains of about 0.1% will
produce a fluid shear stress in the canaliculi of roughly 1 Pa, 20 enough to produce a rapid
response in e.g., endothelial cells. 50,51 Experimental studies in vitro have demonstrated that
osteocytes are also quite sensitive to fluid shear stress of such magnitude. 15-17,19 These results
suggest that the combination of the cellular 3-dimensional network of osteocytes and the ac-
companying porous network of lacunae and canaliculi, acts as the mechano-sensory organ of
bone. The flow of interstitial fluid through the bone canaliculi will have two effects, a mechani-
cal one derived from the fluid shear stress, and an electrokinetic one derived from streaming
potentials. 52,53 Either of the two, or both in combination, might activate the osteocyte. For
instance, streaming potentials might modulate the movement of ions such as calcium across
the cell membrane, 54,55 while shear stress will pull at the macromolecular attachments between
the cell and it's surrounding matrix. 56 Both ion fluxes and cellular attachment are powerful
modulators of cell behavior, and therefore good conveyors of physical information. 57,58
The discovery of an antigen against chicken osteocytes made it possible to specifically study
the mechanosensitivity of osteocytes. 59 With the immunodissection technique three separate
cell populations with a high ( ≈ 90%) degree of homogeneity can be prepared, representing (1)
osteocytes with the typical “spider-like” osteocyte morphology and little matrix synthesis, (2)
osteoblasts with high synthetic activity of bone matrix-specific proteins, and (3) osteoprogenitor
cells (from the periosteum) with a fibroblast-like morphology and very high proliferative ca-
pacity. 59 As the cells are used within 2 days after isolation from the bone tissue, they may well
represent the three differentiation steps of osteoprogenitor cell, osteoblast and osteocyte. By
contrast, the mixed cell cultures derived from bone that are generally used to represent “osteo-
blastic” cells, likely contain cells in various stages of differentiation. Therefore, changes in
mechanosensitivity related to progressive cell differentiation cannot be studied in such cul-
tures.
Using these immuno-separated cell populations, osteocytes were found to respond far stronger
to fluid flow than osteoblasts, and these stronger than osteoprogenitor cells. 15-17,19 Pulsating
fluid flow (PFF) with a mean shear stress of 0.5 Pa (5 dynes/cm 2 ) with cyclic variation of plus
or minus 0.2 Pa at 5 Hz, rapidly stimulated the release of nitric oxide (NO) and prostaglandin
E 2 and I 2 (PGE 2 and PGI 2 ) from osteocytes within minutes. 15,17 Osteoblasts showed less re-
sponse, and osteoprogenitor cells (periosteal fibroblasts) still less. Intermittent hydrostatic com-
pression (IHC) of 13,000 Pa peak pressure at 0.3 Hz (1 sec compression followed by 2 sec
relaxation) needed more than 1 h application before prostaglandin production was increased,
again more in osteocytes than osteoblasts, suggesting that mechanical stimulation via fluid flow
is more effective than hydrostatic compression. 16 One hour treatment with PFF also induced a
sustained release of PGE 2 from the osteocytes in the hour following PFF treatment. 17 This
sustained PGE 2 release, continuing after PFF treatment had been stopped, could be ascribed to