The objective of this investigation was to elucidate the sensitivity to transients in fluid shear stress in bone remodeling. Bone remodeling is clearly a function of the local mechanical environment which includes interstitial fluid flow. Traditionally, load-induced remodeling has been associated with low frequency (1-2 Hz) signals attributed to normal locomotion. McLeod and Rubin, however, demonstrated in vivo remodeling events associated with high frequency (15-30 Hz) loading. Likewise, other in vivo studies demonstrated that slowly applied strains did not trigger remodeling events. We therefore hypothesized that the mechanosensitive pathways which control bone maintenance and remodeling are differentially sensitive to varying rates of applied fluid shear stress

Frangos, John A.

English

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NASA-CR-204942
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NASA Grant NAGW-4256 Final Progress Report
Pulsatile Fluid Shear in Bone Remodeling '_
Abstract:
The objective of this investigation was to elucidate the sensitivity to transients in fluid shear
stress in bone remodeling. Bone remodeling is clearly a function of the local mechanical
environment which includes interstitial fluid flow. Traditionally, load-induced remodeling
has been associated with low frequency (1-2 Hz) signals attributed to normal locomotion.
McLeod and Rubin, however, demonstrated in vivo remodeling events associated with
high frequency (15-30 Hz) loading. Likewise, other in vivo studies demonstrated that
slowly applied strains did not trigger remodeling events. We therefore hypothesized that
the mechanosensitive pathways which control bone maintenance and remodeling are
differentially sensitive to varying rates of applied fluid shear stress.
Using nitric oxide (NO), prostaglandin E 2 (PGE2) and c-fos as markers, we have
investigated the role of transients in fluid shear stress in bone remodeling. We report:
1. Flow-induced NO production is biphasic. Transients associated with the
start of flow activate distinct mechanosensitive pathways relative to steady
and sustained flow.
2. Impulse flow stimulates c-fos activation 2 fold relative to ramped flow to
the same magnitude.
3. Mechanosensitive pathways activated by step changes in flow are calcium
and G-protein dependent.
4. Mechanosensitive pathways associated with sustained flow are calcium and
G-protein independent.
https://ntrs.nasa.gov/search.jsp?R=19970022809 2020-06-16T02:18:33+00:00Z
Introduction:
Bone is a porous and compressible material, which is subjected to a rapid and substantial
interstitial fluid (ISF) flow. This flow is driven by a gradient between the venous pressure
in the intramedullary canal and the lymphatics at the periosteum. Mechanical loading (in
particular, axial or bending loads) induces pulsatile presssure gradients which cause
transients in the ISF flow. The objectives of this investigation were to develop an in vitro
model to demonstrate the characteristics of transients in fluid flow-induced shear stress
versus steady shear stress.
Utilizing a parallel plate flow chamber and either syringe or head driven fluid flow, we
subjected primary rat calvarial cells to well defined fluid shear stresses. Nitric oxide and
prostaglandin E 2, both osseoactive humoral agents, were used as markers. Northern blot
analyisis of c-fos expression was also performed as an indicator of mitogenic activity.
Results:
Using an in vitro flow model with primary rat calvarial cells and a parallel plate flow
chamber, we reported that fluid flow-induced shear stress is a potent stimulus of NO
release in osteoblasts, with a production rate 16 fold greater than that associated with
cytokine stimulation (Figure 1).
*J'q 1_
o 50 s.
o
o
.w-i
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---," I _" I I I _Ir I _,_
@ 0 10 20 30 40 50 60 70
Z Z
40
30
20
10-
160 -
140-
"_ 120-
¢ 100-
8o
_ 6o
_ 40
o 2O
_ 0
0 2 4 6 8 10 12
Time (hours) Time (hours)
ao ho
FIGURE la. Cytokines (o) produces continuous NO release in primary rat calvarial cells ( .6
nmols/mg/hr) after a 12 hour lag phase of zero production. Figure lb. Flow (o) (6 dynes/cm 2)
induces an immediate and sustained release (9.8 nmols/mg/hr), suggesting a constitutively present NOS
isoform. 1001.tM L-NAA (*) inhibits NO production. Static controls (O) (A) produce negligible NO
(From Johnson, McAllister and Frangos 1996).
Utilizing a chemiluminescent NO analyzer with picomolar sensitivity, we delineated the
response to the start of flow (0-.5 hr) from steady flow (.5-6 hr). Flow-induced NO
response demonstrates a biphasic response which we hypothesized was due to activation of
distinct pathways which were sensitive to transients in shear versus steady shear. To
further investigate this biphasic response, we treated cells with GDPI3S, a G-protein
inhibitor, and quin 2/AM, an intracellular calcium chelator. In both cases, the response to
the step change in flow was significantly attenuated, while the response to sustained flow
was unchanged (Figure 2).
t,.,
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[]
g
o
z
1
' T
8
" T
4
2
0
0 - .5 hr .5 - 6 hr
0
"0
0
I-.i
g_
0
Z
20-
T
15-
0 1 2 3 4 5 6
Time (hrs)
Figure 2. Fluid flow (8 dynes/cm 2) (') versus flow + quin 2/AM (301.tM) ( • ) and flow + GDP[3S
(900BM) (¢). Inset. Production rates for flow (black), quin 2/AM (white) and GDP[3S (gray). Flow
induced NO response demonstrates a biphasic response. Chelating intracellular calcium with quin 2/AM (.5
hr preincubation) significantly inhibits initial flow response (0-.5hr) but does not affect sustained
production (.5-6 hr). GDP[3S ( 3 hr preincubation) inhibited initial flow response but did not attenuate
sustained production rate.
Calciumindependencein sustainedproductionwasfurtherdemonstratedby theadditionof
calciumionophoreA23187to staticcultures.While calciumionophorewasableto
stimulateamoderateimmediateresponse(0-.25hr.), nosustainedproductionwas
observed(Figure3).
25
2O
O
E
O
o_
O
©
Z
15
10
41.5-
E
O
E
,_= 1
o
.0.5
O
0
Z 0
CI Static
0-15 minute Production Rate
0 1 2 3 4 5 6
Time
Figure 3. A23187 treatment (ll.tM) (.)does not stimulate sustained NO production relative to control
(vehicle only) (*) but stimulates a burst in NO production within 15 minutes (inset).
To further identify the subclass of G-proteins involved in the immediate NO response, cells
were treated with pertussis toxin. PTx treated cells were not significantly different than
flow alone (Figure 4). PTx did, however, significantly attenuate PGE 2 production as we
had previously reported, demonstrating that the efficacy of the drug.
o
°_
25
20-
1000
750
500
o°.O"
250-
0_ i
o 3 6
Time (hrs)
-z.Ip_ o°°•°'°°°°'°°°°°°
"2-
.,;..""
0 ,ip I I I I I I
0 1 2 3 4 5 6
Time
Figure 4. Pertussis toxin [ I p.g/ml ) (*) does not affect NO production rates relative to flow + vehicle (').
Inset Ptx does inhibit flow-induced PGE2 production.
To further demonstrate that this biphasic response was due to differences in the flow
profile, we investigated c-fos expression as a marker of mitogenic activity. Impulse flow
stimulated a 2 fold increase in c-fos expression relative to ramped flow to the same level of
shear stress (Figure 5).
A
28S-
18S-
a b c d e
c-fos
B
28S-
18S-
GAPDH
40
c 30
,_o 20
d_
10
0
a b c d e
Figure 5. Ramp and step flow elicit c-fos expression in cultured osteoblasts (umr-106). Cells were
subjected to: static control (lane a); ramp flow, where flow was smoothly increased from 0-16 dyne/cm z in 5
minutes, then held at 16 dyne/cm 2 for 25 minutes (lane b) or 30 minutes (lane c); step flow, where flow
was instantaneously increased from 0-16 dyne/cm2 and held at 16 dyne/cm2 for 30 minutes (lane d); and a
positive control where cells were starved for 48 hours then stimulated with 100_tM ATP for 1 hour.
Isolated RNA was analyzed by Northern blot with 1.0 kb c-fos cDNA (panel A) and 0.8 kb GAPDH cDNA
as the probe (panel B). c-fos expression determined by densitometry and normalized by GAPDH mRNA is
shown in panel C.
Previous investigations utilizing pulsatile flow have been limited by the undefined nature of
the flow profile. We have therefore developed an in vitro model to investigate frequency
response under well defined pulsatile shear stress (Figure 6).
032 In
Couette Viscometer Media In
Stationary Outer
Inner Cylinder Rotates
0
0 0
Peristaltic Micropump
Cells grown on inside surface
Media Out
Media Sampling
Port
Figure 6. Schematic of couette viscometer. Cells are grown to confluence on the inner surface of the larger
cylinder. Shear stress is applied by rotating the inner cylinder with a computer controlled motor while
holding the outer cylinder stationary. Shear stress is proportional to fluid viscosity, gap width, and relative
rotational velocity. Cells are grown in a rotating system so that they are preconditioned to a flow
environment. Media is recirculated longitudinally by a micro-peristaltic pump for gas exchange and media
sampling, with one volume exchange every 6 minutes. Lobed cores give a well defined pulsatile shear
stress.
Conclusions:
Using nitric oxide (NO) and prostaglandin E2 (PGE 2) and c-fos as markers, we have
investigated the role of transients in fluid shear stress in bone remodeling. We report:
°
Flow-induced NO production is biphasic. Transients associated with the
start of flow activate distinct mechanosensitive pathways relative to steady
and sustained flow.
2, Impulse flow stimulates c-fos activation 2 fold relative to ramped flow to
the same magnitude.
.
Mechanosensitive pathways activated by step changes in flow are calcium
and G-protein dependent.
. Mechanosensitive pathways associated with sustained flow are calcium and
G-protein independent.
Publications:
Johnson, D.L., McAllister, T.N., and J.A. Frangos.
and continuous release of nitric oxide in osteoblasts.
E208.
1996. Fluid flow stimulates rapid
Am. J. Physiol. 271(34):E205-
Reich, K.M., McAllister, T.N., Gudi, S. and J.A. Frangos. 1997. Activation of G
proteins mediates flow-induced prostaglandin E 2 production in osteoblasts. Endocrinology
138:1014-1018.
Nulend, J.K., Van der Plas A., Semeins, C., Ajbui, N.E., Frangos, J.A., Nijweide P.J.,
and E.H. Burger. 1995. Sensitivity of osteocytes to biomechanical stress in vitro.
FASEB Journal. 9:441-445.
Hillsley, M.J. and J.A. Frangos. 1996. Osteoblast hydraulic conductivity is regulated by
calcitonin and parathyroid hormone. Journal of Bone and Mineral Research. 11:114-124.
Hillsley, M.V. and J.A. Frangos. 1994. Bone tissue engineering: the role of interstitial
fluid flow. B iotechnology and Bioengineering. 43:573-581.
Hillsley, M.V. and J.A. Frangos. 1997. Alkaline phosphatase in osteoblasts is down
regulated by pulsatile flow. Calcif. Tissue Int. 60:48-53.
McAllister,T.N., Moffarah,M. andJ.A.Frangos.Fluid flow-inducedsignaltransduction
inosteoblasts:Theroleof G-proteinsandcalciumin steadyandtransientfluid flow. In
preparation.


Pulsatile Fluid Shear in Bone Remodeling

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