U.S. patent application number 09/731430 was filed with the patent office on 2001-04-12 for mechanical loading system.
Invention is credited to Jones, David, Smith, Evertt L..
Application Number | 20010000195 09/731430 |
Document ID | / |
Family ID | 21978569 |
Filed Date | 2001-04-12 |
United States Patent
Application |
20010000195 |
Kind Code |
A1 |
Smith, Evertt L. ; et
al. |
April 12, 2001 |
Mechanical loading system
Abstract
A mechanical testing device has a rigid frame and a piezo
translator connected to the frame. A Wheatstone bridge is connected
to the translator to produce an electrical signal related to the
compression of the translator, wherein a sample positioned between
the piezo translator and the frame is subjected to loads by the
movement of the translator. A sensor detects the force applied to
the sample by the piezo translator, and produces a signal
indicative of the force. A computer receives the Wheatstone bridge
electrical signal and the signal indicative of the force applied to
the sample. The computer controls the advancement of the translator
to allow the application of precise amounts of compression to the
sample.
Inventors: |
Smith, Evertt L.; (Madison,
WI) ; Jones, David; (Margburg, DE) |
Correspondence
Address: |
LATHROP & CLARK LLP
740 REGENT STREET SUITE 400
P.O. BOX 1507
MADISON
WI
537011507
|
Family ID: |
21978569 |
Appl. No.: |
09/731430 |
Filed: |
December 6, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09731430 |
Dec 6, 2000 |
|
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09110503 |
Jul 6, 1998 |
|
|
|
6171812 |
|
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|
60052587 |
Jul 15, 1997 |
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Current U.S.
Class: |
73/862 |
Current CPC
Class: |
G01N 3/10 20130101; G01N
3/08 20130101; A01N 1/02 20130101; G01N 2203/0051 20130101; G01N
2203/0089 20130101; A01N 1/0247 20130101; G01N 2203/0623
20130101 |
Class at
Publication: |
73/862 |
International
Class: |
G01L 005/00 |
Claims
We claim:
1. A mechanical testing device comprising: a rigid frame; a piezo
translator connected to the frame; a Wheatstone bridge connected to
the translator to produce an electrical signal related to the
compression of the translator, wherein a sample positioned between
the piezo translator and the frame is subjected to loads by the
movement of the translator; a sensor which detects the force
applied to the sample by the piezo translator, and produces a
signal indicative of said force; a computer which receives the
Wheatstone bridge electrical signal and the signal indicative of
the force applied to the sample, the computer controlling the
advancement of the translator to allow the application of precise
amounts of compression to the sample.
2. The mechanical testing device of claim 1 wherein the translator
is controlled to allow the application of compression to the sample
in increments at least as small as 200 nm.
3. A method for testing a sample comprising: preparing a sample to
have parallel top and bottom end surfaces; placing the sample
within a test apparatus having a frame, a piezo translator
connected to the frame, the translator having an output signal
indicating the compression of the sample, and a force sensor
between the sample and the frame; applying a voltage to the piezo
translator to advance the translator to apply a desired level of
compression: detecting the output signal indicating level of
compression of the sample, receiving and recording the outputs from
the force sensor corresponding to the desired level of compression,
and calculating material properties from said signal and said
outputs.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
1. This application is a divisional of U.S. application Ser. No.
09/110,503, filed Jul. 6, 1998, which claimed priority based on
U.S. provisional application No. 60/052,587, filed Jul. 15, 1997,
the disclosures of both said applications being incorporated by
reference herein.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
2. Not applicable.
BACKGROUND OF THE INVENTION
3. The present invention is in the field of biotechnology and
specifically relates to the study of the physiological and
physiochemical processes which govern and underlie the formation,
growth and resorption of human and animal bone. In particular the
invention provides novel means for the study of responses of the
mammalian musculoskeletal system to stress and potentially may lead
to the discovery of novel substances produced by bone during these
responses. The instant system may lead to a better understanding of
diseases such as osteoporosis and the perfusion chamber means
provides means for the study of the effects of drugs and other
substances added to the perfused medium.
4. It has been known for over 150 years that bone responds to
mechanical loading. Although the effects of exercise and mechanical
loading on the musculoskeletal systems have been well documented,
the actual mechanisms by which mechanical loading acts at the
cellular level in the maintenance of skeletal integrity are not
completely understood. Although greater attention is being given to
exercise and nutrition as a means of preventing and/or treating
osteoporosis, the regulatory mechanisms that control skeletal
response to mechanical loading, growth factors and nutrition are
not yet delineated.
5. There is speculation about the biophysical structure and
properties of the sensory and biochemical and molecular biological
mechanism of mechano-transduction. When controlled loads of a given
magnitude and frequency are applied, in vivo, either in an isolated
wing preparation or a rat tibia, bone mineral density is known to
increase to an extent which is approximately proportional to the
load applied. However, according to the prior art, it is not
possible to assess quantitatively the bone-specific regulatory
control product and their mechanisms nor to monitor the bone
production of local growth factors and cytokines, in these in vivo
preparations.
6. Whilst cell culture preparations do permit an investigator to
quantify second messengers, cytokines and local growth factors,
they do not permit one to monitor the responses of bone cells to
mechanical deformation of the bone matrix which are so important in
maintaining and/or remodeling of the skeletal system.
7. Although growth factors have been shown to enhance the
development of new bone, clearly and without the presence of
mechanical loading, under these circumstances, the new matrix is
not formed along lines of strain and it is that feature, in life,
which induces maximum integrity of the new bone so formed. The
present authors have been associated with previous work in which
the viability of osteoblasts from 2 to 4 week old pigs was
successfully maintained, in culture, for 68 days. Careful
consideration of these findings led to the hypothesis that, in a
suitable novel system, which would permit continuous perfusion and
mechanical loading of suitable explanted samples of trabecular bone
from mature pigs, viability might be maintained for 10 to 12 days
or longer. If this were to be achieved, such a time frame would
permit measurements of the rate of bone formation and resorption of
the trabecular bone, not available using the systems, apparatus and
methods of the prior art. Further, such a novel system would be
applicable to the study of human bone.
8. Up to now, prior art apparatus and systems for investigating
bone have either comprised cell culture apparatus of a variety of
well-known types or mechanical means for applying three point and
four point bending forces to a biological test subject. An example
of the three point type is disclosed in U.S. Pat. No. 5,406,853 to
Lintilhac and Vesecky and an example of the four point type is
disclosed in U.S. Pat. No. 5,383,474 to Recker and Akhter.
9. The present authors are not aware of any prior art system or
apparatus which provides means for simultaneous, contemporaneous
and continuous study of axially loaded viable mammalian bone
undergoing concurrent continuous perfusion and the effluent medium
therefrom.
References
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OUTLINE OF THE PRESENT INVENTION
40. In the instant system, apparatus means is provided for the
perfusion and axial mechanical loading of an explanted sample of
mammalian trabecular bone which has been prepared in an appropriate
manner. During use, a prepared trabecular bone biopsy core is
placed within the apparatus and is then loaded mechanically to
induce tension and/or compression to the bone matrix. The bone
explant perfusion and loading apparatus of the instant system is
provided with means for maintaining an environment with stable
oxygen, carbon dioxide, nutrients and systemic hormones.
41. Prepared bone biopsies are in the form of trabecular bone
cores, 10-12 mm in diameter and 3 to 5 mm thick. These are
surgically extracted, under sterile conditions, from suitable long
bones of the subject. This procedure is carried out with care and
precision, using suitable cutting means and cooling means, to
ensure that the resultant bone disk samples are not subjected to
temperature rises during cutting and that extreme dimensional
accuracy and disk flatness are achieved.
42. Cutting means are in the form of a surgical hand saw used to
cut gross samples, a diamond tipped keyhole saw to remove bone
cores from the gross samples and an ultra high precision band saw
with a diamond tipped bladed, operated in conjunction with jig
means, to cut bone disk. Trabecular bone sample disks produced are
flat (.+-.100 nm) and have parallel end surfaces (.+-.2-5 .mu.m).
Cooling means comprise suitable phosphate buffered saline (PBS) at
6.degree. C. which is used to flood the work piece during cutting.
Each trabecular bone sample disk, so prepared, is intended to
supply about 3,500,000-11,000,000 cells (based on an estimate of
10,000-20,000 cells per cubic mm of bone (Mundy 1990; Parfitt
1983). Extracted bone disk samples are perfused and maintained with
suitable circulating medium, Hepes and fetal calf serum.
43. The apparatus of the instant invention provides means for
concurrent mechanical loading of the prepared bone explant sample
disk, located within a novel perfusion chamber, in a controlled
manner. The maximum compressive strain applied to each sample is
0.5% (5,000 .mu.E) generally at 1 Hz, with the capability of using
steeper rise times, if desired. These figures translate to a
maximum compression, in each sample, of 20 .mu.m, at a rate of
50,000 .mu.E sec.sup.-1. Further, the apparatus applies to the bone
sample disks, controlled deformations of 200 nm. The apparatus
applies forces of up to 800N, at frequencies in the physiological
range, of up to 15 Hz and maximum strain rates of between 10,000
.mu.E sec.sup.-1 and 50,000 .mu.E sec.sup.-1. These data are
appropriate to samples of spongy mammalian bone in which Young's
modulus varies between 400 MPa and 1200 Mpa.
44. The apparatus of the instant system also provides an
environment in which many factors can be investigated. Because
whole tissue is used, bone cells can be studied in a near-natural
environment of bone matrix and bone marrow. The apparatus provides
means for the user to monitor cellular response but additionally
and in a novel manner, to monitor the architecture, strain
characteristics and strength of the bone disk and changes
therein.
45. The bone explant perfusion and mechanical loading apparatus of
the present invention preserves the hard matrix of the bone sample
and permits the collection of second messengers and growth factors
in the perfusion medium. The instant system thus has many of the
advantages of cell culture, whilst retaining the bone matrix
encountered in vivo.
46. Means provided within the instant system permit recording of
changes in the explanted trabecular bone core sample and further
permit the calculation of strain, load and Young's modulus for each
such sample. Thus, the instant system permits not only the
monitoring of second messengers, cytokines and growth factors but
further permits study of how these factors, in conjunction with
mechanical loading, will maximize skeletal response to varied
stimuli both alone and in combination.
47. In the instant system there are provided perfusion loading
apparatus means, power means, control means, computer hardware
means, software means and sampling and analysis methods.
48. The perfusion loading apparatus comprises frame means,
adjustable biasing pre-loading means, translator loading means,
force sensor means and perfusion chamber means. Most components are
substantially cylindrical and are accurately machined in corrosion
resistant metal, conveniently stainless steel.
49. Frame mounting means are in the form of a relatively massive
cylindrical frame housing, comprising a base, a lower frame
section, an upper frame section and a cap, each adapted to fit
together. These components are secured together with a series,
conveniently of 6, partially male-threaded hardened steel bolts
which pass through the frame components and are each tightened down
with a female-threaded nut. The frame is about 150 mm high and
about 80 mm in diameter. The lower part of the frame is
substantially solid and has an axial cylindrical hole to accept a
ceramic stacked piezo translator which is secured in place by
virtue of a close fit in the lower frame and also by screw means
through the base.
50. The top part of the frame provides mounting means for
adjustable biasing pre-loading means provided by adjustable screw
means located axially in and through and the frame cap and secured
thereto by threaded means. Within the adjustable biasing
pre-loading means there is provided locating and bearing means for
force sensor means in the form of an annular quartz crystal force
sensor in a precision welded housing.
51. The perfusion chamber assembly is located axially and centrally
in the upper section of the frame and comprises a stainless steel
bottom bearing cap which provides mounting means for a perfusion
chamber body made in durable biologically inert, non-leaching
plastics, preferably polycarbonate. A piston, conveniently made in
stainless steel, is provided with sealing means in the form of an
`O` ring, made from resilient and biologically inert material,
preferably neoprene, engages with the upper part of the perfusion
chamber body and under the influence of the pre-loading and loading
entities, bears down upon a cylindrical explanted trabecular bone
sample placed therein. Fluid pathways formed in the perfusion
chamber body are disposed so as to ensure that perfusing fluid
reaches all parts of the bone sample. Spigots provide connecting
means for suitable tube means for delivering perfusing fluid to the
assembled perfusion chamber and for collecting effluent from
it.
52. The upper and lower components of the perfusion chamber are
provided with locating and compression centering means and the
assembly is located axially above and upon the translator loading
means and directly beneath and in contact with the adjustable
pre-load means which drive through push rod and ball bearing
coupling means.
53. The piezo translator is provided, via cable connecting means,
with a suitable control interface having a microprocessor
controlled digital to analogue converter, low voltage driver,
controller and power supply, a high voltage amplifier and display
unit, all having performance and operating characteristics
appropriate to the functional applications of the instant
system.
54. The force sensor is provided, via cable connecting means, with
a suitable force amplifier having an appropriate power supply and
display unit, all having performance and operating characteristics
appropriate to the functional applications of the instant
system.
55. It will now be apparent that frame means, in co-operation with
adjustable biasing pre-load means having force sensor means,
translator loading means and perfusion chamber means, as
hereinbefore described, constitute perfusion means and instrumented
axial press means for the perfusion and mechanical loading of an
explanted human or animal bone sample.
56. An explanted trabecular bone sample, prepared as hereinbefore
described, is placed within the perfusion chamber, which is then
assembled to the frame and loading apparatus. With connections
established, power on, and perfusing fluid flowing, the adjustable
biasing pre-loading sub-system is adjusted to remove lost motion
from and to apply a biasing force to the load train. The biasing
force is applied using a large load adjustment knob situated above
the frame which drives the adjustable biasing pre-loading means via
fine-threaded screw means. A suitable biasing pre-load may also be
established using electro-mechanical means via regulator loop means
provided in the translator controller. Establishment of a biasing
force allows system integrity to be checked. The desired working
load or linear translation for the experiment in hand may then be
effected using the translator and translator controller.
57. Serial samples of effluent may be collected and assayed for one
or more selected factors. Voltage outputs from the translator and
charge output from the force sensor are processed and displayed
visually. These are used for input to a suitable standard personal
computer employing a standard operating system and running a
bespoke software program for manipulating data. The program
provides software means which produce outputs, via a standard
interface, to the system for set-up, configuration, calibration and
control of hardware as well as for calculation of relaxation and
Young's modulus. Numerical and graphical results may be output to a
suitable monitor and printing device connected to the computer.
58. The instant system allows assessment of bone cellular response
to specific stimuli, under controlled conditions. An understanding
of these mechanisms will allow their manipulation which may
possibly lead to the alleviation or control of osteoporosis and
other deleterious skeletal changes. The instant system advances the
state art in permitting investigators to study physiological
responses of bone tissue under specified conditions. The instant
system also advances the state of the art in permitting study of
human bone biopsies in a controlled environment. It provides means
for identifying morphologic changes occurring in different bone
diseases and potentially, for the determination of the physiologic
and genetic determinants in such diseases.
59. It is thus a first and most important object of the present
invention to provide a novel system for continuous perfusion in
conjunction with mechanical loading and for collecting and
monitoring second messengers, cytokines and growth factors produced
by a viable explanted bone sample in order to study skeletal
response to varied stimuli both alone and in combination.
60. It is a second important object of the present invention to
provide novel means within the instant system for recording changes
in thickness of an explanted bone sample during mechanical loading
and further for the calculation of strain, load and Young's modulus
for each such sample.
61. It is a third important object of the present invention to
provide novel apparatus means for concurrent perfusion and axial
mechanical loading of an explanted sample of mammalian bone,
prepared in an appropriate manner, for an extended period during
which the bone is to be kept viable.
62. It is a fourth object of the present invention to provide
suitable control and recording means for novel apparatus means for
concurrent perfusion and axial mechanical loading of an explanted
sample of mammalian bone.
63. The instant system will now be described in more detail in
conjunction with the following drawings.
DESCRIPTION OF THE DRAWINGS
64. In order that the present invention may be more readily
understood, reference will now be made to the following drawings in
which:
65. FIG. 1, is a diagrammatic front view of the assembled
mechanical and electro-mechanical components of a bone explant
perfusion and mechanical loading system, according to the present
invention.
66. FIG. 2, is a diagrammatic exploded upper perspective axial view
of the mechanical and electro-mechanical components of FIG. 1.
67. FIG. 3, is a diagrammatic exploded inverted perspective axial
view of the mechanical and electro-mechanical components of FIG.
1.
68. FIG. 4, is a diagrammatic exploded section of the components of
the perfusion chamber assembly and a prepared bone sample.
69. FIG. 5, is an underplan view of the perfusion chamber body of
the present invention.
70. FIG. 6, is a side section of the assembled components of the
perfusion chamber assembly with a prepared trabecular bone sample
located therein.
71. FIG. 7, is a schematic diagram of the instant system
particularly illustrating electronic control equipment used in
conjunction with the electro-mechanical equipment of the present
invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
72. With general reference to FIGS. 1-7, there is described a
preferred embodiment of a novel, combined perfusion and mechanical
loading system for explanted bone, generally designated by the
numeral 10.
73. Referring to the FIGS. 1-7, there are shown the principal
assemblies of a perfusion and mechanical loading system 10,
comprising metal frame housing means 100, adjustable biasing
pre-loading means 400, translator loading means 600, force sensor
means 700, perfusion chamber means 800, and electronic control
means 900.
74. As may best be seen with reference to FIGS. 2 and 3, metal
frame housing means 100, are in the form of a substantially
cylindrical and relatively massive metal frame, preferably made
from solid stainless steel and comprising several accurately
machined parts. In this embodiment, a frame base 102, is
substantially circular, conveniently having a diameter and general
thickness of 78 mm and 15 mm, respectively. Additionally, frame
base 102, has a substantially circumferential rim 104, conveniently
4 mm wide and extending somewhat less than 5 mm above general upper
surface 106, of 102. A central circular through hole 108, in frame
base 102, conveniently 4 mm in radius, has a countersunk recess
110, on the under surface 112, of 102. A central circular recess
114, provided in upper surface 106, of frame base 102, is co-axial
with central hole 108. Circular recess 114, has a radius and depth
conveniently of 12.5 mm and 5 mm, respectively. A parallel sided
recess 116, conveniently 9 mm wide, extends from recess 114, to the
edge 118, of frame base 102, interrupting rim 104, thereof. The
depth of parallel sided recess 116, is substantially similar to
that of circular recess 114. A plurality of clearance fixing holes,
120-130, have centers disposed at equal angles around a pitch
circle 132, shown as a center line, conveniently of radius 25 mm
and concentric with 108. Fixing holes, 120-130, are each adapted by
the provision of a counter bore 134-144, on under surface 112, of
frame base 102. Frame base 102, is provided with a counter bore
146, disposed in its upper surface 106, having a diameter and
depth, conveniently, of 6 mm and 7 mm, respectively. Counter bore
146, is centered on a line bisecting the angle between holes 124;
126, substantially midway between pitch circle 132, and rim 104 of
102.
75. A lower frame section 148, is substantially of similar radius
to frame base 102. Lower frame section 148, conveniently has a
height of 61 mm and is provided with substantially circumferential
right angled rebates of 5 mm, top and bottom, indicated at 150 and
151, respectively. A central circular through-hole 152 and a series
of fixing holes 154-164, are provided which have substantially
similar diameters to and relative spatial dispositions
corresponding with 114 and 120-130, respectively, of frame base
102. Similarly, a parallel sided recess 166 and a counter bore 168,
are provided in the underside 170, of lower frame section 148,
having dimensions and positions which correspond with 116 and 146
of 102. Counter bore 168, is adapted by the provision of female
thread means 172. A locating stud 174, adapted by the provision of
partial male thread means 176, threadedly engages with the female
thread means 174, of counter bore 168. Lower rebate 151 and
locating stud 174, of lower frame section 148, together with rim
104 and counter bore 146, of frame base 102, constitute adaptations
for mutual location and secure positioning means.
76. An upper frame section 178, has a height, conveniently, of 65
mm and is of similar radius to 102 and 148. Additionally and also
similarly, upper frame section 178, is provided with a series of
fixing holes 180-190, which have substantially similar diameters to
and relative spatial dispositions corresponding to 154-164, of
lower frame section 148 and 120-130, of frame base 102. A central
circular hole 192, has a diameter, conveniently, of 37 mm.
Observation means are in the form of a parallel sided fenestration
slot 194, conveniently 30 mm wide, extending from 192, through wall
196, of upper frame section 178, from its upper surface 198, to a
depth, conveniently, of 50 mm. Fenestration slot 194, is
symmetrically disposed about fixing hole 184 and has a deep chamfer
200, extending out to a width, conveniently, of 53 mm at the outer
surface 202, of 178. Lower surface 204, of upper frame section 178,
is adapted by the provision of a machined recess 206, to form a rim
208, so dimensioned as to constitute engagement location means for
positioning upon upper rebate 150, of lower frame section 148. A
further parallel sided slot 210, has substantially the same width
and is in a corresponding position to recesses 116; 166, of frame
base 102 and lower frame section 148, respectively. Slot 210,
extends from central hole 192, through wall 196, to outer surface
202 and extends to a depth, conveniently of 25 mm, from upper
surface 198, of the 178. A counter bore 212, in upper surface 198,
of 178, is provided with female thread means 214. A locator stud
216, is provided with partial male thread means 218, for threaded
engagement with female thread means 214, of counter bore 212.
Counter bore 212 and locator stud 216, are of substantially similar
dimensions and are similarly spatially disposed to 168 and 174, of
lower frame section 148. Upper surface 198, of upper frame section
178, has a circumferential rebate 220, substantially similar to
150, of lower frame section 148.
77. A frame cap 222, has a similar diameter and thickness to frame
base 102. Frame cap 222, is provided with fixing holes 224-234,
substantially similar in diameter and disposition to, those of
frame base 102. Fixing holes, 224-234, are each adapted by the
provision of a counter bore 236-246, in upper surface 248, of 222,
substantially similar to 134-144, in 112, of frame base 102.
Additionally, frame cap 222, has a rim 250, disposed
circumferentially about its lower surface 252, substantially
similar to rim 104, of frame base 102. A counter bore 254, is
adapted and positioned to receive locator stud 216, of upper frame
section 178. Rim 250 and counter bore 254, of frame cap 222,
together with rebate 220 and locating stud 216, of upper frame
section 178, constitute adaptations for mutual location and secure
positioning means. A central circular through-hole 256, adapted by
the provision of female thread means 258, has a diameter,
conveniently, of 30 mm. A central circular recess 260, in upper
surface 248, of frame cap 222, is conveniently 40 mm in diameter
with a filleted circumference to its full depth, conveniently of 4
mm. Rim 250, is partially interrupted by a shallow cut out 262, so
sized and positioned as to correspond with fenestration slot 194,
of 178, in order to extend observation means in metal frame housing
100.
78. Metal frame housing 100, is assembled by mutually locating and
positioning 102, 148, 178 and 222 and securing them together with
mutual threaded securing means. These means are in the form of a
series of bolts, preferable made in hardened steel and indicated in
FIGS. 2 and 3, by example bolt 264. Each bolt has a male-threaded
portion, shown on 264, at 266 and extending through corresponding
fixing holes 120-130, in 102; 154-164, in 148; 180-190, in 178 and
224-234, in 222. Bolt heads, indicated on 262, by example bolt head
268, locate in corresponding counter bores 236-246 of 222. Bolts,
exemplified by bolt 264, are each secured by threaded nut means, in
the form of nuts, preferably made of hardened steel and indicated
in FIGS. 2 and 3, by example nut 270, such that each is located in
corresponding counter bores 236-246 of frame cap 222. Nuts and
bolts are evenly tightened using a torque wrench.
79. Adjustable biasing pre-loading means 400, are in the form of a
plurality of substantially cylindrical, accurately machined
components preferably made of stainless steel. A housing 402, is
conveniently 37 mm in maximum diameter and of 65 mm height. A lower
portion 404, is conveniently, 15 mm in height with a diameter of 30
mm and is adapted by the provision of male-threaded means 406, to
threadedly engage from above with female thread means 258, of axial
circular hole 256, of frame cap 222. An axial lower counter bore
408, having a diameter conveniently of 20 mm, extends upwards from
lower surface 410, of 402, conveniently for 30 mm. A through hole
412, conveniently of 4 mm diameter, extends radially through the
wall 414, of lower male-threaded portion 404, of 402, into lower
counter bore 408, thereof and is adapted by the provision of
female-threaded means 416, throughout its length. An axial upper
counter bore 418, having substantially the same diameter as 408,
extends downwards from upper surface 420, of 402, for 15 mm. A
further and larger axial upper counter bore 422, in 420,
conveniently having a diameter of 27 mm and a depth of 10 mm, is
adapted by the provision of female thread means 424. Upper counter
bores 418; 422 and lower counter bore 408, are united by an axial
circular through hole 426, conveniently 10 mm in diameter.
80. An adjusting axial screw 428, conveniently has a length of 60
mm. A lower portion 430, has a diameter conveniently of 10 mm and a
length of 25 mm and is adapted by the provision of male thread
means 432, conveniently extending 22 mm, the remaining 3 mm,
indicated at 434, being plain. An upper plain portion 436, of 428,
conveniently has a diameter of 10 mm and a length of 30 mm. Upper,
plain portion 436, is provided with radial plain blind hole 438,
conveniently situated half way along its length and having a
diameter of 4 mm and extending 2 mm in depth. A central plain
portion 440, of 428, conveniently has a diameter of 20 mm and a
length of 5 mm.
81. A locking collar 442, is conveniently 27 mm in diameter and 10
mm in depth and is adapted on its outer surface 444, by the
provision of male thread means 446, to provide threaded engagement
means for receival by the female-threaded means 424, of counter
bore 422, of pre-load adjuster housing 402. Locking collar 442, is
further adapted by the provision of an axial through hole 448,
having a diameter sufficient to provide sliding engagement means
for the upper plain portion 436, of pre-load adjusting axial screw
428. Upper surface 450, is provided with a pair of counter bores
452; 454, disposed on the same diameter of 442, either side of and
equidistant from axis 456. Counter bores 452; 454, each
conveniently have a diameter of 3 mm and extend to a depth of 5 mm
and constitute tightening drive means for 442.
82. A knob 458, conveniently has a diameter of 60 mm and a maximum
depth of 20 mm. An upper portion 460, having the full diameter of
458, is conveniently 10 mm deep and has a knurled outer surface
462. A lower portion 464, conveniently has a diameter of 30 mm.
Knob 458, is adapted by the provision of an axial through hole 466,
having a diameter such as to provide, in co-operation with upper
plain portion 436, of load adjusting axial screw 428, easy push-fit
means. Knob 458, is further adapted by the provision of a slot 468,
conveniently 12 mm wide and extending through the full depth of
upper portion 460. Slot 468, has a semi-circular inner margin 470,
adapted by the provision of a small central hole 472, extending
radially into central through hole 466 and is further adapted by
the provision of female thread means 474, for the receival of a
grub screw 476. Grub screw 476, constitutes engagement locking
means between pre-load adjuster knob 458 and pre-load adjusting
axial screw 428.
83. An actuator 478, has external dimensions such that, in
cooperation with lower counter bore 408, of pre-load adjuster
housing 402, these elements provide fully engageable sliding
push-fit means. The upper surface 480, of 478, is provided with an
axial counter bore 482, conveniently having a depth of 23 mm,
adapted by the provision of internal female thread means 484, for
threaded engagement with lower male-threaded portion 430, of
pre-load adjusting axial screw 428. Counter bore 482, is further
adapted, at its lower end 486, by the provision of countersink
means, the position of which is indicated by arrow 488, in FIG. 2.
Countersink means 488, are for the receival of the head 490, of
countersunk screw means 492, conveniently of 4 mm diameter. Lower
surface 494, of 478, is provided with a central counter bore 496,
conveniently having a diameter of 6 mm and which communicates with
counter bore 482. External surface 498, of 478, is provided with
vertical, parallel sided groove 500, of such a width and depth as
to co-operate, in the assembled condition, with a blunt-nosed grub
screw 502, provided with partial male thread means 504, received
threadedly in and through hole 412, of housing 402, to provide
engagement guiding and rotation restraining means. It will now be
understood that with 502, engaged in 500, the latter will be
prevented from rotating when knob 458, is used to turn adjusting
axial screw 428 and that instead, actuator 478, will be driven up
or down, within lower counter bore 408, of housing 402, according
to the direction in which knob 458, is turned. It will be further
understood that this arrangement together with the threaded
engagement between internal female thread means 484, of counter
bore 482, of 478, with male-threaded portion 430, of pre-load
adjusting axial screw 428, constitute drive means for setting or
altering biasing pre-load.
84. A push rod 506, has an upper portion 508, so sized as to
co-operate, slidingly, both with counter bore 496, in lower surface
494, of actuator 478 and also with force sensor 700, hereinafter
described. Upper portion 508, of 506, is adapted by the provision
of a central counter bore 510, provided with female thread means
512, for the threaded receival of countersunk screw means 492,
which is introduced down upper counter bore 482, actuator 478. Push
rod 506 also has a lower portion 514, having a greater diameter
than 508. Lower surface 516, of 514, is provided with a small
peripheral chamfer 518 and is adapted by the provision of a
central, substantially hemispherical, recess 520, providing
receival means and compression, locating means for a ball bearing
522, conveniently having a diameter of 6 mm. Ball bearing 522,
provides part of means for transmitting and centering loads applied
to perfusion chamber means 800, hereinafter described. Upper
surface 524, of lower portion 514, provides shoulder bearing means
for push rod 506, against force sensor 700.
85. Translator loading means are in the form of a ceramic stacked
piezo translator 600, incorporating multiple strain gauge means and
having a maximum translational range of 40 .mu.m. Piezo translator
600, may conveniently be a commercial product such as a P-239.30,
incorporating an optional module P-177.10, having four strain
gauges (Physik Instrumente GmbH, Germany). Piezo translator 600, is
substantially cylindrical in form and has a base portion 602, for
which circular recess 114, in upper surface 106, of frame base 102,
provides gentle push-fit location means. Translator base portion
602, has a central counter bore 604, which is provided with female
thread means 606. Securing means between 600 and 102, comprise
countersunk male-threaded screw means 608, having a male-threaded
shank 610. Shank 610, passes through central hole 108, in frame
base 102 and engages, threadedly, with 606. Countersunk head 612,
of 608, is tightened against countersunk recess of 110, of under
surface 112, of 102.
86. Piezo translator 600, has a main body portion 614, having such
a diameter that it engages central hole 152, of lower frame section
148, with a sliding push-fit. Main body portion 614, is of such a
height that, when fully engaged in assembled metal frame housing
100, its upper surface 616, is substantially level with the bottom
of fenestration slot 194, of upper frame section 178. Upper surface
616, of piezo translator 600, is adapted by the provision of an
axial counter bore 618, adapted by the provision of female thread
means 620, for the threaded receival of a small, substantially
cylindrical drive pin 622, having a shank 624, provided with male
thread means 626. Drive pin 622, may conveniently be a commercial
product such as a P-239.95 (Physik Instrumente GmbH, Germany)
described by the manufacturer as a `top piece`. Body portion 628,
of drive pin 622, constitutes boss mounting means for an upper
portion 630, which is substantially hemispherical and has a
diameter conveniently the same as ball bearing 522, of push rod
506. It is to be understood that 630, provides the remainder of
means, hereinbefore described with reference to ball bearing 522,
means for transmitting and centering loads applied to perfusion
chamber means 800, hereinafter described.
87. When assembled to metal frame housing 100, connecting means for
piezo translator 600, in the form of cable means are so disposed as
to lie in parallel sided recesses 116, in upper surface 106, of
frame base 102 and 166, in lower surface 170, of 148, providing
aperture means constituting access means for cable means to
connectors 632 and 634. Piezo translator 600, is provided, via
connectors 632 and 634, with electronic control means 900, all
having performance and operating characteristics appropriate to the
functional applications of the instant system best seen and
described hereinafter, with reference to FIG. 6.
88. Force sensor means are in the form of a quartz crystal force
sensor 700, housed in an extremely rigid, precision welded,
substantially cylindrical housing 702, having dimensions,
conveniently, of outside diameter 14.5 mm, inner diameter 6.5 mm
and height 8 mm. Force sensor 700, may conveniently be a commercial
product such as a model 9011A device (Kistler AG, Winterthur,
Switzerland). Force sensor 700, has an axial through hole 704, for
smooth sliding engagement with upper portion 508, of push rod 506.
In the assembled condition, lower surface 706, of housing 702,
located on 508, bears directly upon upper surface 524, of lower
portion 514, which provides shoulder bearing means for push rod
506. Upper surface 708, of 702, is borne upon by lower surface 494,
of actuator 478. During use of the instant system, compression of
702, between 506 and 478, provides reactive force means for
operation of force sensor 700. Force sensor 700, is provided with
connecting means in the form of cable means which, in the assembled
condition, pass through parallel sided slot 210, of upper frame
section 178. Parallel sided slot 210, constitutes aperture means in
178, for access means for cable means to cable connector 710. Cable
connecting means extend from cable connector 710, to electronic
control means 900, best seen in and hereinafter described with
reference to, FIG. 6.
89. Perfusion chamber means 800, best seen in FIGS. 4, 5 and 6,
comprise three principal, substantially cylindrical components, a
bottom bearing cap 802 and a piston 804, both machined in suitable
grades of stainless steel and a perfusion 806, preferably machined
from a block of suitable biologically inert, non-leaching plastics,
preferably polycarbonate and provided with connection means for
perfusion fluid. The choice of plastics is very important since
many materials leach substances which are toxic or lethal to cells.
Minimally different embodiments may be made in which 806, may be
made from suitably biologically inert stainless steels. The
preferred embodiment confers the advantage, by virtue of
fenestration slot 194, in upper frame section 178, of observability
of perfusion during use of system 10.
90. Bottom bearing cap 802, has a lower portion 808, conveniently
having a radius of 25 mm and a depth of 5 mm. Lower surface 810, of
808, is adapted by the provision of an axial, substantially
hemispherical, recess 812, providing receival means and
compression, locating and centering means for upper hemispherical
portion 622, of drive pin 618, of piezo translator 600. Upper
portion 814, of 802, conveniently has a diameter of 15 mm and a
depth of 5 mm. The upper surfaces 816; 818, of 808 and 814,
respectively, are precision ground to flatness and finished by
polishing. Upper portion 814, of 802, is provided with male thread
means 820.
91. Piston 804, conveniently has a diameter of 12 mm and a height
of 8 mm. Upper surface 822, of 804, is adapted by the provision of
an axial, substantially hemispherical, recess 824, providing
receival means and compression, locating and centering means for
ball bearing 522, of push rod 506. Lower surface 826, of piston
804, is precision ground to flatness and finished by polishing.
Circumferential wall 828, of 804, is adapted by the provision of an
upper and a lower annular groove, indicated at 830 and 832,
respectively and mutually disposed apart in a parallel manner to
upper and lower surfaces 822 and 826, respectively. Circumferential
wall 828, is finished by micro-fine machining and polishing. Lower
annular groove 832, constitutes an adaptation for the receival of a
sealing means in the form of an `O` ring 834, made from inert
resilient sealing material, preferably neoprene.
92. Perfusion chamber body 806, conveniently has an outer diameter
of 25 mm and a height of 15 mm. Lower surface 836, of 806, is
provided with an axial counter bore 838, of such a depth and
diameter as to provide, in conjunction with suitable female thread
means 840, receival and sealing means for upper portion 814, of
bottom bearing cap 802. Lower surface 836, of 806, is precision
ground to flatness and is adapted to cooperate with upper surface
816, of lower portion 808, of bottom bearing cap 802 and suitable
biologically inert non-leaching adhesive means, to provide
additional sealing means between the two components. Lower surface
836, of 806, is further adapted by the provision of a machined
annular channel 842, conveniently having a semi-circular
cross-section of 2.5 mm diameter and lying on a pitch circle,
conveniently 19 mm in diameter and indicated with center line at
844, in FIG. 5. Two small counter bores 846 and 848, having the
same diameter as 842, are each centered on the intersection of a
diameter of 842 and center line 844, one either side of central
axis 850. Small counter bore 846, extends to a depth somewhat less
than that of the main lower axial counter bore 838. A radial hole
852, having the same diameter as counter bore 846, extends through
wall 854, of 806, so as to meet 846, at right angles, forming
substantially continuous lumen means. An upper axial counter bore
856, is so sized and adapted that it may receive an explanted
trabecular bone sample 858, prepared as hereinafter described, as a
sliding fit and also may engage the greater part of piston 804, as
an easy push fit. Lower annular groove 832, of piston 804 and `O`
ring 834, located therein, are particularly included in the
engagement between piston 804 and perfusion chamber body 806. Inner
surface 860, of upper axial counter bore 856, is adapted by the
provision of a parallel sided, annular channel, 862, conveniently 4
mm wide and about 2 mm deep. The position of 862, is such that it
substantially surrounds the outer margin or wall 864, of explanted
trabecular bone sample 858, when this is inserted in 856, of 806.
Second small counter bore 850, extends upwards into 806, to a depth
somewhat greater than the depth of main lower axial counter bore
838, such that it terminates at a point substantially level with
the mid point of the height of inserted explanted bone sample 858.
A diameter hole 866, having the same diameter as small counter
bores 846; 848, extends through wall 854, of 806, following the
line of radial hole 852, hereinbefore described, intersecting
annular channel 862 and also intersecting second small counter bore
848, at right angles, at the limit of its depth, forming further
substantially continuous lumen means. Spigots 868 and 870,
conveniently fabricated in stainless steel, are adapted to engage,
respectively, with radial hole 852 and that portion 872, of
diameter hole 866, which lies on the same side of 806, as 852, with
a forced, sealing, press-fit. A small cylindrical plug 874, of the
same material as 806, is adapted to engage with diameter hole 866,
on the opposite side to radial hole 852, with a press-fit in
conjunction with suitable biologically inert, non-leaching,
adhesive means to provide sealing means between the two components.
Plug 874, is of such a length that it extends up to but does not
substantially encroach into, second small counter bore 848.
93. Annular channel 842, of lower surface 836, of perfusion chamber
body 806, in cooperation with the upper surface 816, of lower
portion 808, of bottom bearing cap 802 and adhesive sealing means;
small counter bores 846 and 848, annular groove 862, of upper
counter bore 856, radial hole 852 and plugged portion 876, of
diameter hole 866, constitute substantially continuous fluid
pathway means for perfusing fluid. Spigots 868 and 870, constitute
connecting means for suitable tube means in the form of tubes
conveniently made of silicone rubber and indicated at 878 and 880,
for delivering perfusing fluid to the assembled perfusion chamber
and for collecting effluent from it for monitoring and
analysis.
94. It will now be understood that the substantially cylindrical
elements of frame means, in co-operation with adjustable biasing
pre-loading means having force sensor means, translator loading
means having electronic control means and connection means and
perfusion chamber means having connecting means for perfusing
fluid, as hereinbefore described, constitute instrumented perfusion
and axial press means for the perfusion and mechanical loading of
an explanted trabecular bone sample.
Performance and Function of Piezo Translator. Force Sensor and
Electronic Control Means
95. With particular reference to FIG. 6, as well as continuing
reference to FIGS. 2 and 3, electronics control means 900,
comprises a rack 902, in which are mounted several major
components. A 220 V AC power supply 904, also houses a display
module 906, which gives readings of high voltage or compression
values. A high voltage amplifier 908, provides the high operating
voltage (-1000 V) to drive piezo translator 600. A controller
module 910, includes a compression signal amplifier (not seen) and
regulator loop (not seen), to force piezo translator 600, to a
required position, within its translational range of 40 .mu.m,
corresponding to a given value of high voltage or compression. This
range is satisfactory for applications involving explanted bone
samples in the instant invention. Controller module 910, may
conveniently be a commercial product such as E-255 PZT Interface
and Controller (Physik Instrumente GmbH, Germany) which
incorporates a digital-analogue converter (DAC). Controller module
910, is linked by cable means (not seen) to low voltage driver and
controller 912, which may conveniently be a commercial product such
as LVPZ Driver and Controller E-809 (Physik Instrumente GmbH,
Germany). Controller module 910, is also linked by cable means (not
seen) to a force signal amplifier 914, which is a charge amplifier
for amplifying output from force sensor 700.
96. A personal computer 916, is equipped with a microprocessor of
at least 386 rating and is provided, internally, with an additional
plug-in card (not seen) which provides a control interface between
an analogue-digital converter (ADC) and DAC of 910. Cable 918,
connects the additional plug in card of 916, to a compression
signal amplifier output provided on low voltage driver and
controller 912. Cable 920, connects the additional plug in card of
916, to force signal amplifier 914. Cable 922, connects between a
communications port COM-1 (not seen) of 916, to the
digital-analogue converter of 910. Cable 924, connects between a
communications port COM-2, (not seen) of personal computer 916 and
a mouse 926. Personal computer 916, is also provided with a local
printer terminal port (not shown) for the connection of a suitable
printer (not shown). Personal computer 916, is also equipped with a
graphics monitor 928, functioning to EGA, VGA or higher standard to
which it is connected by a monitor cable 930. A suitable operating
system, such as DOS.TM. 3.2 or higher or Windows 3.1.TM. or Windows
95.TM., is installed on personal computer 916, together with custom
software which provides means for coordinating and calibrating the
electro-mechanical elements of the system as well as for
collecting, collating and displaying data and making calculations
thereon and displaying the results thereof.
97. In FIG. 6, frame means 100 and adjustable biasing preloading
means 400, are shown in side view to reveal connecting means for
cable means. Cables 932 and 934, connect high voltage amplifier
908, to piezo translator 600, at connectors 632 and 634,
respectively. Cable 936, connects force sensor 700, to controller
module 910, at connector 710.
98. Piezo translator 600, incorporates four strain gauges (not
seen) attached internally to the ceramic stack and arranged in a
full Wheatstone bridge circuit. The multiple strain gauge
arrangement may conveniently be in the form of an optional
commercial module P-177.10 (Physik Instrumente GmbH, Germany). In
conjunction with controller module 910, the bridge arrangement
allows a positioning accuracy of 0.2% of the nominal expansion of
piezo translator 600, to be achieved.
99. Force sensor 700, is a quartz crystal force sensor for
measuring dynamic and quasi-static forces, having a range of 15 kN,
a very high resolution of 0.01N under any pre-load, sensitivity of
z.apprxeq..sub.---4.3pC/N, modulus of 3.6 GPa and very high
rigidity of .apprxeq..sub.---1.8 kN/.mu.m. These characteristics
are satisfactory for applications involving explanted bone samples
in the instant invention.
Experiment 1
Calibration And Validation of Loading Elements Of The System
100. The instant system was validated and characterized by the
following methods:
101. a. determination of any errors in the system
102. b. identifying deformation accuracy, force application,
frequency of loading and calculation of E (Young's modulus) on
known materials and determining the physical compliance in the
system.
103. Calibration and validation was accomplished by comparing
nondestructive test results of the instant mechanical loading,
translator and force sensor elements of the instant system to
identical tests run on an MTS (Bionix) servohydraulic test machine.
Homogeneous materials, with moduli that span the expected range of
cancellous bone, were used (e.g. nylon, aluminum, teflon). These
materials had strain gauges applied to the vertical surfaces.
Strain was monitored on the same materials in both systems and the
results were compared. In addition, a precision extensometer was
placed between the platens on the MTS machine to provide specimen
deformation, as well as load and thus compute the strain. The
current required to achieve similar deformations, strains and loads
was recorded. The systems were compared with ramp and sinusoidal
wave forms. Hysteresis was noted together with time dependent
responses in the materials and test system. The system was the
materials and test system. The system was tested quasistatically
and at increasing frequencies up to 10 Hz (a functional limit for
the MTS system). The system was also tested throughout the range of
functional deformation rates available with the piezo crystal
translator. Similar specimens were taken to failure and the total
material behavior curves of the MTS system and the instant system
using the piezo crystal translator, were compared.
104. Correlation of a very high order was established, validating
the prospective deployment of the novel mechanical loading system,
in conjunction with the novel perfusion means of the instant
invention in explanted trabecular bone samples.
105. It was determined that the mechanical and electro-mechanical
elements of the instant system are capable of applying controlled
deformations, accurate to 200 nm, and applying forces of up to 800
N, at frequencies in the physiological range of up to 15 Hz and
maximum strain rates of between 10,000 .mu.E sec-1 and 50,000 .mu.E
sec-1. Young's modulus for trabecular bone varies from E=400 MPa
to, typically, E=1200 MPa in the adult pig.
Experiment 2
Perfusion
106. Preparation of Explanted Bone Samples
107. Features considered when determining the optimal size of the
bone sample for the instant system were:
108. 1. The practicality of using cow and pig bone samples in the
first instance and the feasibility of using human bone samples,
subsequently, having the same dimensions.
109. 2. The volume of bone and means for achieving adequate
perfusion through it.
110. 3. The amount of tissue which would be necessary to produce
the desired biochemical markers, in quantities sufficient to make
the required measurements.
111. The selection of pig and cow trabecular bone was based on
earlier studies by the present authors and other workers. In
particular the studies of an associate, Dr. Kit Mui Chiu whose
observations were recorded in a doctoral thesis at the University
of Wisconsin, presented in 1996 and entitled "The effect of camitin
dehydroepiandrosterone sulfate on young senescent osteoblast-like
cells", were important. In these studies pig osteoblasts were kept
viable, in culture, for 68 days. Careful consideration of these
findings and other prior art, led to the conclusion that, in a
suitable novel system, providing continuous perfusion means and
suitable loading means, viability might be maintained for a
worthwhile period of study which could be up to 14 days or
more.
112. The bone cores for our experiments were obtained from the
trabecular bone of distal ulnae or femurs of 2 to 3 year old cows
or femora or humeri of 2 to 3 year old pigs. Under sterile
conditions throughout, the limb is first excised and then a 2.5
cm.times.2.5 cm.times.4.5 cm (proximo-distal dimension) sample of
trabecular bone is cut from the central region of the proximal or
distal metaphysis of the bone with a surgical hand saw and the
proximal end is marked. The specimen is visually inspected under a
dissecting microscope at 10X to assure that no growth plate scars
are present.
113. Following isolation of the gross sample, 6.times.5 mm thick
subspecimens are cut from it, under running sterile PBS at room
temperature, using a band saw having a diamond tipped blade (Exact,
Germany). Six bone core disks are then drilled in the
proximo-distal direction, under sterile PBS, from each of the
sub-specimens, using a 10 mm or 12 mm diamond tipped keyhole drill
(Exact, Germany). The 6 bone cores from each 5 mm sub-specimen are
randomized.
114. Each bone core disk is immediately marked on the proximal
surface and placed in serum free medium for 20 minutes prior to
placing it in the perfusion chamber apparatus of the present
invention. Each sample is placed in the perfusion chamber such that
it will be loaded from proximal to distal. The sample is then
allowed 48 hours in the perfusion chamber in order to adapt, prior
to any intervention. Thus, all experiments conducted using this
protocol extend over 16 days, comprising 2 days for core adaptation
and 14 days of intervention.
115. Using this method, bone disks may be cut with the necessary
extreme precision to a flatness of .+-.0.2 microns and a
parallelism of .+-.0.1 microns. The dimensions were selected in
order to produce samples of a practical size for perfusion and in
order to supply between 3,500,000-11,000,000 cells, based on an
estimate of 10,000-20,000 cells per millimeter cube of bone (Mundy
1990; Parfitt 1983), which was considered sufficient to provide an
adequate yield of markers for study.
116. Disk samples of trabecular bone, prepared according to the
method immediately hereinbefore described, were perfused and
maintained with circulating medium. The medium used was Ham's F10
containing 1%-5% FCS, 2 mg glutamine, streptomycin and penicillin G
at 50,000U/1, vitamin C 10 mg/ml, 0.12 g/l of NaHCO.sub.3 and 10 mM
Hepes. The medium was maintained at 37.degree. C. and a pH of
7.1-7.3 for the total 14 days of the perfusion. The perfusion rate
was 0.1 ml/minute and the medium was perfused using a 12 channel
pump (Ismatec). The medium was changed at 12 hour intervals. The
pH, PCO.sub.2 and PO.sub.2 were measured hourly for the first 5
hours then 12 hourly thereafter.
117. A series of FCS batches was tested for biological effect on
the trabecular bone cores using alkaline phosphatase, cell
viability and osteocalcin production. A sufficient quantity was
retained from the most suitable batch of FCS to maintain a
reproducible medium for the performance of the experimental program
contemplated by the investigators. It is important to note that
frozen FCS (-80.degree. C.) has a maximum storage life of 3
years.
118. The flow rate through the explanted bone sample must be fast
enough to maintain cell viability but not so fast that a shear
force greater than 3 dynes/cm.sup.2 is induced. When the flow rate
is too slow, cells are inadequately oxygenated and lactate builds
up. When the flow rate is too fast, the shear force, itself, causes
increases in PGE2 and IGF-1. The flow rate of 0.1 ml/minute
selected was determined as optimal by prior experiment with
differing flow rates in order to provide sufficient effluent medium
volume for sampling and analysis of PGE2, cAMP and IGF-1 and also
in order to maintain PO.sub.2 and PCO.sub.2. PO.sub.2 was monitored
at each flow rate in these experiments to ensure adequate
oxygenation of the cells in the bone explant perfusion/loading
system.
Experiment 3
Injury Response Time (Establishment of Rest Period)
119. Cells placed in culture require time to adapt to their changed
environment and this time period varied with the type of cell and
the type of research we conducted. The necessary rest period for
explanted trabecular bone samples in the instant system was
determined. In our preliminary experiments, trabecular bone samples
were perfused with culture medium plus 10% FCS. Under these
conditions, IGF-1 increased from 5 to 14 hours and appeared to
decline in the 15th hour, at the time the experiment was
terminated.
120. Based on those preliminary data, a rest period of at least 48
hours was accepted as appropriate for IGF-1 to return to baseline
level, before any intervention (mechanical loading, hormones, etc)
was imposed on the bone explant organ culture. However, the
adaptation time required was then documented over a series of full
24 hour periods to determine when the cells had recovered from the
surgical trauma in order to determine the stable baseline condition
from which intervention could be started.
121. Studies have provided the equivalent data for each of the
second messengers, IGF-1 and certain other growth factors.
Experiments To Investigate Cell Viability And Biomarkers Under
Varying Conditions
122. Experiments were designed to investigate a variety of load
magnitudes and frequencies, growth factors and applied active
substances.
123. Specifically it was considered necessary to provide for the
investigation of markers including the release of prostaglandin E2
(PGE2), cyclic-AMP (cAMP), inositol 1,4,5-trisphosphate (IP3) and
insulin-like growth factor (IGF-1), in the perfusion effluent from
explanted bone samples. These entities were to be studied during
responses to stimuli including varying conditions of mechanical
load and further, under the influence of biochemical stimulus with
hormones, growth factors or drug substances.
124. The markers, produced by stimuli, immediately hereinbefore
described, are important in the regulation of bone modeling and
remodeling, at every age and nutritional level, in the adaptive
response of the skeleton to such challenges.
Cell Viability
125. a. Cell viability in samples of cow trabecular bone was
determined at rest, at a maintenance load (the load at which the
bone neither atrophies or hypertrophies) and at microstrains which
ranged from 500 to 5000.
126. b. Oxygen utilization of the bone explant perfusion/loading
model was determined at rest, at varied flow rates, at a
maintenance load and at microstrains ranging from 500 to 5000.
127. Having established the flow rate limits for the instant
perfusion chamber, experiments were conducted to verify cell
viability. Percent viability at various time intervals was assessed
in order to determine the number of cells still alive at any given
time.
128. Two methods are commonly used to assess cell viability in cell
culture. Alamar Blue Assay indicates succinate dehydrogenase
activity in the cells. It incorporates an oxidation-reduction
indicator that causes the Redox indicator to change from oxidized
(non-fluorescent, blue) form to reduced (fluorescent, red) form in
response to the cell metabolism in the culture medium. This assay
is a general indicator of the metabolic function of the system but
it does not allow quantification of cell viability, that is,
calculating the percentage and distribution of viable cells. The
use of MTT (sigma, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide), does permit measurement of the number or
percentage of live cells. In this assay, active mitochondrial
dehydrogenases convert the water soluble MTT into an insoluble
purple formazan by cleaving the tetrazolium ring. Cells with intact
mitochondria will show a dark reddish/purple stain when tissue is
viewed under a light microscope. Thus, mitochondrial staining is
indicative of live cell function at the time MTT is administered.
Since MTT is toxic to the cells, it can be used only at the end of
an experiment. We used this method to determine the viability of
cells after 14 days. At the end of perfusion runs, samples were
perfused with MTT (30 mg/ml) for 6 hours then stabilized to
4.degree. and sectioned to between 100 and 180 microns thick, using
a diamond saw (EXACT, Germany), in order that viability throughout
the sample could be investigated.
129. Somewhat less than 5% of the cells in the cow trabecular bone
core, taken from the distal ulnae of 24 month old cows, died
because of the surgical extraction and disk preparation procedure
and of the remaining cells, more than 95% remained active
throughout the 14 day studies.
Histological Assessment
130. Before loading the explanted bone samples, it was necessary to
verify whole explant tissue viability over time.
131. MTT (30 mg/ml) was used as a cell viability marker. In one
series of our cell viability experiments, four 14-day runs were
conducted. In the first two runs, sample cores were processed with
MTT every two days after a baseline core had been run for 8 hours
and then removed. All cores were compared to the baseline core. 12
bone cores were perfused at a rate of 0.1 ml/minute. A baseline
positive control viability sample was obtained 8 hours after the
start of the experiment by perfusing a core with 30 mg/ml of MTT
for 6 hours. The baseline sample and all other samples were
perfused with serum free medium for the first 24 hours in order to
collect 1 ml of medium for IGF-1 and PGE2 analysis. At the end of
the 6 hour perfusion with MTT, the bone core was maintained at
4.degree. C. at which time sections were cut using a diamond band
saw. Sample sections were cut to a thickness between 100 and 180
microns in order that cell viability could be determined throughout
the sample.
132. The base line sample (8 hours) was used as a positive control
for viability. The number of viable cells in the 14 day sample
showed no difference when compared to the positive control which
had 95% viable cells. The sample sections taken from the top to the
bottom of the sample demonstrated no difference in the number of
cells showing the presence of MTT and the centers of all of the
cores were found to be fully stained. However, there were a few
trabecular areas that demonstrated cell death with no MTT present.
It was felt that the diamond tipped keyhole drill used to excise
the bone samples may have resulted in some damage in the outer few
trabecular segments, resulting in tissue damage and cell death. It
is clear from our results in this study that the bone cores
obtained using this method and using the perfusion chamber
apparatus of the present invention, can be maintained in a viable
for 14 days.
Bioassay
133. Medium from the perfusion chamber to be used for the bioassay
was sampled at varied time intervals according to the biomarker we
chose to investigate. Pig osteoblasts obtained from Crenshaw (U of
WI Madison) were characterized by alkaline phosphatase, collagen
type 1 and the ability to produce bone nodules. Cells were plated
out in 96-well, Nunclon, cell-culture grade, assay plates at a
density of 45,000 cells per cm.sup.2 in 100 ml per well of one of
the following media:
134. Dulbecco's MEM
135. Dulbecco's BGJ (as used for the organ culture)
136. Ham's F-10
137. HI growth enhancement medium (Gibco)
138. The specific medium was chosen through trial and error
depending on the best response of the markers we investigated (e.g.
good for alkaline phosphatase and collagen). To the selected basic
medium was added 10% FCS, ascorbic acid-2phosphate at 5 mg/l plus
L-glutamine (or the stable analogue) for the first 24 hours. For
the assay, the FCS is reduced from 10% to 1%, for 24 hours before
the medium is replaced with medium from the perfusion culture. The
control is unused medium used for the perfusion culture. Eight
replicate wells were used for each sampling point. The cells were
grown for 48 hours and then assayed for growth using the MTT method
to measure succinate dehydrogenase activity. The MTT methods were
calibrated against a known number of cells in a similar growth
state; this was a control experiment using an agar plate and
counting the cells with a cell counter. The presence of growth
factors released from the perfusion culture were then assayed.
Loading
139. In loading experiments, the maximum compressive strain applied
was 0.5% (5,000 .mu.E) at 1 Hz sine wave. This equates to 20 .mu.m
compression at up to 50,000 .mu.E sec.sup.-1.
140. The bone explant perfusion/loading system we have developed
has allowed us to assess bone cellular response to specific stimuli
under controlled conditions. An understanding of these mechanisms
allows for their manipulation and in turn may lead to the possible
alleviation or control of osteoporosis and other skeletal changes
which result in the loss of skeletal integrity and function. The
instant system provides investigators, for the first time, with
effective means to study morphological changes in the skeletal
tissue. In addition, the instant system permits the study of the
physiological responses of the bone tissue under clearly defined
and specified experimental conditions that can be set up to reflect
the human activities of daily living and life style. The present
invention also for the first time, permits the study of human bone
biopsies in a controlled environment. This will not only enable
investigators to identify morphologic changes that occur with
different bone disease but will also permit the determination of
the physiologic and possibly genetic determinants in such
conditions.
141. It will be apparent to those skilled in the art that numerous
modifications or changes may be made without departing from the
spirit or the scope of either the present invention or its method
of use. Thus the invention is only limited by the following
claims.
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