Testing before death and that all humans

Testing and modeling the changes
that occur in human intervertebral discs can be a difficult and time consuming
process.  Reviewing four different
studies testing ex-vivo vertebral specimens from both human cadavers and
mice/rats, we will note the associated positives and negatives. The four
experiments mentioned use dynamic and static methods of compressive and tensile
loading.   

In the human
spine, intervertebral discs separate the vertebral discs and help protect the
spine, brain, and nerves by absorbing energy transferred through the
spine.  The annulus fibrosus and the
nucleus pulposus make-up the intervertebral discs.  The annulus fibrosus surrounds the nucleus
and is strong structure made up of collagen, water and proteoglycans.  Like the annulus fibrosus, the nucleus
pulposus is also made of collagen, water, and proteoglycans, though the nucleus
pulposus contains a higher concentration of water and proteoglycans.  The nucleus pulposus differs from the annulus
fibrosus due to its gel-like composition. 
The gel-like structure of the nucleus pulposus resists compression
forces in the spine (Bridwell,
Rodts, 2017).

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               The
first study was a static compressive ex-vivo spinal experiment was reported on
by Keller, Spengler, and Hansson.  The
first study researched involved 9 specimens of human lumbar spinal sections.
The study used the T11-L5 vertebrae from 9 humans.   The spinal sections were collected from
humans varying in age, however the average age was 65 years old.  It is important to note that humans spent
less than 3 weeks in bed before death and that all humans experienced an acute
illness before passing.  The spinal
specimens were kept frozen until testing, prior to testing the specimens spent
between 16 and 20 hours at room temperature to allow for thawing.  After thawing, the specimens were “sectioned
into spinal motion segments consisting of two intact vertebrae and an
intervening disc,” stated Keller,
Spengler, and Hansson (1987, p.468).  
After segmenting into motion segments, a saw was then used to section
the bodies of the motion segment to ensure that the superior and inferior
surfaces had parallel motion segments. 
This process helps to ensure reduce the amount of “nonuniformities in
the distribution of the axial load” stated by Keller, Spengler, and Hansson (1987, p. 469).   Before loading, measurements were taken
using dial calipers.  The measurements
were taken towards the front and rear of the outer regions of the vertebral
body.  The static compressive loads were
then applied to the 18 specimens.  The
compressive loads for each specimen was determined by the respective human’s
weight above each respective vertebrae. 
The compressive load was applied at a constant rate for a time of 30
minutes.    It is easy to see that one of
the pitfalls of this sort of testing is the time investment involved.  To complete 18 tests at 30 minutes a piece,
the testing would easily take over 9 hours after you include the time involved
with setting up the machine for each test. 
The measured displacement during the experiment was measured with a
linear variable differential transformer. 
The axial displacement under the compressive load was plotted as a
function of time (Keller,
Spengler, Hansson, 1987).

The data
resulting from the experiment clearly proves that intervertebral disc has both
viscous and elastic properties.  During
the loading of the vertebrae plastic flow occurs and when unloaded the
vertebrae almost fully returns to its original state as long as the loading is
not greater than the elastic limit.  The
mathematical solution to the changes occurring in the vertebrae under loading
and the recovery, can be mechanically modeled with springs and dashpots.  Both springs and dashpots must be used due to
the viscoelastic nature of the vertebrae. 
The simplest mechanical model that is used is the Kelvin body.    Looking at the figure below, a spring and
dashpot in parallel is then connected with another spring in series, make up
the Kelvin body. 

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. I. Creep analysis during static
compressive loading. Journal of Orthopaedic Research,5(4),
467-478. Copyright 1987)

The three mechanical components
help to illustrate the reaction of the vertebrae.  Spring E2 shown above allows for immediate
deformation proportional to a sudden loading and the dashpot shown will provide
an infinite resistance.  As the load is
maintained, the resistance provided by the dashpot will decrease, which will
cause the loading on the spring E1 to become greater.  As you can see from the graph below, the rate
of displacement with respect to time decreases as the displacement
increases.  The displacement eventually
reaches equilibrium. 

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. I. Creep analysis during static
compressive loading. Journal of Orthopaedic Research,5(4),
467-478. Copyright 1987)

Keller, Spengler, and Hansson (1987, p.
471) stated, “to analyze the creep data of the human lumbar motion
segments used in this study, a linearization method based on a Taylor series
expansion of the strain solution for the three-parameter model was carried out
on the experimental strain-time data.”  Strain
is the ratio between deformation and the average disc height.  The three-parameter model discussed above produced
a theoretical strain-time response that closely matched the response produced
from the experimental data.  The
resulting average percent error between the strain-time response produced from
the model and the strain-time response produced from the experiment was less
than 0.74% (Keller, Spengler,
Hansson, 1987).   

               Following testing, all 18 motion
segments were examined to determine how the compressive load effected the
intervertebral discs.  The segments could
be classified in four different groups, normal appearance or grade I, slight
degeneration or grade II, moderate degeneration or grade III, and severe
degeneration or grade IV.  The testing
resulted in 9 grade I motion segments, 4 grade II segments, and 5 grade III
segments.  There were no motion segments
with grade IV degeneration (Keller,
Spengler, Hansson, 1987). 

While this method
does illustrate the degeneration of intervertebral disc, it is also very time
consuming.  Constant static compressive
loads are not as common as a more dynamic form of testing.  Even a student wearing a heavy backpack while
walking to and from class has a dynamic or cyclical loading pattern.  Due to needing a form of testing that can be
done under brief time constraints, this form of testing would not be ideal (Keller, Spengler, Hansson, 1987).

               A creep-recovery method of
testing was conducted by Jeffrey MacLean, Julia Owen, and James Latridis.  The testing required the use of 44 deceased
rats, and from those rats four different groups of spinal sections were
used.  Group 1 was a motion segment, made
up of an intervertebral disc between two vertebrae, 11 samples were collected.  Group 2 was 11 samples of permeable platens,
and group 3 was 11 samples of impermeable platens.  Group 4 was 11 samples of single vertebras (Maclean, Owen, Iatridis, 2007).

               The
first three groups of spine were subjected to a series of loadings.  Part 1 of the loading applied 0.04 MPA of
compressive force for 4 hours, part 2 included 0.2MPA creep for a period of
4h.  The 3rd part was the 1st
recovery portion, 0.4MPA was applied for 6h. Part 4 included 1 MPa at creep for
another 4 hours and finally the 5th part was a recovery portion with
0.04 MPa applied for 6h.  The stresses
applied during testing was based on the disc cylindrical cross-section.  The 4th group of specimens only
went through part 1 and 4 of the testing.

 
(Note. Reprinted from Role of endplates in contributing to compression
behaviors of motion segments and intervertebral discs. Journal of
Biomechanics,40(1), 55-63. Copyright 2007)

Where, ‘t’ is
time, ‘d0’ is instantaneous displacement, ‘d?’
is the equilibrium displacement, ‘?’ is the time constant, and ‘?’
is the stretch parameter.  The variables
‘?’
and ‘?’
are the average creep time constant and the ” ‘slope’ of the creep curve”
stated Maclean, Owen, and Latridis (2007, p.4), respectively. (Maclean, Owen, Iatridis, 2007)

               The following charts show the
displacement with respect to time for a typical explant (graph a) and a typical
motion segment (graph b).  You can
clearly see in graph B that some segments show slight transient recovery past
the given 6 hour recovery time frame. 
Additional testing where 24 hours of recovery time was given, only very
little amounts of additional recovery time was needed 
(Note. Reprinted from Role of endplates in contributing to compression
behaviors of motion segments and intervertebral discs. Journal of
Biomechanics,40(1), 55-63. Copyright 2007)

Noting the table below, we see the
permanent deformations that occurred during the testing.

(Note. Reprinted from Role of
endplates in contributing to compression behaviors of motion segments and intervertebral
discs. Journal
of Biomechanics,40(1), 55-63. Copyright 2007)

Below, we see the experimental data
and stretched exponential model for the disc explant (graph a) and the motion
segments (graph b).  The graph shows the
relationship between displacement and time.

(Note. Reprinted from Role of
endplates in contributing to compression behaviors of motion segments and
intervertebral discs. Journal of Biomechanics,40(1),
55-63. Copyright 2007)

The following
graphs show the respective Tau (?) and Beta (?) values for creep (graph A
and B) and recovery (graph C and D) (Maclean,
Owen, Iatridis, 2007).

(Note. Reprinted from Role of
endplates in contributing to compression behaviors of motion segments and
intervertebral discs. Journal of Biomechanics,40(1),
55-63. Copyright 2007)

               In conclusion, the authors listed
four major findings from the testing. 
The first being, that even under low compressive forces, the vertebrae
and intervertebral discs showed significant amounts of deformation.  “Second, we found that cutting the annular
fibers that anchor into the vertebrae to create explant specimens had minimal
impact on equilibrium deformations of disc explants as compared to disc
deformations occurring in the motion segments provided that vertebral deformations
were accounted for”, stated Maclean,
Owen, and Latridis (2007, p. 5).   Third, the creep and recovery time of
explants was lengthened by reducing the permeability of the porous platen.  Finally, even under both high and low axial
compressive loading, the disc explants and motion segments had permanent
deformation.  It is easy to note that
creep-recovery testing allows a user to observe many changes in intervertebral
discs, although it is important to note that this testing is not ideal for a
short time-frame (Maclean, Owen,
Iatridis, 2007).

A dynamic loading
method can also be used to study how the intervertebral disc is affected under
loading conditions.  The particular
experiment used specimens from 17 lumbar spines.  These specimens were previously used in the
static compressive loading experiment described above.  The specimens were exposed to sinusoidal
loading that had a frequency of 1/2 Hz.  
A load-displacement graph plotted the resulting data.  The testing on each individual specimen was
continued for until fracturing occurred or 1000 loading cycles was
completed.  Again it is easy to notice
the possibility of lengthy testing.  If
all 17 specimens were to undergo 1000 cycles, testing time would take close to
9.5 hours to complete.  Fracturing could
be determined during the testing process by either an audible “popping” and/or
axial displacement would increase suddenly (Keller, Holm, Hansson, Spengler,1987).

Post-testing
examination of the specimens allowed for the classification of the specimens
based on levels of degeneration.  All 17 motion
segments were examined, 9 of the segments were classified as normal (grade I),
2 segments were described as being slightly degenerated (grade II), and 7 were
described as being moderately degenerated (grade III).  There were no motion segments that had severe
degeneration.  The motion segments had
only few degrees or less of flexion and there was no noticeable binding or
loading in the motion segments.  Some
disc bulge was noted in the motion segments (Keller, Holm, Hansson, Spengler,1987).

The relationship
between axial stiffness and the loading cycle in Grade 1 motion segments can
easily be observed by looking at the chart below.

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. II. Fatigue strength during
dynamic compressive loading. Journal of Orthopaedic Research,5(4),
479-487. Copyright 1987)

From the chart above, you can
clearly see a rapid increase in stiffness in the early stages of cycling, the
stiffness then increases at a less rapid rate until a state of
equilibrium.  It is also noted that
stiffness decreases rapidly around the time of fracturing.  The stiffness in grade II and III classified
discs was noted to reach an equilibrium state more quickly.  The second graph to be observed, illustrates
the hysteresis shown by the intervertebral disc during the experiment.  The graph shows the hysteresis loops at a
state of equilibrium and during fracturing of grade I,II, and III discs.  The loops were plotted on a deformation-load
chart (Keller, Holm, Hansson,
Spengler,1987).  

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. II. Fatigue strength during
dynamic compressive loading. Journal of Orthopaedic Research,5(4),
479-487. Copyright 1987)

               Compressive
stiffness coefficients were calculated from the resulting experimental
data.  The average compressive stiffness
coefficients at the first cycle and at equilibrium were 1.37 (SD 0.48) MN/m and
3.04 (SD 0.94) MN/m, respectively.  Disc
height and area was factored in to normalize the stiffness value, this was done
to account for discrepancies in disc morphology.  The following chart shows both the measured
axial stiffness and normalized axial stiffness at grade I, II, and III disc
degeneration (Keller, Holm,
Hansson, Spengler,1987). 

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. II. Fatigue strength during
dynamic compressive loading. Journal of Orthopaedic Research,5(4),
479-487. Copyright 1987)

               It
is also worth noting the resulting fatigue strength shown by the intervertebral
discs during the testing.  The stress
applied to the discs during the testing ranged from 0.091 MPa-2.848 MPa with
the mean applied stress being 1.416 MPa. 
The max fatigue strength shown during the testing was around 950
cycles.  There was one motion segment
that did not fracture for all 1000 loading cycles.  The relationship between disc degeneration
and fatigue strength.  You can see the
relationship in the following graph.  It
appears that fatigue strength is reduced at higher levels of degeneration.  You can also see how cadaver age also affects
fatigue strength.  The graph mentions two
age groups, group 1 has a mean age of 52 years and group 2 has an average age
of 78.3 years (Keller, Holm,
Hansson, Spengler,1987).

(Note. Reprinted from
Mechanical behavior of the human lumbar spine. II. Fatigue strength during
dynamic compressive loading. Journal of Orthopaedic Research,5(4),
479-487. Copyright 1987)

               Finally,
looking at the fracturing in the motion segments, we notice that three
different types of fracturing occurred. 
The three types mentioned by Keller, Holm, Hansson, and Spengler
(1987,p.484) are “the node of Schmorl and Junghanns (type 1), central endplate
(type II), and a crush or burst fracture (type III).  Crush or burst fracturing occurred in all
segments that fractured in the first cycle. 
Type I fracturing occurred in segments that had only normal or slight
disc degeneration, whereas type II fracturing occurred in segments that had
moderated disc degeneration.  The segment
that did not fracture, was listed as a type IV. 
Following testing, the segments were observed for lesions and
circumferential clefts and lesions were observed in the outer fibers of the
annulus fibrosus. However, in degenerated disc, there were minor
circumferential clefts found in the central annulus (Keller, Holm, Hansson, Spengler,1987). 

               The
resulting data, charts, and graphs from the dynamically loaded cadavers allows
us to analyze the changes that occur in the intervertebral discs.  We can easily see the effect of degenerated
discs on the disc’s ability to withstand sinusoidal loading.  We can conclude that the stiffness of
intervertebral discs become less stiff as the disc degenerates and the
stiffness increases rapidly during the beginning of load cycling until an
equilibrium state is met.  The
experimental results also show how the type of fracturing seems to be dependent
on the amount of cycles the motion segment was subjected to.  This form is of testing appears to illustrate
how the intervertebral discs change under loading, this experiment has the
possibility to take a lengthy amount of time, though not as lengthy as the
static-compressive loading experiment discussed previously (Keller, Holm, Hansson, Spengler,1987).

               Looking at a final testing
method, described by authors Merceron, Mangiavini, Robling, Wilson, Giaccia,
Shapiro, and Rosebud, involved the dynamic biomechanical testing of ex-vivo
mice vertebras.   The experiment was
designed to test the mechanical effects of Foxa2-Cre-induced HIF-1?
deletion on intervertebral discs.  The
experiment required the 4th and 5th lumbar segment from
12 dead mice, 6 of the lumbar segments was from a control group and the other 6
were from a mutated group.  The test
specimens were subjected to an oscillating loading cycle that applied both 1.5
N of force in compression and tension. 
The loading cycle consisted of 19 sinusoidal waveforms.  During the loading, the frequency was increased,
starting at 0.5 Hz and was then increased by 0.5 or 1 Hz after each cycle.  The max frequency reached during the testing
was 15 Hz.  All 12 lumbar segment
underwent two complete rounds of the dynamic testing.  Displacement, force output, and command
signal were all collected from the experimental testing.  Two resulting equations for force (f) and
displacement (L) were created, shown below (Merceron, Mangiavini, Robling, Wilson, Giaccia, Shapiro,
Risbud, 2014).

 

(Note.
Reprinted from Loss of HIF-1? in the Notochord Results in Cell Death and
Complete Disappearance of the Nucleus Pulposus. PLoS ONE,9(10). Copyright
2014)

               The
particular study conducts a second set of sinusoidal testing mimicking the
first round, except this time, the control and mutant groups of mice are only 4
months old.  Below shows the resulting
graphs that illustrate the applied force sine wave with respect to time for
both the control and mutant group, and the other two charts show the phase
shift angle and energy dissipation both with respect to the applied
frequency. 

(Note. Reprinted from Loss of
HIF-1? in the Notochord Results in Cell Death and Complete Disappearance of the
Nucleus Pulposus. PLoS ONE,9(10). Copyright 2014)

It can be noted
that in the control group, as the sine wave frequency increases the phase shift
angle tends to increase at a slight rate. 
Inversely, in both the control and mutant group the energy dissipation
decreases as the sine wave frequency is increased (Merceron, Mangiavini, Robling, Wilson, Giaccia, Shapiro,
Risbud, 2014).

Due
to the nature and goals of the above experiment, little was mentioned about the
disc degeneration of the intervertebral post-testing.  One of the noticeable positives to this form
of testing is the lack of time required compared to the other two forms of
testing.  One of the key difference and
possible positive to this form of testing compared to the other mentioned type
of dynamic testing is the use of both tension and compressive forces.  My own personal experiences while driving
sprint cars (a form auto racing, that usually races on dirt ovals) is that my
spine is can be subjected to both compressive and tension forces during normal
racing conditions and during serious accidents. 
Knowing this, this particular form of testing could be particularly
useful to understand the changes in the intervertebral discs of humans that
spend large amounts of time driving or operating vehicles in rough or non-ideal
terrain, such as racecar drivers (Merceron,
Mangiavini, Robling, Wilson, Giaccia, Shapiro, Risbud, 2014).

               Simply
deciding which form of testing is the best or most accurately represents
changes in intervertebral discs is not an easy or possible task due to the
loading differences illustrated by the three above methods.  The choice should ultimately be made based on
what form of human activity you are trying to replicate.  In physical activities such as weight lifting,
in particular squats, the static compressive loading described by Keller, Spengler, and Hansson would be
appropriate.  The creep-recovery
testing described by Maclean,
Owen, and Latridis provides an example of how the intervertebral discs can
permanently deform under loading, even after periods of rest, the caveat to
this testing is the length of time require.   Whereas, the dynamic loading described by Hansson, Keller, and Spengler may
be more comparable to common day-to-day activities exhibited by a human.  While the final form of testing mentioned
might be applicable when modeling activities that alternate between tensile and
compressive forces on the spine.  After
the extensive research and time spent comparing and contrasting testing
methods, I believe the best option to test the changes that occur in the
intervertebral discs with strict time constraints would be the dynamic loading
described by Hansson, Keller, and
Spengler.  The dynamic compressive
loading by sinusoidal wave balanced the needs testing under short time
constraints, while still showing various types of degeneration in
intervertebral discs.  The dynamic
loading method mentioned, is presented more in real-life activities than static
compressive loading. 

 

 

 

 

 

 

 

Bridwell, K., MD, & Rodts, M., DNP.
(17, February 23). Intervertebral Discs. Retrieved December 10, 2017, from
https://www.spineuniverse.com/anatomy/intervertebral-discs

Hansson, T. H., Keller, T. S., & Spengler, D. M. (1987).
Mechanical behavior of the human lumbar spine. II. Fatigue strength during
dynamic compressive loading. Journal
of Orthopaedic Research,5(4),
479-487. doi:10.1002/jor.1100050403

Keller, T. S., Spengler, D. M., & Hansson, T. H. (1987).
Mechanical behavior of the human lumbar spine. I. Creep analysis during static
compressive loading. Journal of
Orthopaedic Research,5(4),
467-478. doi:10.1002/jor.1100050402

Maclean, J. J., Owen, J. P., & Iatridis, J. C. (2007).
Role of endplates in contributing to compression behaviors of motion segments
and intervertebral discs. Journal of
Biomechanics,40(1),
55-63. doi:10.1016/j.jbiomech.2005.11.013

Merceron, C.,
Mangiavini, L., Robling, A., Wilson, T. L., Giaccia, A. J., Shapiro, I. M., . .
. Risbud, M. V. (2014). Loss of HIF-1? in the Notochord Results in Cell Death
and Complete Disappearance of the Nucleus Pulposus. PLoS ONE,9(10).
doi:10.1371/journal.pone.