NASA Technical Reports Server (NTRS) 19920020154: In vitro measurement of nucleus pulposus swelling pressure: A new technique for studies of spinal adaptation to gravity

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NASA Technical Memorandum 103853
In Vitro Measurement of Nucleus
Pulposus Swelling Pressure:
A New Technique for Studies
of Spinal Adaptation to Gravity
A. R. Hargens, Ames Research Center, Moffett Field, California
M. G. Glover, M. M. Mahmood, S. Gott, and S. R. Garfin, University of California,
San Diego, California
R. Ballard and G. Murthy, Ames Research Center, Moffett Field, California
M. D. Brown, University of Miami, Miami, Florida
Janua_ 1992
N/k.qA
NationalAeronautics and
Space Administration
Ames Research Center
MoffettField,California94035-1000

In Vitro Measurement of Nucleus Pulposus Swelling Pressure: A New
Technique for Studies of Spinal Adaptation to Gravity
A. R. HARGENS, M. G. GLOVER,* M. M. MAHMOOD,* S. GOTT,* S. R. GARFIN,* R. BALLARD, G. MURTHY, AND
M. D. BROWNt
Ames Research Center
Summary tions. This method requires several (3 to 5) tissue slices
of 20 to 100 mg each for a single measurement, and 24 to
Swelling of the intervertebral disc nucleus pulposus is 48 hours for them to equilibrate with the polyethylene
altered by posture and gravity. We have designed and
glycol solutions into which they are placed. In this paper
tested a new osmometer for in vitro determination of a new osmometer is described that allows determinations
nucleus pulposus swelling pressure. The functional prin- using a reduced amount of nucleus pulposus tissue (a sin-
ciple of the osmometer involves compressing asample of gle 5to 10 mg sample) and a 10- to 30-minute equilibra-
nucleus pulposus with nitrogen gas until saline pressure tion time. The accuracy and precision of the osmometer
gradients across a 0.45 micron Millipore filter are elimi- measurements were evaluated using the equilibrium dial-
nated. Swelling pressures of both pooled dog and pooled ysis technique as the standard for comparison.
pig lumbar disc nucleus puiposus were measured on the
new osmometer and compared to swelling pressures
Materials and Methods
determined using the equilibrium dialysis technique. The
osmometer measured swelling pressures comparable to
Osmometer Technique
those obtained by the dialysis technique. This osmometer
provides arapid, direct, and accurate measurement of The osmometer consists of an acrylic chamber which
swelling pressure of the nucleus pulposus. houses a semipermeable Millipore 0.45-micron, pore size
membrane with a watertight seal (fig. 1). The membrane
is secured against a fixed volume chamber filled with
Introduction
normal saline that adjoins the diaphragm of a Hewlett-
Packard 1280C low volume displacement pressure trans-
The mechanism of disc degeneration and its role in the
ducer calibrated to+100 mm Hg. Five to 10 mg speci-
etiology of lower back pain is unclear. However, there is
mens of nucleus pulposus are placed in the sample well at
much indirect evidence implicating the discs of the lum-
room temperature on the side of the membrane opposite
bar spine in a significant percentage of lower back pain.
the saline column. A threaded cap to the sample well
The swelling pressure of the disc nucleus pulposus may
chamber has connections to a nitrogen source for com-
be an important factor indisc degeneration and/or disc
pressing the disc sample. Pressure is recorded to the near-
herniation. This swelling pressure iscaused by the high
est 0.I psi on aHeise precision pressure gauge (50 psi
negative charge of the proteoglycans, creating a negative
maximum) connected in series to the nitrogen source and
fluid pressure across the anulus or end plate such that
osmometer. Transmembrane pressure gradients are con-
fluid is taken up until the swelling pressure is counter-
tinuously measured by a strip-chart recorder.
balanced by the external load on the disc. This pressure
maintains disc height and is proportional to the fixed The curve in figure 2 illustrates the mechanics of measur-
charge density and proteoglycan concentration within the ing swelling pressure using the osmometer. After cali-
nucleus puiposus. During disc herniation, fluid may be bration, the initial pressure on the saline column, as
taken up by this tissue and which causes compression of recorded by the pressure transducer, is zero because no
neurovascular structures outside of the disc. Current data pressure gradient exists across the membrane. First a
on swelling pressures have been obtained by using the syringe attached to a flexible tube and then tissue paper
technique of equilibrium dialysis, where the swelling are used to remove all the saline from the surface of the
pressure is interpolated within the osmotic pressures of Millipore membrane and the sample well. This results in
polyethylene glycol solutions of different concentra- a plateau of negative pressure between -10 and
-20 mm Hg. The nucleus pulposus is placed in the sample
*University of California, San Diego, California. well on top of the membrane opposite the saline column.
tUniversity of Miami, Miami, Florida. The sample begins to swell as it imbibes saline from
i i
i
N2
Torecorder
Figure 1. New compression-type osmometer formeasuring swellingpressure ofnucleuspulposus. Left:P/egg/as
osmometer mounted on stand withnitrogengas inletattop. Right:Cross-sect/on ofosmometer with sealing ofmembrane
(M) by crimp rings (CR) on thescrew-down Plex/g/as plate. Nucleuspulpceus isplaced inthesample we//(SW) on top
ofthemembrane. Pressure gradients across themembrane are transmittedby thesaline fluidcolumn (S) and monitored
bythepressure transducer (PT) fittedtightlyto thebottomofthe osmometer byan O-ring (OR).
®
+100
® / t
|e °
EV
0
E
o -lOO
® ®
Figure2. Procedure formeasuring swellingpressure ofnucleuspulposus.
1)Theosmorneter iscalibrated for+100,O,and-lO0 mm Hg.
2) Thezero point ischecked again with afew microllters ofsaline atop themembrane.
3) Syringe attached toflexibletube and Klmwlpesare used toremove all saline, which results inaplateau between -10
to-20 mm Hg.
4)Nucleus pu/posus sample isplaced onthemembrane.
5) Thepree_ure isallowed togo tothebottomofthescale and nitrogenpressure is applied. Thezero isovershot and the
nucleus pulposus sample adjuststothecompression.
6)If equilibriumisnot reached, a negative pressure reading resultsand thepressure isallowed again togo tothebottom
ofthescale.
7) Theabove steps 5)and 6)are repeated untilequilibriumisestablished.
8) Equilibriumisdefined as a zeropressure.transducer recording forat least60 seconds.
beneath the membrane, which causes pressure below the wet sample and then drying to constant weight in a
membrane to fall precipitously. The pressure is allowed 125°C drying chamber. The mixed nucleus pulposus of
to fall to the bottom of the scale (below -100 mm Hg). each species was divided into samples for osmometer and
To counteract the negative pressure in the saline column, equilibrium dialysis determinations of swelling pres-
nitrogen pressure is applied to the sample of nucleus pul- sure. Five separate samples were tested and compared.
posus until zero on the transducer is "overshot" and the Additionally, samples of the pooled pig nucleus were
nucleus pulposus' matrix is, therefore, compressed. Dur- measured repeatedly ten times to test the reproducibility
ing this time the pressure transducer senses total tissue of the osmometer.
pressure (combination of pressures from fluid and solid
components of the sample). After a short period, the
Comparison of Osmometer to Equilibrium Dialysis
pressure measured by the transducer begins to fall again.
Technique
Nitrogen pressure is increased and these steps are repeated
until the pressure stabilizes at zero for 60 seconds. This Sections of cut 2000-MW (molecular weight) pore size
"stable" reading is recorded as the equilibrium swelling dialysis tubing were prepared by tying off one end with
pressure. At this point, the applied nitrogen pressure bal- 2-0 nylon monofilament suture and were then weighed. A
ances the swelling pressure of the nucleus putposus and small sample of nucleus pulposus was then placed into
nullifies the pressure gradients across the Millipore the tube and the tube was weighed again (the sample
filter. approximated 47% of the total weight). The change in
weight represented the weight of the enclosed nucleus
pulposus. The open end was then tied with 2-0 nylon
Study Method
suture after evacuation of air, and the composite weighed
Lumbar spines were obtained from afreshly-killed 35-kg after trimming the ends (fig. 4). A size matched control
mongrel dog and two 35-kg mini-pigs and frozen at was also prepared, differing only in that itcontained no
-70°C. When needed, the frozen spine was cut trans- nucleus pulposus. Both were placed in molal solutions of
versely through each vertebral body on aband saw. The 8000 MW polyethylene glycol (PEG) at the following
posterior elements were also removed. The intact discs concentrations: 0.05, 0.1,0.15, 0.2, 0.3, 0.4, 0.6, and
and end-plates were then partially thawed in a 100% 0.8 gPEG/ml water. The manufacturer of our 8000 MW
humidity environment. The spine segments were incised PEG (Sigma, Inc.) indicated that their previously-
and nucleus material was extracted while still partially prepared 6000 MW PEG was actually 8000 MW based
frozen (fig. 3). For the dog, five levels were pooled and upon recent, better techniques for measuring MW. Hence,
mixed thoroughly. Six levels were collected and pooled we used the same virial coefficients for our PEG as those
for the pig. Following mixing, wet and dry weight provided by Edmond and Ogston (1968). The PEG solu-
determinations were made for each species by weighing a tions containing the dialysis tubes were placed in the
nucleus
Curpeuttlipnogsusof
Figure 3. The intervertebral disc plus the adjacent end-plates are isolated on a band saw and the frozen anulus incised.
The frozen nucleus pulposus is removed with acurette and returned to the freezer at -70 °Celsius.
DIALYSISSAC 400
35O
Nucleuspulposus
3OO
Polyethyleneglycol
10o
Figure 4. The sample of nucleus pulposus isplaced in the
2000-MW cutoffdialysis tubing andall excess air is o
expelled. Weights are determined after each step of
.50I" (a), I _ I ,
preparation. The composite is placed in a solution of
0 .2 .4 .6 .8
polyethylene glycol for 48 hours.
refrigerator at 4°C (277°K) for 48 hours. One hour after 200-
removal from refrigeration, each tube was carefully
blotted dry and weighed until five consecutive weighings
150
differed by less than 0.005 grams. The mean of the five
weights was calculated and used for further comparisons.
Calculations of the equilibrium swelling pressure con- | 100
+e-
sisted of determining the pre- and post-PEG solution
exposure weights of the nucleus pulposus minus water 5O
content due only to the dialysis tube itself (determined
from the size matched control). From this the percent
0
weight change in the nucleus was calculated for each PEG
I i I l I i " I
concentration and the results plotted (fig, 5). A log- (b)
arithmic curve fitting procedure was employed and the -50 I
0 .05 .10 .15 .20
X-intercepts were calculated for the pig and dog. These
ConcentrationofPEGbath(9/mlwater)
values were used in the virial expansion equations for
macromolecular solutions to determine the osmotic Figure 5. Thepercent change inweight of the
pressures of the PEG solution that produced no net dog(a) andpig(b) nucleus pulposus when allowed to
swell in thedialysissac immersed in PEG isplotted
weight change in the nucleus pulposus. This osmotic
against theconcentration ofthe PEG bath.
pressure was taken as equal to the swelling pressure of
the nucleus pulposus. The swelling pressure values
obtained with equilibrium dialysis were compared to the The ten samples, measured repeatedly in the osmometer,
mean values obtained with the new osmometer. yielded amean swelling pressure of 0.53 + 0.01 atm with
a variance of 0.0005 for pig nucleus (table 2). This demon-
strates the high reproducibility of the osmometer. An
Results
individual swelling pressure measurement in the osmom-
Mean pressures of 2.3:1:0.3 atm and 0.5 + 0.02 atm were eter required 10to 30 minutes which depended upon the
obtained for the dog and pig nucleus pulposus, respec- magnitude of swelling pressure for a given sample.
tively, as measured with the osmometer (table 1and
fig. 6). This agreed well with the equilibrium dialysis
Discussion
technique which gave pressures of 2.03 atm and 0.49 atm
for the dog and pig pooled nucleus pulposus specimens, Proteoglycans are the major nonaqueous components of
respectively (both corrected to a room temperature of the nucleus pulposus of the intervertebral disc. They are
296°K). In figure 6, values are presented as means +S.E. complex structures with variable molecular weights up
for osmometer results and calculated values for the equi- to 108 daltons (average 6x105) (Bushell et al., 1977)
librium dialysis determination. The dog disc material had whose components include chondroitin sulphate, keratin
a lower water content than that of the pig (81% vs. 90%, sulphate, hyaluronic acid, core protein, and link protein
respectively).
Table I. Swelling pressures of dog and pig nucleus pulposus using osmometer
Dog disc
Trial No. Swelling pressure (atm)
2.1
2.8
2.9
1.2
2.4
Mean (+ S.E.) 2.3 + 0.3
Water Content = 81%
Pig Disc
Trial No. Swelling pressure (atm)
0.5
0.5
0.4
0.5
0.5
Mean (+ S.E.) 0.5 (+ 0.02)
Water Content = 90%
3.0 n
I_] Osmomeler
2.5-- Equilibriumdialysis
vm 2.0--
19
r, 1.5 --
i.
OI
_= 1.0 --
t_ 0.5-- iI //// I M
I f/-.//,!
0 I /i//
Dogsamples Pigsamples
Figure 6. Results of theosmometer technique and equilibrium dialysis technique for measuring the swelfing pressures of
the dog and pig nucleus pulposus.
(Muir, 1979). The negative charges associated with chon- Tanford, 1961). Much work in this area has been per-
droitin sulphate and keratin sulphate are largely respon- formed by Maroudas and Urban, who found that equilib-
sible for the swelling pressure generated by the nucleus rium hydration studies correlate best with the effective
pulposus. These negative charges maintain hydration of osmotic pressure of nucleus pulposus material
the nucleus pulposus in the face of high external loads. (Maroudas and Urban, 1980). Equilibrium dialysis, as a
Also, proteoglycan macromolecules are closely packed, method for determining swelling pressures, is based on
and thus impart a low hydraulic permeability and retard the concept of calculating osmotic pressure of simple
fluid loss from the nucleus pulposus (Maroudas and macromolecular solutions and then using these solutions
Urban, 1980). as a standard against which water loss or gain of nucleus
pulposus samples contained within dialysis tubing can be
Measurement of swelling pressure (Ps) has been a diffi-
compared. Determination of the concentration of a refer-
cult task because of practical and conceptual problems
ence solution that will cause no net gain or loss of water
(Maroudas and Urban, 1980) and many methods have been
allows one to interpolate the swelling pressure of the
employed (Charnley, 1952; Nachemson, Elfstrom, 1970;
Table 2. Reproducibility of osmometer using pig nucleus pulposus
'Trial No. Swelling pressure (atm)
1 0.59
2 0.54
3 0.51
4 0.52
5 0.53
6 0.53
7 0.53
8 0.53
9 0.52
10 0.53
Mean +S.E. 0.53 +0.01 atm
Variance 0.00048
material enclosed in the dialysis tube. Calculation of the samples needed. Because we used the virial coefficients
reference solution pressures is based on the virial expan- derived by Edmond and Ogston (1968), we were obliged
sion for osmotic pressure (re): to dilute our PEG solutions in a manner identical to
theirs (i.e., by dilution with water) in order for us to
rt = RTc/MW(! + Ac/MW + ...)
employ their virial coefficients correctly. Maroudas and
where Urban diluted their PEG solutions with 1.5 and
0.15 M NaC! and found relatively large differences in
R Universal gas constant
osmotic pressure between these two media (Maroudas
T Absolute temperature (°K) and Urban, 1980). However, at ionic strengths lower than
0.15 M, differences between the two media would be
c Grams of macromolecule/gram solvent
expected to be less. Despite the differences between the
MW Molecular weight of macromolecule external media in contact with the nucleus puiposus sam-
ples across the membrane, we obtained similar results for
A Second virial coefficient (moles 2)
the direct osmometric and indirect equilibrium dialysis
The second virial coefficient consists of three indepen- techniques. Whenever possible, it is better to measure a
dent terms: the Donnan term, the excluded volume term, colligative property of a solution or gel directly; this can
and the interaction between the charged macromolecule be done with our new osmometric technique. Despite the
and electrolyte plus a self-attraction term of the elec- differences in the direct (osmometer) and indirect (equi-
trolyte itself. The Donnan term is a measure of the librium dialysis) techniques in terms of bathing media,
imbalances in the concentrations of diffusible elec- membrane pore size, and errors inherent in weighing
trolytes resulting from the fixed charge groups on the small tubes and using empirically-derived virial coef-
macromolecule. In other words, the polyelectrolytes on ficients, we find the agreement between the two tech-
one side of the membrane co-exist with a relatively niques quite remarkable.
lower concentration of small ions of like charge and a
Our purpose was to find a method for measuring the
relatively higher concentration of small ions of opposite
swelling pressure of the nucleus pulposus that gave rapid
charge (Tanford, 1961).
results, used small samples, and provided a directly mea-
We used equilibrium dialysis swelling pressures as a sured rather than calculated or interpolated pressure. As
standard against which to compare our results. We also demonstrated, we have accomplished this with an
employed polyethylene glycol (PEG) as a reference osmometer, modified so that nitrogen gas is used to com-
macromolecule (Maroudas and Urban, 1980). A PEG of press and directly cancel the swelling pressure of the
8000 MW was used rather than Maroudas and Urban's nucleus pulposus sample. The nitrogen pressure over-
20,000 MW because we were able to obtain a more uni- comes problems associated with high negative pressures
form average MW with the 8000 MW PEG. The equilib- in the saline column beneath the membrane, namely cavi-
rium dialysis technique is useful, reliable, and accurate. tation due to the large swelling pressure of the nucleus
However, this technique has practical limitations such as pulposus. This also obviates the problem of measuring
time required for a single determination, its indirect high negative pressures with standard, readily-available
approach, and relatively large amount and number of pressure transducers that are usually nonlinear and
inaccurabteelow-100mmHg.Whentheapplied
pressures were 2.3 atm versus 2.0 atm and the pig nucleus
nitrogepnressuarechieveesquilibriumacrostshe
pulposus was 0.5 atm versus 0.5 atm, respectively, for
membranteh,ereisnegligiblsealinemovemebnettween
osmometer and dialysis techniques. The osmometer tech-
thenucleupsulposussamplaendtheopposinsgaline
nique isreproducible (variance of 0.0005) and is much
columnH.oweveirn,approachienqguilibriumit,is faster than the equilibrium dialysis technique, requiring
possibltehatoursamplewseredilutedsomewhbayt one-half hour or less per observation compared to
salinetranspofrrtomthepressure-transdsuidceeorfthe 48 hours for equilibrium dialysis. Osmometry allows
membranTeh.iswouldi,neffectl,owertheswelling direct pressure measurements with a single 5 to 10 mg
pressuorefthesampleH.owevewr,ewereverycarefutlo sample as opposed to a calculated pressure determination
drythemembranweellbeforeapplyintghesampleW.e
requiring multiple 20 to 100 mg samples.
haveminimizeddryingandlossofmatrixconstituents
bythesamploefnucleupsulposubsy(I)minimizintghe
References
timetoreachequilibrium(10to30minutes()2,)usinga
deepandnarrowsamplweellinsideaclosecdhambearn, d
Bushell, G. R.; Ghosh, P.; Taylor, T. F. K.; and Akeson,
(3)checkinwgetanddryweightosfhomogenized
W. H.: Proteoglycan Chemistry of the Interver-
sample(ssomeplacedontheosmometaenrdothernsot)
tebral Disks. Clinical Orthopaedics and Related
andfindingthatthedry/wertatioisnotaffectebdythe
Research, vol. 129, 1977, pp. 115-122.
measuremeFnutr.thermorweh,ereacsonventional
osmometearlslowsalinetranspoirnttothesamplteoset Charnley, J.: The Imbibition of Fluid as a Cause of
upanegativperessuorenthetransducseidreofthe Herniation of the Nucleus Pulposus. The Lancet,
membranoeu,rosmometmerinimizesduchsaline Jan. 19, 1952, pp. 124-127.
transpobrtycompressioofnthesamplaetaleveel qual
Edmond, E.; and Ogston, A. G.: An Approach to the Study
toitsswellingpressurNe.ucleupsulposusswelling
of Phase Separation in Ternary Aqueous Solutions.
pressurmeeasuremeonbttsaineudsingtheosmometer
Biochem. J., vol. 109, 1968, pp. 569-576.
correlatewdellwiththeosmotipcressurvealues
determinebdyequilibriumdialysisofthecorresponding Maroudas, A.; and Urban, J. P. G.: Swelling Pressures of
sampleTs.heresultfsordogandpignucleuisnthisstudy Cartilaginous Tissues. Studies in Joint Disease I, A.
showgoodcorrelatioanndprovideevidencoefthe Maroudas and E. J. Holborow, eds., Pitman Medical
accuracayndreproducibiloityftheosmometienr Limited (Kent), 1980, pp. 87-116.
measurinsgwellingpressureTsh.eabsolutvealueosf
Muir, I. H. M.: Biochemistry of Articular Cartilage. In:
swellingpressurfeortheseanimalasrerelativelylow,
Adult Articular Cartilage, 2nd ed, M. A. R.
probablbyecaustheedogsandpigwereanesthetizfoerd
Freeman: Pitman Medical Publishing Co. Ltd,
severahlourspriortotissuecollectionT.heprecisioins
Tunbridge Wells, 1979, pp. 145-213.
demonstratbeydthelowcoefficienotfvariation(0.041)
foraserieosfmeasuremeonntpsortionosfapooled Nachemson, A.; and Elfstrom, G.: Intravital Dynamic
sample. Pressure Measurements in Lumbar Discs. A Study of
Common Movements, Maneuvers, and Exercises.
Scandinavian Journal of Rehabilitation Medicine
Conclusions Supplement No. 1, 1970, pp. 5-40.
The new osmometer accurately and directly measures the Tanford, C.: Physical Chemistry of Macromolecules,
swelling pressure of nucleus pulposus. The pressurized John Wiley and Sons, New York, 1961,
osmometer provides results that correlate well with the p.225.
equilibrium dialysis technique. Dog nucleus pulposus
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4. TIT'LE AND SUBTITLE ' 5. FUNDING NUMBERS '
In Vitro Measurement of Nucleus Pulposus Swelling Pressure: A
New Technique for Studies of Spinal Adaptation to Gravity
199 14 12 04
6. AUTHI_RiS)
A. R. Hargens, M. G. Glover,* M. M. Mahmood,* S. Gott,* S. R.
Garfin,* R. Ballard, G. Murthy, and M. D. Brown t
i i ii i
7. PdRFORI_IN(_"ORGANIZATION NAME(S) AND AODRESS(ES) 8. PERFORMING ORGANIZATION
REPORT NUMBER
Ames Research Center
Moffett Field, CA 94035-1000 A-91108
9. 'SPONSORI=N'G_I_IONITOR;hG 'AGENCY NAME(S) AND ADDRESS(ES') 10. SPONSORING/MONITORING'
AGENCY REPORT NUMBER
National Aeronautics and Space Administration
NASA TM- 103853
Washington, DC 20546-0001
i i i ii ii i i
11. SUPPLEMENTARY NOTES
Point of Contact: Alan Hargens, Ames Research Center, MS 239-11, Moffett Field, CA 94035-1000
[phone] or Frs 464-5746
*Un!versit_, of California rSan Die,or, California +Universit_ of Miami rMiami rFlorida
12b.DISTRIBUTIOCNODE
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Unclassified-Unlimited
Subject Category - 51
,13,1 ABSTiiRA•CiiT|1 (Maximumi 200 words) i ii ii i ii
Swelling of the intervertebral disc nucleus pulposus isaltered by posture and gravity. We have designed
and tested a new osmometer for invitro determination ofnucleus pulposus swelling pressure. The functional
principle of the osmometer involves compressing a sample of nucleus pulposus with nitrogen gas until saline
pressure gradients across a0.45 micron Millipore filter are eliminated. Swelling pressures of both pooled dog
and pooled pig lumbar disc nucleus pulposus were measured on the new osmometer and compared toswelling
pressures determined using the equilibrium dialysis technique. The osmometer measured swelling pressures
comparable to those obtained by the dialysis technique. This osmometer provides arapid, direct, and accurate
measurement of swelling pressure of the nucleus pulposus.
i i ii i ii • i ii i i lB. ;;U"BEROFPZa_'s
14. SUBJECT TERMS
8
Nucleus pulposus, Swelling pressure, Intervertebral disc, Spinal pain lB.'PRICECOt3E
A02
i i i i ii iiii L I i i l I i
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