Mechanical e ect of an induced ovine lumbar intervertebral …1.2 Anatomy of intervertebral disc The...

Mechanical e�ect of an induced

ovine lumbar intervertebral

disc degeneration model

Floor Lambers

Student no. 496002

BMTE 06.06

Supervisors:

Marije van der Werf

Keita Ito

Patrick Lezuo

February 7, 2006

Contents

1 Introduction 5

1.1 Low back pain . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2 Anatomy of intervertebral disc . . . . . . . . . . . . . . . . . 61.3 Disc degeneration . . . . . . . . . . . . . . . . . . . . . . . . . 81.4 Causes of disc degeneration . . . . . . . . . . . . . . . . . . . 81.5 Stress pro�lometry . . . . . . . . . . . . . . . . . . . . . . . . 101.6 Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Methods 13

2.1 Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Defect in endplate . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Loading pro�le . . . . . . . . . . . . . . . . . . . . . . . . . . 142.4 Needle Pressure Transducer . . . . . . . . . . . . . . . . . . . 17

2.4.1 Preliminary tests . . . . . . . . . . . . . . . . . . . . . 172.4.2 Main tests . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.5 Data acquisition . . . . . . . . . . . . . . . . . . . . . . . . . 182.6 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3 Results 20

3.1 Preliminary tests . . . . . . . . . . . . . . . . . . . . . . . . . 203.2 Main tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.2.1 Comparison of before and after pressure pro�les . . . 213.2.2 Comparison of before and after nucleus values . . . . . 213.2.3 Comparison of horizontal and vertical pressure pro�les 233.2.4 Absolute Relative normalization . . . . . . . . . . . . 24

4 Discussion 25

A Speci�cation of the spines and motion segments 32

B Calibration of needle pressure transducer 38

1

List of Figures

1.1 A) The spine and location of the lumbar spine. Figure from Bogduk [14].

B) The structure of the disc with the alternating lamellae. Figure from

Errington et al. [22]. . . . . . . . . . . . . . . . . . . . . . . . . . 61.2 Pictures of degenerated discs A) Grade 1 B) Grade 2 C) Grade 3 D)

Grade 4. Figure from Thompson [48]. . . . . . . . . . . . . . . . . . 91.3 Pressure pro�les of di�erently graded discs. A) A healthy disc B) A

grade 2 degenerated disc C) A grade 3 degenerated disc. Adapted from

Adams [2]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1 Photo of embedded motion segment without ligaments, muscles and

transverse processes. . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Simpli�ed drawing of the embedding system. . . . . . . . . . . . . . 142.3 Images of the uoroscope while making the defect in the endplate. A)

The position is marked with a bone chisel. B) The position of the sawing

blade is checked. C) The position of the sawing blade is checked while

halfway in. D) A sawing blade in both defects. . . . . . . . . . . . . 152.4 A) A top-view picture of the defect in motion segment 28, L1. B) A

side-view picture of the defect in motion segment 28, L1. . . . . . . . . 152.5 The loading pro�le for the main tests. M1 = measurement 1, and M2 =

measurement 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.6 The needle pressure transducer inside the intervertebral disc, the indent

shows the position of the sensing element. . . . . . . . . . . . . . . . 182.7 Schematic of how the needle was connected to the input and 2 output

systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.1 The pressure pro�les of the preliminary tests of one disc. The range

shows the value of the �rst measurement plus or minus the accuracy of

the pressure transducer needle. A) The �rst and second horizontal mea-

surement before sawing. B) The �rst and second vertical measurement

before sawing. C) The �rst and second horizontal measurement after

sawing. D) The �rst and second vertical measurement after sawing. . . 203.2 Comparison of before and after pressure pro�les of experimental and

control discs. The lines are the mean values, and the error bars the

standard deviation. A) Experimental horizontal B) Control horizontal

C) Experimental vertical D) Control vertical . . . . . . . . . . . . . 213.3 The normalized pressure values in the nucleus. All values are mean val-

ues � standard deviation. A) Experimental horizontal B) Experimental

vertical C) Control horizontal D) Control vertical E)After horizontal

F)After vertical . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2

3.4 The pressure pro�les averaged over horizontal and vertical values. A)

Experimental B) Control . . . . . . . . . . . . . . . . . . . . . . . 233.5 Relative-absolute normalization. A)Horizontal B)Vertical . . . . . . . 24

4.1 The pressure pro�les averaged over horizontal and vertical values. A)With

all motion segments B)Without motion segment 41 . . . . . . . . . . 26

A.1 Loading pro�le of di�erent pilot tests for preconditioning. The time in

between the red arrows (x) is 15min, the time between the green arrows

(y) is 10 min, and the time in between the purple arrows (z) is 35 min. . 33

B.1 The calibration curve of the �rst needle pressure transducer. . . . . . . 38B.2 A) The bioreactor in which the second needle pressure transducer was

calibrated. B) Insertion of needle . . . . . . . . . . . . . . . . . . . 39B.3 The calibration curve of the second needle pressure transducer. A) Before

measurements B) After measurements . . . . . . . . . . . . . . . . 40

3

List of Tables

1.1 Approximate load on L3 disc in a person weighing 70 kg in di�erent body

positions. Adapted from Nachemson [36]. . . . . . . . . . . . . . . . 71.2 A grading scheme for intervertebral disc degeneration developed by Nachem-

son [35]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3.1 The change between before and after nucleus value. . . . . . . . . . . 223.2 The change between horizontal and vertical nucleus pressure. . . . . . 23

A.1 Overview of spines . . . . . . . . . . . . . . . . . . . . . . . . . . 32A.2 Overview of motion segments . . . . . . . . . . . . . . . . . . . . . 34A.2 Overview of motion segments (continued) . . . . . . . . . . . . . . . 35A.2 Overview of motion segments (continued) . . . . . . . . . . . . . . . 36A.2 Overview of motion segments (continued) . . . . . . . . . . . . . . . 37

4

Chapter 1

Introduction

1.1 Low back pain

Back pain is a major health problem. It has been estimated that nearly85% of the people will recall an episode of back pain by the age of 50 [13].The annual costs are estimated to be 20 billion in the USA [18]. Back painis often thought to be related to intervertebral disc degeneration [2]. Itseems likely that back pain originates from the combined e�ects of mechan-ical damage and biological response to that damage [5]. Because the outerannulus is innervated, pain can arise when intervertebral discs narrow, andstress concentrations occur on the outer annulus [24, 33]. Patients with se-vere back pain have been found to have an increase in sensory nerve �bers inthe endplates and underlying vertebral bodies [15]. It was speculated that achemotactic response to products of disc breakdown was responsible for theproliferation of vascularity and sensory nerves [15]. Under high compressiveloads the endplate bulges into the vertebral body [40], when these are moreinnervated this might cause pain. Bulging of the intervertebral disc can alsolead to disc herniation and pain [9]. Back pain can also arise from the trunkmuscles, when the exion-relaxion phenomenon is absent. Chronic low backpain patients activate the lumbar erector spinae persistently, probably inan attempt to stabilize injured spinal structures and to protect them fromfurther injury and pain [19]. Anders et al. [6] found a relationship betweenacute low back pain and disturbed muscular function as well. Low back paincan also originate from zygapophysial joint pain [42]. Similar to all these�ndings Adams and Dolan state that severe back pain most often arises fromintervertebral discs, apophyseal joints, and sacroiliac joints. They also statethat physical disruption of these structures is strongly but variably linkedto pain [3].

High risk factors for low back pain or sciatica are smoking, obesity, alco-hol consumption, parity, psychologic distress, and poor general health [25].Individual di�erences like age, sex, disc level, and degree of degenerationoften do not have signi�cant e�ect on mechanical behavior of the interverte-bral disc [37]. Heli�ovaara et al. [25] found that these individual di�erencesare not a determinant for low-back pain or sciatica either.

5

1.2 Anatomy of intervertebral disc

The human spine consists of 7 cervical, 12 thoracic, 5 lumbar, 5 fused sacraland 3{5 fused coccygeal vertebrae (see �gure 1.1 A). Intervertebral discs aresituated between the vertebrae of the spine. The endplate is the transitionbetween the vertebral body and the intervertebral disc.

A B

Figure 1.1: A) The spine and location of the lumbar spine. Figure from Bogduk [14].B) The structure of the disc with the alternating lamellae. Figure from Errington et al.[22].

The intervertebral discs consists of 3 anatomical regions [22]:

1. The nucleus pulposus; a gel like structure with proteoglycans thatmaintain the water content. Towards the outside of the disc the watercontent decreases. The cells are initially derived from the notochord,but in humans these cells are replaced with �brochondrocyte-like cellswith age [38]. The cells are chondrocyte-like and spherical with veryshort stumpy processes [22]. The major components of the nucleus areaggrecan and collagen II [22]. Type II collagen accounts for 80% ofthe total collagen content and type I collagen is absent [17].

2. The inner annulus �brosus; a �bro-cartilaginous structure. The in-ner annulus is the transition zone between the outer annulus and thenucleus [7, 30, 47]. It is a lamellar structure like the outer annulus,but contains more proteoglycans. The cells of the inner annulus are�brochondrocyte-like and round with processes shorter than seen inthe nucleus pulposus [22]. Among these cells are large bodied cellswith long processes with no directional preference between the lamel-lae [16]. Cells express mostly type II collagen [7].

3. The outer annulus �brosus; a highly �brous structure with highly ori-ented densely packed collagen �bril lamellae. The angle of the alter-nating lamellae is about 30� with respect to the disc, see �gure 1.1 B.The cells of the outer annulus are �broblast-like and elongated in the

6

direction of the collagen �brils. The processes of the fusiform shapedcells extend along and perpendicular to the major cell axis [22, 16].Between lamellae the cells have a attened, disc-shaped morphology[16]. Cells express mostly type I collagen [7]. Type I collagen accountsfor 80% of the total collagen content and type II collagen is absent[17].

Intervertebral discs are the largest avascular structures in the human body[2, 31]. The outer annulus is supplied by capillaries and this forms a minorroute of nutrient supply [11, 31]. The main nutritional pathway is throughdi�usion [27]. Small nutrients such as oxygen and glucose are supplied fromthe surrounding capillaries in the endplate to the disc's cells by di�usion [49].The permeability in the central regions is signi�cantly higher than in theperiphery [27, 31, 39]. Rates of oxygen consumption, lactic acid production,and glucose consumption are concentration dependent [12]. Although cellsonly form 1% of the disc by volume, their role is vital. The cells produce thematrix constituents and the agents responsible for matrix breakdown andare thus responsible for tissue composition and turnover [11]. It is suggestedthat the processes of the cells serve to sense mechanical strain [22] or toassist the nutritional pathways [16]. Collagens and proteoglycans are themain components of the intervertebral discs and important in load bearing.Collagens give the tissue its tensile strength and proteoglycans give thetissue sti�ness and resistance to compression [20, 43]. Because the nucleusis incompressible, compressive forces are converted to circumferential forcesthat act on the annulus [20]. In 1960, Nachemson showed that the nucleuspulposus behaves hydrostatically [35]. Later he showed the in uence ofdi�erent body positions on the load subjected to the L3 disc [34, 36] (table1.1). Under exion and torsion the load is increased the most.

Table 1.1: Approximate load on L3 disc in a person weighing 70 kg in di�erent bodypositions. Adapted from Nachemson [36].

Action Load on intervertebraldisc (lumbar) (N)

Supine, awake 250

Supine, arm exercise 500

Upright sitting, without support 700

Sitting with lumbar support, back rest inclina-tion 110�

400

Standing, at ease 500

Coughing 600

Forward bend 20� 600

Forward bend 40� 1000

Forward exed 20� and rotated 20� with 10 kg. 2100

Lifting 10 kg, back straight, knees bent 1700

Lifting 10 kg, back bent 1900

7

The most important functions of the intervertebral discs are [17]:

1. Stabilizing the spine by anchoring the vertebral bodies to each other.

2. Giving the spine exibility to allow movement.

3. Distributing and absorbing the loads that are applied to the spine.

1.3 Disc degeneration

Intervertebral disc degeneration can start in the annulus, nucleus or end-plate [4]. High interlaminar shear stresses are associated with propagationof circumferential tears in the annulus [28]. Fazzalari et al. [23] showed thatconcentric tears lead to mechanical changes in the disc and to progressivedamage. Excessive loading in combination with exion or torsion can dis-rupt the nucleus. Minimal damage to an endplate can lead to disruption ofthe adjacent discs [4]. Also Holm et al. [26] found disc degeneration dueto endplate injury. For all cases counts that in a degenerated disc the mor-phology has changed. Nachemson [35] has developed a grading scheme fordisc degeneration. This scheme grades the degeneration of discs into fourgrades (table 1.2). In �gure 1.2 pictures of intervertebral discs with thesedegrees of degeneration are shown.

Table 1.2: A grading scheme for intervertebral disc degeneration developed by Nachem-son [35].

Grade Disc Morphology

Grade I A shiny gelatinous nucleus pulposus is easily delimited fromthe annulus �brosus, which is free of macroscopic rupturesor discoloration

Grade II Macroscopic changes are present in the nucleus pulposus,which is somewhat more �brous, but still clearly distinctfrom the annulus �brosus

Grade III Macroscopic changes are present in both nucleus pulposusand annulus �brosus. The nucleus pulposus is more �broticbut still soft. The boundary between nucleus pulposus andthe annulus �brosus is no longer distinct. Isolated �ssuresare found in the annulus �brosus

Grade IV Severe macroscopic changes exist in both the nucleus pul-posus and annulus �brosus. Fissures and cavities arepresent in both the nucleus pulposus and annulus �brosus.Marginal osteophytes are often found on adjacent vertebrae

1.4 Causes of disc degeneration

Although intervertebral disc degeneration is of signi�cant clinical impor-tance, its etiology and pathogenesis largely remain unknown. Intervertebraldisc degeneration is associated with mechanical damage, loss of nutritional

8

A B

C D

Figure 1.2: Pictures of degenerated discs A) Grade 1 B) Grade 2 C) Grade 3 D) Grade4. Figure from Thompson [48].

pathways and biological degradation [28]. Heredity has a dominant role indisc degeneration. Bati�e et al. [8] found in an exposure-discordant monozy-gotic twin study that physical loading speci�c to occupation can not beassociated with disc degeneration and saw similarities between degenerateddiscs in co twins. They stated that heredity explains 74% of the varianceseen in disc degeneration. Similarly Sambrook et al. [41] found that heri-tability was 74% at the lumbar spine and that genetic in uences are mostapparent on disc height and structural changes.

Degeneration often involves biological cell-mediated changes; mostly seenin the nucleus, and structural changes; mostly seen in the annulus and end-plate [4]. Rates of oxygen consumption, lactic acid production, and glucoseconsumption a�ect cellular activity [12]. Matrix degeneration results from afailure of the cells to maintain and repair the matrix. A shortage of oxygenhas adverse e�ects on the disc; proteoglycan and protein synthesis decreasemarkedly at low oxygen supply [11]. Similar Shah et al. [44] hypothe-sized that biochemical changes could lead to alteration in the biomechanicalproperties of the intervertebral disc and exacerbate tissue damage and thesechanges themselves could again result in biochemical changes [44]. Perhapsthe most dramatic changes in the intervertebral disc with degeneration arethe loss of uid pressurization, hydration, altered biochemical composition,and matrix structure for the nucleus pulposus [43].

Weakening of spinal tissues, due to ageing, can also cause disc degeneration[3]. Both biochemical environment and loading history in uence the weak-ening. Buckwalter [17] suggested that alterations in proteoglycan structureand loss of proteoglycans may be one of the earliest events in the devel-opment of disc degeneration. Loss of proteoglycans results in loss of watercontent. In a less hydrated intervertebral disc the nucleus is more �brous,therefore the load cannot be transferred as well and the nucleus becomesdecompressed and stress concentrations occur in the annulus [2]. Not only

9

proteoglycan content, but also cell density decreases with age, resulting inless responsiveness to mechanical environment [3]. Loss of disc height andalterations in disc composition can a�ect spinal mobility and later the align-ment and loads applied to the facet joints, spinal ligaments and paraspinousmuscles [17]. The major cause of age-related disc degeneration howeverseems to be impaired nutrition. Maroudas et al. [31] hypothesized thatpoor nutrition may account for disc degeneration. Urban et al. [49] showedthat indeed loss of nutrient supply can result in cell death, loss of matrixproduction leading to increase in matrix degradation and thus to disc de-generation. Urban et al. [50] hypothesized that calci�cation of the endplatecould be responsible for a less permeable endplate and therefore for limiteddi�usion for nutrition. Benneker et al. [10] hypothesized that with a de-crease in numbers of openings in the endplate, exchange of solutes from thecapillary buds would be diminished. They showed that there is a correla-tion between opening density in the endplate and morphologic degenerationgrade of the intervertebral disc. They also showed a negative correlationbetween age and density of openings in the endplate.

The intradiscal pressure in the nucleus is signi�cantly lower in the degen-erated disc than in the healthy disc [26]. Similarly, Adams found that notonly the hydrostatic pressure in the nucleus is lowered, but also the regionof the nucleus is reduced [1, 2, 4, 5]. When a sudden high compressive forceor when an enduring compressive force together with repetitive exion isapplied to the intervertebral disc this can result in a nuclear extrusion, i.e.herniation or annular extrusion, i.e. disc bulging [29]. Similarly Adams [1]found that compressive overload can damage the endplate and this can leadto inward collapse of the annulus. Also bending and compression togethercan cause extreme outward bulging of annulus. Flexion and full hydrationare primary risk factors in the disruption of the nucleus [45]. When mechan-ical stress is inappropriate the matrix turnover might result in degradativepathways [11]. Mechanical factors like magnitude, frequency, and durationof static and dynamic compressive load regulate cellular responses in theintervertebral disc and may govern the initiation and progression of disc de-generation [43]. Mechanically induced injury might initiate the degenerativecascade [46].

1.5 Stress pro�lometry

Stress pro�lometry is a technique often used to assess the mechanical prop-erties of an intervertebral disc. A transducer with a stress-sensing elementis mounted into a needle, when this needle is pulled through a disc a pro�leof compressive stress is obtained. For a healthy disc the pressure in thenucleus is constant, and peaks in the pressure can be seen in the annulus[5, 32]. Edwards et al. [21] also found that for all loading conditions thehighest stress occurred in the annulus. McMillan et al. [32] showed thatif pro�les with a pressure transducer needle were repeated the di�erencesbetween pro�les were less than 20 percent. They also showed that when theneedle is smaller, more peaks in the annulus can be seen, thus these peaks

10

are smoothed out by the larger transducer. Adams et al. [1, 2, 3, 5] showedhow the stress pro�le of a degenerated disc di�ers from a healthy one. Thepressure inside the nucleus drops and the region with hydrostatic propertiesis reduced. In �gure 1.3 the stress pro�les of healthy and degenerated discsare shown.

Figure 1.3: Pressure pro�les of di�erently graded discs. A) A healthy disc B) A grade2 degenerated disc C) A grade 3 degenerated disc. Adapted from Adams [2].

11

1.6 Project

It has been hypothesized that during ageing the endplates calcify and causeimpaired nutrition [49]. It has also been hypothesized that impaired nutri-tion leads to disc degeneration [31, 49, 50]. However, the causality betweenlimited nutrition and disc degeneration has never been demonstrated. Inthe overall project the aim was to prove this causality. In the overall projectcalci�cation of the endplates was mimicked in an ovine disc degenerationmodel. In this model the major nutritional route was partially blocked inthe sheep lumbar spine [51]. The blockage was obtained by making a defectclose and parallel to the endplate and inserting a titanium foil into the cre-ated defect. The disrupted vascularity, due to the defect, should decreaseperfusion and therefore partially inhibit di�usion and solute transport toand from the disc. The foil was inserted to keep the vascular buds fromgrowing back. The objective is to see whether this blockage causes discsdegeneration.

However, this defect could have an in uence on the mechanical propertiesof the intervertebral disc. And as shown by Adams [4] minor damage to theendplate leads to progressive structural changes in the adjacent interverte-bral discs. To be able to exclude this in uence, it had to be investigatedwhat the e�ect of the defect is on the mechanical properties.

The objective of this study was to investigate if making a defect close andparallel to the endplate has an in uence on the mechanical properties of theintervertebral disc. In this study stress pro�lometry [1, 5, 32, 35, 36] wasused to investigate this e�ect.

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Chapter 2

Methods

2.1 Specimens

20 sheep lumbar spines were used for this study. From each spine 2{3 motionsegments were obtained. All 53 motion segment had no ligaments, muscles,and transverse processes, but the pedicles and spinal processes still attachedas shown in �gure 2.1. Tables in the appendix A show the speci�cations ofthe spines and the motion segments. For the preliminary tests 4 motionsegments were used. For the main test 21 motion segments were tested, ofwhich 14 were experimental, and 7 control. However one of the experimentaldiscs had to be excluded due to overloading.

Figure 2.1: Photo of embedded motion segment without ligaments, muscles and trans-verse processes.

The motion segments were embedded in poly-methylmethacrylate (PMMA)using an embedding system as shown in �gure 2.2. The plates in this de-vice could be adjusted to ensure the intervertebral disc was perpendicular tothe loading direction and centered. During the whole process, paper toweldrained with ringer solution was wrapped around the intervertebral disc toprevent dehydration. Because the temperature of the PMMA rises up to90 degrees while hardening, a temperature sensor was put in the interverte-bral disc of one specimen; the embedding did not have an in uence on thetemperature inside the intervertebral disc.

13

Figure 2.2: Simpli�ed drawing of the embedding system.

2.2 Defect in endplate

The defects were made on both sides of the intervertebral disc close andparallel to the endplate with a surgical saw. The starting position wasmarked with a bone chisel. The uoroscope was used to check the positionof the bone chisel and the sawing blade while making the defect. When the20 mm deep defects were made on both sides, a picture was taken with sawblades in both slots. In �gure 2.3 pictures of this process are shown.For the mechanical test a titanium foil was inserted into the slots as shownin �gure 2.1. To check afterwards if the slots were in the right positionthe motion segments were cut open. The disc was removed �rst, then theendplate was sawn o�, and sawn perpendicular to the plane of the defect.Top and side view pictures were taken to see if the defect completely coveredthe nucleus and was close to the endplate. In �gure 2.4 titanium foils showthe position of the slots.

2.3 Loading pro�le

First, pilot tests were done to see what kind of preconditioning would benecessary. These test are shown in appendix A. From these test it wasdecided to do preconditioning of holding a load for 15 min. This way of pre-conditioning has also been recommended by Adams [1]. Preliminary testswere done to see if measurements were repeatable. For these tests a loadingscheme similar as shown in �gure 2.5 was used. For the preliminary teststhe loading cycles were 10 min instead of 7 min. In the second and thirdloading cycle both horizontal and vertical measurements were made, fourmeasurements in total.For the main tests the loading pro�le shown in �gure 2.5 was used. In the�rst loading cycle one measurement was taken and in the second loadingcycle also one measurement was taken. For half of the samples the measure-ments were always horizontal in the �rst loading cycle and vertical in thesecond loading cycle, and for the other half the other way around.

14

A B

C D

Figure 2.3: Images of the uoroscope while making the defect in the endplate. A) Theposition is marked with a bone chisel. B) The position of the sawing blade is checked. C)The position of the sawing blade is checked while halfway in. D) A sawing blade in bothdefects.

A1 CM

B 1 CM

Figure 2.4: A) A top-view picture of the defect in motion segment 28, L1. B) A side-viewpicture of the defect in motion segment 28, L1.

15

150

N

100

N

15 m

in7

min

1 m

in2

s

M 1

M

2

20 N

Figure

2.5:Theloadingpro�leforthemain

tests.

M1=

measurement1,andM2=

measurement2

16

2.4 Needle Pressure Transducer

The pressure inside the intervertebral disc was measured with a needle pres-sure transducer. For the preliminary tests another needle pressure trans-ducer was used than for the main tests, because the �rst needle pressuretransducer broke, probably due to water leakage inside the tip of the needle,because the membrane of the sensing element was not well sealed. The out-put of the needle was voltage, but because the needle had been calibrated,the voltage could be converted to the pressure. The calibration of the needlepressure transducers is described in appendix B.

2.4.1 Preliminary tests

The needle pressure transducer had a diameter of 1.3 mm and a blunt tip.The length was 10 cm. A needle with a diameter of 1.2 mm was used tomake a hole in the intervertebral disc before inserting the needle pressuretransducer. In order to start measuring at the outer rim of the posteriorouter annulus, the needle had to be inserted all the way through the disc,as the pressure sensor was located a few mm from the tip of the needle.When the load was 20 N the needle was inserted. For the preliminary teststhe needle was either pulled or turned every 10 seconds. After the �rst10 seconds in which the horizontal measurement was made the needle wasturned to the vertical direction, after the second 10 seconds the needle waspulled out one mm, after the third 10 seconds the needle was turned back tothe horizontal direction and so on through the whole disc. Millimeter markswere made on the needle to know how much to pull out the needle.

2.4.2 Main tests

The needle pressure transducer had a diameter of 1.3 mm and a blunt tip.The length was 14 cm. Before inserting the needle pressure transducer intothe intervertebral disc a sharp needle with a diameter of 1.4 mm was used tomake a hole in the intervertebral disc. When the load was 20 N the needlewas inserted all the way through the disc. In order to start measuring atthe outer rim of the posterior outer annulus, the needle had to be insertedall the way through the disc, as the pressure sensor was located a few mmfrom the tip of the needle. For the main tests the horizontal and verticalmeasurements were taken separately. When the load was at 150 N theneedle was pulled out in steps of 1 mm (anterior to posterior). For everystep the needle was kept in place for 9 seconds, while for moving 1 second wasaccounted. For the main tests a ruler was taped onto a clamp parallel to theneedle. Two pieces of tape on the needle, one for horizontal measurementsand one for vertical measurements marked the position at the ruler.For both the preliminary and main tests the position of the needle inside thedisc was checked with the uoroscope at the start of the measurement andarbitrary at other positions throughout the measurement. Also the markingat the ruler was written down. In a schematic is shown how the needle wasinserted in the disc (�gure 2.6). The indent is the position of the sensingelement.

17

Figure 2.6: The needle pressure transducer inside the intervertebral disc, the indentshows the position of the sensing element.

2.5 Data acquisition

The input voltage of the needle pressure transducer was 15 V. During themeasurements the output voltage was recorded with 2 systems simultan-iously; a potentio multimeter connected to Metex and an adwin gold data-system. The sample frequency of the multimeter was 4 Hz, and that of theadwin-system 40 Hz. For the preliminary tests the multimeter data wasused, because the adwin system was not grounded. For the main tests thedata of the adwin system was used, and the data of the multimeter servedas a back up. In �gure 2.7 a scheme of how the needle was connected to thedata systems is shown.

Figure 2.7: Schematic of how the needle was connected to the input and 2 outputsystems.

2.6 Data analysis

A Matlab R routine was used to translate the output data into pressure. Forevery step the pressure was averaged out over 6 seconds. The pressure pro�lewas normalized over the distance. The pressure values were interpolated toobtain the values for 0, 0.1, 0.2 etc. up to 1 of the normalized distance.Zero is posterior and one is anterior. Then the data was normalized to thenucleus value. The nucleus value was taken separately for each disc. Inthe pressure pro�les the location at which the horizontal and vertical beforemeasurement were close to each other was assumed the nucleus. In the

18

middle of the nucleus the average value of horizontal and vertical was takenas the nucleus value to normalize to. The normalized pressure pro�les werecompared and the normalized nucleus values were compared. The horizontaland vertical measurements were compared to see if the measurements showedhydrostatic behaviour in the nucleus. Another way the data was normalizedwas an absolute-relative way in which (before value{after value) was dividedby before value.

19

Chapter 3

Results

3.1 Preliminary tests

For all the preliminary tests the second measurement was generally withinthe accuracy of the pressure transducer needle of the �rst measurement. In�gure 3.1 the data is shown for one experimental disc. The data of the otherdiscs showed similar results. These data showed that the measurements wererepeatable.

A B

C D

Figure 3.1: The pressure pro�les of the preliminary tests of one disc. The range showsthe value of the �rst measurement plus or minus the accuracy of the pressure transducerneedle. A) The �rst and second horizontal measurement before sawing. B) The �rst andsecond vertical measurement before sawing. C) The �rst and second horizontal measure-ment after sawing. D) The �rst and second vertical measurement after sawing.

20

3.2 Main tests

3.2.1 Comparison of before and after pressure pro�les

The normalized pressure pro�les of before and after measurements were com-pared for experimental and control discs. The pressure pro�les are shown in�gure 3.2. The variability of the before and after measurements of both theexperimental and control disc overlapped. However the after measurementswere always lower than the before measurements. For all points except forhorizontal experimental at 0.2, and for horizontal control at 0.6 and 0.7 thechange was within the error of the pressure transducer needle.

A B

C D

Figure 3.2: Comparison of before and after pressure pro�les of experimental and con-trol discs. The lines are the mean values, and the error bars the standard deviation.A) Experimental horizontal B) Control horizontal C) Experimental vertical D) Controlvertical

3.2.2 Comparison of before and after nucleus values

The normalized nucleus values of before and after measurements were com-pared for experimental and control discs (�gure 3.3 A-D). The after mea-surements were in all cases lower than the before measurements. Also thevertical values were lower than the horizontal values. In table 3.1 it canbe seen that the change between the averaged pressure in the nucleus be-fore and after sawing was larger than the change between before and afterwaiting. Also in both the experimental and control group the change in theaveraged vertical measurements was larger than for the averaged horizontalmeasurements. However the change in pressure in the nucleus before andafter sawing or waiting was in all cases smaller than the accuracy of the

21

pressure needle, which is 0.13 MPa, so there was no signi�cant change inpressure. In �gure 3.3 E and F the after values of experimental and controldiscs are compared.

Table 3.1: The change between before and after nucleus value.

Horizontal Vertical

Experimental 0.0462 0.0915

Control 0.0208 0.0350

A B

C D

E F

Figure 3.3: The normalized pressure values in the nucleus. All values are mean values� standard deviation. A) Experimental horizontal B) Experimental vertical C) Controlhorizontal D) Control vertical E)After horizontal F)After vertical

22

3.2.3 Comparison of horizontal and vertical pressure pro�les

To see whether the pressure inside the nucleus was hydrostatic the horizontaland vertical pressure values were compared. The di�erences between hori-zontal and vertical measurements in the nucleus were small and within theaccuracy of the pressure transducer needle, see table 3.2, thus the nucleusbehaves hydrostatically.

Table 3.2: The change between horizontal and vertical nucleus pressure.

Before (hor-ver) After (hor-ver)

Experimental 0.0189 0.0642

Control 0.0215 0.0358

When values of the whole pro�le were compared the di�erence was within theaccuracy of the pressure transducer needle in the nucleus. The di�erence waslarger than the accuracy at 0.7 for both experimental and control discs. Thedi�erence was also larger than the accuracy of the needle at 0.1, 0.6 and 0.8 ofthe control. When the horizontal and vertical pressure pro�les were averaged(see �gure 3.4) the di�erence between before and after measurements werewithin the accuracy of the pressure transducer needle, except for 0.6 of thecontrol.

A B

Figure 3.4: The pressure pro�les averaged over horizontal and vertical values. A)Experimental B) Control

23

3.2.4 Absolute Relative normalization

Absolute relative normalization was done to see whether the relative changesbetween before and after measurements were consistently smaller for ex-perimental or control discs. In �gure 3.5 it is shown that the relativechanges between before and after measurements of the control were smallerthan changes of the experimental discs for the vertical measurements. Forthe horizontal measurements neither the experimental nor the control discsshowed a consistent smaller change. The measurements had to stay within(0.81+0.13)/0.81 and (0.81-0.13)/0.81 to be within the error of the needle.For both horizontal as well as vertical measurements the values stayed within1.16 and 0.84, so there was no signi�cant change between before and aftermeasurements for the experimental and control group. Also the variabilityof the experimental and control discs overlapped and the changes were notconsistently lower or higher.

A B

Figure 3.5: Relative-absolute normalization. A)Horizontal B)Vertical

24

Chapter 4

Discussion

This study showed that the change between before and after measurementsfor control discs as well as for experimental discs stayed within the error ofthe needle. For both control and experimental discs the pressure was lowerfor the after measurements. For both the control and experimental discsthere was more variation in the horizontal measurements than in the verticalmeasurements. For the horizontal measurements as well as for the verticalmeasurements the change between before and after measurements was notconsistently lower or higher for control or experimental discs. Because therewas no signi�cant di�erence between the before and after measurements thee�ect of the defect on the mechanical properties of the intervertebral disc inthe ovine disc degeneration model can be excluded.

The preliminary tests showed that the measurements are repeatable. Thisis according to the �ndings of McMillan et al.[32]. At a load of 150 N theaverage pressure in the nucleus of an ovine intervertebral disc was 0.81 MPa,this is in the physiological range of pressure that is subjected daily to theintervertebral discs of human and sheep [34, 52, 53]. Also because the �rstpressure transducer needle was accurate only up to 1.3 MPa it was chosen torather stay in a lower pressure range than higher, to still be able to measureall the pressure values. Also the variability of the area of the disc caused thepressure inside the discs to di�er largely under the same load. When testswere done to set the load for the �nal test to obtain a pressure inside thedisc of 1 MPa, the disc areas were probably smaller than average, resultingin a load that was estimated too low.

The vertical measurements showed less variability than the horizontal mea-surements in the annular region. Maybe this can be explained by the factthat the sensing element in the tip of the pressure transducer needle was in alittle indent. Maybe the load pressured the lamellae into the indent, makingit more di�cult to move the needle through the lamellae, and causing theneedle to sense the pressure peaks in the annulus. Whereas for the verticalmeasurements the indent was not in the same direction as the loading direc-tion and therefore did not complicate the moving of the needle through thelamellae. It can also be explained that the horizontal measurements sensedmore of the pressure peaks in the annulus due to the di�erent location of

25

the needle in the lamellae. Adams [1] also found small di�erences betweenhorizontal and vertical pressure pro�les in the annulus.

Generally, the variability in the anterior region was larger than in the pos-terior region, and the smallest in the middle region. This is probably dueto the fact that the nucleus is not centered and that the anterior annulusis often thicker than the posterior annulus. The nucleus was hydrostatic,horizontal and vertical measurements did not show signi�cant di�erences inthis region. In the annulus the di�erence between horizontal and verticaldid not stay within the accuracy of the needle. When motion segment 41was left out of the data still the change between horizontal and vertical wasnot within the accuracy of the needle at 0.2 and 0.7. For normalization tothe nucleus value the values at the points horizontal control at 0.6 and 0.7were not within the error of the pressure transducer needle. However whenmotion segment 41 was left out of the data, all the points normalized to thenucleus were within the accuracy of the pressure transducer needle. Thisdi�erence is due to the fact that the pressure in the annulus is not hydro-static, and that 0.7 and 0.2 of the control discs are in the annulus. In �gure4.1 is shown how the averaged pressure pro�les change when specimen 41is left out. Maybe motion segment 41 showed a strange pressure pro�le,

A B

Figure 4.1: The pressure pro�les averaged over horizontal and vertical values. A)Withall motion segments B)Without motion segment 41

because the spine was taken out of the freezer later than the other spines,and therefore did not have as much time to defrost. However specimen 40and 42 came from the same spine and did not show a strange pressure pro�leand specimen 40 was tested before specimen 41. For this project the changeof pressure in the nucleus was of much higher importance than changes inpressure in the annulus, because the measured pressure in the annulus canbe caused by pressure peaks in the lamellae.

The accuracy of the needle pressure transducer was not very high, but forthese test the accuracy was high enough. According to the observations ofMcMillan et al. [32] the measurements were repeatable if they stayed within20%, but in this study the measurements stayed within 16%. One way toincrease the accuracy of the needle is to lower the input voltage, but thepower has to stay the same, thus the current to the ampli�er also has to beadjusted, but this was too di�cult to adjust in time and too expensive.

26

It would have been better if the needle pressure transducer had a sharp tip.Making a hole with another needle and moving in and out several timescould damage the disc. Even thought the disc was kept moist throughoutthe whole process, the disc dries out more than in the body. Since the test-ing procedure was very long, this might cause the disc to dehydrate andbecome sti�er. The dehydration is probably the reason for why the afterpressure pro�les in the control and experimental discs were lower.

A major complication of needle pressure transducer was that the membraneto the sensing element is not well sealed and that uid might leak in, causingthe needle to give errors. Since the disc is a moist environment the changethat uid leaks in is possible. However, for the second pressure transducerneedle that was used there were no complications with uid leakage. Toprevent from uid in the needle, it was put in a dry environment betweenthe tests.

For the preconditioning a load was kept on for 15 min as recommended byAdams [1]. To completely remove super hydration e�ects by precondition-ing, the load should have been kept on for 2-6 hours. This was impossiblein these tests, because the testing process would then become too long. Inthis case probably the dehydration of the disc would have more e�ect thanthe super hydration had in the tests.

For all the tests the defect was at a similar position close and parallel to theendplate, but it could not be standardized. However the small changes inposition probably do not in uence the pressure pro�le and also in the ovinedisc degeneration model the position of the defect is not always exactly thesame.

The pressure pro�le was made by steps of 1 mm. More accurate mighthave been to use a device to pull out the needle at a certain rate, so a moreprecise pressure pro�le throughout the whole disc is obtained. However sucha procedure would probably not signi�cantly increase the accuracy of thepro�le, because the pressure in the nucleus is constant and with a moreaccurate pro�le, the pro�le will stay the same. Only in the annulus morepressure peaks might be detected, but these peaks were not of interest forthis project, thus steps of one milimeter were accurate enough. A device topull out the needle at a constant rate holding the needle tight could probablydamage the needle. The wires inside the needle are very fragile, thereforethe needle cannot be clamped with too much force, and might loosen duringthe pulling out, causing more inaccuracy in the testing procedure.

In conclusion, despite some possible improvements of the pressure needletransducer and the testing procedure this study is valid. No signi�cantchange was seen in the pressure pro�les between before and after measure-ments for experimental as well as control discs. The defect in the ovine discdegeneration model does not e�ect the pressure inside the disc on the shortterm signi�cantly.

27

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31

Appendix A

Speci�cation of the spines

and motion segments

Table A.1: Overview of spines

Spine no. Motion seg-ment no.

Date ofbirth

Data of eu-thanasie

Sheep no. and treat-ment

1 1,2,3 - - -

2 4,5 1-11-98 11-12-03 293, uorochrome

3 6,7,8 - 2-12-03 587

4 9,10,11 - 3-1-03 361

5 12,13,14 9-4-99 27-11-03 582

6 15,16,17 10-11-99 27-11-03 589

7 18,19 - 30-10-02 424

8 20,21 2-3-97 28-11-02 167, keracup

9 22,23,24 4-2-00 10-12-03 551, peristruct uo-rochrome



12 none 3-2-00 10-12-03 349, uorochrome

13 31,32,33 9-1-99 8-7-05 4021, reosdef

14 34,35,36 1-5-00 10-9-05 4053, reosdef

15 37,38,39 1-9-99 19-7-05 4047, reosdef


17 43,44,45 11-12-97 18-11-02 169, keracup

18 46,47,48 21-10-98 6-3-03 266

19 49,50,51 16-4-99 16-5-05 4011, reosdef

20 52,53,54 - - -

32

Pilots were done to see which preconditioning would be most applicablefor the tests. In �gure A.1 the 5 di�erent ways that were tested are shown.In table A is referred to these tests. In pilot 1 the �rst 3 loading cycles were

A

Figure A.1: Loading pro�le of di�erent pilot tests for preconditioning. The time inbetween the red arrows (x) is 15min, the time between the green arrows (y) is 10 min,and the time in between the purple arrows (z) is 35 min.

the preconditioning, the last 2 loading cycles were for measuring. The loadwas applied in 5 s, and also the unloading to 50 N was in 5 s. In pilot 2preconditioning was 10 cycles at 0.5 Hz between 50 N and 250 N. The last2 loading cycles were for measuring. In pilot three the preconditioning wasapplying a load of 300 N for 15 min. Pilot 4 is similar to pilot 2, exceptfor that there are 20 cycles at 0.5 Hz, and there is one loading cycle extrabefore measuring. In pilot 5 the preconditioning is the same as in pilot 3,but there is no unloading for measuring. When in the table is referred to�nal test, this is the test described in the methods, �gure 2.5.

33

Table A.2: Overview of motion segments

Motion seg-ment no.

Lumbar no. Date spineobtained

Data spinewas dis-sected(d),embedded(e),tested(t)

Sort test

1 - 3-5-05 4-5-05 (d) 11-5-05 (e,t)

Measure temperature duringpotting. Practice sawingw/o uoroscope.

2 - 3-5-05 4-5-05 (d) 8-6-05 (e) 9-6-05(t)

Practice sawing w/o uoro-scope.

3 - 3-5-05 4-5-05 (d) 8-6-05 (e) 9-6-05(t)

Practice sawing w/o uoro-scope.

4 L1/L2 12-5-05 13-5-05 (d) 6-6-05 (e,t) 10-6-05 (t)

For instron machine expla-nation and for uoroscopeexplanation on the 2nd test-ing date.

5 L3/L4 12-5-05 13-5-05 (d) 8-6-05 (e) 10-6-05 (t)

Practice sawing with uoro-scope.

6 L5/L6 10-6-05, infridge 11-6-05

13-6-05 (d,e,t) This specimen broke in theinstron machine, probablydue to wrong load cell (1kN)and wrong settings (PID).

7 L3/L4 10-6-05, infridge 11-6-05

13-6-05 (d,e,t) Pilot 2; but cycling between200 N and 400 N and re-peating the measurement af-ter 10 min at 50 N.

8 L1/L2 10-6-05, infridge 11-6-05

13-6-05 (d,e,t) Pilot 1; repeating the mea-surement after 10 min at 50N.

9 L1/L2 10-6-05, infridge 12-6-05

14-6-05 (d,e,t) Pilot 1; repeating the mea-surement after 10 min at 50N.

10 L3/L4 10-6-05, infridge 12-6-05


11 L5/L6 10-6-05, infridge 12-6-05


12 L5/L6 21-6-05 23-6-05 (d,e,t) Pilot 1

13 L4/L3 21-6-05 23-6-05 (d,e,t) Pilot 2

14 L2/L1 21-6-05 23-6-05 (d,e,t) Pilot 2

15 L1/L2 22-6-05 24-6-05 (d,e,t) Pilot 1

16 L3/L4 22-6-05 24-6-05 (d,e,t) Pilot 1

17 L5/L6 22-6-05 24-6-05 (d,e,t) Pilot 2

18 L4/L5 26-6-05 28-6-05 (d,e,t) Pilot 3

19 L2/L3 26-6-05 28-6-05 (d,e,t)7-7-05 (t2)

Pilot 4. With needle: Load-ing for 15 min at 120 N and3 times loading at 180 N for5 min and 1 min in between.Repeated with 100 and 170N.

34

Table A.2: Overview of motion segments (continued)

Motion seg-ment no.



Sort test

20 L3/L4 27-6-05 29-6-05 (d,e,t)5-7-05 (t2)

Pilot 3. With needle: Load-ing at 250 N for 30 s,then 3times loading at 350 N for100 s with 15 s in between.Repeated same test with 30s in between and 150 s atloading.

21 L1/L2 27-6-05 29-6-05 (d,e,t)5-7-05 (t2)

Pilot 4. With needle: Step-wise loading at 100, 200,300, 350 N.

22 L1/L2 27-6-05, infridge 28-6-05

30-6-05 (d,e,t)7-7-05 (t2)

Pilot 3. With needle: 15 minat 100 N and 3 times loadingat 170 N for 5 min. and 1min in between. Repeatedwith 150 N at loading.

23 L3/L4 27-6-05, infridge 28-6-05

30-6-05 (d,e,t)4-7-05 (t)

Pilot 4. With needle: Step-wise loading at 0 N, 100 N,and 200 N.

24 L5/L6 27-6-05, infridge 28-6-05

30-6-05 (d,e,t) Pilot 5. After test I putin di�erent needles and tookpictures with the uoro-scope.

25 L5/L6 7-7-05 8-7-05 (d,e,t) With needle: Final test. Ad-win was not connected toground, false values. Con-trol disc for preliminarytests.

26 L3/L4 7-7-05 8-7-05 (d,e,t) With needle: Final test. Ad-win was not connected toground, false values. Exper-imental disc for preliminarytests.

27 L1/L2 7-7-05 8-7-05 (d,e,t) With needle: Final test.Pressure pro�les of horizon-tal and vertical were takenseparately. Control disc forpreliminary tests.

28 L1/L2 12-7-05 14-7-05 (d,e,t) With needle: Final test.Pressure pro�les of horizon-tal and vertical were takenseparately. Experimentaldisc for preliminary tests.

29 L3/L4 12-7-05 14-7-05 (d,e,t)15-7-05 (t) 18-7-05 (t)

After the �rst measurement,the needle stopped workingdue to uid inside the nee-dle. Test was done on 15-7 to see if this was thecase, and on 18-7 to see ifit was working again, butthen the wires of the nee-dle burned, probably due toshort-circuiting.

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Motion seg-ment no.



Sort test

30 L5/L6 12-7-05 14-7-05 (d,e) The segment is put in freezeron the 14th, fridge on 15thand back in freezer on 18th.And in the fridge on the25th. The sample could notbe used.

31 L1/L2 15-8-05 16-8-05 (d,e,t) The load cell was not work-ing properly. After start-ing the load went up to 1.2kN. The sample could not beused.

32 L3/L4 15-8-05 16-8-05 (d,e,t) The load cell was not work-ing properly. After start-ing the load went up to 2kN. The sample could not beused.

33 L5/L6 15-8-05 16-8-05 (d,e)17-8-05 (t)

Tested with new load cell.Not used for data.

34 L1/L2 16-8-05 17-8-05 (d,e,t) Experimental. First hori-zontal, second vertical.

35 L3/L4 16-8-05 17-8-05 (d,e,t) Experimental. First verti-cal, second horizontal. Thebutton of control pannel gotstuck and the load becameto high. The sample couldnot be used.

36 L5/L6 16-8-05 17-8-05 (d,e,t) Control. First horizontal,second vertical.


38 L3/L4 18-8-05 19-8-05 (d,e,t) Experimental. First verti-cal, second horizontal.

39 L5/L6 18-8-05 19-8-05 (d,e,t) Control. First vertical, sec-ond horizontal.










36


Motion seg-ment no.



Sort test

49 L1/L2 25-8-05, tookout freezer 28-8-05

29-8-05 (d,e,t) Control. First vertical, sec-ond horizontal.


29-8-05 (d,e,t) Experimental. First hori-zontal, second vertical.


29-8-05 (d,e,t) Experimental. First verti-cal, second horizontal.



54 L1/L2 30-8-05 31-8-05 (d,e,t) The needle did not go fromthe spinal cord into the discor the other way around.Probably the endplate wastoo bony or too convex atposterior side. The samplecould not be used.

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Appendix B

Calibration of needle

pressure transducer

McNally kindly calibrated the �rst needle pressure transducer for us between0 and 13 bar at a power supply of 15 V DC. In �gure B.1 the curve of thecalibration is shown.

Figure B.1: The calibration curve of the �rst needle pressure transducer.

When the �rst needle broke a new needle pressure transducer had to beordered from Gaeltec. This needle was calibrated by Patrick Lezuo, thetechnician of the lab. The needle was inserted into a bioreactor, in whichthe pressure could be controlled. This bioreactor is shown in �gure B.2. Forthe calibration the pressure was increased in steps of 0.2 MPa from 0.5 MPaup to 2.5 MPa and back. For every step the pressure was kept constantfor 2 minutes. The output voltage of the needle was recorded during thewhole process with the multimeter and connected to metex. The calibrationwas done before the measurements and repeated after the measurementshad been �nished. For the �rst calibration the needle was sealed into thebioreactor, but there was some water leakage. For the second calibration

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A

B

Figure B.2: A) The bioreactor in which the second needle pressure transducer wascalibrated. B) Insertion of needle

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the device was adapted, and no water could leak out of the bioreactor. In�gure B.3 the calibration curve of this needle pressure transducer is shownbefore and after the measurements. The di�erence in voltage for the �rstand the second calibration was was in the order of Pa, and therefore not sig-ni�cant. To calculate the pressure for the main tests the curve of the secondcalibration is taken, because there was no water leakage in this calibration.

A

B

Figure B.3: The calibration curve of the second needle pressure transducer. A) Beforemeasurements B) After measurements

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Mechanical e ect of an induced ovine lumbar intervertebral …1.2 Anatomy of intervertebral disc The...

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