Intervertebral Disc Degeneration

Intervertebral Disc Degeneration: Studies in the Loaded Disc Culture System C.P.L. Paul

Intervertebral Disc Degeneration: Studies in the Loaded Disc Culture System

C.P.L. Paul

Colophon

Research funding: The research presented in this thesis was supported by the Dutch Government ZonMw Program ‘‘Alternatives for life animal testing’’: grant number 11400090. The development of the Loaded Disc Culture System (LDCS) was supported by the government led consortium for the development of BioMedical Materials, BMM: grant number P2.01 IDiDAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the thesis or individual manuscript.

Publication of this thesis was supported by: Amsterdam Movement Sciences Nederlandse Orthopaedische Vereniging Graduate school, AMC Stichting Beroepsopleiding Huisarts (SBOH) Stichting Proefdiervrij Dutch Spine Society Anna Fonds | NOREF The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the thesis or individual manuscripts.

Cover illustration:

“Kneeling Atlas” by FMP Westerhuis – Paul © 2017, Gouda, The Netherlands. All rights reserved.

Lay-out and printing:

Loes Kema, GVO drukkers & vormgevers B.V. Ede, The Netherlands.

ISBN:

978-94-6332-293-5

Digital version available@: http://hdl.handle.net/11245.1/9411a201-59b9-479d-b777e60eaf0e42af

Copyright © 2017 CPL Paul, Amstelveen, The Netherlands. All rights reserved. No part of this publication, cover illustration, text or figures may be reproduced, stored or transmitted in any poart or means, without written permission of the author.

Intervertebral Disc Degeneration: Studies in the Loaded Disc Culture System ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op 7 februari 2018, te 14:00 uur door Cornelis Pieter Louis Paul geboren te Beverwijk

Promotiecommissie: Promotor:

Prof. dr. ir. T.H. Smit

AMC-Universiteit van Amsterdam

Copromotoren:

Dr. M.N. Helder

VU medisch centrum

Dr. M.G. Mullender

VU medisch centrum

Prof. dr. G.M.M.J. Kerkhoffs


Prof. Dr. Ir. G.J. Strijkers


Dr. M.J.B. van den Hoff


Prof. dr. W.F. Lems

Vrije Universiteit Amsterdam

Dr. A.D. Bakker

Vrije Universiteit Amsterdam

Overige leden:

Faculteit der geneeskunde

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“All thanks to sis’, I can do that!” Mike Cass (Charles McGown), A chorus line, 1985

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Contents

Page 10

Chapter 1

General introduction

Chapter 2

Simulated-physiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture.

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Chapter 3

Dynamic and static overloading induce early degenerative processes in caprine lumbar intervertebral discs

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Chapter 4

Static axial overloading primes lumbar caprine intervertebral discs for posterior herniation

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Chapter 5

Changes in Intervertebral Disc Mechanical Behavior during Early Degeneration

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Chapter 6

Quantitative MRI in early intervertebral disc degeneration: T1rho correlates better than T2 with biomechanics and matrix content

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Chapter 7

General discussion

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Chapter 8

Summary

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Addendum

Dutch summary / Nederlandse samenvatting Acknowledgements LDCS articles Curriculum Vitae

216 236 239 241

Chapter 1. General Introduction

C.P.L. Paul

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Chapter 1

Low-back pain and intervertebral disc degeneration Low-back pain (LBP) is the most common medical complaint in Western society (1), encompassing immense ensuing socio-economic costs (2-4). It is widely recognized that multiple factors underlie the complex and broad spectrum of pathologies linked to LBP (5-7). When regarding somatic causes, intervertebral disc (IVD) degeneration, or degenerative disc disease (DDD), is strongly associated with both the presence and severity of LBP complaints (8-11). Multiple large general population-based studies in the last decade have provided strong evidence for their correlation (12-15). Presently, there are no curative therapies for patients with symptomatic DDD. Treatment strategies are directed at symptom relieve and comprise exercise programs and physical therapy (16), pain medication (17) and acupuncture (18-23). For patients with severe, and severely symptomatic DDD, surgical salvage procedures are the only option. These involve removal of the disc followed by fusion or arthroplasty of the motion segment, which results in only moderate outcomes (24-27).

IVD anatomy and function The intervertebral disc is the central part of the spinal motion segment. The disc functions to permit limited motion and flexibility of the spine, while maintaining segmental stability and absorbing and distributing external loads on the vertebral column. Its structure is complex and consists of several tissue types. The IVD is the largest avascular structure in the body and is subjected to substantial loading conditions (28). The central structure of the disk, the nucleus pulposus (NP), is a gellike substance comprising predominately of collagen type 2 and proteoglycans (PG’s), mostly glycosaminoglycans (GAGs). Radially confining the NP is a highlyorganized sheets of fibrils arrangement in laminae, which is called the annulus fibrosis (AF) (29). The laminae, usually around 20 to 25, are composed mainly of collagen type 1 fibrils and elastin, ideal to resist tensile forces (30;31). The IVD is one of the most sparsely cellular tissues in the body, with cell densities at maturity around 4·106 cells/cm3 in the NP and 9·106 cells/cm3 in the AF. The role of the chondrocyte-like and fibroblast-like cells residing in the NP and AF matrix is not completely understood; whether they serve an active role to maintain extracellular 10

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matrix (ECM) content or are the passive remnants or senescent cells of their embryologic predecessors, (the notochordal and) mesenchymal cells, is still subject of research and debate (28;32-34). Covering the IVD proximally and distally are the cartilage (CEPs) and vertebral (VEPs) endplates (35). The CEP is a layer of hyalinelike cartilage that is thought to be responsible for most of the nutrients exchange with the adjacent vertebral body (VB). One IVD and two VB constitute the motion segment (MS), which is the functional unit of the spine. The motion-segment has shock absorbing properties mainly due to the high intradiscal pressure (36-39). The osmotic pressure is build up by the GAGs ability to retain or imbibe water and is both constrained by the annulus and enabled by the AF’s porous structure allowing fluidexchange. The viscous-osmotic features of the NP, together with the porous-elastic characteristics of the AF give the healthy IVD its unique poro-elastic properties (40;41). These poro-elastic properties give the IVD a direct stiffness at an initial axial load and extra recuperating ability afterwards, making it capable of coping with peak loads as well as longer lasting mechanical burdens (42-44).

Current concept of DDD etiology and the role of mechanical loading Intervertebral disc degeneration is considered to be a multifactorial process (45), in the physiological sense euphemistically referred to as “natural ageing of the spine” (46-50). Many vastly different risk factors have been identified that may aggravate this “natural process”, such as genetic predispositions (51-57), trauma (5860) and infections (61-63) of the spine, loss of nutrient supply (64-66) to the disc due to atherosclerosis and stenosis of lumbar arteries (67-69), cardiovascular disease (70;71), high cholesterol (72), obesity (73) and diabetes (67;74). The degenerative cascade of the IVD starts in the nucleus pulposus (NP) where numerous incentives can cause a loss of proteoglycans (PGs, mostly glycosaminoglycans; GAGs) from the extracellular matrix (ECM), causing a decrease in the discs’ capability to retain or imbibe water (75). This affects its biomechanical function (i.e. poro-elastic behavior), as it hampers the disc’s ability to recover from daily loading conditions. Subsequently, creep (e.g. irreversible deformation) with loss in disc height and other gross morphological changes will occur over time (76;77). 12 11

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Chapter 1

Mechanical loading is considered to be the major extrinsic cause of intervertebral disc degeneration (78-80). The mechanism by which loading causes IVD degeneration (e.g. instigates loss of PGs) is subject of debate and somewhat paradoxical when considering that load bearing is the primary function of the IVD (81;82). Intervertebral discs are continuously under considerable pressure even during rest. Moreover, mechanical loading is known to be a natural stimulus to chondrocytes and regarded to be essential for maintenance of the homeostasis in cartilaginous tissue by facilitating fluid flow and distribution of nutrients towards and waste products from the cells (78;83;84). Furthermore, it has been reported that threshold values for beneficial or detrimental effects of static and dynamic loading differ between disc regions (nucleus, inner- and outer-annulus) (85-93).

Disc region specific response to loading; a link to lumbar herniation Lumbar disc protrusion and herniation are age-related phenomena, that coincide with degenerative disc disease (DDD) (94-102). The potential connection is unclear, as a recent article by Lama et al. underlines (103). In their clinical study on human herniated discs of working age adults, it was shown that herniated discs needing surgery had only mild to moderate degeneration on the Pfirrmann score, and the herniated nucleus pulposus (NP) tissue did not show signs of degeneration (no significant loss of proteoglycans or water compared to controls). Herniations of the disc occur more frequently in the lower lumbar spine with a predilection of the annulus tear in the posterolateral corner (104). Spinal loading conditions have been identified to be major risk factors for developing a lumbar hernia (105-108). In fact, recent observational studies in the general adult population found that physical loading (109-111) and sitting hours (a static axial load on the spine) (112) are the most important extrinsic risk factors for developing a lumbar hernia. However, it is still unclear why some people develop (symptomatic) degenerative disc disease and debilitating hernias, while others have “uncomplicated” ageing and degeneration of their lumbar IVDs (94;113;114). Studies on human and various types of animal (lumbar) IVDs have shown a correlation between high mechanical forces applied to the disc, and degenerative changes (89;115-119). Both high static and high dynamic

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General introduction axial overloading have negative effects on disc cells and matrix, but effects can vary by region, both nucleus and annulus, as well as anterior versus posterior region

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(87;117;120-127). All factors considered, mechanical loading is a factor of interest, as it is one of the few factors that can be manipulated. However, we need to better understand its protective and harmful effects on the IVD. Mechanical loading conditions might be a determining factor whether lumbar discs age naturally, degenerate or herniate. If so, some regions of the IVD might be more prone to degenerate due to a certain overloading condition than others.

Recognizing early intervertebral disc degeneration In order to therapeutically act on IVD degeneration, we need to understand its etiology better; how does the mechanical environment interact with the IVDs cell and matrix and can overloading cause a loss of GAGs from the NP, that starts the degenerative cascade towards DDD? How does early intervertebral disc degeneration present when looking at the biomechanical properties of the disc? And how can we better visualize and quatify the changes in the IVD involved with early stage degeneration. If we can identify the first degenerative changes, preferably before the disc has suffered irreversible damage, we could intervene to potentially slow down or halt the detrimental effects. The functional changes in the IVDs biomechanics that lead to irreversible height loss are poorly understood. And pain as well as IVD mechanical behavior cannot be seen on a still image (128). In order to identify early DDD, we need to find ways to more reliably measure changes involved with its onset. Novel parameters should be tested for their discriminative capabilities to recognize early and region-specific degeneration (129;130). Focusing on how the disc behaves during loading and recovery, could prove more meaningful in early identification of DDD than assessing relative height loss alone. The biomechanical behavior of the IVD has been characterized in experimental settings with parameters such as stiffness and relaxation time-constant (131-133). However, the matrix’ poro-elastic properties give the IVD a non-linear subsidence and recovery behavior, which results in a large variation in calculated material stiffness, depending on the moment chosen to assess them (134;135). The possibilities to characterize disc behavior in a quantitative manner using modeling 14

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Chapter 1

with stretch exponential function fitting, which is less sensitive to timing, has been studied previously (38;44;136;137). However, the fit parameters have not yet been tested for their ability to distinguish the biomechanical changes occurring with loss of GAGs, as the primary phase of disc degeneration. T2-weighted appearance (signal intensity) of the disc is the resultant of the number of protons (mostly from water) in the nucleus and annulus fibrosis. T2relaxation time based MR techniques cannot provide information on the nature of the water loss or gain in the disc (138;139). Whether the water loss is due to daily activity and diurnal rhythm (circadian; which is known to influence both water and height parameters) (140-145), or a water content drop in the ECM due to a loss of PGs from the matrix and therewith the (in)ability to retain water cannot be distinguished (146148). T2 images is thus an indirect measure of potential matrix degeneration and can be obscured by the influx of fluids during an inflammatory process, by the presence of free water in the lacunas of degraded ECM (149) or even more systemic aberrancies in the motion segments blood flow (67;69). Also, when clinically significant height and T2 signal loss of affected disc levels have occurred, the degenerative cascade will already have reached a stage in which matrix integrity has been lost to a degree that IVD mechanics is failing permanently and the IVD is deemed unrepairable with anti-inflammatory or regenerative modalities (150). Novel quantitative MR techniques such as T1rho (T1r) and apparent diffusion coefficient (ADC) have been suggested to be more accurate in detecting true matrix changes in cartilaginous tissue such as the IVD (151;152). However, these parameters have yet to be directly compared to gold standard quantitative T2 mapping, histological degeneration grading and biomechanical function parameters for IVD degeneration in their ability to distinguish early DDD better and in a region-specific manner (149;153).

Approaches to study the IVD There is a long and continued effort to develop preventive or curative therapies for symptomatic DDD. Efforts are hampered by the complex multifactorial nature of both LBP and DDD (154), as well as a more basal lack of understanding of the mechanisms behind degenerative disease of cartilaginous structures such as the IVD (155-157). As our fundamental knowledge on the etiology expands, therapeutic 14

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General introduction targets can be identified and new treatment strategies developed to potentially slow down or halt the progression of DDD (158-162). Mechanical loading has been

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extensively studied and evidence has gathered that it is a major extrinsic factor in preservation of disc homeostasis on the one hand and the onset of degeneration of the IVD on the other hand (83). More detailed knowledge is needed on exactly how mechanical conditions influence the disc (163). Various experimental models are utilized to get answers to specific questions on the role of particular factors involved in DDD. Human cadaveric spines and IVDs would theoretically be the ideal specimens for such investigations. However, this material is only available in small quantities and are never fresh (more than 8 hours post-mortem strongly decreases IVD (cell) viability) due to legislation. Moreover, human cadaveric material is mostly from old individuals, who usually have severely degenerated spines. This rules these specimens out for studies on the etiology of IVD degeneration. Post-mortem cooling or even freezing and the use of fixation techniques (i.e. formaldehyde) render specimens unusable for any type of experimental application, such as mechanical or (cell) biological (164-169). It is very difficult to study the effects of loading in in vivo (animal) models, because they lack direct control and monitoring of mechanical conditions of the IVD. In addition, in vivo methods are costly and raise ethical concerns (170-172). In vitro cell culture models are less appropriate because these cannot mimic the specific tissue composition and exceptional physical conditions of the IVD (hyperosmotic and extremely low concentrations glucose, oxygen and other nutrients) (172;173). Several organ culture (ex vivo) models with IVDs of various animal species have been introduced to study disc mechanical function and the role of mechanical loading in DDD (174-178). These models vary in their relevance to the human situation with regard to IVD dimensions (small animals like mice and rats) (179), biomechanical properties of the animal IVD (bovine tail, lumbar porcine and sheep) (180-186), and cellular and matrix composition (rabbit lumbar disc) (172;187-189). Ideally, an ex vivo model would implement a large species lumbar IVD, comparative in biological and mechanical properties to the human IVD [41], as a precursory platform to an in vivo DDD model for follow-up studies. Natural mechanical loading conditions of the caprine spine resemble those in the human erected spine, despite being a quadruped and therewith the predominately horizontal orientation of the spinal motion segments (190), as in vivo measurements 16

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Chapter 1

have shown (191). The geometry of the lumbar caprine IVD (76;192;193), biomechanical properties (39;43;44;135;194;195) and matrix content are more comparative to human IVDs (132) than discs from small animals (rats, rabbits), or tail discs from cows, pigs or sheep (196-200). Importantly, as in human IVDs, the caprine IVD lacks notochordal cells, which also makes the goat IVD comparable from an embryological and cell biology perspective (187;188;201). It is thought that due to the lack of notochordal cells, like the human IVD, the goat IVD has naturally occurring degeneration with ageing (202;203). Together with our well established goat in vivo IVD degeneration (188;204) and herniation model (205), the caprine lumbar IVD is an excellent candidate for ex vivo studies on disc degeneration and regeneration. Such a translational study platform could be utilized to answer fundamental questions in a more comparative to human model, and separate the wheat from the chaff regarding newly proposed diagnostic and therapeutic options.

Overall aim of this thesis The general goal of the current thesis is to get a better understanding of how mechanical loading conditions can influence the IVDs status using an ex vivo IVD culture model. We want to know to what extent loading can be considered a stimulus and at what point it becomes detrimental to the IVD. We will examine different types of loading conditions (no load, simulated-physiological and overloading) and how they affect IVD cells, extracellular matrix and mechanical properties. We will study the IVDs response in more detail to gain knowledge on disc region specific responses and whether a certain type of mechanical loading can be linked to herniation of the IVD. Furthermore, we will utilize our ex vivo model to examine the potentials of a biomechanical evaluation method and novel MRI techniques in their ability to detect early degenerative changes in the IVD.

Specific aims and questions Chapter 2: Is long-term ex-vivo culture of a large lumbar IVD feasible? 16

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General introduction The objective of the study in chapter 2 is to test the feasibility of ex vivo culture of caprine lumbar IVDs in the Loaded Disc Culture System (LDCS) over a

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21-day period. The LDCS is a custom-build bioreactor (figure 1), designed to culture an entire large lumbar IVD. By culturing a complete IVD with cartilaginous endplates intact, we hope to conserve the IVD cells in their native environment. The LDCS allows for precise control and monitoring of oxygen- and nutrient supply, as well as (axial) mechanical loading conditions via a force-feedback loop. If culture is feasible, the LDCS will provide a platform to study the interaction between disc loading and IVD biology. Moreover, an ex vivo model may serve as a prescreening platform of future diagnostics and therapeutics prior to in vivo testing on life animal and clinical trials. We hypothesize that applying a diurnal regime of sinusoidal mechanical axial loads with adequate magnitude, along with other specific culture conditions will simulate in vivo physiological conditions and therefore maintain the goat IVD properties in culture.

Is there a need for axial loading to maintain native IVD properties in culture? What is the effect of unloading or low dynamic loading when compared to a simulatedphysiological load? We aim to characterize the IVDs response to ex vivo culture with and without loading on a biomechanical, cellular and extracellular level. We hypothesize that without an adequate axial force applied to the disc and therewith no stimulate of fluid-flow due to a lack of disc deformation, the unloaded or low dynamic loading condition will have a detrimental effect on the IVDs status.

Chapter 3: Can mechanical overloading cause IVD degeneration? The main function of the IVD is to transfer high magnitude axial forces, while maintaining flexibility of the spine. Loading is therefore a natural stimulus for the IVD and is even thought to be essential for maintenance of cell viability and matrix biology (206). Conversely, excessive mechanical loading evokes catabolic cellular behavior, which may trigger a cascade towards disc degeneration, i.e. loss of proteoglycans and water from the disc, with subsequent changes in mechanical 18

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Chapter 1

properties of the disc and further matrix breakdown (82). Whether mechanical loading is a positive stimulus or induces damage to the IVD, is dependent on the type of load applied, its magnitude, duration and frequency (120;123;207). Furthermore, it has been reported that threshold values for beneficial or detrimental effects of static and dynamic loading differ between disc regions (89;115-119). We aim to improve our understanding of the mechanobiology involved in load-induced IVD degeneration and thereby provide more integral insight in the early degenerative process. We hypothesize that both static and dynamic overloading lead to disc degeneration, resulting in changes in the biomechanical behavior of the discs cell survival, gene expression, and matrix structure and content.

What is the effect of dynamic and static overloading on the nucleus and annulus region? We will investigate whether dynamic and static overloading have different degenerative effects on the nucleus and annulus of caprine lumbar discs. We want to know how the biomechanical response changes over time and how this is connected to cell and matrix response of the different IVD regions. We hypothesize dynamic and static overloading will have different detrimental effects on the discs’ nucleus and annulus.

Chapter 4: Is there a region (anterior, lateral and posterior) specific response to dynamic and static overloading that could explain the posterolateral predilection of lumbar hernias? The shape of the disc, annulus thickness and the presence of the posterior longitudinal ligament (PLL) have been described as factors contributing to the posterolateral predilection of hernias (104). However, all humans have the same lumbar disc morphology and a PLL, but not all discs herniate. Furthermore, it does not explain why many herniations in patients occur without a clear inciting moment such as heavy lifting or the combination of flexion and torsion in which a weak spot in the disc might tear, but during seemingly arbitrary loading conditions (208).

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General introduction If dynamic and static overloading have different effects on the nucleus and the annulus region, there may also be a difference in effect to the anterior, lateral and

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posterior disc (outer annulus), which could explain the posterolateral predilection of hernia's. We will conduct a series of experiments with the LDCS to investigate the influence of strictly axial dynamic or static mechanical overloading on the various regions of the IVD. We address the question of possible location-dependent effects by analyzing the regional biomechanical response (height loss and pressure distribution) in the disc to axial loads during culture and how this influences the cells and matrix in the various disc regions over time. We hypothesize that strictly axial static overloading (as a simulation of a sedentary life-style) will affect the cells and matrix in the posterolateral region more strongly than in other regions of the intervertebral disc.

Chapter 5: How do the poro-elastic properties of the IVD change in early intervertebral disc degeneration? Intervertebral discs have to cope with considerable (axial) pressure even during rest. They are able to do so in a healthy state via a hyper-hydrated pressurized state of the nucleus within the constraints of a semi-porous annulus ring. The disc deformation in response to axial (un)loading resembles both a fluid-flow and solidelastic dynamic, resulting in the unique poro-elastic behavior of the IVD as displayed during subsidence and recovery. The unique intradiscal matrix properties which enable this behavior, also rely on these same mechanical and fluid-flow dynamics to maintain their own matrix and cell properties, as it protects the disc from high peak loads or too fast deformation and facilitates nutrient supply to the cells and diffusion of waste products out of the IVD (78;83;84). The hyper-hydrated state of the nucleus is enabled by the abundant presence of GAG molecules in the nucleus, which actively attract and hold high amounts of water in their negatively charged molecular structure. It is widely accepted that the cascade of degeneration of the IVD starts when the IVD loses GAGs from its nucleus (209). In time, this causes the disc to loose height, but must affect disc behavior prior to that, in order for height loss to occur. How the poro-elastic behavior is affected will be further elucidated in this chapter. To this end we will use our bioreactor and 20

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Chapter 1

injection of Chondroïtinase ABC (Cabc; GAG cleaving enzyme) to create standardized degeneration in the IVDs, as a simulation of the primary phase of disc degeneration. The LDCS will allow standardized loading and culture conditions with continuous monitoring of IVD displacement (76;210). We will assess the changes in the IVD poro-elastic behavior and use histological grading, matrix content measurements and standard MR imaging to evaluate the effects of injection on the IVDs.

Can we use exponential fitting to identify dysfunctional disc behavior before height and water are permanently lost? We will characterize the deterioration of the poro-elastic properties of the IVD in response to the Cabc injection, by fitting the recovery displacement curves with a stretched exponential function. The disc behavior will be expressed by the three parameters from the fits and we will evaluate them on their capability of identifying early DDD. Outcomes will be compared to absolute displacement data (height loss) and correlated to histological grading, matrix water and GAG content measurements and Pfirrmann score. We hypothesize that the stretched exponential parameters will closely reflect the various changes in biomechanical and matrix properties.

Chapter 6: How do quantitative T2, T1rho and ADC maps change with mild IVD degeneration? We will use our ex vivo culture model and injection of two different concentrations of Cabc to create graded disc degeneration of the lumbar caprine IVD. High-resolution (9T) images will be obtained before and after the culture experiment. The differences between T2, T1r and ADC pre- and post-scans will determine which sequence is most sensitive to detect the various grades of IVD degeneration. We hypothesize that T1r and ADC, due to a higher spatial resolution, will have stronger signal changes when compared to T2.

Which MRI technique is superior in quantifying early (regional) IVD degeneration?

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signal changes when compared to T2.


Which MRI technique is superior in quantifying early (regional) IVD degeneration? We will correlate quantitative T2- and T1rho and ADC mapping results with

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measured biomechanical parameters of disc behavior, histological appearance21 (Rutges scale for degeneration) and the absolute amount of water and PGs in the matrix after culture, to depict which quantitative MRI sequence is the best candidate to detect early degenerative matrix changes in the disc. We hypothesize that T1r will correlate strongest to actual biomechanical and matrix changes, because it is most sensitive to changes in protein-bound proton content (e.g. loss of GAGs).

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Brinckmann P. Disruption of bovine tail discs resulting from compressive testing dependent on flexion, state of hydration, and stress rate. Spine (Phila Pa 1976 ) 2002 Jan 15;27(2):217-9.

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Risbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Crit Rev Eukaryot Gene Expr 2011;21(1):29-41.

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Paul CP, Zuiderbaan HA, Zandieh DB, van der Veen AJ, van de Ven PM, Smit TH, et al. Simulatedphysiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture. PLoS One 2012;7(3):e33147.

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Chapter 2. Simulated-physiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture

C.P.L. Paul, H.A. Zuiderbaan, B. Zandieh Doulabi, A.J. van der Veen, P.M. van de Ven, T.H. Smit, M.N. Helder, B.J. van Royen, M.G. Mullender

Adapted from: PLoS One. 2012;7(3):e33147 36

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Abstract Low-back pain (LBP) is a common medical complaint and associated with high societal costs. Degeneration of the intervertebral disc (IVD) is assumed to be an important causal factor of LBP. IVDs are continuously mechanically loaded and both positive and negative effects have been attributed to different loading conditions. In order to study mechanical loading effects, degeneration-associated processes and/or potential regenerative therapies in IVDs, it is imperative to maintain the IVDs’ structural integrity. While in vivo models provide comprehensive insight in IVD biology, an accompanying organ culture model can focus on a single factor, such as loading and may serve as a prescreening model to reduce life animal testing. In the current study we examined the feasibility of organ culture of caprine lumbar discs, with the hypothesis that a simulated-physiological load will optimally preserve IVD properties. Lumbar caprine IVDs (n=175) were cultured in a bioreactor up to 21 days either without load, low dynamic load (LDL), or with simulated-physiological load (SPL). IVD stiffness was calculated from measurements of IVD loading and displacement. IVD nucleus, inner- and outer annulus were assessed for cell viability, cell density and gene expression. The extracellular matrix (ECM) was analyzed for water, glycosaminoglycan and total collagen content. IVD biomechanical properties did not change significantly with loading conditions. With SPL, cell viability, cell density and gene expression were preserved up to 21 days. Both unloaded and LDL resulted in decreased cell viability, cell density and significant changes in gene expression, yet no differences in ECM content were observed in any group. In conclusion, simulated-physiological loading preserved the native properties of caprine IVDs during a 21-day culture period. The characterization of caprine IVD response to culture in the LDCS under SPL conditions paves the way for controlled analysis of degeneration- and regeneration-associated processes in the future.

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Simulated-physiological loading preserves IVDs

Introduction Low-back pain (LBP) is the most common medical complaint in Western society, possibly leading to an incapacitating condition and encompassing considerable ensuing socio-economic costs (1). It is widely recognized that multiple

2

factors underlie the complex pathology of LBP. Intervertebral disc (IVD) degeneration, or degenerative disc disease (DDD), has been associated with LBP (25) and recent large population based studies provide strong evidence for their correlation (6). Presently, the only options for patients with symptomatic disc degeneration are conservative treatments, such as physical therapy (7), pain medication (8) and acupuncture (9), or surgical salvage procedures involving removal of the disc followed by fusion or arthroplasty (10, 11). Various new treatment strategies are being developed to halt the progression of degeneration or even to regenerate the intervertebral disc. This is challenging as DDD itself is considered a multi factorial process (12). Many risk factors have been identified such as trauma to the spine (13, 14), aging (15-17), loss of nutrient supply to the disc (18), and genetic predispositions(19-23). Mechanical loading of the intervertebral disc is considered to be a major extrinsic cause of intervertebral disc degeneration (24-27). Yet, load bearing is the primary function of the IVD, with discs continuously being under considerable pressure even during rest. Moreover, mechanical loading is a natural stimulus to chondrocytes and regarded to be essential for maintenance of the cartilaginous matrix (12, 24, 28-32). In order to develop therapies against DDD more detailed knowledge is needed on the influence of loading on the preservation, degeneration and regeneration of the IVD (4). This cannot be adequately investigated in cell culture models, because these cannot mimic the specific tissue composition and exceptional physical conditions of the IVD. In vivo animal models such as described in earlier studies from our group, lack close control and monitoring of mechanical conditions of the IVD. Several organ culture models with IVDs of various animal species have been introduced to study disc function and the role of different etiological factors involved in DDD (33-37). These models vary in their relevance to the human situation with regard to IVD dimensions, biomechanical properties, and cellular and matrix composition (38). 38

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Ideally, an ex vivo model would implement a large species lumbar IVD comparative in biological and mechanical properties to the human IVD (39), as a precursory platform to an in vivo DDD model for follow-up studies. As we have shown in recent publications, the goat IVD closely resembles the human IVD with respect to mechanical properties (40). Moreover, as in human IVDs, the caprine IVD lacks notochordal cells, which also makes the goat IVD comparable from a biological perspective. Together with our well established goat in vivo IVD degeneration and herniation model, the caprine lumbar IVD is an excellent candidate for ex vivo studies on disc degeneration and regeneration (41-44). Therefore we have developed a bioreactor, the Loaded Disc Culture System (LDCS), designed to culture entire large IVDs (i.e. IVD with cartilaginous endplates), conserving IVD cells in their native environment. The LDCS allows for precise control and monitoring of oxygen- and nutrient supply, as well as mechanical loading conditions via a force-feedback loop. Providing a platform to study the interaction between disc loading and IVD biology. Moreover, an ex vivo model may serve as a prescreening platform of future therapeutics prior to in vivo testing on life animal. The objective of the current study is to test the feasibility of ex vivo culture of caprine lumbar IVDs in the LDCS over a 21 day period. More specifically, we aim to characterize the IVDs response to culturing with and without loading. We hypothesize that applying simulated-physiological loading will be appropriate to maintain the goat IVD properties.

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Material & Methods Specimens and Culture Conditions Cadaveric lumbar spines (n = 12) from healthy skeletally mature female (3-5 year-old) slaughter goats (Capra aegagrus hircus, sub breed Dutch white milk goat)

2

were obtained from an abattoir (no approval of ethical board required). Within 3 hours of slaughter, IVDs with adjacent cartilaginous endplates (L1-L6) were dissected under sterile conditions using an oscillating surgical saw. Maximal width, depth (midsagittal), and height of the IVDs with endplates (EP) were measured with a caliper. The cross-sectional IVD area was calculated assuming an elliptic shape: IVD area ≈ 1/4π × width × depth From each spine, two IVDs (Th13-L1 and L1-L2) were used as baseline reference for the parameters measured. The remaining IVDs were cultured over 7, 14, or 21 days in individual culture chambers of the LDCS, which is housed in an incubator at 37 ºC, 95% humidity, and 5% CO2. Discs were cultured in standard DMEM (Gibco, Paisley, UK) with 10% FBS (HyClone, Logan, UT), 4.5gr/L glucose (Merck KGaA, Darmstadt, Germany), 50 µg/ml ascorbate-2-phosphate (Sigma Aldrich, St. Louis, MO), 25 mmol/L HEPES buffer (Invitrogen), 10,000 u/ml penicillin, 250 µg/L streptomycin, 50 µgr/mL gentamicin and

1.5µgr/mL

amphoterizin B (all from Gibco). Medium (40 ml per culture system) was circulated continuously (3ml/hr) using a peristaltic pump and was exchanged every 48 hours and checked for pH (7.2-7.4) and osmolarity (360-380 mOsm; measured by cryoscopy).

Loaded Disc Culture System An overview picture of the LDCS actuators, a schematic picture of a single actuator and a detailed cross-section image of an actuator are given in figure 1. The LDCS consists of two large incubators (Forma Steri-cult, Thermo Scientific, Asheville, NC), each housing twelve actuators which are individually controlled and monitored by a Labview-based custom built software program. Each actuator delivers force-controlled axial loading (clamping module EV63, Festo Corporation, Hauppage, NY) to a culture chamber, which is regulated via a feedback-loop system. IVD 40

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loading and displacement are continuously measured (Kam-e load cell, Bienfait, Haarlem, The Netherlands; oadm12 optoelectric sensor, Baumer, Berlin, Germany), signals are digitized (100Hz) and stored in a PC for further analyses. The custom designed three piece culture chamber comprises of two similar top and bottom parts made of polycarbonate, with a central in- and output channel for the culture medium. A thin semi-transparent silicon membrane connects top and bottom halves. The IVD is placed in the center of the culture chamber with rigid titanium filters on each endplate. The center axial screw is set to make contact with the top part of the culture chamber, thereby adjusting for the individual height of the IVDs. This is done during real-time load measurement, making sure the screw is fitted without applying load to the disc (between 0-5 Newton maximum). Culture medium is pumped over the bottom endplate into the culture chamber, immersing the IVD. Medium exits over the top endplate into a high-surface, low-volume medium reservoir for optimal gas exchange with filter sterilized air.

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Figure 1. The Loaded Disc Culture System (LDCS; upper left), a single actuator (upper right) with culture chamber and reservoir, and a schematic cross-section (bottom) of an actuator with IVD.

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Loading Protocols and Mechanical Properties Mechanical loading of the IVDs was strictly axial. Loading magnitudes and frequency were derived from in vivo pressure measurements in a lumbar segment of a goat during different activities (e.g. lying down, walking and jumping on a haystack) (45). Firstly, all discs were subjected to a low sinusoidal load (Low Dynamic Load, LDL; 0.09-0.11 MPa; 1Hz) during the first 8 hours of culture to investigate the response of the disc to initial low axial loading (subsidence behavior). IVDs were assigned to one of three experimental culture groups: 1. without loading (unloaded), 2. continuous low dynamic load (LDL; 0.1-0.2 MPa, 1Hz) or 3. diurnal simulated physiological load (SPL consisting of a sinusoidal load (1 Hz) alternating in magnitude every 30 minutes (between 0.09-0.11 MPa and 0.1-0.6 MPa) for 16 hours per day, followed by 8 hours of LDL (Fig 2). The LDL and SPL loading conditions are approximations of the measured pressures during respectively lying down and walking. For standardization, the LDL and SPL regimes were preceded by a low dynamic load (sinusoidal; 0.1-0.2MPa; 1Hz) during the first 8 hours of culture, the SPL regime also ended with 8 hours of LDL loading. The mean displacement at the end of each daily loading cycle was analyzed to assess overall disc height changes over time. In dynamically loaded discs, IVD stiffness was calculated from the load deformation curves of the ascendant part of 5 consecutive sine waves at consistent time intervals using regression.

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Figure 2. Scheme of the daily simulated-physiological loading (SPL) regimes. On the Y-axis the axial load (MPa) as applied on the IVDs. All IVDs started with 8 hours of low dynamic load (LDL) around 0.1 MPa, after which a 16 hour SPL loading regime was applied as indicated in the caption, this diurnal regime is repeated daily.

Histology and quantitative cell biology Directly after dissection from the spine (baseline control) or culture in the LDCS, selected IVDs were fixed in 4% formaldehyde for 48 hours and decalcified for 10 days using standard Kristensen’s fluid. Paramidsagittal tissue slices (3mm thick) were cut from the IVD specimen with a scalpel and embedded in paraffin. With a microtome, 3 micrometer (μm) thin sections were cut and stained with safranin-O (proteoglycans) and Masson’s trichrome (collagen). Cell viability was assessed in the nucleus pulposus (NP), the inner (iAF) and outer annulus fibrosis (oAF). We removed one endplate and incubated IVDs (n≥6 for each group and time point) in a 6-well plate in serum-free medium containing 2µM Celltracker Green (CTG; Chloromethylfluorescein, Molecular Probes, Eugene, OR) and 2µM propidium iodide (PI; Sigma) under free-swelling conditions. After incubation for 1 hour, IVDs were washed in PBS, flash-frozen and 10 µm transverse cryosections were cut with a cryostat. Images (10481342 pixels) were taken at 10 magnification (surface area ~1mm2) using fluorescent light on an inverted microscope 44

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(Leica DM6000, Wetzlar, Germany; filters: I3 S450-490nm and N2.1 S515-560 nm). (Leica DM6000, Wetzlar, Germany; filters: I3 S450-490nm and N2.1 S515-560 nm). The total number of cells per area (cell density) and the percentage of live cells (100% The total number of cells per area (cell density) and the percentage of live cells (100% (# live cells / # total cells)) were determined using 10 images per region for each IVD. (# live cells / # total cells)) were determined using 10 images per region for each IVD. Co-labelled cells were counted for the cell density measurement, but were excluded Co-labelled cells were counted for the cell density measurement, but were excluded from the analysis of cell viability. A fresh (day 0) IVD was used as positive control. from the analysis of cell viability. A fresh (day 0) IVD was used as positive control. As a negative control, a thoracic IVD, which underwent a freeze-thawing cycle three As a negative control, a thoracic IVD, which underwent a freeze-thawing cycle three times prior to staining was used. times prior to staining was used. RNA isolation, cDNA synthesis and RT-qPCR RNA isolation, cDNA synthesis and RT-qPCR Nucleus and outer annulus tissue samples were homogenized with ceramic Nucleus and outer annulus tissue samples were homogenized with ceramic beads in a lysis solution (MagnaLyser, GmbH, Roche Diagnostics, Brussels, Belgium) beads in a lysis solution (MagnaLyser, GmbH, Roche Diagnostics, Brussels, Belgium) with 4 runs of 30 seconds at 6500 rpm with in-between cooling. Total RNA was with 4 runs of 30 seconds at 6500 rpm with in-between cooling. Total RNA was isolated with the MagnaPure robot using the RNA isolation kit III (both Roche isolated with the MagnaPure robot using the RNA isolation kit III (both Roche Diagnostics). cDNA synthesis was performed using Superscript Vilo® (Invitrogen, Diagnostics). cDNA synthesis was performed using Superscript Vilo® (Invitrogen, Merelbeke, België) and real-time PCR reactions were performed using the Merelbeke, België) and real-time PCR reactions were performed using the SYBRGreen reaction kit (Roch Diagnostics) both according to the manufacturer’s SYBRGreen reaction kit (Roch Diagnostics) both according to the manufacturer’s instructions in a LightCycler 480 (Roche Diagnostics). IVD cell gene expression was instructions in a LightCycler 480 (Roche Diagnostics). IVD cell gene expression was assessed for a range of anabolic (collagen types 1, 2 and 6, aggrecan, biglycan, and assessed for a range of anabolic (collagen types 1, 2 and 6, aggrecan, biglycan, and Sox9), catabolic/remodeling (MMP (matrix metalloproteinase) 1, 13 and 14, Sox9), catabolic/remodeling (MMP (matrix metalloproteinase) 1, 13 and 14, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) 4 and 5, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) 4 and 5, TIMP (tissue inhibitors of metalloproteinase) 1 and 3) and inflammatory-related genes TIMP (tissue inhibitors of metalloproteinase) 1 and 3) and inflammatory-related genes (c-JUN, COX (cyclooxygenase) 2 and IL (interleukin) 6). The primers used for the (c-JUN, COX (cyclooxygenase) 2 and IL (interleukin) 6). The primers used for the gene expression analyses are shown in Table 1. Stability of expression of gene expression analyses are shown in Table 1. Stability of expression of housekeeping genes YWHAZ (tyrosine 3-monooxygenase/tryptophan 5housekeeping genes YWHAZ (tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein) and 18S (ribosomal RNA) was calculated by monooxygenase activation protein) and 18S (ribosomal RNA) was calculated by geNorm software (http://medgen.ugent.be/genorm). As the expression of all genes geNorm software (http://medgen.ugent.be/genorm). As the expression of all genes was within a 3-fold range of YWHAZ expression levels, this housekeeping gene was was within a 3-fold range of YWHAZ expression levels, this housekeeping gene was used as normalization factor. Absolute expression of all genes was quantified with used as normalization factor. Absolute expression of all genes was quantified with fitpoint calculation (Lightcycler software) using the standard curve method, based on fitpoint calculation (Lightcycler software) using the standard curve method, based on serial dilution of standards for each gene. Relative gene expression is shown as the serial dilution of standards for each gene. Relative gene expression is shown as the ratio between absolute expression of the gene of interest divided by the absolute ratio between absolute expression of the gene of interest divided by the absolute YWHAZ expression of the same sample. Samples with no detectable RNA YWHAZ expression of the same sample. Samples with no detectable RNA concentration of the target gene, but with detectable gene concentration of the 44

45 housekeeping genes (Ct 0.001) when compared to changes in the PBS group (58.5ms (p < 0.001). Post-experiment T2 and T1rho values in the nucleus region were ±10.4). T1rho decrease was also significantly lower in both the 0.25 Cabc (83.3ms significantly lower compared to pre-experiment signal (p < 0.001) in all three ±30.1; p = 0.001) and the 0.5 Cabc (74.3 ±29.6; p > 0.001) group when compared to experimental groups (Fig 4). More specifically, T2 nucleus values dropped changes in the PBS group (107.4 ±32.1) (Fig 4A and B). T2 and T1rho changes for significantly more in the 0.25 Cabc group (47.7ms ±9.8; p = 0.04) and the 0.5Cabc the inner- and outer annulus regions in the Cabc groups did not differ significantly group (46.1ms ±8.9; p > 0.001) when compared to changes in the PBS group (58.5ms when compared to the PBS control group (data not shown). For all AF regions ±10.4). T1rho decrease was also significantly lower in both the 0.25 Cabc (83.3ms changes were less consistent and none significantly different in comparison with the ±30.1; p = 0.001) and the 0.5 Cabc (74.3 ±29.6; p > 0.001) group when compared to PBS control group (Fig 4). changes in the PBS group (107.4 ±32.1) (Fig 4A and B). T2 and T1rho changes for Withand ADC the 0.5 Cabc group a larger drop in (in thesignificantly anterior the innerouter annulus regionsdisplayed in the Cabc groups didsignal not differ and outer-annulus) 0.25group Cabc (data and not PBSshown). group. However, whenposterior compared to the PBS than control For all AFwhen regions compared with PBS were significant different in any region (Fig 4C, D and E). changes were less none consistent and none significantly different in comparison with the

PBS control group (Fig 4). With ADC the 0.5 Cabc group displayed a larger drop in signal (in the anterior and posterior outer-annulus) than 0.25 Cabc and PBS group. However, when

6

compared with PBS none were significant different in any region (Fig 4C, D and E).

Figure 4. Bar graphs showing mean ± SD difference (paired %post-experiment values to pre) for all three experimental groups. Results are shown of the T2 (a) and T1rho (b) values in the 175 nucleus and ADC in the outer-annulus; anterior (c), lateral (d) and posterior (e). PBS clearly had the least influence on quantitative MRI values for all three parameters, whereas 0.25 and 0.5 Cabc had a dose-dependent loss of signal loss in the nucleus. Only 0.5 Cabc also showed significant change in the anterior and posterior outer-annulus when compared to the PBS-treated controls. Asterisk (*) indicates p-values below 0.05.

175 Correlation of MRI with recovery behavior:

173

Figure 4. Bar graphs showing mean ± SD difference (paired %post-experiment values to pre)

when compared to thegroups. PBS control group (data notT2shown). For all AF regions for all three experimental Results are shown of the (a) and T1rho (b) values in the

Chapter 6

nucleus and ADC the outer-annulus; anterior (c), lateral (d) and posterior (e). PBS clearly changes were lessin consistent and none significantly different in comparison with the had least influence on 4). quantitative MRI values for all three parameters, whereas 0.25 and PBSthe control group (Fig

0.5 Cabc had a dose-dependent loss of signal loss in the nucleus. Only 0.5 Cabc also showed With significant the anterior and posterior outer-annulus when compared to the ADCchange the 0.5inCabc group displayed a larger drop in signal (in the anterior PBS-treated controls. Asterisk (*) indicates p-values and posterior outer-annulus) than 0.25 Cabcbelow and 0.05. PBS group. However, when

compared with PBS none were significant different in any region (Fig 4C, D and E). Correlation of MRI with recovery behavior: Changes in recovery behavior due to injection of PBS or Cabc are shown in Table 2. As expected, Cabc injection had a dose-related effect on the IVD’s poroelastic behavior. The tau (time-constant) increases significantly more compared to PBS in the 0.25 Cabc (p = 0.024) and 0.5 Cabc (p < 0.001), and 0.5 more than 0.25 Cabc (p = 0.005). The beta (stretch-constant) decreased significantly in the 0.5 Cabc group when compared to PBS (p = 0.006). The delta infinite (height loss) also changed significantly in the 0.5 Cabc groups when compared to PBS (p < 0.001) and 0.25 Cabc (p = 0.02).

Table 2. Stretched exponential fit parameters for the recovery curves at day 20 Exp. group

175

τ (tau)

β (beta)

δ∞ (delta infinite)

PBS

3.08 ± 0.61

1.01 ± 0.12

-4.6 ± 0.8

0.25 Cabc

3.74 ± 0.62

0.87 ± 0.14

-5.2 ± 0.9

0.5 Cabc

5.54 ± 1.41

0.70 ± 0.24

-6.4 ± 1.2

Table 2. Descriptive parameters (mean ± SD) from the stretched-exponential fits of the recovery deformation curves during the last LDL loading at day 20 for all three experimental groups.

The measures of disc biomechanics during recovery at day 20 are correlated to the post-experimental MR scan values (Fig 5). T2 and T1rho nucleus values have an 176

174

T1rho to detect early IVD degeneration

equal negative moderate correlation with tau (r = -0.675 and r = -0.691, respectively; not significantly different). For beta both have a moderate positive correlation. For the ADC neither the separate outer-annulus region values nor a mean outer-annulus value showed any correlation (R-value above 0,3) with the day 20 tau, beta or delta infinite values (data not shown).

6

Figure 5. Correlation of post-experiment T2 (top row) and T1rho (bottom row) nucleus values with day 20 stretched-exponential parameters tau, beta and delta infinite.

In Fig 6 the correlations are shown between the MR post-experiment values and the histological score for degeneration. The Rutges score in the PBS group was the lowest; 2 to 5 (mean score=3,6), corresponding with no or mild degenerative changes. Both Cabc groups had scores ranging between 4 and 9 (0.25 Cabc mean score=5.8; 0.5 Cabc mean score=6.2), showing mild to moderate degenerative changes. T1rho values correlate strongly with the degeneration score (r = -0.854), significantly stronger than T2 and ADC nucleus values (p = 0.005 for T1rho compared to T2 and p < 0.001 for T1rho compared to ADC). 177

175

Chapter 6

Figure 6. Correlation of post-experiment MR T2, T1rho, and ADC nucleus values with Rutges histological degeneration score.

The relation between matrix-content in the nucleus (water and GAG) as measured after the experiment and the post-experiment MR values in show that for T2 had a strong positive correlation with water-content (r = 0.863), significantly stronger than T1rho (r = 0.634; p = 0.01) and ADC (r = 0.692; p = 0.047) (Fig 7). T1rho showed the strongest positive correlation with GAG-content (r = 0.872), significantly stronger than T2 (r = 0.807; p = 0.03) and ADC (r = 0.620; p = 0.01).

Figure 178 7. Correlation of post-experiment MR T2, T1rho, and ADC nucleus values to GAG and water content.

176


Discussion In the current study, we demonstrated that quantitative MR mapping, and T1rho in particular, is sensitive to small degenerative changes in the IVDs matrix. To our knowledge, this is the first study to directly compare the correlation of quantitative high-resolution T2, T1rho, and ADC maps with actual disc recovery behavior. These correlations combined with histology and GAG-content in a lumbar IVD large-animal model and region-specific results, demonstrate that T1rho nucleus values correlate with GAG content, histological degeneration, as well as disc mechanical properties to a higher degree than T2 and ADC. With the use of a 9.4T MRI we were able to image the lumbar caprine IVD in high anatomical detail. We quantitatively mapped the IVD’s 5 distinct regions on T2, T1rho, and ADC images and found significant differences between the nucleus, innerannulus, and the anterior, lateral and posterior outer-annulus (Fig 2 and Table 1). These regions have previously been described to be distinct histologically and in

6

biochemical composition of the matrix in both human and caprine IVDs (45, 46, 48, 54-57). The MR baseline values found for these IVD regions in the pre-experimental scans closely resemble values found in similar regional measurements of human lumbar discs (58-62). Furthermore, pre-loading values correspond to those found for Pfirrmann grade 1-2 (healthy) human lumbar IVDs, while post-loading data is similar to grade 2-4 mild degenerative IVDs (59, 61, 63, 64). This further validates our Cabcinduced degeneration model in lumbar caprine discs as a comparative model for early human IVD degeneration. T1rho values pre- and post-experiment showed a larger range of values than T2 (Fig 3 and 4). This was expected as it is in part inherently due to the physical differences between T2 and T1rho measures, since T1rho has a larger dynamic range than T2 (65-68). However, the differences found in the correlation of T2 and T1rho and disc mechanical properties, as expressed by the stretched-exponential parameters (Table 2 and Fig 6), show that T1rho values are also more closely linked to actual disc function. Both T2 and T1rho correlated to the Cabc dose-dependent tau increase. T1rho’s higher correlation to beta, is most likely due to T1rho’s stronger correlation to GAG-content (Fig 7). The R-values found in the current study are comparative to T1rho-GAG correlations reported in human (65, 69). The stretch-constant beta 180

177

Chapter 6

deviates further from 1 (to zero) when poro-elastic properties are lost in favor of more linear (solid-elastic) material behavior (52). In the case of the IVD, this has been shown to be caused by loss of GAGs (and therewith water) from the NP (70) and structural damage to the disc (50). Taken together, when lower T1rho values are found, this is representative for the biomechanical deterioration of the poro-elastic properties of the IVD, which is the first step in the degenerative cascade of DDD. Our data on T2- and T1rho in correlation with mechanical properties of the disc, e.g. recovery behavior (Fig 4 and 5 and Table 2) are in agreement with numerous other reports that studied similar correlations. In reports from Mwale et al. (bovine tail IVD, 2008) and Antoniou et al. (human lumbar IVDs, 2013) T2 and T1rho values were found to correlate (r-values between 0.6-0.7) with compressive modulus and hydraulic permeability of NP and AF tissue samples of various degenerative states (71, 72). On human subjects with in vivo discography measured “opening pressure” (OP), Borthakur et al. (2011) reported lower T1rho values and pressures in painful discs with moderate correlation (r = 0.54) of T1rho and OP (73). Various other reports have shown similar correlations of T1rho to intradiscal pressure (39, 74). In addition, an important finding in the current study is that in the same IVD samples, T1rho also correlates strongly to the histological degeneration score (r = 0.854) and significantly better than T2 and ADC (Fig 7). The Rutges degeneration score is an adaptation of the traditional Thompson and Boos scores (53). Besides sagittal and transvers H&E stained sections, it includes a safranin-O and alcian-blue staining of transverse IVD sections. The latter two are both PG-content sensitive staining techniques (75). Therefore, we feel the found correlation further exemplifies T1rho’s strong affinity with actual GAG-content in the IVDs matrix. All groups, even the PBS injected IVDs, showed signal loss of T2 and T1rho after culture (Fig 4). Culture medium is hyperosmotic, but comparable to reported osmotic pressures in vivo (76-79). This may still have caused slight efflux of water during culture and loading, explaining the overall loss. However, the LDCS culture conditions do not seem to have distorted T2 values and the correlation to watercontent (r = 863; Fig 7), as these are in line with reports in literature for both goat and human lumbar IVDs (80-83).

178

181


In the current study the pre- and post-experiment scans were performed while IVDs were unloaded for >8 hours and submerged in PBS. This can be regarded as both a strength and limitation of this study. All discs are hereby enabled to return to their maximum hydrated and height recovered state (48). Healthy discs will respond to this environment differently than degenerated discs (79, 84). This could potentially exacerbate the effects of Cabc-induced degeneration or diminish water-content loss under axial compression in degenerative discs. We observe a slightly higher overall water-content (76-89%) in the IVDs when compared to data from our previous reports (70-85%) (45, 46, 48). However, we can still quantify T2 and water-content differences between the experimental groups (Figs 4 and 7), which refutes the latter. Conversely, the much higher correlation of T2 (r = 0.863) with water-content than T1rho (r = 0.634) could be a resultant of an exacerbating effect of the scanning circumstances (i.e. unloaded and submerged in PBS) (Fig 7), although found correlations are consistent with those found in other studies (39, 69, 81, 83, 85, 86). This underlines a very important message, namely that T2 results can be distorted by influences of the IVD environment, more particular the surrounding medium, whereas

6

T1rho –correlating more to GAG content– is less affect by these circumstances. In regards to the influence of unloaded or compressive state of IVDs to MR measures, a study from Manac’h et al. (2013) reports no influence of compression of IVDs during scanning on T1 and T2 parameters (25). However, the influence of the unloaded hydrated equilibrium of IVDs on ADC is unknown. The relatively small changes found in the ADC signal between groups in the current study might be explained (in part) by this unnatural equilibrium (Fig 4, 6 and 7). ADC is a measure for (direction of) diffusion and will likely measure less difference between various degrees of degeneration, when disc height recovery and intradiscal osmotic pressure have reached a static equilibrium to their surroundings. Other reports on the relation between IVD degeneration and ADC values have found significant correlations in both nucleus and annulus (71, 87, 88). The studies distinguishing outer-annulus regions all report ADC to be sensitive to anisotropy. This could also explain why in the current study we did not find effects of degeneration on the ADC values in the lateral outer-annulus, as this was the region most influenced by anisotropy due to our standardized IVD orientation in the magnetic field (Fig 2 and 3).

182

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Chapter 6

In summary, quantitative MRI and novel MRI techniques such as T1rho and ADC provide objective and more accurate information on the degenerative status and mechanical behavior of intervertebral discs than current routine T1 and T2 imaging (23, 89-91). Selective implementation of these tools, e.g. when (early) DDD is expected but T2 images do not confirm the suspicion, T1rho and ADC could aid in observing degenerative changes with more certainty (92). Potentially, with improved patient selection at an early stage, early intervention using therapeutics such as antiinflammatory-, disease-modifying- or regenerative drugs might yield more success in relieving symptoms and preventing progression of the IVDs degenerative cascade.

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Wu N, Liu H, Chen J, Zhao L, Zuo W, Ming Y, et al. Comparison of apparent diffusion coefficient and T2 relaxation time variation patterns in assessment of age and disc level related intervertebral disc changes. PLoS One. 2013;8(7):e69052.

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Lerer A, Nykamp SG, Harriss AB, Gibson TW, Koch TG, Brown SH. MRI-based relationships between spine pathology, intervertebral disc degeneration, and muscle fatty infiltration in chondrodystrophic and non-chondrodystrophic dogs. Spine J. 2015;15(11):2433-9.

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C.P.L. Paul

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Discussion and conclusions Life expectancy has increased in the past 200 years from 40 years to 80 years, while in the last 2 million years our anatomy and physiology has changed little (1). Our intervertebral discs are subject to ageing and degeneration, and much like any castle, they will crumble given enough time (2). But as one would attempt to extinguish a fire in a castle, we should also attempt to stop painful processes accelerating the deterioration of the IVD, especially when this pain causes grave burdens and loss of quality of life. We must therefore rely on our ingenuity (and therewith science) to compensate and aid our hopelessly ageing bodies to cope with our “unnaturally” increased life expectancy (3). The combined work in this thesis shows that it is possible to culture a large lumbar IVD. The LDCS is a bioreactor capable of maintaining caprine lumbar IVDs native properties and live cells for up to three weeks in culture. The LDCS is a good alternative for life animal testing, as we are able to answer important basal and translational questions regarding IVD physiology and pathology on cadaveric lumbar IVDs, rather than with in vivo experiments on live goats. The LDCS can be used to get a better understanding of the role of mechanical load in IVD homeostasis. In addition, we were able to implement this model to study the mechanical changes in early DDD and the potentials of quantitative MRI to identify the associated matrix changes.

The LDCS model: “All models are wrong, but some are useful” (George Box, 1976)” Clearly, the LDCS is a good and very useful model, but not perfect in simulating in vivo conditions. In chapter 2 we show that with the addition of a substantial amount of dynamic axial load in a diurnal rhythm we are able to maintain caprine lumbar IVDs native properties for up to three weeks. However, even with optimal culture

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remodeling or even catabolic response and some creep in the matrix (4). The necessity of some dynamic loading for the vitality of the IVD is a very important finding, and many other IVD culture models have shown beneficial effects of some form of moderate loading and detrimental effects of the absence of loading (5-11). In contrast, a study from Gawri et al. (2011) reports the maintenance of an intact human lumbar IVD in a culture chamber without loading for up to 4 weeks (12). However, only cell viability (and not cell density) and gross matrix content were analyzed. The LDCS is excellent for answering basal questions regarding IVD (biomechanical) function, studying the processes in the onset of DDD and stringent analyses of the interaction between mechanical load and the cells and matrix of the IVD. Even short-term effects of therapeutics can be studied using the LDCS. However, like any model, the LDCS has multiple limitations. Firstly, it is not suitable for maintaining a large lumbar IVD over a longer culture period than three weeks. Studying processes that would require long-term (>3 weeks) follow-up cannot be performed in the LDCS. Regenerative therapeutics, such as (stem)cell therapy and anti-catabolic agents, which need long follow-up to produce any measurable effect, especially if they require systemic recruitment of excipients, cannot be studied to their fullest potential in the LDCS (13-15). Furthermore, currently the LDCS is only

7

capable of axial loading on the IVDs. The role of torsion and bending, which may influence nutrient exchange, cellular responses and have region-specific response cannot yet be taken into account in our system. Some important research questions cannot be answered in an ex vivo setting (16). Inherently, since the IVD is outside the body (ex vivo) in a bioreactor, systemic responses, such as immunological processes, recruitment of additional nutrients and cells from the vertebra bone-marrow, migration of vessels and nerves into the endplate and IVD, pain perception etcetera and other collateral effects of various (detrimental) circumstances cannot be simulated or studied in the ex vivo setting (17, 18). Also, culture of IVDs in the LDCS is laborious, expensive and not without complications such as (load)system malfunction, leakage and infection of specimens. Taken together, the LDCS is a good and useful substitute for some in vivo studies, but it cannot fully replace life animal testing. 192

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Mechanical overloading and degeneration: In chapter 3 we showed that compared to the SPL condition, high dynamic and static overloading cause degenerative changes in the nucleus and annulus of the disc (19). How this type of accelerated load-induced degeneration compares to the in vivo situation in the human spine is difficult to determine (20-22). All the degenerative responses described in our loading studies have also been reported in the human degenerated IVD (23-33). However, we may have only simulated one single exacerbated subtype of disc degeneration, where there are probably numerous pathways by which the human IVD can deteriorate and become symptomatic DDD. Also, we can only speculate whether the reported dosage of overloading would result in disc degeneration in the caprine spine in vivo. In the culture set-up of the LDCS, the strictly axial load is delivered solely on the IVD, without the vertebrae attached and without the ligamentous tissues, the motion segments posterior elements, such as the facet-joints and connecting transverse and spinous processes with spinal musculature and even the additional support of the abdomen. This is both a strength and a limitation of the model; it allows for strict study of the IVDs responses to certain (loading) circumstances, and for exact control and monitoring of that force on the IVD: on the other hand, it might impair its validity to the in vivo situation, as the surrounding spinal elements will have their influences on motion segment response. However, the IVD is continuously axially loaded in the LDCS, and therefore in its neutral zone. Effects of surrounding elements would likely only have a greater role if flexion or torsion moments would be introduced. Loading on the spine in vivo is not strictly axial but multidirectional, magnitude and frequency of loading vary greatly with motion of the spine (34-37). This means that our simulated axial overloading and its effects might not directly correspond with the situation in a living and moving subject. Furthermore, in our ex vivo model the cells in the IVDs cannot recruit additional nutrients or protective excipients (anti-inflammatory, anabolic etc.) in a period of higher stress, simply because the ex vivo condition lacks this systemic feedback regulation (18).

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With regard to the loading condition, many factors could be altered which may have varying results. For instance, the cumulative detrimental effects might have been less profound if the period of rest following the overloading had been longer. The overall effect of overloading on the IVD might be relative to the resting period an IVD gets after a period of high loading (38, 39). In addition, varying the frequency and the shape (in our studies always a constant 1 Hz sinusoidal load) of (over)loading might also influence the outcome (40-42). For culture conditions, there is clear evidence that pH, osmotic pressure and glucose concentrations influence outcome of both cell response and IVD mechanics (43-55). Many other culture conditions have been tested in other models, and many additional excipients have been added to culture media (different pH buffers, NSAIDs, (cartilage) substrate and growth factors) in an attempt to improve or alter IVDs’ response to culture (11, 56-59). We have always stuck with one single simple culture medium recipe, because we wanted to focus on the influence of mechanical conditions. The potentially protective or detrimentally effect of changing any of these factors has not been tested, and although in other models results have been reported, we can only speculate on any effect in our bioreactor (57-61). In the light of the presented data in chapter 3 we can conclude that axial overloading compared to SPL loading of a caprine IVD will result in detrimental

7

changes in biomechanics with loss of non-linear poro-elastic recovery behavior, inflammatory and catabolic responses on a cellular level, and loss of matrix structure and content. This essentially confirms that mechanical overloading can indeed induce degeneration of a healthy intervertebral disc.

Static axial overloading and hernia’s: In chapter 4 we studied the potential relation between static overloading and herniation of the annulus. We found that the posterolateral corners suffers the most damage when the IVD is overloaded with a static axial load (62). Whether the posterolateral part of the annulus is by concept the weakest mechanical site of the outer-annulus or if this weakness is an attainment of a regional degenerative cell response to overloading as accumulated during life, cannot be deduced. What influence the individual changes, e.g. due to ageing, adaption to the mechanical environment and regional specific degenerative changes within the disc, have in the 194

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development of DDD and herniations remains subject to further study (63, 64). We also expect the sagittal posture, the posterior spinal elements and many other factors present in the in vivo situation to have some (protective) effect (65-67). We can only conclude that an isolated lumbar caprine IVD loaded with strictly axial static loading will accumulate the most damage in the posterolateral outer-annulus and leave the nucleus relatively spared. Catabolic cell behavior at this site would indeed prime the IVD for herniation (68-71).

Biomechanical properties in early DDD: In chapter 5 we described that a minor loss of GAG (less than 10%) from the nucleus due to Cabc-injection, resulted in early stage mild IVD degeneration with significant changes in the poro-elastic properties of the disc. These degenerative changes were most apparent in Cabc treated discs, and less in PBS injected IVDs. We showed that the (changes in) recovery behavior of the IVD could be well characterized by the parameters of a stretched-exponential fit. We observed that the depressurization from a small GAG loss already affects the mechanical behavior of the discs during physiological range loading (SPL regime). The decreased intradiscal pressure (due to loss of osmotic pressure in the nucleus) accelerates subsidence during load and slows down recovery during unloading (72, 73). Disc degeneration causes a typical change in the stress-strain curve of the IVD, moving from a typical exponential towards a more linear pattern. We propose this change (disappearance of exponential disc height gain and loss) is the hallmark of degeneration, and the distinctive biomechanical characteristic between healthy and degenerated intervertebral discs. It marks the transition of the poro-elastic behavior of the healthy disc (a resultant from both GAG pressurized fluid in the NP region together with the elastic properties of the intact annulus) to the more solidelastic function of the degenerated disc (in which the porous and fluid-pressurized dampening effects have diminished). This functional difference due to GAG loss is described by the difference in the stretch-constant beta (chapter 5 Fig. 6 and Table 1), which is the major advantage of the stretched exponential fit compared to traditional stiffness and time-constant measures. The changes of the beta parameter with

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increasing degeneration found in the current study concur with earlier studies using stretched-exponential fitting for IVD degeneration (74-77). Other exponential fits have been utilized to describe these highly non-linear IVD biomechanics. In recent studies, the double-void exponential fit has been advocated as well, as it is better able to fit any mechanical “state” of the intervertebral disc (regardless of (de)hydration, osmotic pressurization, degenerative state) (74, 78). This is a clear limitation of the stretched-exponential function as used in chapter 5: if disc behavior becomes too solid-elastic (too linear due to degeneration), the stretchedexponential function will not fit properly. However, the goal of our study was to describe the changes in biomechanical behavior occurring with early degeneration; the initial loss of non-linear poro-elastic behavior. The stretched-exponential fit proved to be able to do so, and due to its simplicity, the parameters in the model can be easily related to actual matrix changes.

Quantitative MRI to detect early DDD: In chapter 6 we demonstrated that quantitative MRI and MRI techniques such as T1rho and ADC provide objective and more accurate information on the matrix

7

status of intervertebral discs than current routine T1 and T2 imaging (79-82). T1rho is particularly sensitive to small degenerative changes in the IVDs matrix. Enzymatic induction of IVD degeneration resulted in dose-dependent changes in biomechanical parameters (height loss and recovery behavior), loss of GAGs, and mild to moderate degeneration on histological sections. To our knowledge, this is the first study to directly compare the correlation of quantitative high-resolution T2, T1rho, and ADC maps with actual disc recovery behavior, combined with histology and GAG-content in a lumbar IVD large-animal model. Our region-specific results demonstrate that T1rho nucleus values correlate with GAG content, histological degeneration, as well as disc mechanical properties to a higher degree than T2 and ADC. Although T1tho is most sensitive in detecting small degenerative changes in the matrix, on the basis of our findings we cannot conclude that it will be superior to T2 on an individual patient level in identifying early DDD. Whether the statistically 196

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significant differences to T2 are clinically relevant has to be further explored. Interestingly, in recent clinical studies T1rho has been found to correlate to the Oswestry and SF-36 pain- and disability scores, significantly better than T2 (83, 84).

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Future direction The combined results in chapters 2 through 4 clearly show that diurnal mechanical loading is required for a healthy condition of the intervertebral disc and that overloading of the IVD will detrimentally affect the discs’ cells, matrix and biomechanics. The apoptotic/necrotic, remodeling and inflammatory cell-responses found, could be at the heart of the degenerative cascade of the IVD or could merely be the gene-expressive last sigh of dying or senescent cells. To examine the significance of the found cell-response to overloading, one could condition the culture medium with anti-inflammatory agents to see if this would block the cells’ response and in turn, would protect the IVD matrix from further degradation. However, as we have already discussed, LDCS culture is not without some remodeling effects, especially in long-term culture. Furthermore, the detrimental cell-response to overloading is very broad; many cellular inflammatory, remodeling- and apoptotic pathways seem to be activated simultaneously. This would imply that when attempting to block this adverse cell response, one would have to implement a non-specific or a combination of several specific anti-inflammatory agents to halt these activities or employ stem cells to secrete trophic factors. In addition, the potential protective effects on the matrix would be small during a feasible culture period in the LDCS and therefore very difficult to measure. Stretched-exponential parameters can distinguish the mechanical behavior of healthy from degenerated IVDs as we have shown in chapter 5. In vivo “functional” imaging of the spine with simultaneous (biomechanical) measurements have been conducted; the influence on disc height of sagittal posture, bending and torsion, with and without heavy lifting have been studied using either serial x-rays, CT or MRI (85-91). Combining serial imaging of the spine with measurement of IVD height could yield a functional in vivo assessment of the IVDs health. One could propose a standardized MR imaging protocol of the recovering spine (in the sagittal plane) during resting / sleeping after a day of standardized heavy lifting. Subsequent fitting of the recovery curves of individual IVDs with a stretched-exponential function, could potential provide insight in the function of (lumbar) IVDs. A practical challenge would be the relatively long time-constant of the human lumbar IVD and the requirement of the stretched-exponential fit to have at least a multitude of times that time-constant to fit properly. Depending on the degenerative state of their IVDs, this could require subjects to sleep / lie down for 12 or even 16 hours, which might render this option impractical.

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In addition to such “functional” assessment of IVDs biomechanics, T1rtho with its superior ability to assess ECM content and detect small degenerative changes in the matrix, could be used to measure IVD status accurately and provide a matrix related explanation for the found functional differences. Implementation of T1rho in clinical MR scanners requires only the addition of a new scanning protocol in the software, hardware changes (coils, detectors) are not needed and scan time for structure like the lumbar spine, knee and ankle are comparative to T2. However, as long as the finding of early DDD in symptomatic patients does not have therapeutic consequences, i.e. same conservative treatment regardless, no radiologist, spine surgeon or rheumatologist will be interested in implementing these superior techniques on a routine basis clinically. Potentially, if randomized controlled trials on the (medicinal) treatment of patients with symptomatic early DDD will be undertaken, these MRI techniques could be of interest for patient-selection and follow-up. If certain disease-modifying, and/or (selective) inflammation-suppressants (biologicals) and/or regenerative medicine strategies are found to positively influence the course of symptomatic (early) DDD, the advantages of an exact functional assessment of IVDs and MR sequences like T1rho will be of practical use. If indeed such a treatment regime is found to be successful, in the light of the findings in this thesis, we can speculate on a better treatment regime for especially young patients with early discogenic low-back pain. With the detrimental processes better identified, there are several ways to improve current “wait-and-see” and “self-management” conservative treatment protocols. There are many parallels between rheumatoid arthritis (RA) and degenerative disc disease to deny some relation. The exact same cytokines that message joint destruction in RA are found in DDD and osteoarthritis. The same collagenases and (metallo)proteinases (ADAMTS and MMPs) that eat away the cartilage and periarticular bone in the synovial joints in RA, also destroy the IVD and its endplates. And like RA, DDD and multi-joint osteoarthritis are strongly genetically predisposed. I would therefore advocate to consider DDD and multijoint osteoarthritis as a sort of lingering form of RA. Not necessarily in the strict sense an autoimmune driven process like RA, but at least for DDD also on the basis of genetic “imperfections” combined with detrimental extrinsic factors like overloading, trauma and inadequate nutrition of the IVD. DDD is a far slower form of joint destruction than RA, but like

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RA also has symptomatic episodes as an exacerbation of this otherwise lingering inflammatory process, which temporarily accelerates degeneration of the disc or joint. By perceiving symptomatic DDD more like an RA disease entity, one can easily make the parallel to an according treatment protocol; when a general practitioner is presented with a (young) patient with LBP complaints that could be discogenic, the patient is quickly referred to a rheumatologist for additional diagnostics. When T2 and T1rho MRI show early signs of disc degeneration and other causes of LBP are ruled out, early symptomatic DDD can be diagnosed. Like RA, initial treatment could start (within 6 months of disease onset) with a combination of relatively high dosage disease-modifying-(anti-rheumatic)-drugs (DMARDs) and antiinflammatory agents to get the disease in remission, i.e. radiologically (PET / T1rho) and clinically (pain and stiffness free). After remission is achieved, dosage and combination of medication can be slowly decreased and at times of exacerbation be increased. Intensive monitoring of disease activity and medication side-effects is imperative for treatment success and safety (92). A physiotherapist should have a role in ensuring that healthy daily physical activities are optimized: patients will need to get educated in the role of extrinsic factors like loading on the spine, posture during daily activity and the necessity of adequate rest (93). Obesity, high cholesterol, diabetes and a sedentary life-style should also be dealt with (94-98). In conclusion, are still many fundamental andresearch clinical questions research questions to be In conclusion, there arethere still many fundamental and clinical to be answered, before we will be able to make our intervertebral discs compatible with the burdens answered, before we will be able to make our intervertebral discs compatible with the burdens of our prolonged life-span. The LDCS is a useful tool in this quest. of our prolonged life-span. The LDCS is a useful tool in this quest.

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de Vries SA, Potier E, van DM, Meij BP, Tryfonidou MA, Ito K. Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue stimulates matrix production by canine nucleus pulposus cells and bone marrow-derived stromal cells. Tissue Eng Part A. 2015;21(5-6):1077-84.

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Gooyers CE, McMillan EM, Noguchi M, Quadrilatero J, Callaghan JP. Characterizing the combined effects of force, repetition and posture on injury pathways and micro-structural damage in isolated functional spinal units from sub-acute-failure magnitudes of cyclic compressive loading. Clin Biomech (Bristol , Avon ). 2015;30(9):953-9.

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Belavy DL, Adams M, Brisby H, Cagnie B, Danneels L, Fairbank J, et al. Disc herniations in astronauts: What causes them, and what does it tell us about herniation on earth? Eur Spine J. 2016;25(1):144-54.

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Lama P, Le Maitre CL, Dolan P, Tarlton JF, Harding IJ, Adams MA. Do intervertebral discs degenerate before they herniate, or after? Bone Joint J. 2013;95-B(8):1127-33.

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Laws CJ, Berg-Johansen B, Hargens AR, Lotz JC. The effect of simulated microgravity on lumbar spine biomechanics: an in vitro study. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society. 2016;25(9):2889-97.

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Belavy DL, Adams M, Brisby H, Cagnie B, Danneels L, Fairbank J, et al. Disc herniations in astronauts: What causes them, and what does it tell us about herniation on earth? European spine journal : official

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Emanuel KS, Vergroesen PP, Peeters M, Holewijn RM, Kingma I, Smit TH. Poroelastic behaviour of the degenerating human intervertebral disc: a ten-day study in a loaded disc culture system. Eur Cell Mater. 2015;29:330-40.

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Berry GC, Plazek DJ. On the use of stretched-exponential functions for both linear viscoelastic creep and stress relaxation.

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Mulligan KR, Ferland CE, Gawri R, Borthakur A, Haglund L, Ouellet JA. Axial T1rho MRI as a diagnostic imaging modality to quantify proteoglycan concentration in degenerative disc disease. Eur Spine J. 2015;24(11):2395-401.

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Zuo J, Joseph GB, Li X, Link TM, Hu SS, Berven SH, et al. In vivo intervertebral disc characterization using magnetic resonance spectroscopy and T1rho imaging: association with discography and Oswestry Disability Index and Short Form-36 Health Survey. Spine (Phila Pa 1976 ). 2012;37(3):214-21.

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van Deursen LL, van Deursen DL, Snijders CJ, Wilke HJ. Relationship between everyday activities and spinal shrinkage. Clin Biomech (Bristol , Avon ). 2005;20(5):547-50.

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Wilke HJ, Rohlmann F, Neidlinger-Wilke C, Werner K, Claes L, Kettler A. Validity and interobserver agreement of a new radiographic grading system for intervertebral disc degeneration: Part I. Lumbar spine. European spine journal : official publication of the European Spine Society, the European Spinal

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Keller TS, Colloca CJ, Harrison DE, Harrison DD, Janik TJ. Influence of spine morphology on intervertebral disc loads and stresses in asymptomatic adults: implications for the ideal spine. Spine J. 2005;5(3):297-309.

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Rousseau MA, Bradford DS, Hadi TM, Pedersen KL, Lotz JC. The instant axis of rotation influences facet forces at L5/S1 during flexion/extension and lateral bending. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society. 2006;15(3):299-307.

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Ostelo RW, van Tulder MW, Vlaeyen JW, Linton SJ, Morley SJ, Assendelft WJ. Behavioural treatment for chronic low-back pain. Cochrane Database Syst Rev. 2005(1):CD002014.

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Hangai M, Kaneoka K, Kuno S, Hinotsu S, Sakane M, Mamizuka N, et al. Factors associated with lumbar intervertebral disc degeneration in the elderly. Spine J. 2008;8(5):732-40.

95.

Wilder DG, Pope MH, Frymoyer JW. The biomechanics of lumbar disc herniation and the effect of overload and instability. J Spinal Disord. 1988;1(1):16-32.

96.

Jhawar BS, Fuchs CS, Colditz GA, Stampfer MJ. Cardiovascular risk factors for physician-diagnosed lumbar disc herniation. Spine J. 2006;6(6):684-91.

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Longo UG, Denaro L, Spiezia F, Forriol F, Maffulli N, Denaro V. Symptomatic disc herniation and serum lipid levels. Eur Spine J. 2011;20(10):1658-62.

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Kanayama M, Togawa D, Takahashi C, Terai T, Hashimoto T. Cross-sectional magnetic resonance imaging study of lumbar disc degeneration in 200 healthy individuals. Journal of neurosurgery Spine. 2009;11(4):501-7.

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Summary With clinical evidence emerging on the relationship between intervertebral disc (IVD) degeneration and low-back pain (LBP), and a growing body of knowledge on the fundamental mechanisms involved in IVD degeneration, the need increases for translational platforms to test theories on etiology, diagnostics and possible treatments of degenerative disc disease (DDD). The primary purpose of the present thesis was to investigate the feasibility of ex vivo culture of a large lumbar intervertebral disc (IVD). We pursued to establish a bioreactor model in which a live intervertebral segment can be sustained under controlled mechanical loading conditions for a prolonged period of time (the Loaded Disc Culture System; LDCS). With the use of this model we aimed to answer fundamental research questions regarding intervertebral disc degeneration on a ‘representative-for-human’ lumbar IVD. With close control of culture conditions and applying a diurnal simulated-physiological loading, we were able to maintain lumbar caprine IVDs in culture for up to three weeks. This model allows us to study biomechanical, cellular and extracellular processes in the IVD simultaneously. Furthermore, it enables us to distinguish between the different regions within the IVD (e.g. nucleus, inner-annulus and anterior/lateral/posterior outer annulus). The secondary objective with the LDCS was to clarify the role of various mechanical loading conditions on the IVDs native properties. We found that the absence of loading, underloading and overloading of the IVDs have negative effects on mechanical properties, cells and matrix, which are comparable to degenerative changes in human DDD. We found that dynamic and static overloading affects the IVDs regions (nucleus, inner- and outer-annulus) differently. We used the LDCS to investigate if the potential relationship between a sedentary lifestyle and lumbar IVD herniation may be explained based on biomechanics of the IVD. We found static axial overloading to be detrimental especially to the posterior annulus. Furthermore, we studied early degenerative processes in the IVD, for which the LDCS is especially well suited.

We observed that for early mild degenerative changes in IVDs

biomechanical function and quantitative MR mapping with T1rho provide better information than, for instance IVD height, water-content and T2-weighted imaging.

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Specific aims and answers: Chapter 2. Is long-term ex-vivo culture of a large lumbar IVD feasible? And is there a need for axial loading to maintain native IVD properties in culture? In chapter 2 we showed that it is feasible to culture lumbar goat IVDs with cartilaginous endplates in the Loaded Disc Culture System, with conservation of their native properties over a 21-day culture period based on our presented data of cell behavior, matrix status and biomechanical properties. Application of a diurnal axial simulated-physiological loading (SPL) regime proved essential for maintenance of caprine IVDs in culture. Ex vivo culture of large IVDs is challenging and many factors have been identified to be critical for maintenance of IVD properties. Although especially NP cells have been reported to be robust and able to withstand harsh environmental conditions, for preservation of cell phenotype and metabolism a narrow optimum range for glucose, pH, oxygen and osmotic pressure has been reported. These environmental conditions have been studied in other models for their effects on the IVD and optimal range for culture have been described. These conditions could all be adequately controlled to maintain the caprine lumbar IVD in the described custom-designed LDCS.

8 What is the effect of unloading or low dynamic loading when compared to a simulated-physiological load? An absence or deficit of axial load on the IVD caused pathological changes in the disc as was evident from a decline in cellular vitality (both viability and density) and changes in gene expression patterns, especially in the NP region. In the unloaded group, cell viability already drops significantly within the first week of culture, without a significant change in cell density. Although cell viability in the unloaded group seems to stabilize with increased culture duration, the drop in cell density reveals that overall disc vitality is still diminishing.

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Continuous low dynamic loading (LDL) could not prevent cell death. Alterations in gene expression in response to different loading conditions were more evident in NP cells than in AF cells. The most pronounced changes could be observed in the unloaded culture group. Absence of mechanical loading led to reduced expression of all anabolic genes, except collagen type 1. Remodeling genes as well as inflammation-related genes are known to be upregulated in an adverse response of cells to loading. Both the unloaded and LDL group showed significant up-regulation of the expression of several of these target genes, especially in the nucleus. Histological sections did not reveal changes in matrix staining between day 0 and day 21 of cultured discs. Longer culture periods may be needed to measure significant matrix content loss. These findings are the resultant of both direct and indirect influences of mechanical loading on the IVD. The observed effects come from a complex system in which cells are within their native matrix environment and interact with loading, cell and matrix deformation and fluid distribution. Cells which in the in vivo situation receive abundant mechanical stimuli from the various forces on the IVD, are deprived of these stimuli in the unloaded culture group. A lack of hydrostatic fluctuations in combination with slightly hypo-osmotic medium (when compared to the osmotic pressure in the NP of the disc), causes a different stress equilibrium compared to the physiological situation. Indirect effects may involve a decrease of fluid flow by a deficit of deformation of the IVD in the unloaded and LDL state. This could impair distribution of nutrients towards and waste products from the NP.

Chapter 3. Can mechanical overloading cause IVD degeneration? The data presented in chapter 3 substantiate the hypothesis that any type of axial overloading will result in degenerative changes in the IVD. With regard to mechanical behavior, we could clearly observe that overall subsidence of the IVDs depended on the amount of loading that the IVD received. IVDs subjected to the static loading regime received on average the highest loading, which was associated with the largest subsidence. Nevertheless, overall deformation was almost as large in the 208

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high dynamic regime. Especially, height recovery was hampered in the overloaded groups. For the high loading regimes, the 8-hour recovery time is too short to regain the water pressed out during the first loading cycle. One may therefore assume that these discs remain in a less hydrated state as compared to the IVDs in the SPL group. This was confirmed by our quantitative matrix measures of water and GAG, which showed loss in NP and AF regions with overloading. On a cellular level, there was significantly more loss in cell viability and cell density, and cell behavior in the overloaded groups shifted towards catabolic/remodeling and inflammatory when compared to the SPL control.

What is the difference in effect of dynamic and static overloading on the nucleus and annulus region? We observed differences in the onset as well as the pattern of damage throughout the tested discs between overloading regimes. With high dynamic loading, all regions are moderately affected after 21 days, whereas with high static loading especially the outer AF was damaged in some cases already after 7 days of culture. We also observe positive staining for GAGs in the inner- and outer-annulus, likely due to loss and diffusion of GAGs from the NP. Analogous degenerative changes occurred at the cellular level. High dynamic loading caused substantial cell death within 7 days in all disc regions, with cell density dropping significantly after 21 days

8

when comparing to baseline and SPL. The decrease in cell viability and density with high static loading was most pronounced in the AF region.

Chapter 4. What is the effect of dynamic and static overloading on the nucleus and annulus region? Is there a region (anterior, lateral and posterior) specific response to dynamic and static overloading that could explain the posterolateral predilection of lumbar hernias? With prolonged axial overloading of caprine lumbar IVD, 1) significant height loss occurs without changes in the exterior pressure distribution over the disc, 2) general cell death and matrix disruption occurs in all disc regions with high dynamic 212

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overloading and 3) static overloading results in a posterior AF region-specific breakdown, with significant cell death and matrix disintegration and relative sparing of the NP region. Therefore, we conclude that the manner of axial overloading (dynamic or static) influences the various regions and structures of the IVD differently. Most importantly, we found that although the mechanical changes due to dynamic and static overloading of the disc are the same (significant height loss without change in pressure distribution), the type of overloading -dynamic versus static- will affect the biological response within the disc and is region-specific. A region-specific mechanically induced degenerative cycle [39] is triggered specifically in the posterior region of the AF with static overloading. The nucleus is relatively spared, staying hydrated and pressurized, while the posterior region by comparison is weakened by apoptotic and necrotic cell response, which will trigger the vicious cycle of degeneration [39]. Therefore, we conclude that prolonged static axial overloading primes the lumbar caprine IVD for posterolateral herniation. Our findings provide a clear biological rational for the observed predilection of hernia's in the posterolateral corner of the lumbar spine in individuals with a sedentary life-style.

Chapter 5. How do the poro-elastic properties of the IVD change in early intervertebral disc degeneration? We found that a minor loss of GAG (less than 10%) from the nucleus due to Cabc-injection, caused early stage mild IVD degeneration as scored on histological sections. This mild degeneration was associated with changes in the poro-elastic properties under physiological range loading (SPL regime). These changes could be well characterized by the parameters derived from stretched-exponential fits of the displacement curves during the recovery phase of the diurnal load. Besides the expected increase in the time-constant tau for recovery behavior (axial displacement), the beta also deviates further from 1 (closer to zero) in the Cabc group. This means the fit requires more correction by its stretch constant beta, as the curve is no longer strictly exponential but increasingly linear. The changes of the beta parameter with increasing degeneration found in the current study concur with observations in earlier 210

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studies with stretched-exponential fitting for load-induced IVD degeneration and are likely correlated to GAG loss from the nucleus.

Can we use exponential fitting to identify dysfunctional disc behavior before height and water are permanently lost? Already very early in the degenerative process, we observed an alteration of the mechanical behavior of the disc. Only a minor loss of GAGs causes a change from a more poro-elastic towards a more elastic behavior of the IVD. This change could be quantified by the parameter beta in the stretched exponential model, which was fitted to the recovery curve. This is a major advantage of the stretched exponential fit function compared to traditional height and stiffness measures. Therefore, we concluded that in the current experimental model, the stretchedexponential fit is capable of identifying early degenerative disc changes, before irreversible height and water-content changes occur.

Chapter 6: How do quantitative T2, T1rho and ADC maps change with mild IVD degeneration? All tested quantitative MR mapping, and T1rho in particular, detected the small degenerative changes in the IVDs matrix (loss of GAG) due to Cabc-injection.

8

With the use of a 9.4T MRI we were able to image the lumbar caprine IVD in high anatomical detail. We quantitatively mapped the IVDs 5 distinct regions on T2, T1rho, and ADC images and found significant differences between the NP, inner-AF, and the anterior, lateral and posterior outer-AF. T1rho values pre- and post-loading showed a larger difference than T2, due to T1rho’s larger dynamic range. We only found moderate effects of the degeneration on the ADC values in the (anterior and posterior) outer-AF. Lack of measured changes are most likely due to the effects of ADC’s sensitivity to anisotropy and the IVD culture conditions.

Which MRI technique is superior in quantifying early (regional) IVD degeneration?

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ADC’s sensitivity to anisotropy and the IVD culture conditions.

Chapter 8

Which MRI technique is superior in quantifying early (regional) IVD degeneration? T1rho nucleus values correlate strongly with the histological degeneration 2 score 214 (R = 0.729) and significantly better than T2 and ADC. T1rho values are more

closely linked to actual ECM content and therewith biomechanical function (recovery behavior; the stretch-constant beta) of the disc. Both T2 and T1rho correlated to the Cabc dose-dependent tau increase. T1rho’s stronger correlation to the stretch constant beta, is most likely due to T1rho’s stronger correlation to GAG-content. The stretchconstant beta deviates further from 1 (to zero) when poro-elastic properties are lost and the disc material shifts towards a more linear (solid-elastic) behavior (54). In the case of the IVD, this has been shown to be caused by loss of GAGs (and therewith water) from the NP (73) and structural damage to the disc (55). Taken together, when lower T1rho NP values are found, this is representative for the biomechanical deterioration of the poro-elastic properties of the IVD, which is the first step in the degenerative cascade of DDD.

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Dutch summary / Nederlandse samenvatting

C.P.L. Paul

Addendum

Lage rugklachten en tussenwervelschijf slijtage Lage rugklachten komen veel voor in de algemene populatie en is de meest frequente reden voor mensen in de Westerse samenleving om medische hulp te zoeken (1). Door ziektelast, werkverzuim, arbeidsongeschiktheid en medische kosten zijn de socio-economische gevolgen van lage rugklachten enorm (2-4). Lage rugklachten kunnen zeer veel verschillende oorzaken hebben (5-7). Kijkend naar lichamelijke oorzaken tot nu toe bekend, is de (versnelde) slijtage, ook wel degeneratie genoemd, van de lumbale (lende/lage) tussenwervelschijven in de werkvelkolom, een veel voorkomende reden. De aanwezigheid en ernst van degeneratieve tussenwervelschijf (discus) ziekte is gerelateerd aan de aanwezigheid en ernst van lage rugklachten (8-11). Meerdere grote Magnetic Resonance Imaging (MRI) studies, uitgevoerd in verschillende werelddelen in een gezonde algemene populatie hebben dit verband aangetoond (12-15). Toch zijn er ook veel mensen met op MRI één of meer versleten tussenwervelschijven, die geen lage rugklachten rapporteren. Wanneer tussenwervelschijf slijtage tot pijnklachten leidt is nog niet volledig opgehelderd. Evenzeer is het onduidelijk waarom sommige mensen eerder, sneller en ernstiger slijtage van de tussenwervelschijven krijgen dan anderen. Momenteel is er ook geen curatieve (genezende) behandeling voor degeneratieve discus ziekte. De therapie bestaat uit symptoombestrijding door middel van pijnstilling (16), oefen/fysiotherapie (17), en eventueel accupunctuur (18-23). Als een patiënt ernstig lijdt onder de lage rugklachten veroorzaakt door ernstige of deformerende degeneratieve discus ziekte, kan er soms chirurgisch ingegrepen worden. In de meeste gevallen zal de versleten tussenwervelschijf verwijderd worden en de aangrenzende wervellichamen samen vastgezet. Uitkomsten van deze ingreep zijn niet zonder meer goed (18-21). Het is daarom van groot belang dat wij meer kennis krijgen over wat slijtage van de tussenwervelschijf is en hoe degeneratieve discus ziekte ontstaat. Als wij weten welke factoren daarop van invloed zijn en hoe, kunnen wij wellicht eerder en beter ingrijpen bij het ontstaan van degeneratieve discus ziekte en hierdoor ziektelast voorkómen.

Anatomie en functie van de tussenwervelschijf De menselijke wervelkolom telt meestal 33 wervels (bot), waarvan de bovenste 24 “articulerend” ofwel bewegelijk zijn, doordat zij niet aan elkaar vast zitten, maar gescheiden worden door een tussenwervelschijf (kraakbeenachtig). De tussenwervelschijven (meestal 23) zitten, zoals de naam al zegt, tussen twee wervellichamen in, geven enige bewegelijkheid aan 216


de wervelkolom en hebben een schokabsorberend vermogen (22). De tussenwervelschijf is opgebouwd uit sterk verschillende weefseltypen (23). In het midden van de schijf zit de nucleus pulposus (NP), die een kleverige gelstructuur heeft en kan opzwellen door water op te nemen. De NP kan goed water aantrekken en vasthouden dankzij speciale suikereiwitten; glycosaminoglycanen, kortweg GAGs. De NP wordt omsloten door een ringvormige structuur, de annulus fibrosis (AF), die is opgebouwd uit vele (20-25) vezelige lagen (lamellen). De AF is zowel poreus als trekvast (24, 25). Hierdoor laten de lamellen enerzijds water door, waardoor de NP goed kan zwellen, maar beperken zij anderzijds deze zwelling waardoor er een (osmotische) zwellingsdruk in de NP ontstaat. Hieraan dankt de tussenwervelschijf zijn schokabsorberende eigenschap (26, 27). De zwelling van de NP wordt aan de boven- en onderzijde beperkt door de dek- (bovenzijde) en sluitplaat (onderzijde). Deze platen bestaan uit een kraakbenige en botachtige laag en verankeren de NP en AF aan de wervels (28-31). De tussenwervelschijven zijn de grootste structuur in het menselijk lichaam zonder bloedvoorziening. De NP en AF bevatten dan ook het laagst aantal cellen per kubieke centimeter (4·106 cellen/cm3 in de NP en 9·106 cellen/cm3 in de AF). Zij komen embryologisch voort uit het notochord (NP) en mesenchym (AF) en hebben respectievelijk kraakbenige en fibreuze eigenschappen. Of deze cellen onder normale omstandigheden nog een functie hebben bij het in stand houden van het weefsel in de NP en AF van de tussenwervelschijf is nog niet volledig opgehelderd (22, 32-34).

Huidige ideeën over degeneratie van de tussenwervelschijf en de rol van mechanische belasting: Momenteel wordt het onder fysiologische (normale, gezonde) omstandigheden slijten van de tussenwervelschijf met het ouder worden eufemistisch omschreven als “een natuurlijk verouderingsproces”. Slijtage van de tussenwervelschijf wordt verder gezien als een multifactorieel proces. Het gaat altijd gepaard met een verlies van water uit de nucleus pulposus door een geleidelijk verlies van GAGs (35). Hierdoor veranderen de mechanische eigenschappen van de tussenwervelschijf, waardoor deze alleen maar meer GAGs en water gaat verliezen. Het slijtageproces lijkt dan ook in de meeste gevallen een zichzelf versterkend vicieus proces (36, 37). Voor de vroege, versnelde slijtage van de tussenwervelschijf die veelal symptomatisch is (pijnklachten geeft), degeneratieve discus ziekte genoemd, zijn meerdere en sterk verschillende risicofactoren bekend. Zo zijn er vele genetische afwijkingen

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geïdentificeerd die samenhangen met vroege en/of ernstige degeneratie van disci (38-44), vormt trauma van de wervelkolom zoals breuken van wervels en scheuren van de annulus een risico (45-47) en treedt ook door infectie van de tussenwervelschijf degeneratie op (48-50). Van groot belang blijkt verder de voorziening van voedingsstoffen naar de wervel en tussenwervelschijf te zijn (51-53). Zo zijn atherosclerose (slagader verkalking) en verstopping van de lumbale slagaderen (54-56), hart- en vaataandoeningen in het algemeen (57, 58), hoog cholesterol (59), overgewicht en obesitas (60) en suikerziekten (54, 61) alle geassocieerd met het ontstaan van discus degeneratie en lage rugklachten. Mechanische belasting van de wervelkolom wordt van oudsher gezien als een belangrijke extrinsieke (buiten het lichaam gelegen) factor, die de tussenwervelschijf eerder en ernstiger kan doen slijten (62-64). Hoe mechanische belasting precies degeneratie van de tussenwervelschijf veroorzaakt is nog onduidelijk. Als men bedenkt dat de tussenwervelschijf juist toegerust lijkt op het verwerken van grote mechanische krachten en het mechanisch rondpompen van vloeistof in de discus nodig is voor de voeding van cellen en het afvoeren van afvalstoffen, lijkt het paradoxaal dat diezelfde dagelijkse krachten zouden leiden tot degeneratie (65, 66). Echter, uit observationele onderzoeken blijkt in de praktijk dat mensen in beroepen waarbij de wervelkolom zwaar belast wordt, onder invloed van vibraties staat (67-70) of juist langdurig statisch belast wordt (zittende beroepen) meer risico hebben op degeneratieve discus ziekte en hernia’s (71). De invloed van mechanische belasting is interessant om te bestuderen, omdat deze een van de weinige factoren is die we gecontroleerd in een testsysteem kunnen beïnvloeden. Kennis over de interactie tussen fysische krachten, de weefsels (de extracellulaire matrix; ECM) en de cellen in de tussenwervelschijf biedt ons mogelijk meer inzicht in het slijtageproces, ook van ander (kraakbeen) weefsel. Begrip van de beschermende en beschadigende invloeden van mechanische krachten op de tussenwervelschijf moeten leiden tot betere therapieën voor tussenwervelschijf degeneratie.

Bestudering van mechanische invloeden op de tussenwervelschijf De invloed van mechanische belasting op de tussenwervelschijf wordt al decennia middels verschillende modellen en experimentele simulaties bestudeerd. Alhoewel geen enkel model perfect overeenkomt met de situatie in het menselijk lichaam, kan het toch op specifieke vragen antwoorden verschaffen. Sterker nog, sommige vragen rondom de invloed van belasting op de tussenwervelschijf kunnen en mogen niet op levende mensen onderzocht worden en 218


behoeven dus experimentele modellen om tot antwoorden te komen. Humaan kadaver materiaal lijkt dan het beste alternatief. Echter, humaan kadaver materiaal is meestal afkomstig van oude mensen, waar de tussenwervelschijven niet meer normaal gezond van functioneren en vaak al fors versleten zijn. Daarnaast is humaan kadaver materiaal nooit “vers” (altijd pas meer dan 8 uur post-mortem beschikbaar), waardoor de NP en AF cellen in de tussenwervelschijf reeds sterk aan vitaliteit hebben ingeboet op het moment van obductie. Hier bovenop komen nog de schadelijke invloeden op de weefseleigenschappen van het koelen van de stoffelijke overschotten, alsmede de invloed van fixatietechnieken (zoals gebruik van formaldehyde), welke de mechanische eigenschappen sterk beïnvloeden en onvergelijkbaar maken met het levende verse weefsel (72-77). Een andere optie zijn levende diermodellen. Alhoewel sommige vraagstukken rondom de invloed van mechanische belasting op tussenwervelschijven met behulp van in vivo (levende) diermodellen beantwoord kunnen woorden, zijn de mogelijkheden beperkt door een gebrek aan controle over de mechanische omstandigheden in de wervelkolom, wat ook geldt voor het betrouwbaar meten van de effecten ervan. Daarnaast zijn in vivo modellen duur en ook door ethische kwesties beperkt in hun toepasbaarheid (170-172). In vitro (in een kweekfles) cel- en weefselkweek modellen zijn niet goed toepasbaar, omdat ze niet de unieke en specifieke orgaaneigenschappen kunnen benaderen: de hoge osmotische en mechanische druk, de lage concentratie van zuurstof en nutriënten, de unieke inbedding van cellen in een matrix van de TWS kunnen niet in celkweek gesimuleerd worden. Hierdoor verandert het gedrag van de cellen in vitro teveel ten opzichte van de in vivo situatie (172;173). Derhalve hebben we een orgaan kweeksysteem (bioreactor) opgezet om zaken te onderzoeken die niet (goed) in vivo of in vitro te bestuderen zijn (174-178). Dit betreft vooral vraagstukken waarbij de functie van de volledige tussenwervelschijf betrokken is en naar het effect van degeneratieve processen op de TWS als geheel moet worden gekeken. De mogelijkheden van de bioreactoren en de gebruikte diersoorten beperken echter vaak de relevantie van de experimentele uitkomsten voor de menselijke situatie. Zo worden veelal kweeksystemen zonder de mogelijkheid van het mechanisch belasten van de tussenwervelschijf gebruikt of kan het kweekmedium niet ververst worden waardoor er dus maar kort gekweekt kan worden. Ook worden (te) kleine diersoorten (muizen en ratten) gebruikt met tussenwervelschijven die mechanisch en door de geringe hoogte qua voedingsvoorziening niet vergelijkbaar zijn met lumbale tussenwervelschijf (afkomstig uit de staart bijvoorbeeld), of worden tussenwervelschijven gebruikt van diersoorten die notochordaal cellen behouden in hun

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NP regio en/of qua samenstelling van hun extracellulaire matrix (ECM) teveel van humaan verschillen (veel meer GAGs en collageen type 2 bijvoorbeeld; varken en schaap). Het ideale ex vivo model zou moeten bestaan uit de combinatie van een veelzijdig kweeksysteem, tezamen met een aan humaan gelijke tussenwervelschijf. De tussenwervelschijf zou afkomstig moeten zijn van een grote diersoort (qua grootte en gewicht in de orde van humaan), met lumbale tussenwervelschijven die qua mechanische eigenschappen en cel- en matrixinhoud vergelijkbaar zijn met humane lumbale disci. Het kweeksysteem zou verschillende typen nauwkeurig aanstuurbare belasting op de tussenwervelschijf moeten toelaten en in een constante kwaliteit van kweekmedium moeten voorzien, waardoor de tussenwervelschijf lang genoeg goed gehouden kan worden om effecten van interventies betrouwbaar te kunnen bepalen. Onze onderzoeksgroep heeft de afgelopen 20 jaar met veel verschillende diersoorten en modellen ervaring opgedaan met chirurgische ingrepen aan de wervelkolom, biomechanische studies van de wervelkolom en met onderzoek naar kraakbeen- en tussenwervelschijf eigenschappen. Uit de verschillende onderzoeken kwam naar voren dat lumbale geiten tussenwervelschijven qua vorm en inhoud goed vergelijkbaar is met de humane lumbale tussenwervelschijven, meer dan met varken en schapen tussenwervelschijven. Uit metingen bleek dat ondanks het feit dat geiten viervoeters zijn, de tussenwervelschijven in hun lage rug bloot staan aan dezelfde krachten als in de menselijke lage rug. De grootte en vorm van de lumbale geiten tussenwervelschijf is goed vergelijkbaar met die van de mens en ook de biomechanische eigenschappen komen sterk overeen. Minstens zo belangrijk is de afwezigheid van notochordaal cellen in de NP van de volwassen geit, waardoor de geit, net als de mens, met ouderdom “natuurlijke” slijtage van de tussenwervelschijf ondervindt. Aangezien onze onderzoeksgroep reeds de beschikking had over een goed in vivo geiten model, zou de aanvulling met een ex vivo model ervoor zorgen dat wij een groot scala aan meer fundamentele onderzoeksvragen ten aanzien van de invloeden van mechanische belasting op de tussenwervelschijf op hetzelfde dier kunnen beantwoorden. Daarnaast zouden we het ex vivo model als translationeel testplatform kunnen gebruiken om de haalbaarheid en effectiviteit van nieuwe therapeutische interventies uit te testen zonder hier direct levende proefdieren voor nodig te hebben.

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Doel van dit proefschrift Het algemene doel van het onderzoek in dit proefschrift was om meer te weten te komen over de invloeden van mechanische belasting op de tussenwervelschijf. Hiertoe wilden wij de haalbaarheid van het kweken van lumbale geiten tussenwervelschijven in een kweeksysteem onderzoeken en de randvoorwaarden van de optimale kweekcondities vaststellen. Met een goed gevalideerd ex vivo model wilden wij vervolgens de effecten van verschillende soorten (over)belasting (dynamische en statische) gaan bestuderen op het niveau van de cel, het weefsel (de ECM) van de tussenwervelschijf en het biomechanisch gedrag van de tussenwervelschijf. Deze invloeden van belasting wilden wij op regio nauwkeurig niveau (nucleus en annulus apart; voor- zij- en achterkant apart) bepalen. Hierbij wilden wij niet alleen de nucleus pulposus van de binnenste- en buitenste annulus onderscheiden, maar ook onderscheid maken tussen het voorste (anterieure), zijwaartse (laterale) en achterste (posterieure) deel van de annulus. Daarnaast hadden wij als doel het ex vivo model te gebruiken om beginnende, vroege discusdegeneratie na te bootsen en nieuwe methoden te testen die milde degeneratie biomechanisch karakteriseren en met MRI visualiseren.

Specifieke vragen en antwoorden per hoofdstuk Hoofdstuk 2: Is ex-vivo kweken van een intacte lumbale geiten tussenwervelschijf mogelijk? Is voor het optimaliseren van de kweekcondities mechanische belasting op de TWS nodig? En wat is het effect van het onbelast laten of te laag belasten van de TWS in vergelijking met gesimuleerd fysiologische belasting? In dit hoofdstuk hebben wij de haalbaarheid van het kweken van een volledige lumbale geiten tussenwervelschijf getest in een kweeksysteem dat speciaal voor dit doel door onze onderzoeksgroep ontwikkeld is. Deze bioreactor, het Loaded Disc Culture System, kortweg LDCS genoemd, bestaat uit een grote incubator (kweekstoof; temperatuur, zuurstof- en CO2gereguleerd) met daarin actuatoren. Deze kunnen zeer nauwkeurig de individuele tussenwervelschijven axiaal belasten en tegelijkertijd het inzakkings- en herstelgedrag van de tussenwervelschijf volgen. Alle tussenwervelschijven zaten in individuele kweekpotjes in hun actuatoren. In de kweekpotjes waren de tussenwervelschijven omringd door een speciaal samengesteld kweekmedium. Deze was qua pH (HEPES gebufferd), osmolariteit, 221

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glucoseconcentratie en andere metabolieten volledig aangepast aan de behoeften van de cellen in de tussenwervelschijf. Het kweekmedium werd continu gecirculeerd en werd iedere 48 uur vervangen om er onder andere voor te zorgen dat de beschikbaarheid van biologisch actieve (verse) vitamine C gewaarborgd werd. Door eerdere drukmetingsexperimenten in de tussenwervelschijven in levende bewegende geiten, wisten wij ongeveer hoeveel belasting (druk) met welke frequentie (Hz) en vorm (sinusoïdaal) de tussenwervelschijf in het kweeksysteem belast moest worden om dagelijkse activiteiten en belasting op de tussenwervelschijf te simuleren. Het dagelijkse belastingregime dat we hieruit geëxtrapoleerd hebben, heeft een dag-nachtritme met een inzakkingsfase gedurende de dag en een herstelfase van 8 uur tijdens de nacht. Dit regime hebben wij de gesimuleerd-fysiologische belasting gedoopt, in het Engels “simulated-physiological loading”, of kortweg SPL. Alle kweekcondities tezamen stelden ons in staat de geiten tussenwervelschijf in het LDCS gedurende een periode van 3 weken te conserveren, dat wil zeggen: de tussenwervelschijf behield zijn native gedrag (zoals gemeten in controle tussenwervelschijven op dag 0) op het niveau van de cellen, de matrix en qua mechanisch gedrag. In afwezigheid van, of bij een gebrek aan mechanische belasting op de tussenwervelschijf maten wij op celniveau negatieve effecten. Met name in de NP stierven cellen al binnen een week en vonden wij minder cellen (dood of levend) terug in de ECM. De nog levende cellen vertoonden verhoogde activiteit in de celkern van gen-afschrijving die veelal bij stress- en ontstekingsreacties optreden of in situaties waarbij de cel zichzelf en zijn omgeving poogt te veranderen. De tussenwervelschijven die laag dynamisch belast werden lieten minder sterfte en andere negatieve reacties zien dan de onbelaste groep. Beide groepen verloren op celniveau meer native eigenschappen in vergelijking met de SPL-groep. In de ECM maten wij geen veranderingen en het mechanisch gedrag bleef ook ongeveer gelijk voor alle groepen gedurende de drie weken van kweek. Wij concluderen hieruit dat geiten tussenwervelschijven in ons kweeksysteem 3 weken te kweken zijn met handhaving van hun native eigenschappen indien zij met SPL belast worden. Onbelast of inadequaat belast ondervinden de tussenwervelschijf negatieve gevolgen, meetbaar op celniveau. Waarschijnlijk berust het positieve effect van mechanische belasting op zowel directe als indirecte invloeden van de belasting. Enerzijds heeft dit waarschijnlijk te maken met de directe mechanische druk op de cellen en matrix, die de normale omstandigheden het beste simuleert en schadelijke zwelling van de cellen of matrix tegengaat. Anderzijds wordt het waarschijnlijk beïnvloed door het indirecte effect van dynamische belasting op 222


vloeistofstromingen in de tussenwervelschijf, die een adequate uitwisseling van vocht en nutriënten mogelijk maakt met het kweekmedium. Hierdoor worden de cellen voorzien van voldoende glucose, zuurstof en andere voedingsstoffen, terwijl hun afvalstoffen de matrix kunnen uitstromen.

Hoofdstuk 3: Kan mechanische overbelasting tussenwervelschijf degeneratie veroorzaken? In dit hoofdstuk onderzochten wij of overbelasting (2 à 3 maal hoger dan SPL) een negatief effect op de tussenwervelschijf heeft en resulteert in discusdegeneratie. Wij vonden dat bij iedere vorm van overbelasting er degeneratieve veranderingen in de tussenwervelschijf optraden in vergelijking met onze SPL controlegroep. Gekeken naar de mechanische effecten, zagen wij dat met overbelasting een tussenwervelschijf verder inzakt en hiervan in de loop der tijd trager herstelt, waardoor het hoogteverlies blijvend en progressief is. Er werd door overbelasting veel celdood geïnduceerd zodat het aantal cellen verminderde. De overlevende cellen vertoonden veel stress-, ontstekings- en katabole activiteiten. In de ECM zagen wij dat zowel de hoeveelheid water als het aantal GAGs afnam. Op gekleurde coupes van de overbelaste tussenwervelschijven zagen wij dat de weefselstructuur was beschadigd. Alles bij elkaar concludeerden wij dat met overbelasting, op ieder gemeten niveau, de tussenwervelschijf significante degeneratieve veranderingen ondergaat in vergelijking met de SPL controlegroep.

Wat is het effect van dynamische en statische overbelasting op de nucleus en annulus regio van de tussenwervelschijf? Als wij de groepen met hoge dynamische overbelasting vergelijken met de groepen met de hoge statistische overbelasting zien wij een aantal regionale verschillen. Hoge dynamische belasting heeft op de gehele tussenwervelschijf, dus zowel NP als AF regio, schadelijke effecten over de 21 daagse kweekperiode. Voor statische belasting geldt dat de schadelijke effecten veel duidelijker zijn in de annulus en dan met name in de buitenste laag. Verder valt op dat deze veranderingen al binnen een week meetbaar zijn. Hieruit concluderen wij dat het type overbelasting, dynamisch of statisch, voor de verschillende regio’s in de tussenwervelschijf, verschillende effecten heeft.

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Hoofdstuk 4: Is er een regionaal verschil (anterieur, lateraal en posterieur) in de reactie op dynamische en statische overbelasting, dat de voorkeurslocatie van lumbale hernia’s in de posterolaterale hoek van de tussenwervelschijf kan verklaren? Van oudsher worden de vorm van de tussenwervelschijf en de verschillen in dikte van de annulus als oorzaak genoemd (of als reden gegeven) voor het ontstaan van hernia’s aan de achterzijde van de tussenwervelschijf. Toch hebben alle mensen dezelfde tussenwervelschijven, maar ontwikkelen niet alle mensen hernia’s. Daarnaast is het zo dat hernia’s niet per se ontstaan tijdens torsie momenten (draaiing van de wervelkolom) en zware belasting van de wervelkolom, maar ook of juist tijdens veelal arbitraire (belastings)momenten. In dit hoofdstuk keken wij daarom specifiek naar de voor-achterwaartse verschillen in de tussenwervelschijf van de effecten van overbelasting. Wij wisten uit hoofdstuk 3 dat statische overbelasting de nucleus relatief spaart, terwijl deze overbelasting de annulus juist verzwakt. Wij hebben hier getoetst of deze verzwakking aan de voorzijde, zijkant en achterzijde van de annulus gelijk is of regionaal verschilt. Wat wij zagen is dat dynamische overbelasting wederom op de gehele tussenwervelschijf ongeveer gelijke schadelijke effecten had en dat er geen voor-achterwaartse verschillen meetbaar waren. Hier stond tegenover dat met statische overbelasting de achterzijde van de buitenste annulus aanzienlijk meer celsterfte kent en op de gekleurde weefselcoupes aldaar beduidend vaker schade liet zien. Hieruit concludeerden wij dat statische overbelasting met name schadelijk was voor de posterieure (achterzijde) van de annulus en dat de nucleus relatief gespaard bleef. Dit creëert een situatie in de tussenwervelschijf die ervoor zou kunnen zorgen, dat de nog goed onder druk staande nucleus, via de verzwakte annulus aan de achterzijde preferentieel herniëert. Onze bevindingen corresponderen met recent onderzoek in een grote werkende populatie van gezonde volwassenen, waarbij als sterkste risico voor het ontwikkelen van een hernia, statische (over)belasting van de rug werd gezien.

Hoofdstuk 5: Hoe verandert het mechanisch gedrag van de tussenwervelschijf in de eerste fase van degeneratie? Door de unieke combinatie van een nucleus die als osmotisch actieve hydrogel fungeert en een annulus die met zijn poreuze, elastische eigenschappen heel trekvast is, vertoont de 224


mechanica van de gezonde tussenwervelschijf een complex poro-elastisch of poro-viskeus gedrag. Dit wil zeggen dat de tussenwervelschijf een sterk niet-lineair vervormingsgedrag laat zien onder belasting en ontlasting. Dit gezonde gedrag kenmerkt zich door een exponentiële verandering van de tussenwervelschijf hoogte in de tijd: hoge initiële weerstand bij belasting en snel terugveren bij ontlasting. In de eerste fase van tussenwervelschijf slijtage verliest de tussenwervelschijf in zijn nucleus kleine hoeveelheden GAG. Hierdoor bevat de nucleus minder water en kan het water minder goed vasthouden. Hierdoor verliest de tussenwervelschijf bij gelijkblijvende krachten meer hoogte en herstelt zij langzamer. Daarnaast zou men kunnen veronderstellen dat door een verlies van een viskeus deel van het mechanische geheel (osmotische druk van de GAGs, water inhoud), de tussenwervelschijf in deze beginfase qua mechanisch gedrag verandert; zich meer als een elastisch (meer lineair verband tussen vervorming en tijd) dan als poro-elastische/viskeus materiaal gaat gedragen. In dit hoofdstuk simuleerden wij de eerste fase van tussenwervelschijf slijtage door een gestandaardiseerde hoeveelheid GAGs via een injectie van een enzym in de nucleus op te lossen. In het LDCS monitorden wij continu het mechanisch gedrag onder belasting- en ontlastingsomstandigheden. Met behulp van een wiskundig model berekenden wij parameters die het mechanisch gedrag van de tussenwervelschijf beschrijven tijdens de herstelfase (moment van mechanische ontlasting waarbij de tussenwervelschijf weer zijn hoogte herwint). In de groep waarbij het enzym was ingespoten, maten we inderdaad significante veranderingen in het mechanisch gedrag ten opzichte van de gezonde controlegroep. Deze veranderingen bevestigden onze hypothese dat in de vroege degeneratieve fase de tussenwervelschijf een meer lineair, elastisch herstelgedrag gaat vertonen.

Kunnen wij een stretched-exponential fit gebruiken om de veranderingen in mechanisch gedrag te kwantificeren met vroege degeneratie, nog voordat de tussenwervelschijf irreversibel hoogte en water heeft verloren? Het wiskundig model dat wij gebruikten en gebruiken om het vervormingsgedrag (herstel van hoogte) van de tussenwervelschijf te beschrijven en kwantificeren, is een zogenoemde stretched-exponential fit. Deze algebraïsche functie beschrijft het gedrag met behulp van een tijdsconstante tau en een stretch-constante beta. Uit onze metingen bleek dat deze parameters samen betrouwbaar de vroege veranderingen in het gedrag van de tussenwervelschijven kunnen objectiveren. Waar tau de verwachte veranderingen beschrijft zoals ook verkregen kunnen worden uit stijfheidsbepalingen, voegt beta informatie toe over het 225

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verlies van de exponentiele component. Dit is een duidelijk voordeel ten opzichte van de vaak gebruikte eenvoudige stijfheidsberekeningen, zeker als het vroege degeneratieve veranderingen betreft. De gedragsveranderingen zoals met de stretched-exponential parameters beschreven tijdens het experiment, waren constant en op alle meetmomenten goed te bepalen. Dit terwijl het gemeten hoogteverlies in de enzym-gedegenereerde groep nog niet irreversibel bleek en de verloren hoeveelheid GAGs nog geen meetbaar verlaagde hoeveelheid water in de nucleus had veroorzaakt. Tevens traden deze veranderingen op, voordat er irreversibel hoogte of water verlies geleden was. Hieruit maakten wij op dat het in de vroege fase van discusdegeneratie belangrijk is te kijken naar mechanisch gedrag, en niet alleen naar direct meetbare factoren zoals het totale hoogteverlies of de water-inhoud van de discus, aangezien dit resultanten van gedrag zijn en dus pas later veranderingen zullen laten zien.

Hoofdstuk 6: Hoe veranderen de waarden van kwantitatieve T2, T1rho en ADC maps met milde tussenwervelschijf degeneratie? Magnetic Resonance Imaging (MRI) wordt vaak gebruikt om afwijkingen van de tussenwervelschijf in beeld te brengen. Het T2 signaal van de discus is de resultante van het aantal protonen (voornamelijk van water) in de nucleus en in mindere mate in de annulus fibrosis. De hoogte van het T2 signaal en de eventuele afname ervan ten opzichte van andere tussenwervelschijven, vertelt de arts iets over de toestand van de tussenwervelschijven in de wervelkolom. Probleem hierbij is dat de hoogte van het T2-signaal onderhevig is aan zeer veel factoren (pre-scan belasting van de wervelkolom, tijdstip op de dag), die de interpretatie ervan kunnen beïnvloeden, zeker in de gevallen waarbij de slijtage subtieler is. Daarnaast heeft de water-inhoud van de tussenwervelschijf wel een relatie met de graad van werkelijke slijtage, maar deze wordt pas sterk in de latere fases van slijtage. Kortom, voor het vaststellen van vroege degeneratie is het T2 signaal geen ideale parameter. Een aantal nieuwere MRI-technieken zijn ontwikkeld en voorgesteld. Daarmee wordt meer gekeken naar de protonen (de watermoleculen) die daadwerkelijk aan de ECM vastzitten (T1rho). Ook zijn er scantechnieken beschikbaar die de richting en/of snelheid van diffusie van de vloeistoffen in de tussenwervelschijf kwantificeren (apparent diffusion coefficient; ADC). Echter, de kwantitatieve mapping mogelijkheden van deze technieken waren nog niet goed onderling en met T2 vergeleken op hun onderscheidend vermogen ten aanzien van vroege discus degeneratie. 226


In dit hoofdstuk gebruikten wij ons ex vivo model om onder gestandaardiseerde omstandigheden milde tussenwervelschijf-degeneratie op te wekken. Door van de tussenwervelschijf vooraf en achteraf kwantitatieve T2, T1rho en ADC maps te maken, konden wij deze technieken goed vergelijken op hun vermogen de eerste degeneratieve veranderingen te detecteren. Alle technieken toonden na het experiment signaalverlies. T1rho bleek sterker te veranderen dan T2 en ADC veranderde juist het minst. Hieruit concludeerden wij dat T1rho gevoeliger is voor kleine veranderingen in samenstelling van de extracellulaire matrix en daarmee beter geschikt is om vroege discus degeneratie op te sporen dan T2 of ADC.

Welke MRI techniek correleert het sterkst met de gouden standaard bepalingen van tussenwervelschijf slijtage? Wij hebben de MRI metingen gecorreleerd aan de histologische graad van degeneratie (zoals semi-kwantitatief vastgesteld op histologische coupes), het mechanisch gedrag van de tussenwervelschijf (zoals beschreven door de stretched-exponential fit parameters) en met de hoeveelheid water en GAG in de ECM. T1rho waarden uit de nucleus regio correleerden significant sterker met de histologische graad van slijtage, de stretch-constante beta en de GAG inhoud, dan T2 of ADC. Hieruit concludeerden we dat de T1rho nucleuswaarden de werkelijke toestand van de tussenwervelschijf beter weergeven dan T2 en ADC in beginnende tussenwervelschijfslijtage.

Samenvattende conclusies Samenvattend hebben de studies in dit proefschrift ons meer kennis gebracht over de interactie tussen mechanische belasting en de cellen en matrix in de tussenwervelschijf. De stimulerende of beschadigende invloed van belasting op de tussenwervelschijf laat zich omschrijven als een optimum curve; te weinig belasting is niet goed en teveel is eveneens schadelijk. De homeostase (evenwicht) in de tussenwervelschijf kent verder vele extremen (weinig zuurstof en nutriënten, hoge osmotische druk, grote mechanisch krachten) die alle gewaarborgd dienen te worden voor handhaving van de native eigenschappen van de cellen, matrix en daarmee de biomechanische eigenschappen, om niet te vervallen in de vicieuze cirkel van degeneratie. Met de experimenten in dit proefschrift hebben wij gedetailleerde informatie gekregen over de regionale verschillen binnen de tussenwervelschijf van de effecten van verschillende typen overbelasting (dynamisch en statisch). We hebben de biomechanische

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veranderingen die optreden tijdens de eerste fase van discus degeneratie in meer detail beschreven. Daarnaast laten wij zien dat kwantitatieve MRI mapping met T1rho superieur is aan traditionele T2 en het nieuwere ADC om de eerste kleine veranderingen in de nucleus van de tussenwervelschijf met beginnende degeneratie te detecteren. Met de nieuwe kennis over de schadelijke invloeden van belasting op de tussenwervelschijf is de ontwikkeling van betere, specifiekere therapieën gericht op het stoppen of remmen van de vicieuze cirkel van degeneratie een stap dichterbij gekomen. De nieuwe kennis over de kenmerken van vroege discusdegeneratie

kan

mogelijk

in

de

toekomst

worden

ingezet

om

vroege

tussenwervelschijfslijtage eerder en betrouwbaarder op te sporen in bepaalde patiënten met lage rugklachten. Uit het gezamenlijke werk in dit proefschrift kunnen verder de volgende zaken worden geconcludeerd: 1. Het is mogelijk een intacte lumbale geiten tussenwervelschijf in een kweeksysteem (het LDCS) gedurende 3 weken te conserveren. Hiervoor is een adequate hoeveelheid axiale belasting nodig (SPL regime). 2. Onbelast of met te weinig axiale belasting ondergaat de tussenwervelschijf degeneratieve veranderingen op cellulair niveau over een kweekperiode van drie weken. 3. Overbelasting van de tussenwervelschijf leidt tot degeneratieve veranderingen op cellulair, extracellulair en biomechanisch niveau. 4. De degeneratieve effecten van dynamische en statische overbelasting zijn voor nucleus, binnen- en buiten annulus verschillend. 5. Statische overbelasting van de tussenwervelschijf is met name schadelijk voor de cellen en ECM aan de posterieure zijde van de buitenste annulus, terwijl het de nucleus regio relatief spaart. Deze situatie zou een verklaring kunnen zijn voor de posterolaterale voorkeurslokatie van lumbale hernia’s. 6. De invloed van statische overbelasting op de tussenwervelschijf zou een verklaring kunnen zijn voor het verhoogde risico op lumbale hernia’s bij mensen met een zittende levensstijl. 7. Bij beginnende discus degeneratie veranderen de biomechanische eigenschappen van de tussenwervelschijf direct als reactie op GAG verlies, en gaan van non-lineair poro-elastisch naar meer lineair vast-elastisch gedrag.

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8. De veranderingen in de poro-elastische eigenschappen van de tussenwervelschijf laten zich beter kwantificeren door de tau en beta parameters van de stretchedexponential fit, dan door hoogteverlies- of stijfheidsmetingen. 9. Kwantitatieve mapping van de tussenwervelschijf met T1rho is sensitiever dan T2 en ADC in het detecteren van kleine GAG verliezen in de nucleus. 10. T1rho waarden correleren beter met histologische discus degeneratie scores, biomechanische parameters en GAG inhoud van de matrix en zou daarom de voorkeur verdienen in de kliniek boven T2 en ADC in het diagnosticeren van vroege discus degeneratie.

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Acknowledgements C.P.L. Paul

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“Know when to hold ‘em, know when to fold ‘em. Know when to walk away, know when to run” The Gambler, Kenny Rogers, 1978

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Acknowledgements

Acknowledgements The combined research in this thesis was completed over a period of ten years, involved people from 5 different universities and over two dozen clinical and research departments. Through the years we have examined the caprine lumbar intervertebral disc on a molecular biological, cell biological, (immune)histochemical, biochemical, biomechanical and radiological level. This would not have been possible without the numerous fruitful collaborations with the many co-authors and other researchers of all these different institutes and their various scientific backgrounds. And although this work might just be only a small step for science, it represents a giant leap in my personal life-time, carrier and development. Clearly, it is impossible to acknowledge everybody on name basis, but I am grateful to all the wonderful colleagues of all the various institutes and departments I had the opportunity to collaborate with on this work. My promotor, Professor Theodoor Smit, I would like to acknowledge personally, simply because this thesis would not have seen the light of day, if it weren’t for his support. Theo, during difficult times, you reached out and helped me get back in the research game, even as odds were turning for you. Your unconditional support in finishing this work and continuous efforts to improve the final product, always kept me inspired to do the same. I am very grateful to have had you as my mentor on this journey. My co-promotors, Dr. Marco Helder and Dr. Margriet Mullender, I would also like to acknowledge personally for their continuous support throughout the years. You

A

too made this thesis possible and I am thankful for your guidance, abundant efforts, and immense patients in completing this work. I would also like to thank the members of the promotional committee, Prof. dr. G.M.M.J. Kerkhoffs, Prof. Dr. Ir. G.J. Strijkers, Dr. M.J.B. van den Hoff, Prof. dr. W.F. Lems, Dr. A.D. Bakker for the time and effort they have invested in reading this thesis and my defense of it. I am grateful for their comments and discussions. Their involvement with this thesis, with their various backgrounds, will hopefully spark further collaborations on translational and clinical research projects.

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LDCS articles

LDCS international publications: 1. Paul CP, de Graaf M, Bisschop A, Holewijn RM, van de Ven PM, van Royen BJ, Mullender MG, Smit TH, Helder MN. Static axial overloading primes lumbar caprine intervertebral discs for posterior herniation. PLoS One, 2017 2. Vergroesen PA, Emanuel KS, Peeters M, Kingma I, Smit TH. Are axial intervertebral disc biomechanics determined by osmosis? J Biomech., 2017. 3. Vergroesen PP, van der Veen AJ, Emanuel KS, van Dieen JH, Smit TH. The poro-elastic behaviour of the intervertebral disc: A new perspective on diurnal fluid flow. J Biomech., 2016. 4. Peeters M, van Rijn S, Vergroesen PP, Paul CP, Noske DP, Van der Top PW, Wurdinger T, Helder MN. Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture. Scientific Reports, 2015 5. Vergroesen PP, Kingma I, Emanuel KS, Hoogendoorn RJ, Welting TJ, van Royen BJ, van Dieën JH, Smit TH. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage, 2015. 6. Emanuel KS, Vergroesen PP, Peeters M, Holewijn RM, Kingma I, Smit TH. Poroelastic behaviour of the degenerating human intervertebral disc: a ten-day study in a loaded disc culture system. Eur Cell Mater., 2015. 7. Castro AP, Paul CP, Detiger SE, Smit TH, van Royen BJ, Pimenta Claro JC, Mullender MG, Alves JL. Long-Term Creep Behavior of the Intervertebral Disk: Comparison between Bioreactor Data and Numerical Results. Front. Bioeng. Biotechnol., 2014.

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8. Vergroesen PP, van der Veen AJ, van Royen BJ, Kingma I, Smit TH. Intradiscal pressure depends on recent loading and correlates with disc height and compressive stiffness. Eur Spine J, 2014. 9. Paul CP, Schoorl T, Zuiderbaan HA, Zandieh Doulabi B, van der Veen AJ, van de Ven PM, Smit TH, van Royen BJ, Helder MN, Mullender MG. Dynamic and static overloading induce early degenerative processes in caprine lumbar intervertebral discs. PLoS One, 2013. 10. Paul CP, Zuiderbaan HA, Zandieh DB Albert J. van der Veen AJ, van de Ven PM, Smit TH, Helder MN, van Royen BJ, Mullender MG. Simulatedphysiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture. PLoS ONE, 2012 244

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Curriculum Vitae

Curriculum Vitae

C.P.L. (Kees-Pieter) Paul

Education, experience, interests:

https://nl.linkedin.com/in/kees-pieter-paul-79293a2b

Scientific profile:

https://www.researchgate.net/profile/Cornelis_kees_Pieter_Paul

Articles available at:

https://www.ncbi.nlm.nih.gov/pubmed

International publications 1. Paul CP, de Graaf M, Bisschop A, Holewijn RM, van de Ven PM, van Royen BJ, Mullender MG, Smit TH, Helder MN. Static axial overloading primes lumbar caprine intervertebral discs for posterior herniation. PLoS One, 2017. 2. Peeters M, van Rijn S, Vergroesen PP, Paul CP, Noske DP, Van der Top PW, Wurdinger T, Helder MN. Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture. Scientific Reports, 2015.

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3. Castro AP, Paul CP, Detiger SE, Smit TH, van Royen BJ, Pimenta Claro JC, Mullender MG, Alves JL. Long-Term Creep Behavior of the Intervertebral Disk: Comparison between Bioreactor Data and Numerical Results. Front. Bioeng. Biotechnol., 2014. 4. Paul CP, Schoorl T, Zuiderbaan HA, Zandieh Doulabi B, van der Veen AJ, van de Ven PM, Smit TH, van Royen BJ, Helder MN, Mullender MG. Dynamic and static overloading induce early degenerative processes in caprine lumbar intervertebral discs. PLoS One, 2013.

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5. Bisschop A, Kingma I, Bleys RL, Paul CP, van der Veen AJ, van Royen BJ, van Dieën JH. Effects of repetitive movement on range of motion and stiffness around the neutral orientation of the human lumbar spine. J. Biomech., 2013. 6. Bisschop A, van Dieën JH, Kingma I, van der Veen AJ, Jiya TU, Mullender MG, Paul CP, de Kleuver M, van Royen BJ. Torsion biomechanics of the spine following lumbar laminectomy: a human cadaver study. Eur. Spine J., 2013. 7. Bisschop A, Kingma I, Bleys RL, van der Veen AJ, Paul CP, van Dieën JH, van Royen BJ. Which factors prognosticate spinal instability following lumbar laminectomy? Eur. Spine J., 2012. 8. Paul CP, Zuiderbaan HA, Zandieh DB Albert J. van der Veen AJ, van de Ven PM, Smit TH, Helder MN, van Royen BJ, Mullender MG. Simulatedphysiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture. PLoS ONE, 2012. 9. Paul CP, Everts M, Dent P Paul CP, Everts M, Glasgow JN, Dent P, Fisher PB, Ulasov IV, Lesniak MS, Stoff-Khalili MA, Roth JC, Preuss MA, Dirven CM, Lamfers ML, Siegal GP, Zhu ZB, Curiel DT.Characterization of infectivity of knob-modified adenoviral vectors in glioma. Cancer Biology & Therapy, 2008.

Submitted for publications 10. Paul CP, Emanuel K.S., Kingma I., van der Veen A.J., Holewijn R.M., Vergroesen P.P.A., van de Ven P.M., Mullender M.G., Helder M.N., Smit T.H. Changes in intervertebral disc mechanical behavior during early degeneration. Submitted to Journal of Biomechanical Engineering, 2017. 11. Paul CP, Smit T.H., de Graaf M., Holewijn R.M., Bisschop A., van de Ven P.M., Mullender M.G., Helder M.N., Strijkers G.J. Quantitative MRI in early intervertebral disc degeneration: T1rho correlates better than T2 with biomechanics and matrix content. Submitted to PLoS ONE, 2017.

National (Dutch) publications 1. Paul CPL, Lems WF. Capita Selecta; Moderne behandeling van Rheumatoïde Arthritis. Nederlands Tijdschrift voor de Geneeskunde, Studenteneditie. 2008 2. Paul CPL, Lems WF. Intensieve monitoring bij Rheumatoïde Arthritis. Nederlands Tijdschrift voor de Rheumatologie. 2007

International presentations

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Paul CPL, Strijkers G, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. Quantitative MRI of degenerated lumbar caprine discs correlates with biomechanical and histological changes. International Cartilage Repair Society annual meeting, Montreal, 2012. Paul CPL, HA. Zuiderbaan, T. Schoorl, B. Zandieh Doulabi, AJ. vd Veen, TH. Smit, MN. Helder, BJ. van Royen, MG. Mullender. Mechanical Overloading Induces Early Degenerative Processes in the Lumbar Intervertebral Disc. Orthopedic Research Society annual meeting, San Francisco, 2012. Paul CPL, HA. Zuiderbaan, B. Zandieh Doulabi, AJ. vd Veen, TH. Smit, MN. Helder, BJ. van Royen, MG. Mullender. The Loaded Disc Culture System; Ex Vivo Organ Culture and Loading of Large Species Intervertebral Discs. International Society for Studies of the Lumbar Spine (ISSLS), Annual meeting, Gothenburg, 2011. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. An Ex Vivo Organ Culturing and Loading System for Large Species Intervertebral Discs; a Feasibility Study With Caprine Lumbar Discs. International Cartilage Repair Society, Barcelona 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Effects of Short Term Loading on Intervertebral Disc Cell Viability in the Loaded Disc Culture System. Eurospine, Wenen, 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. A feasibility study of Ex Vivo Culture and Loading of Large Species Intervertebral Discs; Validation of the Loaded Disc Culture System. Biospine 3, Amsterdam, 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Short Term Loading of Intervertebral Discs Effects Cell Viability and Gene Expression in the Loaded Disc Culture System. Biospine 3, Amsterdam, 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Validation of the Loaded Disc Culture System; a Novel Bioreactor to Study the Effects of Loading on Large Species Intervertebral Discs. European Orthopaedic Research Society, Davos 2010.

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National (Dutch) presentations Paul CPL, HA. Zuiderbaan, B. Zandieh Doulabi, AJ. vd Veen, TH. Smit, MN. Helder, BJ. van Royen, MG. Mullender. Effecten van Verschillende Typen Mechanische Belasting op Geiten Tussenwervelschijven. Nederlandse Orthopedie Vereniging Jaarvergadering, Groningen, 2011. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. The Loaded Disc Culture System; a Feasibility 247

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Study for Culturing and Loading of Goat Intervertebral Discs in the Loaded Disc Culture System. BMM-TeRM annual meeting, Ermelo, 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. The Loaded Disc Culture System: Characterization of Goat Disc Response to Loading. NBTE annual meeting, Lunteren, 2009.

Poster presentations Paul CPL, Strijkers G, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. Changes in biomechanical, histological and quantitative MRI parameters in lumbar caprine intervertebral discs subjected to chondroïtinase-induced degeneration. World Forum for Spine Research, Helsinki, 2012. Paul CPL, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. The posterior region of the caprine lumbar intervertebral disc is most sensitive to static overloading. Eurospine, combined meeting Spineweek, Amsterdam, 2012. Paul CPL, Strijkers G, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. Correlation of quantitative MRI with biomechanical and histological changes in enzymatically-degenerated lumbar caprine discs. International Society for Studies of the Lumbar Spine (ISSLS), combined meeting Spineweek, Amsterdam, 2012. Paul CPL, Strijkers G, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. Rapid Dose-Dependant Degenerative Changes in Lumbar Intervertebral Discs after Chondroïtinase Injection Quantified by Biomechanical, MRI and Histological Parameters. Orthopedic Research Society annual meeting, San Francisco, 2012. Paul CPL, Graaf M. de, Bisschop A., Veen AJ. van der, Smit TH, Helder MN, Royen BJ. van, Mullender MG. Regional Variations in Sensitivity of Cells and Matrix to Mechanical Overloading of Lumbar Intervertebral Discs. Orthopedic Research Society annual meeting, San Francisco, 2012. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. The Loaded Disc Culture System; a Novel Bioreactor for Culture of Intervertebral Discs. Orthopedic Research Society annual meeting, Long Beach 2011. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Effects of Short-Term Loading on Intervertebral Disc Cell Viability in the Loaded Disc Culture System. Orthopedic Research Society annual meeting, Long Beach 2011.

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Curriculum Vitae

Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Loading Effects Intervertebral Disc Cell viability in the Loaded Disc Culture System. International Cartilage Repair Society, Barcelona, 2010. Paul CPL, Zuiderbaan HA, Zandieh Doulabi B, Veen AJ van der, Smit TH, Helder MN, Royen BJ van, Mullender MG. Effects of Short Term Loading on Intervertebral disc Cell Viability in the Loaded Disc Culture System. European Cell & Materials, Davos, 2010.

Grants and scholarships:

Fellowship Saal van Zwanenbergstichting, Organon 2006-2007 Student research grant KWF Kankerbestrijding Student research grant Hersenstichting Nederland

Scientific awards:

Winner best poster presentation, European Cells & Materials meeting, Davos, Switzerland, 2010. Poster title: “Effects of short-term loading on the cell viability of intervertebral disc in a loaded disc culture system”. Winner best Master research project, VU University Medical Center, Amsterdam 2006. Article title: Characterization of infectivity of knob-modified adenoviral vectors in glioma. Paul CP, Everts M, Dent P et al. Cancer Biology & Therapy. 2008 Presentation title: “Verkoudheidsvirus nieuwe hoop tegen hersentumoren”.

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