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Review Article

Current understanding of cellular and molecular eventsin intervertebral disc degeneration: implications for

therapyA. J. Freemont*, A. Watkins, C. Le Maitre, M. Jeziorska and J. A. Hoyland

Musculoskeletal Research Group, The Medical School, Stopford Building, University of Manchester, Manchester M13 9PT, UK

*Correspondence to:

Professor A. J. Freemont,

Musculoskeletal Research Group,

The Medical School, Stopford

Building, University of Manchester,

Manchester M13 9PT, UK.

E-mail: [email protected]

Received: 17 September 2001

Accepted: 12 October 2001

Abstract

Until recently, material removed from the intervertebral disc (IVD) at surgery consisted either

of ‘loose bodies’ from the centre of the IVD or discal tissue displaced (prolapsed) into the

intervertebral root or spinal canals. This material is best regarded as a by-product of disc

degeneration and therefore not representative of the disease process itself. Recent advances in

surgical techniques, particularly anterior fusion, in which large segments of the anterior part of

the IVD are excised with the anatomical relationships between different components intact, have

generated material that can be investigated with modern molecular and cell biological techniques.This is an important area of study because degeneration of the lumbar IVDs is associated,

perhaps causally, with low back pain, one of the most common and debilitating conditions in the

West. ‘Degeneration’ carries implications of inevitable progression of wear-and-tear associated

conditions. Modern research on human IVD tissue has shown that this is far from the case and

that disruption of the micro-anatomy described as degeneration is an active process, regulated by

locally produced molecules. The exciting consequence of this observation is the possibility of being

able to inhibit or even reverse the processes of degeneration using targeted therapy. Copyright #

2002 John Wiley & Sons, Ltd.

Keywords: intervertebral disc degeneration; angiogenic molecules; neurogenic molecules;

cytokines

Chronic low back pain and the IVD

Approximately 70% of the population will experience

low back pain (LBP) during their lives [1] and it

accounts for about 15% of all sickness leave in the

UK [2]. Although LBP constitutes an important public

health issue, little is known of its pathogenesis. The

organs that together make up the spine and its

accessory structures are so numerous and complex

that the causes of LBP are undoubtedly multifactorial.

However, clinical, interventional, and imaging studies

on volunteers and patients [3–5] and therapeutic trials[6] have produced evidence implicating mechanical or

traumatic disorders of the intervertebral disc (IVD) in

up to 40% of cases. In almost every case when IVD

tissue has been examined from patients with LBP, it

has shown the features typical of discal degeneration

(see below).

Structure and function of the IVD

The three components of the IVD are:

(i) The end-plate – This structure consists of a layerof cartilage, resembling articular cartilage that

covers the central parts of the inferior and super-

ior cortical bone surfaces of the vertebral bodies.

Ex vivo vertical loading experiments of isolated

spinal segments show that this is the weakest part

of the spinal column [7].

(ii) Nucleus pulposus (NP) – The space between the

end-plates of adjacent vertebrae is filled by the

nucleus pulposus, consisting of chondrocytes

within a matrix of type II collagen and proteo-

glycan, mainly aggrecan [8]. Surprisingly little is

known of the structure of the NP or the biology of

its cells. The type II collagen fibres are not

believed to give the same level of order to the

structure or the same degree of mechanicalstability to the matrix as in articular cartilage.

The proteoglycans are hydrophilic, causing the NP

to swell. The swelling pressure of the NP proteo-

glycans is constrained by the end-plates above and

below and the annulus fibrosus (see below) around

the periphery. On H&E sections, the NP appears

homogeneous and pale lilac-blue, consistent with

its complement of proteoglycans. In polarized

light, it exhibits little birefringence.

(iii) Annulus fibrosus (AF) – This comprises dense

sheets of highly orientated collagen fibres (mainly

type I but also types II and III) in which are cells

with the morphology and phenotype of fibro-blasts. Our experience is that when cultured in

alginate they can be transformed into chondro-

cytes. Functionally, the annulus fibrosus is a very

Journal of Pathology

J Pathol 2002; 196: 374–379.

Published online 11 February 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1050

Copyright# 2002 John Wiley & Sons, Ltd.



strong ligament binding together the outer rims of

adjacent vertebrae. On H&E sections, it exhibits

fairly uniform eosinophilia and in polarized light,

its constituent alternating bands of highly orien-

tated collagen fibres are clearly seen.

The IVD is a joint and, as such, allows movement

between bones, in this case adjacent vertebral bodies.In its normal fully hydrated state, each IVD permits

small degrees of flexion, extension, lateral bending, and

twisting, but is resistant to compressive loads. When

summated, the small movements at each IVD permit

great mobility of the entire spine.

‘Degeneration’ of the IVD

It is believed that many of the disorders of the IVD are

mechanical in origin. The erect spine is subjected to a

range, rate, and degree of loading that arguably it is

poorly designed to sustain. As a consequence, it suffers

mechanical damage, which, in a high proportion of

individuals, results in a pattern of morphological and

histological changes, well characterized from autopsy

studies, which together constitute discal degeneration.

With the exception of the outer third of the AF, the

normal adult IVD is avascular and aneural; there is a

relatively clear demarcation between the AF and the

NP; and the end-plate forms an intact layer of cartilage

overlying cortical bone. In degeneration, the NP is

disrupted, with changes in the proportion and types of

proteoglycans and collagens (making the NP more

eosinophilic), a reduction in the total number of lacunae containing viable chondrocytes, but the for-

mation of chondrocyte clusters in many lacunae, just

as in osteoarthritis of articular cartilage, which may

even increase the total number of cells present in the

diseased tissue. In addition, there is breakdown of the

matrix with the formation of permeative ‘slit-like’

spaces. There is often also disruption of the collagen

fibre arrays in the AF, traumatic damage to the end

plate, and vessel and nerve ingrowth into the inner AF

and NP. Current understanding of the biology of

connective tissues would causally implicate alterations

in the function of local cells in these events.Because man is the only obligate bipedal vertebrate,

replicating human disorders of the IVD in other

animals has proved problematic. As a consequence,

there are no satisfactory animal models of human IVD

disease, which has inhibited the study of this group of

disorders. An alternative strategy is to study human

tissue removed from cadavers or from patients at

surgery. This has been facilitated by the development

of modern tissue-probing techniques [9–12] that can be

performed on histological sections of human tissue,

such as immunohistochemistry, in situ hybridization,

in situ RT-PCR, and lectin histochemistry, as well as

by new surgical approaches to the management of IVDdisease. The recent advent of an anterior approach to

the diseased IVD to facilitate disc replacement or

spinal fusion has generated large wedges of discal

tissue for study, often, in the case of spinal fusion,

from multiple levels. These are a valuable source of

material because they consist of primary diseased

tissue in which the components of the IVD are in the

same anatomical relationship to one another as in vivo.

There has also been a trend towards pre-operative

discography, in which IVDs are injected with a radio-opaque medium. This is performed under ‘aware state

anaesthesia’ so that the patient can report if disco-

graphy recapitulates symptoms. Either ‘pain level’ or

‘non-pain level’ (or both) IVDs may be removed

depending on the clinical indications, particularly

in multi-level fusion surgery, and this gives the

pathologist the opportunity to relate findings to

symptomatology.

In scientific terms, studies of random samples of

human tissue cannot be as satisfactory as controlled

experiments. Nevertheless, they have generated useful

data from which has come the realization that the

origins of IVD degeneration are neither as passive nor

as inevitable as the term implies. This has, in turn,

opened the way for a re-evaluation of the management

of IVD disease and LBP.

Recent advances in the understanding of disc degeneration

The problems of controls

Two major issues have dictated the pace and direction

of research into the processes of IVD degeneration: the

adequacy or otherwise of control material and thedifficulty in defining a recognized starting point from

which to assess degenerative change.

Tissue acquisition

Until recently, difficulty in accessing relatively intact

normal human IVD, other than from cadavers, has

meant that a greater emphasis has been placed on

those aspects of the IVD that do not change

significantly in the first few hours after death, such as

matrix chemistry and disc biomechanics. As spinal

surgery, particularly spinal reconstruction for meta-

static malignancy, has advanced, this situation haschanged and with it, opportunities have developed to

obtain an improved understanding of the biology of

the IVD cells in discal degeneration.

Ageing and load

The nature of collagenous tissues means that their

structure changes with time and with the organism’s

age. Continuing post-synthetic changes in the structure

of many matrix molecules and alteration in the

nutritional status of these poorly vascularized tissues

with age lead to modifications in the collagen and

proteoglycan composition of the IVD [13]. As theincidence of discal degeneration also increases with

age, distinguishing ‘normal ageing’ from ‘disease’ [14]

becomes paramount. This is complicated by the high

Current understanding of cellular and molecular events in IVD degeneration 375

Copyright# 2002 John Wiley & Sons, Ltd. J Pathol 2002; 196: 374–379.



frequency of disc degeneration at some spinal levels

(e.g. L3–4, L4–5, and L5–S1), making the definition of

‘normality’ problematic.

In almost every connective tissue there are cells that

respond to differential loading by changing their

biology [15] and the IVD is no different [16]. Lifestyle,

body weight, and age are major factors that influencethe load environment of the normal IVD. In patients

with LBP, pain and stiffness also play a part, through

the natural protective reaction of abnormal prolonged

contraction of specific muscle groups. Such factors

further complicate the starting point from which to

assess IVD degeneration.

Altered matrix composition and integrity

When compared with age- and sex-matched controls,

there are profound alterations in the cell biology of

chondrocytes in degenerate IVD. Normal discal chon-

drocytes are characterized by expression of type IIcollagen and proteoglycans [17] and we have derived

evidence that this is regulated by the ‘master chondro-

regulatory gene’ SOX-9 (unpublished observations). In

discal degeneration, chondrocyte synthesis of matrix

molecules changes differentially with the degree of

degeneration [18], leading to an increase in the

synthesis of collagens I and III and decreased produc-

tion of aggrecan. Exactly what effect these changes

have on IVD function is unknown. Furthermore, the

regulation of matrix turnover is deranged, affecting

both synthesis and degradation [19]. In degeneration,

there is a net increase in matrix-degrading enzyme

activity over natural inhibitors of such activity, which

leads to loss of discal matrix. Particular attention has

been paid to the role of matrix metalloproteinases

(MMPs) in these processes [20–23], making them a

potential target for therapy designed to inhibit discal

degeneration. More recently, following work on the

mechanism of aggrecan degradation in articular carti-

lage, interest has grown in the possible role of

aggrecanases in IVD degeneration [19]. Aggrecan has

two cleavage sites, one acted upon by MMPs and the

other by members of a group of enzymes called the

ADAMs family, after their hybrid function ‘A Disin-

tegrin And Metalloproteinase’. In fact, the aggre-canases are two enzymes, ADAMTS4 and 5, that in

addition to their disintegrin and metalloproteinase

function also have ‘ThromboSpondin’ motifs. From

the pathological perspective, these studies have largely

been undertaken, not by looking for the enzymes

themselves, but through the application of antibodies

targeted at the breakdown product of aggrecan formed

by enzyme action at the specific site [19].

Reduced cell number

The reduction in chondrocytes that typifies IVD

degeneration has been ascribed to apoptosis [24].Indeed, there is some evidence of a ‘dose-dependent’

relationship between apoptosis and excessive load

(lifestyle, body weight, age) [25]. As in other chondroid

tissues, nitric oxide has been implicated in the induc-

tion of apoptosis [26].

Nerve and blood vessel ingrowth

Although the normal adult IVD is avascular and

aneural, nerves [27] and blood vessels [28,29] grow

into diseased IVD. One avenue of investigation hasbeen the local production of angiogenic and neuro-

genic molecules within degenerate IVD. Expression

of the potent angiogenic factor vascular endothelial

growth factor (VEGF) [30] has been demonstrated

within the IVD. We have just completed an investiga-

tion of the active (b) chain of nerve growth factor

(NGF) expression by cells in the IVD. NGF is syn-

thesized by blood vessels in discs showing nerve

ingrowth. Furthermore, the small nerves adjacent to

the vessels express the high-affinity receptor for NGF,

Trk-A. The implication of these data is that while

either angiogenesis or neuronogenesis could be targetsfor therapy, angiogenesis drives nerve ingrowth and

may be the more important from a therapeutic

perspective.

Cytokines as regulators of diseaseprocesses

Recently there has been growing interest in the possible

role of cytokines in regulating the connective tissue

degradation, nerve and vessel ingrowth, and macro-

phage accumulation that characterize IVD degenera-

tion. A number of cytokines have been implicated,including TNF, IL-1, PDGF, VEGF, IL-6, IGF-1,

TGF-b, EGF, FGF, and IL-10, amongst others

[23,30,31]. Of these, IL-1 is particularly interesting,

because in other settings there is evidence that it is

involved in cartilage homeostasis [32], largely through

its ability to switch chondrocytes from anabolism to

catabolism, inducing cartilage breakdown at molecular

and morphological levels [33,34]. It has also been

shown to be a regulator of angiogenesis in joints and

non-articular cartilage, possibly by acting as a promo-

ter of the potent angiogenic factor VEGF [35,36]. In

the IVD, both its isoforms, IL-1a and IL-1b have beenshown [37–39] to increase proteoglycan release and

degradative enzyme production. IL-1b also increased

production of MMPs and pain mediators, such as the

eicosanoid prostaglandin E2, by human IVD cells [40].

IL-1 production by human IVD cells has also been

reported [41]. Although not by any means conclu-

sive, these studies implicate IL-1 both in matrix

degradation and in pain. In addition, there is evidence

that IL-1 could mediate some of the other character-

istics of IVD disease, such as nerve and vessel

ingrowth [42].

If IL-1 is the regulator of cartilage catabolism, the

transforming growth factor beta (TGF-b) superfamilyis the regulator of cartilage anabolism. Although there

have been few experiments investigating the presence

or effects of TGF-b on discal cartilage metabolism,

376 A. J. Freemont et al.




Nishida et al . [43] induced a two-fold increase in

proteoglycan synthesis by rabbit nucleus pulposus cells

following injection of rabbit discs with an adenoviral

vector carrying a human TGF-b transgene. This will

undoubtedly turn out to be a key experiment in this

area.

There is a considerable amount still to be discoveredabout the role of cytokines in discal degeneration.

However, these pioneering experiments hint at possibi-

lities that have not previously been considered, both

for improving understanding of the processes leading

to IVD degeneration and for its management. The

exciting aspect of this work is that the interest in the

cytokine biology of the IVD comes at a time when

advancing technology makes it possible to advance the

study more rapidly and comprehensibly than has

hitherto been possible. For instance, we have under-

taken cytokine microarray analysis of cDNA derived

from RNA extracted from diseased human IVD. Theresults, showing expression of at least ten different

cytokines working through two separate intracellular

signalling pathways, are an indication of the extent of

cytokine activity in the diseased IVD. The question still

remains as to what initiates cytokine-mediated events

and how this might be linked to the association

between load and disc degeneration.

Clinical implications of these studies

Currently, the mainstays of treatment for damaged

IVD are either symptomatic medical therapy or

surgery, both of which have limited success. The

novel data that are starting to accumulate hold out

the possibility of managing IVD degeneration by

normalizing the cell biology of the NP and the AF. A

working hypothesis is that discal degeneration is an

active process, induced by abnormal load and

mediated by cytokines.

We and others are currently examining the possibi-

lity of using genes introduced into target cells to

produce proteins within the degenerate disc that willcreate a chemical environment that is conducive to

restoring cell function towards normality. This is

helped by the relatively immune privileged and

hypovascular nature of the IVD, which allows the use

Abnormal

load

Changed chemical

environment

Normal Degenerate

Slit

Neuronogenic and angiogenic

cytokines

Cytokine

Synthesis

Changed matrix

synthesis

Degradative

enzyme

synthesis

Cytokine

mediated events

Cell

proliferation

NP

Figure 1. The pattern of cellular events that lead from ‘normality’ to ‘degeneration’ as currently understood. Abnormal load (usually

repetitive) sometimes associated with an abnormal chemical environment leads to cytokine synthesis by the chondrocytes within the

nucleus pulposus. Either through direct cytokine-mediated events (e.g. angiogenesis and nerve ingrowth) or through secondary

phenomena, the changes that histopathologists recognize as degeneration [e.g. cell proliferation (and apoptosis), matrix degradation,

and changes in the balance between collagen and proteoglycan synthesis] occur





of viral vectors for gene delivery whilst minimizing the

risk of an immune response against infected cells and

of dispersal of the gene product widely throughout the

body [43,44]. The problem still remains of establishing

a normal load environment within the damaged IVD in

which genetically engineered cells might thrive, but

combinations of physiotherapy and/or modern implanttechnology, with the potential to fashion support

devices with biodegradable smart matrices that act as

scaffold, load normalizer and cell stimulator, make this

an opportune time to be a connective tissue pathologist

with a personal interest in the management of back

pain.

Acknowledgements

We wish to acknowledge the support of the Wellcome Trust

(SHoWCASe awards 057601/Z/99 and 063022/Z/0), the Arthritis

Research Campaign (Project grant F0531, ICAC grant F0551),

the MRC (Co-operative Group Grant G9900933), and the jointResearch Councils (MRC, BBSRC, EPSRC) UK Centre for

Tissue Engineering (34/TIE13617).

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