Literature Review

G r o u p P r o j e c t

D e v e l o p m e n t o f a t r i - a x i a l b i o r e a c t o r f o r

t h e t i s s u e e n g i n e e r i n g o f t h e

i n t e r v e r t e b r a l d i s c

Chapter 1

1.1 Introduction [R.T]

Back pain is usually associated with intervertebral (IVD) degeneration which has a high

occurrence of about 35% in the Western World. In the UK, it has also proven to be quite a

financial burden, as it involves disability benefits and insurance being provided for the

patients, as well as medical costs being taken into consideration. Treatment of this condition

has mostly been unsuccessful due to the lack of sufficient understanding of the

mechanobiology and mechanotransduction pathways in the tissue (Urban & Roberts, 2003).

The following project will discuss the characteristics of the healthy IVD, the pathologies

involved, including the available treatment strategies. Main focus will be placed in tissue

engineering treatment, describing the possible types of cells, scaffolds and bioreactors which

can be incorporated into the treatment process.

1.2 Characteristics of Healthy Intervertebral Disc

1.2.1 Intervertebral Disc Anatomy and Function[R.T]

Intervertebral discs are cartilaginous, viscoelastic tissues located between adjacent vertebrae

with ligamentous tissues connecting them to form a typical functional spinal unit (FSU). The

viscoelastic nature of the disc allows the spine to undergo torsion, extension and flexion

during daily physiological activities (Raj, 2007). They are heterogeneous in nature and form

2

20-33% of the total length of the spine (De Palmer & Rothman, 1970; White & Panjabi,

1990). The disc also has a very low cell density representing approximately 1% of its total

volume (Bibby et al., 2001, Setton & Chen, 2004). These cells are embedded in an

extracellular matrix (ECM) and are isolated from one another. They are responsible for

maintenance and repair of the disc by the production of proteinases for the breakdown and

the production of macromolecules for the synthesis of matrix. A controlled balance in the

matrix production is vital particularly during the repair of damaged disc via cellular repair

responses (Martin et al., 2002; Urban J. P., 2002). However, since the IVD represents an

avascular and aneural tissue and due to its low cell density, it undergoes rapid degeneration

and has limited self repair when damaged, particularly when compared to other vascular

tissues (Setton & Chen, 2004).

The intervertebral disc is divided into three main zones: the outer annulus fibrosus, the

central nucleus pulposus and the intermediate transition zone (De Palmer & Rothman, 1970;

White & Panjabi, 1990). These zones consist of different populations of cells of varying

morphology which are responsible for the production of the various components of the matrix

(Horner et al., 2002).

Figure 0-1: Structure of the intervertebral disc (Raj, 2007).

The IVD reveals a biphasic state with both a solid and fluid phase. The fundamental

constituents of the IVD in its hydrated state include collagen, proteoglycans, non-collagenous

proteins (glycoprotein) and the extracellular enzymes, which contribute to the swelling nature

of the disc (Culav et al., 1999). These constituents form the solid phase of the tissue; where as

the fluid phase consists of interstitial fluid. Table 1-1 below lists the various components of

the IVD and their corresponding proportions in which they are present.

3

Table 0-1: Major components of the intervertebral disc (Le Maitre et al.,2007)

1.2.1.1 Annulus Fibrosis

The annulus fibrosus is divided into two regions, namely, the intermediate transitional region

that is close to the nucleus pulposus and the outer collagenous region. Accordingly, the

composition and properties of the annulus gradually change from the outer region to the

transitional region.

The annulus fibrosus is made up of fibrous collagen strands, which are arranged, in 15-25

concentric lamellae running in the same direction (Urban 1995). The nuclear pulposus is

surrounded by these lamellae of the annulus, which become distinct with the lamellae moving

outward radially. The collagen fibres of the lamellae are obliquely oriented with an angular

orientation of 40-70º to the vertical axis and the obliquity being in opposite directions in

adjacent lamellae. This results in the anisotropic nature of the annulus (Houben et al., 1997;

Stoeckelhuber et al., 2005). The outermost lamellae, called Sharpey’s fibres, attach firmly to

the vertebral body, compared to the inner fibres that are attached to the cartilaginous end

plates. The cartilaginous end plates are made of hyaline cartilage and separate the IVD from

adjacent vertebrae (White & Panjabi, 1990).

The ECM of the annulus has a very low cell density of about 9000 cells/mm3 (Bayliss &

Johnstone, 1992). These cells express properties similar to those of fibroblasts (Raj, 2007),

since they originate from the mesenchyme. The cells are thin, elongated and arranged

perpendicular to the collagen fibres. Such properties include the production of proteoglycans

and collagen types I and III. In addition, these cells are arranged parallel to the collagen

fibrils and are oval in shape (Houben et al., 1997; Stoeckelhuber et al., 2005). The

concentration of collagen fibres in the IVD is greatest in the annulus fibrosus, with its main

function being to support the cells with its tough network and to enmesh the proteoglycans.

4

These collagen fibres include types I, II, V, VI, IX and XI (Bayliss & Johnstone, 1992).

Adjacent to the cells is a region called the pericellular matrix (PCM), which is characterised

by the presence of minute collagen fibres and more proteoglycans compared to any other

region of the disc. As such, the PCM acts as a mechanotransducer for the cells by altering

their mechanical environment (Poole, 1997).

The transition zone which is enclosed by the AF is also predominantly made up of type I

collagen fibres together with a high concentration of PGs unlike the outer annulus. The cells

present in this region of the disc are fibrocartilaginous in nature exhibiting both chondrocytes

and fibroblasts in the ECM (Walker et al., 2004).

The end plates on either sides of the disc are cartilaginous and highly vascularised with an

approximate thickness of 1mm. The collagen fibres located in these plates are parallel to the

vertebrae unlike those of the AF.

1.2.1.2 Nucleus Pulposus

The nucleus pulposus forms the central region of the intervertebral disc which has an ECM

made up of fine collagen fibrils that are suspended in a proteoglycan-water gel. This gel

consists of mucopolysaccharides and enmeshing it is a network of randomly oriented non-

collagenous proteins and collagen fibrils (Urban et al., 1979; White & Panjabi, 1990). The

water content of the NP ranges between 70-90% and its swelling capability. A young IVD is

initially made up of notocordal cells which are then substituted with mesenchymal cells as the

disc ages. The NP has a very low cell density of approximately 4000cells/mm3, with the cells

having a chondrocyte-like nature.

Collagen types I and II are the main types of collagen fibres found in the NP, however, there

are also small quantities of collagen types VI, IX and XI. Its ECM also consists of elastin

fibres, collagen type II fibres together with aggrecan (Bayliss & Johnstone, 1992; Roughley,

2004). In the NP, collagen fibrils are loosely packed in order to allow accommodation of

interfibrillar water, hence giving the NP its high water content (Comper, 1996).

The concentration of PGs in the NP is the highest compared to the other regions of the disc,

which allows the disc to withstand compressive loading. The predominant type of PG present

in the IVD is aggrecan, which consists of the major sGAG chains, namely keratin and

chondroitin sulphate chains. These chains have a negative fixed charged density which

strongly attracts water molecules and positive ions, hence creating osmotic pressure and

5

hydrostatic pressures within the disc. As such, due to their hydrophilic nature, PGs play a

vital role in the mechanical properties of the tissue. Proteoglycan concentration in the IVD is

greatest around the extrafibrillar areas and the cells (Bayliss & Johnstone, 1992).

Degeneration of the disc leads to a decrease in the density of sGAG chains and their length.

Alterations in the length of these sGAG chains are also due to changes in the oxygen levels of

the disc (Roughley et al., 2004).

1.2.1.3 Nutrition

The cells in the IVD depend on simple diffusion of nutrients via 2 pathways since the disc is

avascular in nature. These pathways include: the annulus periphery and end plates, the later

being the main pathway for nutrient supply.

The end plates are selective barriers that allow tiny uncharged molecules to pass through it.

However, with age, the thickness of these end plates decrease and its hyaline cartilage

structure becomes highly calcified inhibiting the transport of nutrient to the IVD (Urban et

al., 2004; Raj, 2007).The NP depends on the diffusion of nutrients from blood vessels located

in adjacent vertebral bodies, through these porous cartilaginous end plates, to the cells located

in the central region of the disc. However, obtaining nutrition for the NP is made difficult due

to its location right at the centre of the disc. Also, since it is located 6-8mm from the closest

blood vessel, it is evident that it acquires low nutrition (De Palmer & Rothman, 1970; Urban

et al., 2004). As such, the cells of the disc acquire a low level of O2 due to similar reasons,

especially those located in the NP. Consequently, they make use of Adenosine Triphosphate

(ATP) from the process of glycolysis resulting in the production of lactate. This production of

lactate results in the lowering of the pH in the tissue to acidic conditions which exceeds on

the application of loads. Synthesis of ECM is inhibited when pH levels getlower than 6.1 and

O2 levels decrease below 5% (Grunhagen et al., 2006).

The presence of oxygen is essential for the generation of chondroitin sulphate chains made of

glucuronic acid. The formation of keratin sulphate on the other hand is not influenced by the

presence of oxygen, provided galactose exists (Roughley et al., 2004).

In the case of the annulus periphery pathway, tiny blood vessels have been found in the AF

which are incapable of penetrating no more than 2mm (Urban et al., 2004).

Since load bearing is accomplished by the osmotic properties exhibited by the disc via the

GAG chains present (as mentioned above), nutrient transport through the end plates is

6

regulated via changes in the proteoglycan content. Other factors that influence the rate of

nutrient transport include the sizes and charges of the molecules diffusing through the ECM

(Roughley et al., 2004).

Figure 2: Figure 0-2: Changes in levels oxygen, glucose and lactic acid from the endplate to the centre of the disc

(Grunhagen et al, 2006).

The high percentage of PGs in the nucleus contributes to the diffusion of nutrients and the

rate at which they diffuse, due to the high diffusion coefficients generated (Bayliss &

Johnstone, 1992; Horner et al., 2002). As such it is worth saying that the ECM itself acts as a

selectively permeable membrane to the diffusion of molecules into it. On removal of the NP,

the diffusion gradient of molecules increases significantly (Urban et al., 2004).

1.2.2 Biomechanics [R.T]

The IVDs, which are viscoelastic in nature, act as cushions in the spine, separating adjacent

vertebrae from one another. As such, the spine can be modelled as a series of semi-

viscoelastic segments (White & Panjabi, 1990).

Loads are always being applied to the functional spinal units (FSUs) due to the daily

physiological activities of the body. When standing, these units typically undergo

compressive loadings initially due to the weight of the body acting above them. In

conjunction with facet joints that are highly significant in the spine, the IVD supports most of

the compressive forces. Indeed, it has been estimated that the facet joints support

approximately 20% of the axial loads (Hirsch & Nachemson, 1954; Prasad et al., 1974).

Further stability of the spine is maintained as the discs and vertebrae in the lower regions of

the spine have an increased cross sectional area, minimizing the stresses acting on them

(White & Panjabi, 1990).

7

1.2.2.1 Loading Modes

Compression

Application of compressive loads on the IVDs reveals a sigmoid type plot of deformation

versus load. This implies that the application of very little compressive forces typically result

in very little resistance of the disc. On the other hand, the application of high loads results in

the high resistance and stability (White & Panjabi, 1990). Such compressive loadings are

unlikely to cause disc herniation, which is thought to result from a more complex loading

pattern (Virgin, 1951; Hirsch & Nachemson, 1954; Markolf & Morris, 1974)

Compressive forces being applied on the IVDs results in complex stresses being generated

within the disc such as an increase in hydrostatic pressure in the NP, causing it to push

adjacent structures away from it, as shown in figure 1-3. In addition, the stresses generated in

the outer lamellae of the annulus are smaller compared to those of the inner lamellae, as

indicated by the magnitude of the lines shown in figure 2B. The fibres of the AF when

oriented at an angle of ±30º, enables the disc to withstand the tensile stresses that are

generated, while the NP withstands the compressive forces (Culav et al.,1999; White &

Panjabi, 1990; Hall, 1999)

Figure 0-3: Non-degenerated disc under compression. (A) Pressure within the nucleus is produced

because of compression. This pressure pushes the disc annulus and the two end-plates outward. The disc

bulges out in the axial direction. (B) In the outer layers the stresses are small. Axial, circumferential and

radial stresses are tensile. In the inner layers of the annulus, the axial, circumferential and radial stresses

are still compressive but their magnitude is larger. The fibre stress is larger and still tensile. (Shirazi-Adl,

Shrivastava, & Ahmed, 1984)

When the disc is subjected to a compressive load, the NP withstands the resulting

compressive stresses while the AF withstands the tensile stresses (White & Panjabi, 1990).

Also, water is expelled from the disc, which leads to an increase in the concentration of PGs

and a slight alteration in the tension of the PG-collagen network. Subsequently, this results in

8

a rise in the osmotic pressure. Water continues to be lost with a simultaneous increase in the

absorption of potassium and sodium, until the applied compression balances out. This is

governed by equation 1. When the disc is relieved of the load, its water content is restored,

leading to a drop in the osmotic pressure (Bader & Lee, 2000; Hall, 1999).

ncompressioappliedflowfluid Pp __ =∆ +fibrosusannulusbytensileP ___

-pulposusnucleusbyswellingP

___ (1)

(Bader & Lee, 2000)

Tension

Tension hardly occurs in the discs during normal physiological activities of the body even

when traction is applied to the spine. However, tensile stresses are generated in the disc in all

directions during such physiological activities. During flexion, when the instantaneous axis of

rotation (IAR) passes through the centre, tensile stresses occur on the posterior region of the

disc. These stresses are also generated during axial rotation at approximately 45º to the disc

plane (White & Panjabi, 1990).

The anisotropic nature of the disc results in high tensile strength and stiffness which helps it

to withstand excessive tensile loads, which are capable of resulting in disc degeneration.

However, the orientation of the specimens usually alters these stiffness values, where the

highest stiffness values are obtained when the specimens are at 15º to the horizontal

compared to those which are 70º to the horizontal (Galante, 1967). High levels of stiffness

also occur during compressive loading compared to tensile loading due to high fluid

pressures generated within the NP (Markolf, 1970).

In order to resist applied tensile loads, normal and shear stresses are generated within the

IVD. The shear stresses generated which are of higher magnitudes are not absorbed by the

annulus fibres, unlike the normal stresses which are of lower magnitudes. As such, it can be

said that the disc is more susceptible to failure on the application of tension compared to

compression (White & Panjabi, 1990; Shirazi-Adl et al.,1984)

Bending (extension, lateral bending and flexion)

When the spine undergoes bending, one side of the IVD experiences compression while the

other undergoes tension. These sides are separated by the instantaneous axis of rotation. On

the tension side, the disc contracts and the annulus fibres are stretched while on the

compression side, the annulus fibres are relaxed and the disc bulges as shown in Figure 1-4

(White & Panjabi, 1990).

9

Biomechanical tests confirm that without the posterior components of the disc, bending alone

is capable of damaging the disc (Brown et al., 1957). Bending causes the disc to bulge

outwards which causes irritation of the nerve roots and, as a consequence, can be one of the

causes of low back pain (LBP) (Breig, 1978; Adams 2004; Hall, 1999).

Figure 0-4: Non-degenerated disc under compression. (A) Pressure within the nucleus is produced

because of compression. This pressure pushes the disc annulus and the two end-plates outward. The disc

bulges out in the axial direction. (B) In the outer layers the stresses are small. Axial, circumferential and

radial stresses are tensile in the inner layers of the annulus, the axial, circumferential and radial stresses

are still compressive but their magnitude is larger. The fibre stress is larger and still tensile. (Shirazi-Adl,

Shrivastava, & Ahmed, 1984)

Torque

Angular displacement usually results from the application of torque on IVDs. Biomechanical

tests yield a sigmoid form of the applied torque versus angular deformation curves, which can

be divided into 3 phases. The initial phase involving minimal torque levels produces an

angular deformation ranging between 0-3º. This is followed by a linear slope of the torsion

and angular deformation in the intermediate zone at deformation values ranging between

3ºand12º. Finally, in the third phase, the healthy disc undergoes failure at a mean angle of

approximately 16º. By contrast, a degenerated disc without a nucleus will undergo failure at

an average lower angle of 14.5º (Farfan, 1973).

The distribution of stresses in the IVD on the application of torque depends greatly on the

stage of degeneration of the IVD and whether the posterior elements of the disc are removed.

In a healthy disc, the tensile stresses in the annulus fibres resulting from the application of

torsion in the direction of the fibres are highest in the anterior half of the IVD compared to

the posterior half and the posteriorlateral regions of the IVD. By contrast, when the posterior

elements of the disc are removed the stresses within the disc are distributed around its

periphery (White & Panjabi, 1990).

10

Shear

The IVD does not only undergo shear forces alone, as they are usually associated with

rotational and flexion movements. From shear tests performed on the disc, it has been

observed that the resistance to shear forces is equal throughout the disc. However, the shear

flexibility varies in the horizontal plane of the lumbar spine. In the posterior region the shear

flexibility is lowest compared to the anterior region (Liu et al., 1975;Tencer et al., 1982).

Although the disc provides less resistance to shear forces compared to axial ones, shear

forces alone are unlikely to lead to the damage of the annulus fibrosus. This is governed by

their high shear stiffness of approximately 260N/mm in the horizontal plane.

The applications of shear forces to the IVD results in the formation of compressive, tensile

and shear stresses within the disc. The shear stresses are generated on the transverse plane of

the spine being of equal magnitudes as the shear force. They are of zero magnitude on the

surface of the disc, increasing towards the centre (Shirazi-Adl et al., 1984).

Viscoelastic Behaviour, Creep and Hysteresis

The spine is known to exhibit a viscoelastic, nonlinear and biphasic behaviour. Properties

such as strain rate dependence, load relaxation and creep exhibited by the FSUs and IVDs are

used in order to characterise the viscoelastic behaviour of the disc during mechanical tests

(Hirsch & Nachemson, 1954). Experiments reveal that by increasing the load applied to the

disc, there is an associated increase in deformations which leads to an enhanced rate of creep

in the disc. It can therefore be concluded that the viscoelasticity of the disc decreases with

deformations as shown in figure 1-5 (Kazarian, 1975; Rolander, 1966).

Figure 0-5: Creep behaviour of the disc. The creep behaviour of a structure is documented by applying a

sudden load and maintaining it. The deformation of the structure as a function of time is recorded. This

behaviour seems to correlate with the degree of degeneration of the intervertebral disc. A sample of the

11

creep curves for discs with different grades of degeneration as shown. The non-degenerated disc (grade 0)

has smaller overall deformation and this deformation is reached over a relatively longer period as

compared with degenerated disc (grade 3). (Kazarian, 1975).

Hysteresis

Hysteresis is one of the most important characteristics of the IVD as it acts as a protective

mechanism for the spine, particularly when it is exposed to excessive loading. The

phenomenon involves the absorption and loss of energy when the spine undergoes loading

and subsequent unloading cycles. However, hysteresis varies depending on the magnitude

and type of the load being applied and the age of the disc. Also, the lower lumbar IVDs have

a higher level of hysteresis compared to those of the upper lumbar and thoracic regions.

Furthermore, it is essential to note that hysteresis is less effective when the load is applied in

a repetitive manner (Virgin, 1951).

1.3 Characteristics of pathological IVD [VT]

The IVD is subjected to high loads, which are distributed across the IVD. However, due to its

heterogeneity in structure this distribution is uneven in nature. This results in a greater

susceptibility to a number of disorders, which can be said to be of mechanical origin.

Therefore a pathological response can be a result of a number of factors, some of which are

largely unknown.

Alterations in the structure and function may not always be due to pathological changes, but

may arise due to trauma sustained in an accident and also with ageing. A major problem,

which occurs due to trauma, is disc herniation, where the disc may be displaced therefore

leading to lower back pain (LBP), which in turn would lead to a degenerative response of the

disc. This degenerative response is increased due to ageing where the NP and AF lose

function. Therefore it can be difficult to differentiate the changes, which occur due to ageing

and pathological changes (Urban & Roberts, 2003).

1.3.1 Disc Degeneration [VT]

Disc degeneration is a major occurrence, which results in chronic pain as well as disability to

a patient, it can be associated with age progression and excessive loading which, in turn, can

cause structural deformation as well as biochemical degradation of the disc (Cheung et al.,

2010).

The structure and function of the IVD is greatly dependent on the composition, organization

and integrity of the ECM. Alterations in the tissue composition of this matrix cause an

12

adverse effect on the function of the IVD, therefore providing a starting point for disc

degeneration. For this reason disc degeneration can be defined as:

‘’ an abnormal cell-mediated response to progressive structural failure (Colombini et al.,

2007)’’

For disc degeneration to occur a structural failure must also occur in the disc, this can be for a

number of reasons mainly being of mechanical origin. These structural failures can be

categorised into 5 groups:

1. Annulus Tears

a. Circumferential

b. Peripheral rim tears

c. Radial Fissures

2. Disc Prolapse

3. Endplate Damage

4. Internal Disc Disruption

5. Spondylosis

a. Disc Narrowing

b. Radial bulging

c. Vertebral Osteophytes

Age related degeneration are caused by a number of factors; declining nutrition, loss of viable

cells, cell senescence, post-translational modification of matrix proteins, accumulation of

degraded matrix molecules, and fatigue failure of the matrix. As a result of degeneration

changes, which occur,include decreased diffusion, decreased cell viability, decreased

proteoglycan synthesis, and alteration in collagen distribution (Kasra et al., 2003)

Together with this structural failure and cell-mediated response disc degeneration affects

12% to 35% of the population. Other non-essential effects occur due to disc degeneration and

structural failures, such as alterations in disc height and mechanics of the spinal column.

These can unfavourably affect the behaviour of other spinal structures, such as muscles and

ligaments. In the long term this can lead to spinal stenosis, a major cause of pain and

disability in the elderly (Urban & Roberts, 2003).

1.3.1.1 Effect on Annulus Fibrosis and Nucleus Pulposus

As ageing progresses with skeletal maturity and growth the boundary between the AF and NP

decreases, however, the two tissues remain distinguishable. The NP tissue loses its gel like

structure and becomes more of a fibrous structure due to dehydration. The negative effect of

this dehydration mechanism is that the NP becomes stiffer, reducing its ability to transfer

13

stresses across the IVD. Due to degeneration and ageing, the disc itself becomes disorganised

with the AF also becoming irregular, due to the collagen network becoming disorganised

(Figure 1-6)(Urban & Roberts, 2003).

Figure 0-6: A normal and degenerated disc (Urban & Roberts, 2003)

It can be seen from Figure 1-6 a cleft formation in the NP region with formation of fissures,

which further disrupts the organisation of the lamellae in the AF. The ECM plays an

important role in this degeneration, as the breakdown of the matrix results in the formation of

fissures (Urban & Roberts, 2003).

During the process of disc degeneration the ECM of the AF and NP are altered, which results

in the loss of function of the IVD. The most significant effect on the ECM is the decrease in

Proteoglycan (PG) formation. As the large aggrecan molecules are degraded, tissue hydration

decreases due to the osmotic pressure (Colombini et al., 2007). It is the loss of GAGs that

leads to the hypo-osmotic cellular environment. The mechanotransduction pathway therefore

alters to maintain the gene expression (Gilbert et al., 2010).

Other than PG formation during disc degeneration, collagen synthesis and composition also

alter IVD function. During early stages it has been found that type II collagen synthesis is

increased in the NP providing evidence of a repair mechanism. As the degeneration

progresses, type II levels increase in the outer AF and type I in the inner AF and NP forming

stronger collagen fibrils. The resulting cleft formation, can be linked to the observation of

type X collagen which indicated abnormal cellular activity (Le Maitre et al., 2007).

Matrix breakdown occurs as the rate of catabolic processes exceeds that of the anabolic

processes. Catabolic processes occur in the presence of MMPs and ADAMTs, enzymes

which are responsible for the breakdown the matrix components, type II collagen and

aggrecan, respectively. The onset of degradation is triggered by the by products of aggrecan

degradation and the decreased capacity to synthesise PG (Urban & Roberts, 2003).

14

The load bearing characteristics of IVD are altered due to loss of PG causing the osmotic

pressure to decrease therefore reducing hydration, under loading the disc may not be able to

maintain hydration, causing a disc bulge. Under this loss in mechanical characteristics of the

IVD means they do not behave hydrostatically under loading, therefore the stress

concentrations are inappropriate (Urban & Roberts, 2003). During physiological loading the

amount of strain acting on the disc is also likely to differ with degeneration due to the loss of

matrix proteins, which can lead to diminished elasticity and increased stiffness of the disc

(Gilbert et al., 2010).

1.3.2 Current Treatment Strategies

Disc degeneration results in major discomfort to the patient and if left untreated can lead to

further disabilities. There are a number of methods available, each with their specific

advantages and disadvantages. These can be classified into the following five approaches,

which are critiques in Table 1-2.

• Discectomy

• Spinal fusion

• Partial and total disc replacement

• Artificial disc implants

• Tissue Engineering

15

Table 0-2: A critical analysis of the five strategies used to treat intervertebral disc degeneration (Mohi

Eldin, 2009) (University of Maryland Medical Center, 2007) (Stenum Spine, 2004)

1.4 Tissue engineering and regenerative medicine [RT, VT, DB]

Tissue Engineering is an interdisciplinary field that applies the principles of engineering and

life sciences toward the development of biological substitutes that restore, maintain, or

improve tissue function or a whole organ (Langer & Vacanti, 1993)

Regenerative medicine is divided into two aspects; the repair of degenerated tissues by stem

cell therapies, which involve the injection of cell suspension or cell engraftment into the

patient, sometimes including the use of scaffolds. The other aspect includes the formation of

grafts or organs in vitro for the ailing of organs in vivo.

Strategy Method Advantages Disadvantages Discectomy

• Surgical removal

of herniated disc

which presses

against the spinal

cord.

• Effective and

low risk due to

the fact that

muscles/nerves

are not involved

• Invasive

• Long recovery

period

Spinal Fusion

• Surgical

technique used to

join 2 vertebrae

together

• Effective Pain

relief

• Promotes bone

growth around

damaged disc

• Invasive

• Fixation device

may be needed

• Limits motion at 2

vertebrae

Artificial Disc

Implants

• Surgical

technique used to

implant a

artificial disc

after a

discectomy is

carried out

• Increased return

of movement to

vertebrae

• Pain relief

• Non essential host

response

• Invasive

• Long procedure

• Longer recovery

period

• Decreased

Biocompatibility

Tissue Engineering

• With the use of

cells cultured in a

bioreactor, these

are seeded onto a

scaffold which

can be implanted

into the patient

• Essential Host

response

• Bio compatible

• Quick recovery

period

• Implantation is

invasive as

involves surgery

16

When applying tissue engineering to the treatment of Disc Degeneration; the repair method is

established by inducing the regeneration of the tissue in vivo by the use of biological

manipulation. Whereas for the replacement of IVD tissue; a functional tissue unit is

developed in vitro and then grafted into the body (O'Halloran & Pandit, 2007). There are

three main components of Tissue Engineering; Cell, Scaffold and Signals (growth factors and

mechanical stress). The most common approach to the development of tissue for regenerative

medicine is by immobilizing the cells, which will be used for tissue growth in biocompatible,

and biodegradable, porous scaffolds. These cells are then cultivated in bioreactors, which

provide the dynamic environment for the cells to grow. The native cells are exposed to forces

such as compression, tension or hydrostatic pressure and they regulate the matrix production

and gene expression. The application of mechanical forces by using a bioreactor may to help

to mimic the in vivo microenvironment of the cells and help them respond in a similar way to

their native behaviour (Sebastine & Williams, 2006).

1.4.1 Cell type [RT]

Cells are the ‘key’ raw materials in the tissue engineering of biological substitutes in order to

restore, maintain or improve tissue function. For tissue engineering to be successful, it is

essential to provide a tissue-like environment for the cells. Also, it is necessary to renew the

components of the ECM, since they are related to the physical properties of the disc (Enderle

et al., 2005).

As mentioned above in section 1.2, the inner AF, the end plates and the NP are made of

chondrocyte-like cells, whereas the outer AF contains fibroblast-like cells. As such, in the

tissue engineering of IVD, the cells selected must closely mimic these cells for the formation

of a functional IVD. In the NP, the biosynthetic activity of the cells is regulated due to the

presence of notocordal cells. Absence of these cells is said to be one of the many factors

responsible for the degeneration of IVD (Aguiar et al., 1999). However, there is a limited

availability of these cells for tissue engineering of IVD. As such, in the tissue engineering of

IVD, the cells selected must closely mimic these cells for the formation of functional IVD.

Also, since IVD disc cells, especially those of the NP have a low cell density when harvested

and also have a low capacity of proliferation, regulation of the disc viability becomes

essential (O'Halloran & Pandit, 2007).

One of the most common methods of tissue engineering of the IVD involves initially

restoring the NP by supplying its vacuole with cells and or cell-seeded scaffolds. On the

17

renewal of the NP, the tension created in the AF due to the swelling pressure in the NP can

provide appropriate stimuli to initiate repair of the AF causes the AF to repair. However, for

such an approach to be successful, the AF must be capable of withstanding the swelling

pressure of the restored NP. In treatment strategies where the AF is compromised, the only

method suitable in such cases is the production of functional IVDs in vitro and implanting

them into the body. These first two strategies of treatment are incorporated when cells of the

NP no longer respond to growth factors for the production of ECM. However, growth factors

can still be used in order to facilitate the production of ECM (O'Halloran & Pandit, 2007)

Certain factors need to be considered in the selection of the appropriate cell type for tissue

engineering of IVD. These include:

• the technique used to harvest the tissue,

• the manner in which the cell is processed and isolated,

• safety testing of the cells,

• activation of cells,

• the development of the medium for the cell culturing,

• the storage and stability of the cells,

• the issues in controlling the quality of the cells and the assurance of the quality of the

cells (Enderle et al., 2005)

The main cell types commonly used in the tissue engineering of IVD include adult

mesenchymal stem cells (MSCs), AF cells, NP cells, chondrocytes and notorchodal cells

(O'Halloran & Pandit, 2007). These cell types can be used individually or in combinations

during cell culture. For example, NP and AF cells can be used together for functional tissue

production. However, the resulting tissues will vary in characteristic properties.

The source of the cell type is another important factor in the selection of the cell type, as the

characteristic properties of these cell types vary with individual sources. For example, cells

can be obtained from a bovine, porcine, rabbit, rat, sheep or human specimens. Syngeneic

stem cell transplants can also include autologous cells. Some of the disadvantages of

allogeneic cell transplants are that the cells are capable of being rejected by the recipient,

since they are not genetically identical. By contrast, autologous cells which are cultured and

manipulated via specific procedures, will not lead to host rejection. Such autologous cells can

only be used for chronic conditions and, by implication will involve the use an adult cell

18

source. However both the use of allogeneic and autologous cells for the tissue engineering of

IVD is limited by cell availability (Seguin et al., 2004). It has been proposed that the ideal

cell type that may be used for tissue engineering and cell therapies in the future is the

‘universal donor’ cell line and sources, which have the least capability of immunogenicity

during transplant procedures (Enderle et al., 2005; Seguin et al., 2004).

By contrast, the use of adult mesenchymal stem cells has led to promising results in the tissue

engineering of IVD. These cells are obtained by puncturing of the bone marrow from which

autologous cells are obtained (Brisby et al., 2004).

Allogeneic Cells [AD]

Various studies have examined the possibility of implanting allogeneic IVD tissue grafts in

the animal models (Mochida & Nishimura, 1998; Matsuzaki et al., 1996). The potential

disease transmission and low availability of these cells proved restrictive influences on the

technique. However, latest advancements in the field of tissue engineering have rediscovered

the potential in allogeneic cell source for tissue regeneration techniques; which implies, ‘off

the shelf’’ accessibility (Pandit & O'Halloran, 2007).

Adult MSC are available in adequate amount in bone marrow and fat tissues, and are

relatively easier to isolate and proliferate in cultures (Tuli R, 2003). Allogeneic implants are

expected to initiate the immune response, but since IVD is an avascular immune-deprived

tissue, these foreign cells may go undetected. Also, compared to mature allogeneic cells,

MSC are less likely to be identified by the host immune, as they are able to skip alloantigen

detection (Lui et al., 2006; Ryan et al., 2005). In a study conducted by Nomura et al., (2001),

the allogeneic implant did not stimulate any inflammatory response. Furthermore, using

allogeneic MCS’s, excludes influence of genetic predilection or reduced potency of ageing

stem cells in the autogenic graft (Noponen-Hietala et al., 2005).In a study, allogeneic MSCs,

implanted in rabbit’s lumber disc remained viable, proliferated and actively produced ECM

for six months (Zhang YG, 2005).While initial results are supportive, possible immune

rejection over long period and disease transmission are still the restraining factor on the use

of allogeneic cell source. (Seguin et al., 2004).

1.4.2 Scaffolds[VT]

When producing a tissue-engineered product the scaffold used is important as it provides

structural support for cell seeding as well as tissue development (Chan & Leong, 2008). In

addition to providing structural support and tissue development the role also includes;

19

delivering and retaining cells and biochemical factors, facilitating diffusion, and also exerting

mechanical and biological influences to modify as well as increase cell

proliferation/expression. For these to take effect the scaffold must follow specific

requirements related to the tissue being produced. These include; porosity for nutrient

transport, biocompatibility, and biodegradation, as well the rate at which this occurs with

respect to matrix formation.

The production of a useful scaffold relies on the material used as well as the type of scaffold.

This can be related to the method used in production, and if not optimised then its

biocompatibility will be compromised. The different techniques in which scaffolds are

produced relate to the type of cells being seeded and tissue being produced. The techniques

used include; fibre bonding, solvent casting and particulate leaching, melt moulding, gas

foaming, freeze drying, electro spinning and extrusion (Lanza et al., 2000).

The intervertebral disc is a specialised structure which supports loading of the spine therefore

it is important for the scaffold used to produce IVD tissue to mirror this complexity (Kim et

al., 2007). With two tissue types in the IVD, the scaffold must be able to accommodate both

cell types in increasing the cell proliferation as well as maintaining cell viability.

1.4.2.1 Hydrogels [AD]

Hydrogels are the most commonly used scaffold material for tissue engineering applications.

Source to most of these hydrogels are naturally occurring polymers, however some limitation

to these hydrophilic molecules have lead to the use of modified or the synthetics gels (Lee &

Mooney, 2001) Most hydrogels have their mechanical and structural properties resembling a

number of tissues (Deming, 1997; Drury & Mooney, 2003).

Design Features [AD]

The type of cells involved in the study, defines the required design features for the

scaffolding gels. These features primarily include physical parameters, its mechanical and

structural integrity; it also includes more critical parameters, like adhesions and

biocompatibility. Biologically incompatible material may trigger inflammation, which may

lead to unfavourable immune response towards the implanted cells and may also damage the

neighbouring tissue (Babensee et al., 1998; Rihova, 2000). The gel is required to have spaces

to compensate for the tissue growth while providing a 3D environment for the cells to attach.

Cell response to its new environment is vastly influenced by the mechanical properties of the

scaffold they are seeded in (Huang & Ingber, 1999). Mechanical properties of the construct

20

are reliant on a number of factors i.e. type and density of cross linking, as well as the swelling

caused by internal repulsion (Mooney et al., 2000). In various tissue engineering application,

hydrogels are requires to degrade with time. Depending on the type of hydrogel and the cells

it is seeded with, either of the following factors proceeds its degradation i.e. hydrolysis,

enzymes or dissolution. It is desirable to have the rate of degradation synchronized with the

rate of ECM synthesis. Although increasing the concentration of hydrogel increases its

degradation time, it also increases its stiffness. However, introduction of defects in the

crosslinking increased the degeneration time with changing its stiffness. Below are some of

the hydrogel used in the tissue engineering of IVD.

Collagen [AD]

Collagen is the naturally occurring scaffold material in almost all the mammalian tissues. It is

biologically compatible and provides structural stability against tension. However, processed

collagen gels have limited physical properties and in most cases are chemically treated with

glutaraldehyde or diphenylphosphorylazide to improve its mechanical characteristics

(Herbage et al., 1999). These treatments can be very costly and may still not ensure the

homogeneity and consistency among the batches (Pulapura & Kohn, 1992). However,

collagens unique composition provides many useful biological features. The amino acids in

the collagen gel can be digested by collagenase produced by the seeded cells. Cells from

many tissue types are acquaintance with the collagen fibre and hence offer ready attachment

to the scaffold. These biological features can be controlled by the introduction of other GAG

molecules, such as fibronectin, chondroitin sulphate or hyaluronic acid. (Srivastava et al.,

1990)

In a study carried out by Alini and colleagues (2003), the nucleus and annulus cells were

found viable for the duration of about 2 months in a collagen-based scaffold, with the calf

fetal serum and various growth factors provided through the culture media. Apart from cell

survival and proliferation, ECM production was also examined, as this is a vital aspect of disc

repair mechanism. It was also found that the matrix produced by annulus cells was

comparable to nucleus pulposus in many ways, implying that annulus cells alone perhaps can

be use used for disc regeneration. Gruber and research team (2004) also looked at the

performance of collagen sponge and hydrogel as a prospective scaffold for IVD tissue

engineering

21

Agarose [AD]

Agarose is a polysaccharide, extracted from the cell walls of agarophyte red algae. Agarose

has a double helical structure, with multiple aggragates on the edges. In contrast to alginate,

agarose is a thermally alterable hydrogel (Lee & Mooney, 2001). The mechanical strength of

agarose gels can be improved by increasing its concentration, which is complemented by

smaller pore size. The opposite is true for the lower concentration constructs. High

concentration gels may provide better structural stiffness but larger pore size may facilitate

cell proliferation and migration (Dillon et al., 1998). These properties brand agarose

appropriate candidate for the IVD tissue engineering. Gruber (1997) and Gokorsch (2004)

used agarose constructs for their studies on IVD tissue engineering.

1.4.2.2 Bi-Phasic scaffolds [VT]

The main objective of a bi-phasic scaffold is to provide the appropriate environments in

which two different tissue types, which have different ECM’s, can be grown in a synergistic

manner. As is the case in IVD both the NP and AF have different ECM therefore the scaffold

types if producing them separately would be different. When using a bi-phasic scaffold both

can be incorporated into one so that both can be grown together and an IVD can be produced.

In theory a bi-phasic scaffold can be said to be two different scaffold types joined together to

make one, which would mimic the tissue being replaced.

A further consideration when tissue engineering an IVD, is that one of its components, is

made up of two separate parts, namely the outer AF and the inner AF, as illustrated in Figure

1-7. Accordingly a bi-phasic construct would be beneficial in constructing the AF

component. (Wan et al., 2008).

Figure 0-7 IVD tissue structure (Wan et al., 2008)

22

A recent study has reported the use of a bi-phasic scaffold to produce a tissue engineered IVD

(Nesti et al., 2008). It consisted of an electrospun, nanofibrous scaffold to mimic the AF

(outer region), which enveloped a hyaluronic acid hydrogel core, which was designed to

mimic the NP. This scaffold would provide structural, biochemical as well as mechanical

functions similar to that of the native ECM, as well as providing a compatible environment

for the seeded cells. The study utilised multipotent adult human mesenchymel stem cells.

The steps, which were carried out to produce the scaffold, are outlined in Figure 1-9. Initially

electro spinning was used to form a nanofibrous scaffold that would enable the cells seeded

to produce an ECM similar to that of AF tissue and permit tissue growth. The seeding of cells

involved a 2-step process, involving seeding cells onto one side of the scaffold and

incubating them at 37oC for 2 hours to enable them to diffuse into the scaffold. This process

in then repeated the other side. Simultaneously during the 4 hour incubation period serum-

containing culture medium is perfused into the scaffold every 30mins to prevent dissection of

the constructs (Nesti et al., 2008). The final step of producing an IVD scaffold involved the

formation of the NP tissue. This was achieved by injecting a mixture of hyaluronic acid and

MSC’s into the pre-prepared scaffold for AF. This would produce a scaffold, which mimics

the IVD, as it is a nanofibrous scaffold, which encapsulates a hyaluronic acid hydrogel

similar to that of AF surrounding NP in IVD.

Figure 0-8: Schematic diagram describing HANFS construction. After isolation and expansion, MSCs are

loaded onto the nanofibrous scaffold NFS (A) to ensure uniform cell distribution (B). A 250 mL aliquot of

23

hyaluronic acid (HA)/MSC slurry is injected into the centre of the NFS (C) to create the NP and tension

the NFS layer to approximate the AF (D).

As described bi-phasic scaffolds can also be used to created AF tissue as in AF itself there is

an inner AF and outer AF (Figure 1-8). A study carried out by Wan and collegues (2008) has

developed this type of scaffold for AF tissue regeneration. This consisted of an outer scaffold

of ring-shaped demineralised bone matrix gelatine extracted from cortical bone; designed to

mimic the type I collagen structure of the outer AF. The inner scaffold consisted of PPCLM

orientated in concentric sheets and seeded with chondrocytes to recapitulate the inner AF,

which is made up of type II collagen and proteoglycans.

The importance of the interactions between cell type and scaffold are critical. These are

summarised for a number of reported combinations in Table 1-3.

Cell Source Method (cell density, Scaffolds Outcome (Advantages) Outcome

(Disadvantages)

Reference

MSCs Adult rat bone

marrow (allogenic

cells)

• Cell density used:

1x107cells/ml

• constructs

inserted into

healthy rat disc

models

15% hyaluronan

scaffold • Resulting tissue was similar

to native NPafter 28 days.

• Decrease in

number of

cells is due to

the

hyaluronansca

ffold being

cytotoxic and

the ejection of

cells from the

disc.

Crevenste

n

MSCs Rabbits (autologous

cells) • Cell density used:

1x106 cells/ml.

• Constructs

inserted into

degenerative

rabbit disc

models.

0.3% type II

atellocollagen • Cell viability maintained post

implantation.

• Signficant PG synthesis was

achieved.

• Degeneration is decelerated.

• Positive effects

after 8 whole

weeks.

Sakai

Notoch

odal

and NP

Cells

Six specimens of 8

month old IVDs • Cell density used:

5x106 cells/ml.

• Tissue culture

rotator was used.

Poly-D, L-lactic

(PDLA) beads,

Demineralised

bone matrix

(DBM),

Gelatinmicrocarrier

s.

• Cells were cultured for 4

weeks.

• Resulting cells had

fibroblast-like morphology

with DBM and gelatine

scaffolds.

• On DBM surface, cells have

a decreased and increased

gene expression for collagen

types II and I respectively,

compared to gelatine

scaffold.

• Lack of cell

adhesion to

PDLA.

Brown

AF

cells

Thompson grade III,

IV and II human

IVDs.

• Cells seeded onto

scaffolds in vitro.

Two types of

collagen sponges,

two types of

collagen gels,

fibrin gels, alginate

and agarose were

• 10 days of culture.

• Collagen Sponges: High

level cell adhesion with

sponges and gene

expressions for aggrecan,

collagen types II and I,

• Collagen Gels: No

considerable

gene

expression and

ECM

Gruber

25

Table 0-3: studies carried out different cell types and scaffolds

used. because of high porosity

level of scaffold and hence

nutrient diffusion.

• Collagen Gels: Cell

proliferation occurred.

production.

• Agarose,

alginate and

fibrin gels: low cell

properties.

NP

cells

Six specimens of

bovine caudal IVD

which are nine

months old.

• Cell density used:

160000 cells/mm.

• Cells were seeded

in vitro on the

upper surface of

the porous

substrate.

Bone substitute

material (calcium

polyphosphate)

with thermoplastic

tubing (Tygon

tubing)

surrounding it.

• After 6 weeks, formation of

1.8mm of continuous tissue.

• Compressive properties of

tissue, increased cell density,

decreased collagen and PG

content are similar to that of

native NP tissue.

Seguin

AF and

NP

cells

2 specimens of 3 year

old bovine steers • At 2x10

6 cells/ml

cells were seeded

onto the scaffolds

in vitro.

A 9:1 ratio of a

composite scaffold

comprising of type

I collagen and

hyaluronan

respectively.

• Very little difference in the

capacity of the AF and NP to

produce PGs, aggrecan and

fibrillar collagen.

• The scaffold

retained the

PGs produced

by the cells

and these PGs

were not

uniformly

distributed.

Alini

IVD

cells

The NP of porcine

IVDs with a high

population of

notochordal cells.

At 1x106cells/ml cells

were seeded onto the

scaffolds in vitro.

Alginate beads • As the culture time increased

there was an increased gene

expression for collagen types

II and I.

• Phenotype of cells remained

constant.

• Culturing was

done for 16

weeks.

• Matrix was not

mechanically

functional.

In addition to what has already been mentioned in section 1.4.2, three predominantly required

functions of a scaffold for IVD are to: provide a three dimensional environment, Nutrient

supply (diffusion and convection) and adequate space for cell proliferation and ECM

synthesis (Langer & Vacanti, 1993) The properties like porosity, pore size, stiffness and

biocompatibility are critical to the scaffold’s performance specific to IVD (Hollister, 2005).

These properties are required to be quantified in order to produce premium scaffolds.

Quantification of these parameters is conducted by experimental data analysis and

computational models (CM) (MacArthur et al., 2004). The importance and part played by

CMs are further discussed in a later section 3.11

A Critical situation often encountered in tissue engineered IVD constructs / tissues is the non-

homogeneous dissemination of cells and ECM (Freed & Vunjak-Novakovic, 1998). The

periphery of the scaffold is often found to have higher cell proliferation and ECM formation

in contrast to the center of scaffold. It is believed that this inconsistent cell/ECM distribution

is a result of anisotropic nutrient penetration. As the primary mode of nutrient transport to

inner cells is diffusion through micro-pores, the supply to inner cells gets increasingly

restricted as the tissue development proceeds. The restriction is a consequent effect of newly

formed tissue cells obstructing the passed to inner cells and utilizing most of the nutrients in

the periphery. The reduced cell number in the center of the scaffold can also be related to the

restriction to waste removal. This problem is severe in hydrogels as the pore size is already

small for large molecules to migrate into the construct. (Pluen et al., 1999)

Depending on the type of scaffold and the cells involved, these factors can be counter acted

by changing the biochemical and biomechanical conditions using a bioreactor. (Bursac et al.,

1996). As the amount ECM is accountable for the mechanical properties of the scaffold, such

non-homogeneous placement of cells and ECM may result in the insufficient structural

stability, producing tissue with undesirable properties (Vunjak-Novakovic et al., 1999). This

makes the use of bioreactor in tissue growth critically vital.

A number of studies have been conducted to investigate the mechanism of nutrient transport.

Constantly reviving nutrient media, use of excessive nutrients or forced perfusion showed

positive effects in the nutrition delivery to the cells at the centre of the construct (Pazzano et

al., 2000; Bancroft et al., 2002). Some bioreactors are specifically designed to enhance

nutrient transport into the construct. They provide forced perfusion and continually media

refreshment (increase diffusion gradient). Bose Biodynamic is one such bioreactor.

27

1.4.3 Mechanobiology[DB]

The mechanobiology of intervertebral disc cells has been an area of significant interest

(Hsieh & Twomey, 2010). Mechanobiology is the research area that studies how cells

respond to different mechanical forces and it also looks at the mechanotransduction

mechanisms which convert these forces into molecular events.

It is known that the cells in every tissue are able to alter their metabolic activity in response to

applied load. Both the level of the applied strain and the dynamic frequency are known to be

important in obtaining a specific response. Figure 1-10 shows the influence of loading in the

biosynthesis activities of chondrocytes. Two fundamental model systems have been adopted

to observe the response of IVD cells, each with their specific advantages and disadvantages.

In one case, IVD explants are used to recreate the in vivo ECM environment for the cells.

Alternatively, 3D model systems are used incorporating isolated IVD cells.

Figure 0-9: Schematic of the influence of loading on biosynthesis activities of chondrocytes (Stoltz,

Dumas, Wang, Payan, Mainard, & Maurice, 2000)

Mechanical loading of the intervertebral disc will result in a complex set of physical changes

that may be transduced as mechanical stimuli to the cells (Setton & Chen, 2006). Researchers

have been extensively studying the response of IVD cells to a variety of physical stimuli that

includes fluid flow, electrokinetic effects, deformation in tension, compression, shear stress

as well as hydrostatic and osmotic pressure. There has been significant effects observed and

these differences in response has shown that cells from different regions will respond

differently under various conditions. (Setton & Chen, 2004; Walsh & Lotz, 2004).

28

1.4.3.1 Physicochemical Conditioning

The physicochemical conditioning of cells is performed by the use of growth factors and is

also influenced by changes in the osmolarity. Several studies performed to understand the

effect of these two factors are now introduced.

Osmolarity [HO]

The intervertebral disc is an osmotic system, its hydration changes with the application of

mechanical loads. A change in hydration of the intervertebral disc during loading alters the

extracellular osmolarity (Wuertz et al., 2007). This, in turn, will lead to changes in tissue

hydration modifies the concentration of the extracellular matrix and the pH within and around

the cells (Chen, et al., 2002 ).

Accordingly, the cells of the intervertebral disc have to adapt to these osmolarity changes in

conjunction with changes in the biosynthesis of the proteoglycans. This is due to the change

in fluid diluting or concentrating the aggrecan, which is comprised of proteoglycans and is a

major constituent of the intervertebral disc cell matrix (Ishihara, et al., 1997).

The response of intervertebral disc cells to altered osmolarity or the role of osmolarity on the

cells developed in vitro has been largely ignored (Takeno et al., 2007). Of the few related

studies, Ishihara and colleagues (1997) examined nucleus pulposus cells taken from bovine

coccygeal intervertebral discs (18-24 year olds), which were cultured in 430mOsm and

28mOsm osmolarity. The study suggested that the high solute concentration caused a

decrease in the cell synthesis rates. A study further investigated the effects of medium

osmolarity of 300, 400 and 500mOsm on the gene expression of nucleus pulposus and

annulus fibrosus cells taken from both human IVDs and 18-24 month old bovine tails

(Wuertz, et al., 2007). Mechanical conditioning of hydrostatic pressure and cyclic strain

(0.25MPa and 4% strain respectively) were also applied to these cell cultures. The study

suggested that the osmotic environment influenced the level of expression and partly

determined whether mRNA expression was stimulated or inhibited with the application of

pressure. As an example a decrease in the osmolarity may result in a significant reduction of

both aggrecan and collagen II expression. This in turn may reduce matrix synthesis and

promote disc degeneration.

In a related study (Chen et al., 2002), the responses of cells from different zones of the IVD

were examined with respect to changes in the osmolairty. IVD cells were harvested from 4-5

29

month old pigs and culturing under iso-osmotic (293mOsm/kg H2O), hypo-osmotic

(250mOsm/ kg H2O) and hyper-osmotic (450mOsm H2O) solutions. The study reported that

under hypo-osmotic conditions, the gene expressions for aggrecan and type II collagen were

up-regulated in the cells from the transition zone(TZ) although a similar response was not

observed for the NP cells. Nucleus pulposus cells under hypo-osmotic and hyper-osmotic

media conditions exhibited no change in gene expression for aggrecan, type I and II collagen.

Under hyper-osmotic conditions, NP and TZ cells exhibited respective decreases and

increases in gene expression for the small proteoglycans, biglycan, and decorin. The results

of this study, as summarised in Table 1-4 suggest that osmotic pressure regulates the

intervertebral disc cell synthesis of matrix molecules, including proteoglycans and collagen,

at the transcriptional level.

Table 0-4: A summary of a study investigating differences in the relative mrna levels in the intervertebral

disc cells subjected to an altered osmolarity. (Based on Chen et al., 2007)

The effect of different osmolarities (270, 370, 470 and 570mOsm) on the cell viability and

GAG production of the nucleus pulposus cells from bovine cells was also studied (Takeno et

al., 2007). Their results indicated that the cell viability was not affected by changes in

osmolarity and the cell viability was 90% for all after 6 days of culture. The largest GAG

production was observed in the group under osmolarity of 370mOsm and lowest was

observed in the group with osmolarity of 270mOsm. The cells cultured at 370 and 470 mOsm

were thus more active and accumulated significantly more GAG than cells cultured at 270

and 570 mOsm.

30

Growth Factors [SP]

Growth factors bind to cell membranes via specific transmembrane receptors, resulting in the

activation of an intercellular signalling cascade. Growth factors exert biological effects, such

as stimulation of cell proliferation, differentiation, migration, and apoptosis. They also

regulate matrix production and repair by various types of cells (e.g., chondrocytes, skin

fibroblasts, and endothelial cells) (Masuda et al., 2004). Growth factors have been studied

extensively in articular cartilage, and have been shown to regulate matrix metabolism and cell

proliferation. Clinical applications of these techniques include the injection of growth factors,

scaffolds, and cell transplantation for the repair of articular cartilage defects. Because of the

similarity of the phenotype of articular cartilage and disc cells, the success of these studies

has led to the evolution of research in disc regeneration.

In the intervertebral disc (IVD) although there is little vascularity, tissue fluids can deliver

growth factors through the endplate by an endocrine mechanism. In the outer layer of the

annulus, neovascularization after injury or degeneration can induce an influx of growth

factors. In addition, the autocrine and paracrine production of growth factors is considered to

be major regulatory mechanisms in IVD tissues (Masuda et al., 2004). In the last decade, new

tissue culture techniques, especially three-dimensional culture methods, including agarose,

alginate, and pellet cultures, were developed or adapted to study the effect of growth factors

on the metabolism of IVD cells (Gruber et al. 1997; Masuda et al. 2004).

Many growth factors have been found to be present in the IVD; these include insulin-like

growth factor (IGF)-1, bFGF, bone morphogenetic protein (BMP)-2, BMP-4, growth

differentiation factor (GDF)-5, platelet-derived growth factor (PDGF), and TGFβ (Masuda et

al., 2004). The following table (Table 1-5) summarises the in-vitro studies of the effects of

growth factors on IVD tissue. The table vividly explains the effects of each growth factor in

terms of cell proliferation, proteoglycan synthesis, Glycosaminoglycan production and cell

viability.

Growth factors obtained from commercial sources have different relative activities and levels

of purity. Also, each growth factor has a unique optimal concentration and duration of

activity. Some growth factors can induce a significant enhancement of biologic activity by

being exposed to the cells for a longer period of time. The simple comparison of activity

based on concentration might not be adequate to address these issues. Different culture

31

conditions (monolayer culture or three dimensional culture) and different sources of cells or

explants (species, age, and location) can affect the results. Different results can also be

obtained depending on the presence or absence of serum or other supplements in the culture

medium. Thus, results should be properly interpreted based on the scope of the study.

Table 0-5: In-vitro studies of the effects of growth factors (reproduced from (Masuda, Oegema, & An,

2004)

The IVD, which is composed of the nuclear pulposus and annular fibrosus, shows a gradual

transition in cell population and phenotype. The differences in cell phenotype within the IVD

tissue and the difficulties in maintaining those phenotypes in vitro have made a detailed

investigation of growth factors difficult.

1.4.3.2 Mechanical Conditioning [HO]

Biophysically stimulating cells in a mechanical environment can have a major impact on the

regeneration of the IVD cells by having a direct influence on the cell proliferation and

metabolic activity of the cells (Zeiter et al., 2005).

32

It is essential to have mechanical loading for intervertebral disc cell matrix homeostasis and

anabolism. The mechanical loading, which the intervertebral disc is subjected to, varies

across the disc. The IVD influences complex physical stimuli such as compression, tensile,

shear stress, hydrostatic pressure, osmotic pressure and fluid flow. The outer annulus fibrosis

is subjected to tensile strains due to the flexion, extension and torsion of the disc whilst the

inner annulus and nucleus influences hydrostatic pressure. The various physical stimuli have

an impact on the matrix synthesis and turnover of the disc. For this reason many research

groups have performed investigations to examine the biological responses of the cells in the

intervertebral disc to various types of loading at a range of frequencies (Neidlinger-Wilke et

al., 2005). Indeed Gilbert and colleagues (2010) suggest that when the frequency of loading is

below normal physiological frequencies, the loading could lead to the degeneration of the

matrix of the IVD.

Physical forces in vivo are the factors that in extreme cases may result in intervertebral disc

degeneration. However the molecular mechanisms associated with this are not fully

understood. (Rannou et al., 2003)

Compression [DB]

Studies have looked at the effect of different loading regimes such as compression, tension

and hydrostatic pressure. It has shown that compression, which is a more physiological load,

causes greater disc shrinkage than any other type of loading. The duration of loading

enhances this effect and hence younger discs have greater height. Therefore compressive

loading and maturation has a vital role in disc degeneration. (Maclean et al., 2004; Wang et

al., 2007 ; Walsh & Lotz, 2004 ; Korecki et al., 2009)

33

Figure 0-10: shows the axial compressive loading of IVD. Compression of the disc induce shearing and

tensile stresses as well as radial expansion that results in compression in both axial and radial directions

(Setton & Chen, 2006)

Variation in the cell morphology of IVD causes the cells from different areas to respond

differently. The NP cells at high frequency compressive loading have been shown to

experience high hydrostatic pressures, but minimal change in volume (Setton & Chen, 2006).

Short-term compression at moderate magnitude has resulted in an increased synthesis of

proteoglycan and collagen in NP and inner AF cells, whereas the outer AF region exhibited

an increase in PG synthesis alone (Ohshima et al., 1995).

The response of motion segments and isolated cells in constructs is frequency dependant

(Walsh & Lotz, 2004; MacArthur et al.,2004; Sah et al. 1989; Korecki et al. 2008; Korecki et

al. 2009). At lower frequency the cells experience a higher matrix deformation and at higher

frequency they experience a lower fluid pressurisation. Therefore, at higher magnitudes and

lower frequencies of dynamic loading there is elevated cell death. Lower magnitude of either

compressive or tensile load has been shown to be beneficial when applied for a short period

of time (Ishihara et al., 1996; Ohshima et al., 1995). Individual studies will be discussed in

more detail in the following sections.

Static compression applied to motion segments in vivo has been associated with premature

onset of intervertebral disc degeneration. In vitro culture of IVD provides a means to study

mechanisms that regulate biological responses to IVD mechanical loading under well-

controlled conditions (Boyd et al., 2006). Waltz and Lotz showed that static loading has

catabolic role on the disc. Static compression for a prolonged period applied in large

magnitude in vivo cause detrimental effects in matrix composition such as cell morbidity,

apoptosis, decrease in the gene expression for aggrecan and collagen as well as an elevated

gene expression for protease primarily in the NP cells (Lotz et al., 2002; Lotz et al., 1998).

They have also shown that dynamic loading at lower magnitude can benefit the synthetic

activity of IVD cells, exemplified by a positive anabolic response. It increases the

proteoglycan and collagen synthesis, elevated gene expression for both types of collagen

(Ohshima et al., 1995; Setton & Chen, 2004). These responses are similar for both NP and

inner AF cells, the outer AF cells, although the latter cells are less responsive to these stimuli.

The change in frequencies of compressive loads applied to inner IVD cells have shown to

have an effect on the gene expression. At lower frequency, there is an increase in the

34

expression of genes encoding types I and II collagen and aggrecan. On the other hand, a

higher frequency has shown to increase the expression of genes encoding the mRNA for

proteases such as ADAMTS-4, MMP-3 and MMP-13 (Walsh & Lotz, 2004; Maclean et al.,

2004).

Research involving disc regeneration has examined the application of static and dynamic

compression on motion segments as well as intervertebral disc cells. Research is mostly

undertaken by loading a rodent model incorporating coccygeal discs, and its relevance to

human IVD cells or explants must be questioned. Therefore, any investigated data should be

repeated on human cells or explants to learn the exact response and not just the trend or

pattern of effects.

The research has revealed that static and dynamic compressions can induce a different

biological response in IVD explants. The study carried out by Wang et al (2007) and a few

others on intervertebral disc under compression is summarised in Table 1-6. Wang et al

showed that there is a catabolic response during static compression and an anabolic response

during dynamic loading. They studied the effect of static and dynamic loading in rabbit IVD

in vitro under unconfined uniaxial compression. Static unconfined uniaxial compression

suppressed the gene expression for collagens and aggrecan in the disc, whereas dynamic

loading showed significant anabolic change with increase in gene expression for type I and II

collagen and aggrecan. (Wang et al., 2007)

Figure 0-11: Schematic diagram the in vitro compressive loading of an IVD explants. (Wang, Jiang,

& Dai, 2007).

SOURCE SAMPLE CONDITIONS RESULT

(Ariga et al., 2003)

Intact IVD mouse

coccygeal

Static compressive stresses

(0, 0.2,0.4, 0.8, and 1.0 MPa)

for 24 hours.

-Loaded discs became bulged, and the disc space became narrow.

-Apoptosis was absent in discs without load, but noticeable in loaded discs

at 1.0 MPa.

-Apoptotic cells increased depending on stress magnitude.

-The two MAPK inhibitors increased the number of apoptotic cells.

(Boyd et al., 2006) Rat tail motion

segments in vivo

Uniaxial compressive loads

(0.1 MPa) for 24hrs

- 23% decrease indisc height.

- For the nucleus, mRNA increased for ADAMTS-4 (4.8-fold) and type I

collagen (2.6-fold), while aggrecan mRNA increased in the annulus only (2-

fold).

- Decreased MMP-3 mRNA (-1.6-fold). mRNA for type II collagen and

collagenase (MMP-13) were stable in all tissues.

(Ching et al., 2003) Rat-tail model in vivo. Static compression and cyclic

loading of 0.5, 1.5, or 2.5 Hz.

Loading was applied for 1 h

each day.

-Changes in the IVD height depended on the frequency of loading

-Decrease in disc height in the static compression group significantly greater

than that in all other groups

-Decrease in the 1.5 Hz cyclic compression group was significantly smaller

than that in all other compression groups.

(Iatridis et al., 2007) Wistar rats caudal disc

in vivo

1.5 hours of loading at 1MPa

and 1Hz

-In the annulus, peak gene expression occurred after 8hrs in TIMP-1 and

TIMP-3; after 24 hrs in agg, ADAMTS-4, and MMP-3; and after 72 hrs in

Col-I, Col-II, and MMP-13.

-No change was observed in MMP-2

-In the nucleus, maximum changes in gene expression occurred after 8hrs in

Col-1 and TIMP-3; and after 24 hrs in aggrecan, TIMP-1

(Korecki et al., 2009) Cells from young (4–6

months) and mature

(18–24 months) bovine

caudal AF and NP.

Seeded into alginate

Dynamic compression for 7

days at either 0.1, 1, or 3 Hz

-DNA content larger in mature NP cell constructs (286.2 ± 65.4 ng)

-In the AF, DNA content increased in young AF cells (181.7 ± 21.5 ng).

- In the NP, expression of collagen types I and II increased significantly in

mature cells.

-In the AF collagen type I expression was significantly affected by aging

and 3 Hz loading

36

(Lee CR et al., 2004) Intact IVD, bovine tails Static compression at 5, 200N

(0.2–0.8 MPa) for 6 hrs

AF and NP: Proteoglycan synthesis compared to 50N load

decreased at 200N

(Lotz et al., 1998) Mouse tail disc in vivo Compression at 0.4, 0.8, and 1.3

MPa for 1wk

-Expression of Type II collagen suppressed at all levels of stress

-Expression of aggrecan decreased at the highest stress levels in apparent

proportion to the decreased nuclear cellularity.

-Discs loaded at the lowest stress recovered annular architecture but not

cellularity after 1 month of recuperation.

-Recovery at higher stress, displaying islands of cartilage cells in the middle

anulus at sites previously populated by fibroblasts.

(Lotz et al., 2002) Webster mice disc in

vivo

Static compression at (0.4, 0.8

and 1.3 MPa) for various

times (3 h±7 days)

-Static loading results in distortion of nucleus cell aggregates and lamellae

of the inner annulus with a concurrent down-regulation of type II collagen

expression within 6 h of loading.

-By 7 days, extensive apoptosis is apparent within the inner annulus and

nucleus.

(Ohshima H et al., 1995) Intact IVD, bovine

coccygeal (2 yrs)

Static compression at 0.5–15

(0.02–0.6 MPa) for 8 hrs

-Outer AF had no change in PG at 0.2–0.4 MPa, decreased at 0.6 MPa,

increased at 0.2–0.6 MPa.

-Inner AF had increased PG at 0.2–0.4 MPa, decreased at 0.6 MPa. NP had

increased PG at 0.2–0.6 MPa

(Quinn et al., 1998) Rat tail disc 72 hours of immobilization (n =

6), 2 hours of dynamic

compression (1 MPa/0.2 Hz) (n

= 8)

-Immobilization and dynamic compression affect anabolic and catabolic

genes, with an overall down regulation of types 1 and 2 collagen and up

regulation of aggrecanase, collagenase, and stromelysin in the annulus.

-Effects of immobilization and compression additive for collagen types 1

and 2 in the anulus, but not in the nucleus, and not for catabolic genes.

(Wang et al., 2007) Rabbit IVD explants Unconfined uniaxial

compression. Static and

dynamic load of 0.5 and 1MPa

at 0.1 and 1Hz for 6hrs

-Static compression suppressed gene expression for collagens and aggrecan

-Dynamic compression increase gene expression for type I and II collagen

and aggrecan.

-Both caused, up-regulation of IL-1_ and TNF-_ expression, and increase in

TUNEL-positive cells in

Table 0-6: studies carried out on ivd under compression

The expression of IL-1and TNF mRNA was maximal at an applied static load of 0.5 MPa.

Although all the loading conditions caused cell death, the maximum was corresponding to a

compressive load of 1MPa. This study showed that the biological response of IVD explants

varied with the frequency, magnitude and duration of loading. Elevated gene expression for

aggrecan was observed for dynamic loading, whereas static loading reduced the aggrecan

expression. There was a similar trend in the expression of Type II collagen mRNA. The

dynamic compression showed significant anabolic changes in the AF, NP and end plates of

the explants. In the AF, there was a significant decrease in the expression of type I collagen

mRNA after static loading, where as there was no significant change during dynamic loading

except at the higher frequency and magnitude. Except for the low frequency low magnitude

dynamic loading, there was an increase in the gene expression in the nucleus at both loading

conditions. (Wang et al., 2007)

Cell death was found in the AF, NP and endplates of the disc, and increased under all loading

conditions. The most significant cell death was noted in the disc loaded at static 1MPa.These

results showed similar trends to those results published by other authors using cartilage

explants and chondrocytes (Burton-Wurster et al. 1993; Gray et al. 1988; Korver et al. 1992;

Larsson et al. 1991; Palmoski & Brandt 1984; Sah et al. 1989). Matrix synthesis was

suppressed at static compression and elevated at dynamic loading. This shows that the

different signals can be generated during mechanical loading that causes the difference in

response.

Mechanical loading has a significant influence in the regulation of IVD extracellular matrix.

Evidence shows that the quality and quantity of this matrix production is altered during

ageing and degeneration. Researchers have been investigating the interaction between

loading and degeneration with respect to gene expression and biosynthesis. For example,

Korecki and colleagues (2009) recently demonstrated that ageing influence the homeostasis

and cell response under dynamic loading. In response to mechanical loading, mature cells

were seen to be less capable of retaining their matrix components than younger cells.

(Korecki et al., 2009).

In general, the 4-6 month cells showed an anabolic gene expression, while the 18-24 month

cells showed a decreased catabolic gene expression involving MMP3 in association with a

reduced GAG production. This showed that, although an increase in age may enhance

anabolic gene expression and reduce the catabolic responses, it may not affect the level of

40

GAG production. Overall in this study, the maturation factor was dominating than the

frequency of loading as the affect of loading showed more significant changes.

Tension[HO]

The biological response of intervertebral disc cells in response to tensile loading is an area

yet to be fully explored. Indeed the number of such studies is limited by the difficulty of

applying tension forces to intervertebral disc. Although the magnitude and response of tensile

loading on the IVD cells are not fully understood, tensile strains are always present in most

type of physiological loading of the intervertebral disc. (Setton & Chen, 2004).

The response of the intervertebral disc cells to tensile strain depends on various factors such

as the frequency, duration and magnitude of the loading. One factor that has an effect on the

cell proliferation and viability is the magnitude of the tensile loading applied to the cells.

Rannou et al (2003) measured how the effect of tensile stretch magnitude on the PG synthesis

in rabbit IVD cells by subjecting them to 1% and 5% stretch at a frequency of 1Hz for 24

hours. These authors concluded that there was no associated cell death induced and that the

two magnitudes did no alter the mRNA aggrecan content which suggests that these two

stretch magnitudes do not reduce the synthesis of PGs. Neidlinger-Wilke et al (2005)

however, concluded that there was no significant change in the gene expression between the

cells subjected to different cyclic tensile strain magnitudes (1-8%) after testing human IVD

cells in 3D collagen gel scaffolds. The cyclic strain was found to increase the gene expression

of the matrix-forming proteins, collagen II and aggrecan, but decrease the matrix-degrading

enzyme MMP-3 of the annulus fibrosus cells. Matsumoto et al (1999) also examined the

response of nucleus pulposus cell cultures from four-week old rabbits under cyclic

mechanical stretch of 20% at 0.05Hz for 48 hours. The study reported that the cell growth

rate increased along with the increase in collagen synthesis. Rannou et al (2000) examined

the effects of cyclic tensile stretch of AF cells from the rabbit IVD. The study reported that

continuous loading for 12 hours (20% maximum strain at 1Hz) did not alter AF cell

phenotype as the loading regime did not induce cell detachment from the substrate. The

annulus fibrosus cells continued to express collagen type II, but collagen type I was not

detected.

Studies have been performed to see the effect of frequency of tensile loading on the

biophysical response of the IVD cells in vitro. For example human IVD cells were used to

investigate the effect cyclic tension at 10% strain on the biophysical response. The cells were

41

subjected to a tensile stretch of at two different frequencies of 0.33Hz and 1.0 Hz for a period

of 20 minutes. At the higher frequency, there was a reduction in catabolism of the AF cells,

whilst at a frequency of 0.33Hz there was a reduced gene expression of type I and II collagen.

Gilbert et al (2010) also suggested that the strain rate and cycle number may also have an

effect on the mechanotransduction pathways and may cause alterations in the kinetics of

these pathways.

An in vitro study by Benallaoua and colleagues (2006) involved varying the magnitude (5%

stretch vs. unloaded) and frequency (0.0Hz, 0.05Hz, 0.1Hz, 1Hz) of tensile stretch applied to

AF cells taken from rabbit IVDs. They determined nitrite concentration at different time

intervals (8hr, 12hr, 24hr). It was concluded that CTS could participate in the regulation of

IVD matrix by decreasing PG synthesis through nitrite (NO) production. This was supported

by the results with unloaded cells, where no significant difference in the production of nitrite

throughout the experiment was reported. However nitrite concentrations within the loaded

cells were shown to decrease until 8hrs and have no significant difference from 8hr to 24hr

(See Table 1-7). Also decrease in frequency of loading resulted in a decrease in NO

production with decreasing frequency.

Table 0-7: shows the change in nitrite production for static controlled and CTS loaded AF cells with

change in duration of loading and frequency of loading. (based on Benallouoa et al., 2006)

Studies showed that tensile loads applied to rodent tail motion segments in vivo yielded a

reduction in the contents of proteoglycan and both types of collagens in the IVD (William C

Hutton et al. 2002; Court et al. 2001). Axial traction stress applied to porcine tissue explants

studied the effect of tensile loading in the synthesis of proteoglycan. It was observed that

extreme traction of spine for a prolonged period could lead to degeneration of the IVD.

42

It was clear that the rate of synthesis of PG in the outer AF was increasingly lowered

compared to that of the NP. These results suggest that a prolonged excessive axial traction

stress induces a decrease in tissue hydration in the annulus fibrosis, and this may lead to an

increase in the fractional volume of solid in the matrix and tissue osmotic pressure, resulting

in diffusion inhibition of solute and suppression of proteoglycan synthesis. (Terahata et al.

1994).

Source Sample Conditions Result

(Rannou et al., 2003)

Rabbit IVD

cells

1% and 5% stretch at

a frequency of 1Hz

for 24 hours

no cell death induced

two stretch magnitudes do not reduce the synthesis of

PGs

(Neidlinger-Wilke et al., 2005)

Human IVD

cells in 3D

collagen gel

scaffold

(annulus

fibrosus)

1%, 2%, 4% and 8%

cyclic tensile strain

increase in the gene expression of collagen II and

aggrecan

decrease in MMP-3

(Matsumoto et al., 1999)

4-week old

rabbit IVD

nucleus

pulposus cells

Cyclic mechanical

stretch of 20%

elongation at 0.05Hz

for 48 hours

cell growth rate increased along with the increase in

collagen synthesis

nucleus pulposus cells division rate increased

(Rannou et al., 2000)

AF cells from

rabbit IVD

20% stretch at a

frequency of Hz for

12 hours

no alteration to AF cell phenotype

no detachment of cells from substrate.

collagen type I expression not detected.

(Gilbert et al., 2010)

human IVD

cells

Cyclic tensile strain

(CTS) of 10% at two

different frequencies

of 0.33Hz and 1.0 Hz

for a period of 20

minutes

1.0 Hz - reduction in catabolism of the AF cells,

0.33Hz - reduced gene expression of type I and II

collagen

(Benallaoua et al., 2006 )

AF cells taken

from rabbit

IVDs

5% stretch at

frequency of 0.0Hz,

0.05Hz, 0.1Hz, 1Hz

3.8-fold increase was observed after 8 h at 5% CTS

frequency decrease to 0.1 or 0.05 Hz resulted in

decrease of the nitrite production

Cyclic tensile stretch decreases PG synthesis through

nitrite (NO) production

Table 0-8: Summary of studies mentioned earlier to understand the effects of tensile loading on IVD cells

Hydrostatic pressure [HO]

Many studies suggest that a physiological level of hydrostatic pressure has an anabolic effect

on the synthesis of ECM, whilst abnormal pressures, lower of higher than the physiological

level may result in the reduction of PG synthesis. (Kasra et al., 2003). Accordingly a number

of studies are of relivance. On e such study examined whether the direct application of

hydrostatic pressure would have an effect on the collagen and aggrecan synthesis of IVD

from male hound dogs (Hutton et al., 1999). These authors reported that a hydrostatic

pressure of 1.0MPa resulted in a decrease of collagen and PG synthesis for the AF cells by

56% and 45% respectively. The equivalent increases for NP cells were 67% and 48%

respectively. It was suggested that there was no significant difference in the cell proliferation

43

with varying magnitude and that a hydrostatic pressure of 1MPa was not the optimal pressure

for the cell metabolism of the annulus fibrosus.

An in vitro study performed by Reza and Nicoll (2007) involved outer annulus, inner annulus

and nucleus pulposus cells harvested from bovine caudal IVDs. These authors showed that a

hydrostatic pressure of 5MPa at a frequency of 0.5Hz, 4 h a day for 12 days, resulted in no

quantitative difference in synthesis of GAG and collagen between the loaded and unloaded

cells. It was reported that this loading regime increased the COL II production for outer

annulus cells, however this statement was not established for the inner annulus cells. The

mechanical stimuli were reported to have no significant effect on the content of COL I.

A similar strategy was adopted by Neidlinger-Wilkeand colleagues (2006), who employed

nucleus pulposus cells harvested from both human disc biopsies and caudal bovine (age < 24

months) discs. It was designed to determine the effect of application of 0MPa (control),

0.25MPa and 2.5MPa (with a frequency of 0.1Hz for 30 mins for a day) of hydrostatic

pressure on the gene expression and cell viability. The study reported that a hydrostatic

pressure of 0.25MPa increased aggrecan and COL I expression and decreased mRNA

expression for the humans discs whilst matrix turnover enzymes MMP1, MMP2, MMP3 and

MMP13 were not influenced. By contrast, at this low hydrostatic pressure bovine cells did

not show a significant change.in addition at the higher hydrostatic pressure of 2.5MPa, there

was an increase in the expression of matrix turnover enzymes for both cell types. The cell

viability was reduced by less than 5% for both stimulated and control cells.

Another factor, which affects the response of cells to mechanical stimuli, is the duration of

the loading. A study performed by Kasraand colleagues (2003) reported that for aduration of

9 days in culture, annulus fibrosus cells in 3-D culture reduced their protein synthesis with an

associated stimulation in their degradation. The study also highlighted the effect of 3

dayculture on both a monolayer of nucleus pulposus cells and a 3-D annulus fibrosus

construct. Results indicated that physiological amplitude of loading protein synthesis was

stimulated in both culture techniques.The findings indicate that short-term application of high

loading amplitudes and frequencies is likely to be beneficial to the disc by stimulating protein

synthesis and reducing protein degradation.

In situ, the intervertebral disc is subjected to dynamic loading, which has a variable amplitude

and frequency. For example Kasra and colleagues (2006) tested porcine IVD cells (10 months

old) to see what effect the frequency has on the content of DNA in the annulus and nucleus

44

cells by applying hydrostatic pressure of 1MPa magnitude with cyclic frequencies of 1, 3, 5,

8 and 10 Hz to the constructs for 3 days. The study stated that with all disc regions there was

a significant decrease in the DNA content at different loading frequencies. The study also

reported that there is a frequency range from 3 Hz to 8 Hz that disrupts the cell function. The

frequency is equivalent to the resonant frequency of the spine.

An in vitro study on rabbit IVD cells showed that high amplitude and frequency of

hydrostatic pressure on 3-D nucleus cells increased protein synthesis and decreased protein

breakdown (Kasra at al., 2003).

The normal hydrostatic pressures existing in the healthy IVD at rest and during physiological

loading will necessarily decrease due to ageing and degeneration of the spine. Hydrostatic

pressure over or below the normal 3MPa has shown to inhibit the synthesis of proteoglycan

as well as increasing the production of nitric oxide and MMP-3 (Liu, et al., 2001)

Finally, there is yet no consensus on the most physiologically relevant loading regime of

hydrostatic pressure on IVD cells’ although many studies have been performed. (Reza &

Nicoll, 2008)

Source Sample Conditions Result

(Hutton, et al., 1999)

IVD from

male hound

dogs

pressure of 1.0MPa AF: decrease of collagen synthesis by 56% and PG

synthesis by 45%

NP cells an increase of collagen synthesis by 67%

and proteoglycan synthesis by 48%.

(Reza & Nicoll, 2008)

AF and NP

from bovine

caudal

IVDs

pressure of 5MPa at

a frequency of

0.5Hz, 4 h a day for

12 days

No quantitative difference in synthesis of GAG and

collagen between the loaded and unloaded cells

(Neidlinger-Wilke , et al., 2006)

NP cells

from

human and

caudal

bovine (<

24 months)

pressure of 0MPa

(control), 0.25MPa

and 2.5MPa (with a

frequency of 0.1Hz

for 30 mins for a

day)

0.25MPa:

• Human cells - increase in aggrecan and COL

I expression. Decrease in mRNA expression.

No change : MMP1, MMP2, MMP3 and

MMP13

• Bovine cells – no change

2.5MPa:

• increase in the expression of matrix turnover

enzymes for both cell types.

Cell viability reduced by less than 5% for both

stimulated and control cells.

(Kasra, Goel, Martin, Wang, Choi, &

AF cells

from

Rabbit

IVDs in 3-

Magnitude of 0.75,

1.5 and 3.0MPa.

Frequency of 1, 10

Examination of the released to total collagen ratio

indicated no significant differences between the

control group and the three groups with 0.75 MPa

amplitudes. There was a signifi- cant reduction in the

45

Buckwalter, 2003)

D culture and 20Hz ratio for the 1.5 MPaamplitude loading conditions,

but no significant differences at this load level across

the three frequency values. There was a further drop

in the ratio for the 3.0 MPa amplitude loading

conditions, and the cultures demonstrated monotonic

decreases with increased frequency

(Kasra, Merryman, Loveless, Goel, Martin, & Buckwalter, 2006)

porcine

IVD cells

(10 months

old)

pressure of 1MPa

magnitude with

cyclic frequencies

of 1, 3, 5, 8 and 10

Hz for 3 days

All disc regions showed significant decrease in the

DNA content at different loading frequencies.

Frequency range from 3 Hz to 8 Hz disrupts the cell

function

Table 0-9: summary of the studies carried out on IVD under hydrostatic pressure

1.4.4 Bioreactors for tissue engineering [S.P]

Bioreactor systems play an important role in tissue engineering, as they enable reproducible

and controlled changes in specific environmental factors. They can provide technical means

to perform controlled studies aimed at understanding specific biological, chemical or physical

effects. Furthermore, bioreactors allow for a safe and reproducible production of tissue

constructs. For later clinical applications, the bioreactor system should be an advantageous

method in terms of low contamination risk, ease of handling and scalability. To date the goals

and expectations of bioreactor development have been fulfilled only to some extent, as

bioreactor design in tissue engineering is very complex and still at an early stage of

development. Considering the current techniques in cell culture, the stimulation of cellular

proliferation and the formation of tissues are widely performed in academic and industrial

research laboratories. However, the formation of cohesive, organized, and functional tissues

by three-dimensional (3D) cell culture is complex. A suitable environment is required, which

is achieved and maintained in a specific bioreactor, a device that reproduces the physiological

environment (including biochemical and mechanical functions) specific to the tissue that is to

be regenerated. Bioreactors can also be used to apply mechanical constraints during

maturation of the regenerating tissue for studying and understanding the mechanical factors

influencing tissue regeneration. Figure 1-12, interprets the role of bioreactors in tissue

engineering.

46

Figure 0-12: A typical paradigm of tissue engineering. Cells, when inoculated in biocompatible scaffolds,

exposed to appropriate signaling molecules, and incubated in an environment that simulates physiologic

conditions, can organize into tissue-like structures in vitro over time

1.5 Bioreactor systems

The design of a bioreactor is a complex task as an understanding of both engineering and

biological backgrounds are required in order to develop such a mechanically controlled

environment for the growth of tissue. A number of criteria establish a blueprint for the design

of a bioreactor. The criteria may change for various tissue types under development, but in

general, a bioreactor must be designed to meet the following requirements:

• control the physiochemical environment

• facilitate monitoring of cell/tissue quality

• ensure the culture of tissue samples occurs under sterile conditions

• establish a substantial level of cellular distribution and attachment to developing

scaffolds

• ensure tissues have sufficient nutrient and waste exchange with their surroundings

(i.e. provide efficient mass transfer to the tissue)

• expose the developing tissue to mechanical forces such as compression and tension,

as well as hydrodynamic forces such as shear stress and pressure

• maintain a high degree of reproducibility

• control the flow of media whether it is steady or pulsatile

• reduce excessive turbulence in the fluid flow

• provide a low volume capacity

• make effective use of growth factors and medium components

47

• ensure that the materials from which the bioreactor is fabricated are compatible with

cells/tissues

• be easy to clean and maintain

• enable the user to easily fix the seeded scaffold in place

• ensure the culture of tissue samples under physiological conditions

• be compact in size to fit in a standard size incubator

• avoid the accumulation of metabolites

The design and functional requirements of the tissue to be engineered determine the specific

design requirements in the bioreactor. In the design of a bioreactor, both the biomechanical

and biochemical controls are essential in the creation of a simulated physiological

environment for cell and tissue growth. Pulsatile forces, pressure, flow rate, compression,

tension, shear stress, frequency, are extremely important design considerations. The

biochemical environment is equally important, with the transfer of nutrients and the removal

of waste products essential aspects of cellular proliferation and healthy tissue development.

1.5.1 Bioreactors and mechanical stimulation

A generalized representation of possible mechanical stimulation systems used in bioreactors

is provided in Figure 1-13. For the engineering of load-bearing tissues, tensile and

compressive loadings are the first regimes that come in mind. Compressive loading can be

further categorized. Uniaxial compression is probably the most frequently used stimulation

method for cartilage tissue engineering. In confined uniaxial compression (Fig. 1-13B), the

tissue is compressed in one direction, while deformation of the tissue in any other direction is

prohibited. This occurs when a sample perfectly fits a container, while a piston at one side

compresses it. In unconfined compression (Fig. 1-13A), the tissue is allowed to expand freely

in the direction perpendicular to the axis of compression. In both compression types,

particularly in a confined setup, the porosity of the container, the supporting plate and/or the

piston are important boundary conditions for the applied mechanical loading. Time-

dependent tissue deformation and fluid pressurization depend on fluid expression from the

tissue during compression and on rehydration of the tissue upon relaxation. In addition to

uniaxial compression, it is possible to compress a tissue from all sides at the same time, for

instance by imposing a hydrostatic pressure via the surrounding fluid (Fig. 1-13C). Finally,

shear loading of cells and tissues has been shown to stimulate cells and to induce formation

of particular tissue types. Grabbing two sides of a construct and sliding the grips in opposite

directions in parallel planes can apply shear. A specific type of such shear loading is applied

48

by rotating the grips. Rather than by grips, however, shear loading on cells in culture is more

frequently induced by forced fluid flow. Drag forces due to the flow of culture medium over

the sample or the cells are known to stimulate cell metabolism.

Figure 0-13: Schematic drawings illustrating the main loading principles used in cartilage bioreactor

systems: unconfined compression (A), confined compression (B), hydrostatic pressure (C) and forced

perfusion (D). Black: bioreactor walls; dark-gray: tissue engineering construct; light-gray: culture

medium; striped: porous (or non-porous) compression and supporting plates. Mechanical force (F), air

pressure or forced fluid flow directions are indicated with arrows (reproduced from van Donkelaar &

Schulz, 2008).

The primary reason for inducing fluid flow in bioreactors for tissue engineering, is often not

mechanically stimulate the cells, but rather to enhance transport of nutrients and other

metabolites.

Limited nutrition in the centre of cell-seeded construct explains the low viability and matrix

synthesis in the centre and enhanced proliferation and matrix synthesis along the edges

(Vunjak-Novakovic et al., 1999). In bioreactors that mix the medium around constructs, for

instance in the well known rotating wall vessel (RWV) bioreactors, nutrient supply is

enhanced and waste-product accumulation is prevented in the deeper areas of the construct.

49

This results in more homogenous tissue distributions. To further enhance nutrient delivery to

cells in the centre of constructs, bioreactors have been designed by which perfusion is forced

through the constructs (Fig. 1-13D). This forced perfusion not only delivers nutrients, but

induces shear forces to the seeded cells as well. Although, the effect of the individual

contribution of these effects is hard to distinguish, both are likely to contribute to the

development of tissue-engineered tissue.

In addition to the method of loading, it is evident that the loading regime, i.e. the frequency

and magnitude, and whether the load is continuously or intermittently applied, is of utmost

importance for the development of the tissue (Chowdhury et al., 2003). The loading

experienced by the cells further depends on the properties of the scaffold material. Many

studies have shown that scaffold stiffness and texture affect cell behaviour immediately, and

that properties such as scaffold porosity and permeability are important in tissue engineering.

The latter has special relevance in relation with forced perfusion and nutrition (Martin et al.,

2004).

1.5.2 CriticalAnalysis

Bioreactors are well established for the cultivation of microbes or mammalian cells under

monitored and controlled environmental and operational conditions (e.g., pH, temperature,

oxygen tension, and nutrient supply) up to an industrial scale. However, as individual cells

are mostly applied, these concepts are inapplicable to 3D tissue constructs. Furthermore, each

type of tissue construct (e.g., skin, bone, blood vessel, and cartilage) will likely require an

individualized bioreactor design (Ratcliffe & Niklason, 2006). Therefore, tissue-specific

bioreactors should be designed on the basis of comprehensive understanding of biological

and engineering aspects. Additionally, typical engineering aspects such as reliability,

reproducibility, scalability and safety should be addressed. Several bioreactor systems have

been developed and usually the expectations are very high (Martin et al., 2004). The question

is, however, whether bioreactors can indeed fulfil these expectations. In this section, key

technical challenges are identified and an overview of existing culture systems and

bioreactors used for tissue engineering are analysed

1.5.3 Bioreactor Validation using Computational Modelling

For the prevention of tissue engineering evolvement reaching its stagnation point,

multidisciplinary research is essential. With recent advancements in the computer algorithm

technology and processing power, new applications of computational engineering have

succeeded in various disciples. The complexity of biological systems has always been the

50

restrictive factor in using computer aids to analyse bio-systems. Computational models are

now employed to model the mechanical response of soft tissues to various loading modalities

as well as exchange of nutrients and waste, ECM synthesis, cell proliferation dynamics, cell-

scaffold interaction (migration, attachment), cell-to-cell interface, forced perfusion and

construct structural analysis. (Sengers et al., 2007). These are given in table 1-10.

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Frank et al., 2000)

Custom made

A versatile shear

and compression

apparatus for

mechanical

stimulation of tissue

culture explants

• Incubator housed

• Biaxial-tissue-loading device

• Axial deformations as small as

1 µm

• Sinusoidal rotations as small

as 0.01º

• Axial linear stepper motor

• Load cell

• Torque cell

• Rotary position table

• 12 sample well plate

• Applies unconfined

compression to samples

• Shear modulus tests

performed in both torsional

(pure shear) and simple shear

modes

• Performs studies of

tissue in shear and

compression

� Material

property testing

� Metabolic

studies

• Simple system

• Multi-sampling

• At strains in excess

of 1.2%, the torque

waveforms showed

distortion

indicative of

slipping between

the tissue and the

platens

• Long term culture

not feasible due to

poor media

refreshment

facility

• Explants were used;

mechanical properties

obtained do not reflect

material properties of

cartilage in-vivo.

• Orientation of collagen

fibres plays an important

role when obtaining

mechanical properties.

• Torque cell and load cell not

attached to individual

explants which means load

and torque obtained is an

average measure.

(Mauck et al.,

2000)

Custom made

Functional tissue

engineering of

articular cartilage

through dynamic

loading of

chondrocyte-seeded

agarose gels

• Dynamic loading

• Unconfined compression

• Peak-to-peak compressive

strain amplitude of 10%, at a

frequency of 1 Hz

• Eccentric circular cam driving

a spring-loaded translating

cam-follower connected to a

loading platen.

• Camshaft connected via a

flexible shaft to a motor

controlled by a signal

generator used for adjusting

the loading frequency.

• Applies a sinusoidal

strain to cell-seeded

hydrogels.

• Multi-sampling

• Low cost

• Friction not

negligible between

the platens and the

agarose disks.

• Microfissures

produced in the

hydrogels due to

dynamic loading.

• Medial cells of the

construct obtain

insufficient

nutrition compared

to those of the

radial due to the

use of impermeable

platens.

• The unconfined nature of

the bioreactor loading

results in higher pressures in

the central region and

greater flow velocities in the

periphery region of the disk.

• The measurements obtained

for the mechanical

properties would be an

average value as one load

cell would be used over a

set of constructs.

52

Bioreactor Characteristics Schematic Advantages Disadvantages • Comments

(Altman et al.,

2002) Custom made

Advanced

Bioreactor with

Controlled

Application of

Multi-Dimensional

Strain For

Tissue Engineering

• Computercontrolled bench-top

bioreactor system

• Applies complex

concurrentmechanical strains

to three-dimensional matrices

• Independently housed in 24

reactor vessels

• Enhanced environmental and

fluidic control.

• Two independently controlled

bioreactorsthat share a

common environmental

control chamber

• Mechanical strains at ‹0.1 µm

for translational and ‹0.1° for

rotational strain

• Two independently

controlled

bioreactorspermit

the concurrent

study of varied

strain rates and

percentage strain

• Individual reactor

vessels can be

added, replaced or

withdrawn at any

time during an

experiment without

disturbingsystem

function

• Reactor vessels, via

the top mount O-

ring and bushing,

provide a barrier to

contamination

• Excessive use of

media

• System is large

• Complex system to

assemble and

operate

• Enhanced flexibility

hasbeen achieved through

this design allowing

concurrent but independent

operation of up to 24 reactor

vessels

• Cell seeding options have

been improved through

enhanced fluidic control

utilizing perfusionthrough

and/or flow around the

matrix

• The complexity of the

system outweighs the

advantages however.

53

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Gokorsch et al.,

2003) Custom made

Stimulating

extracellular

matrix production

by

intervertebral disc

cells

• Three-dimensionalperfusion

culture with cyclic pressure

under defined conditions.

• Cover plate equipped with

compression stem to apply

compressive loading

• Confined compression

• Easy addition of

medium

supplements

• Efficient use of

media

• Autoclaveable

• Long term

cultivation

• Simple system to

assemble and

operate

• Low number of

samples

• Poor monitoring

system

• The bioreactor doesn’t seem

to allow enough perfusion

for healthy growth of cells

• Although this simple system

enables ease of assembly

and use the data obtained is

poor

• The system is designed

purely for metabolic studies.

(Schulz et al.,

2008)

Custom made

Development and

validation of a

novel bioreactor

system for load and

perfusion controlled

tissue engineering

of chondrocyte

constructs

• Cyclic mechanical loading

combined with medium

perfusion over long periods of

time under controlled

cultivation and stimulation

conditions while ensuring

sterility.

• The vertical motion of the

stainless steel mini actuator

with the loading plate is

controlled by a magnetic field

inducedby external permanent

magnets.

• Cyclicloading is applied by

alternating the magnet

orientationabove the

bioreactor.

• Possibility of static

culture

• Embedded

perfusion system

• Simple system to

assemble and

operate

• Small enough to be

housed in a

standard incubator

• Low number of

samples

• No feedback

control system for

culture medium

• Bioreactor in which both

fluid-handling and

compressive loading are

controlled.

• This system combines non-

contact loading with areal-

time measurement of

effective load and

displacement.

• The design of the bioreactor

is intelligent as it allows

both static and continuous

culture of constructs.

54

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Chowdhury et al.,

2010)

Custom made

Biomechanical

modulation of

collagen fragment

induced anabolic

and catabolic

activities in

chondrocyte/

agarose constructs

• The characteristics are same

as the bioreactor above with

the only difference being the

increased sample number.

• Multi sampling

• Simple system to

assemble and

operate

• Small enough to be

housed in a

standard incubator

• No feedback

control system for

culture medium

• The bioreactor was

modified to accommodate

more samples

• The system is still based

on the magnetic actuator

to apply the compressive

strain

• The bioreactor is most

suitable for metabolic

studies related to loading

regimes.

(Lagana et al.,

2008)

Custom made

A new bioreactor

for the controlled

application of

complex

mechanical stimuli

for cartilage tissue

engineering

• Applies hydrodynamic shear

stresses and hydrostatic

pressures (static or cyclic) to

3D constructs

• Shear stresses are imposed by

forcing the culture medium

through the scaffolds

• Hydrostatic pressure is

generated by means of a

pneumatic actuator

compressing the medium.

• Appliesa wide range of

bothhydrodynamic shear

stresses, around the

physiologicallevel of 0.1 Pa,

and hydrostatic pressures up

to 15 MPa, either static or

cyclic at lHz.

• Bioreactor can be

housed in a

standard incubator

• Multisampling

possible(max. 8

samples)

• Long term culture

possible

• Excessive use of

media

• Actuator is

hydraulic therefore

high maintenance

cost

• No monitoring of

the magnitude of

shear stress applied

to the constructs

• Extraction of

constructs is

tedious

• Air bubbles within

the chamber would

have a significant

effect

• The design was based on a

computational model,

although a novel approach

to the application of

hydrostatic pressure on 3D

constructs the measurement

of the mechanical stimulus

is unsatisfactory.

• Reliability and

reproducibility of

experiments is also a

debatable issue.

• However questionable the

design maybe, the metabolic

study would provide useful

information to researchers

incorporating hydrostatic

pressure in the design of

bioreactors.

55

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Wartella &

Wayne, 2009)

Custom made

Bioreactor for

Biaxial Mechanical

Stimulation to

Tissue Engineered

Constructs

• Biaxial bioreactor for 3D

construct stimulation

• An assembly to apply tensile

stresses

• An assembly to apply

compressive stresses

• A chamberto contain the

tissue construct and bathing

media.

• The ability to apply

both mechanical

loads along

different axis to the

construct during

culture period.

• Fits in an incubator

• Does not include

perfusion

• During tensile

loading the tension

grips interfaces

with the tissue

specimen by

clamping the

specimen in place

with the teeth. This

causes high stress

concentration

around the teeth.

• One construct per

experiment

• Excessive use of

media per

construct.

• The bioreactor has a

novel approach to apply

biaxial stimuli and the

assembly is based on

simple principles

• Interpreting and

analysing any results

obtained from biaxial

systems is a new avenue

of research work

• The system is however

inefficient due to the

single construct testing

facility, hence validating

any findings would be

impossible.

56

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Miloša et al.,

2009)

Custom made

A Novel Bioreactor

with mechanical

stimulation for

skeletal tissue

engineering

• Provides dynamic

compression and perfusion.

• Dynamic compression can

be applied at frequencies up

to 67.5 Hz and

displacementsdown to 5

µm

• Regimes of the mechanical

stimulation and acquisition

of load sensor outputs are

directed by an automatic

control system using

applications developed

within the LabView

platform.

• The

bioreactorwas

shown to be

biocompatible

and to support

packed bed

cultures of

chondrocytes

immobilized in

alginate

microbeads.

• Can be housed in

an incubator

• Simple system to

assemble and

operate

• Load sensor is

calibrated so to

measure average

loads imposed on

tissue samples

(no individual

loading).

• Another bioreactor that

applies compression with

perfusion using

displacement control as a

mode of measurement.

57

Table 0-10: critical analysis of the current bioreactors

Bioreactor Characteristics Schematic Advantages Disadvantages Comments

(Naing et al., 2009)

(abstract)

Made in

collaboration with

Bose Corporation

ElectroForce

Systems Group

Towards a

Physiologically

Informed

Bioreactor:

Engineering

Challenges &

Compromises

• a multi-axial bioreactor was

designed and developed to

deliver the following

dynamic mechanical

stimulation conditions:

- Hydrostatic pressure

- Pulsatile perfusion flow

- Uniaxial compression.

• This arrangement allows

cyclic triaxial mechanical

stimulation and

simultaneous mechanical

characterisation of samples,

simulating the conditions

experienced by the nucleus

pulposus in vivo.

• Compact Size

• No contamination

• Ideal for low force

testing(no-friction).

• Easy autoclaving

• WinTest system is

user friendly

• Data (feedback) is

automatically

obtained

• Fluid flow

controlled in

different phases of

stimulation

Determination of

rate of degradation

of the certain

scaffolds – sensors

• Provision of

sufficient nutrients

to sample

• Pulse volumes and

pressures can be

controlled through

individual samples

• Virtually

maintenance free

• Strain is measured

by a digital video

extensometer.

• Expensive

• Incorporates only 4

samples per

chamber

• The Bose BioDynamic

test instrument is a

complex system that

provides a variety of

loading modalities.

• The highlight of the

system is the linear motor

which provides the

precision of the

mechanical stimulus

• However the cost to

features ratio is high.

• The trademark has

become the selling point

of this bioreactor rather

than its actual

functionality.

58