CARTILAGE
Cartilage is a key body component, performing important structural and functional roles.
Cartilage cells develop from mesenchymal stem cells.[1] Developing as early as the 5th week
of embryonic growth, it forms 1% of total adult tissue volume and is also closely involved in
the development of bone.[2]
In humans, cartilage is found in many forms in various locations around the body. Its major
role in adults is to provide mechanical support as well as a range of secondary functions.
These functions provide; a smooth surface for articulation of bones, growth of long bones and
strengthening of tendons such as at the pubic syphysis. The different forms are divided into 3
main groups (See Table 1).
TABLE 1: TYPES OF CARTILAGE AND FUNCTION
Type of Cartilage
Key Locations
Hyaline
Tracheal Rings, Joint surfaces of Bone
Elastic
External Ear, Epiglottis
Fibrocartilage
Intervertebral Discs, Pubic Syphysis
The different functions of cartilage require a variety of micro structures, which relate to the
functional role it is undertaking. All of the different types of cartilage are made up of similar
components. Through different levels of gene expression and synthesis, of these components,
the structural differences of cartilage are exhibited.
COMPONENTS OF CARTILAGE
Cartilage is made up of 3 components; chondrocytes (cartilage cells), extracellular matrix
(ECM) and interstitial fluid. Cartilage is avascular, alymphatic and has no innervation.
CHONDROCYTES
Chondrocytes are the sole constituent cell of cartilage. Cells average only 2% of total
cartilage volume,[3] a situation almost unique within the human body. They are sparsely
distributed and isolated from neighbouring cells by the ECM. The chondrocytes role is to
produce and organise the ECM, which carries out the functions of cartilage.
Chondrocytes are rounded or polygonal in shape, and on average have a diameter of 13µm.[2]
They have an abundance of rough endoplasmic reticulum and glogi apparatus (FIGURE 1), as
expected for a cell producing such large quantities of extracellular material. Chondrocytes
also have relatively few mitochondria as a result of their low oxygen/avascular environment.
In fact the whole cells metabolism is geared to low oxygen tensions.
In the deep layers of
cartilage oxygen tensions may be less than 1%.[4]
FIGURE 1: AN ELECTRON MICROGRAPH OF A CHONDROCYTE
From “The Chondrocyte”[4]
Chondrocytes are regulated by a poorly understood combination of mechanical, electrical and
physiochemical signals. These are thought to be transmitted by the ECM to the cell through
membrane receptors and direct binding.
The chondrocytes respond to these stimuli by
regulating their metabolic activity through gene expression.[5] This area will be discussed in
more detail later with regard to articular cartilage.
COMPONENTS OF THE EXTRACELLULAR MATRIX (ECM)
COLLAGEN
Collagen acts as the organised structure or ‘endoskeleton’ of cartilage. It represents up to
50% of the dry weight of cartilage.[6] It provides the tensile strength,[7] and resistance to
forces.
Collagen also provides resistance to the osmotic pressure exerted on the
proteoglycans which indirectly attach to the collagen framework.
Collagen is composed of repeat amino acids sequences, with side chains. There are more than
20 types of collagen. They are broadly classified into fibrillar and non-fibrillar. The 4 major
collagens in cartilage are outlined below (See Table 2). Collagen forms the ‘pillars’ and
4
‘beams’ which give cartilage its inherent structure however it is the ‘filling in’ of these spaces
that give cartilage its strength against compression.
TABLE 2 : TYPES OF COLLAGEN AND FUNCTION
Collagen
Fibrillar / Non-Fibrillar Function
II
Fibrillar
Structural/ Mechanical Fibres
VI
Non-Fibrillar
Peri-cellular Adhesion
IX
Non-Fibrillar
Point of attachment on fibrils for cross-linking
X
Non-Fibrillar
Small Network to facilitate calcification
Adapted from: Skeletal Tissue Mechanics.[7]
Collagen is formed from chains of amino acid. These chains combine to form the collagen
units (see table 2), these units are either fibrillar (fibrous) in form or not. Finally these fibrils
combine along with non-fibrillar units to form larger units known as fibres.
Collagen II is the key molecule that provides tensile strength. It consists of three identical α1
chains arranged in helical form. This forms the majority of the fibrils (FIGURE 2) in the ECM.
In certain zones it accounts for 80-90% of the dry weight of cartilage.[8] The fibrils are
strengthened further by cross linking.
Collagen VI forms highly branched filaments that intertwine with collagen II.[9] It is found
almost exclusively in pericellular regions and is thought to be involved in attaching the
chondrocyte to the ECM. It also binds to hylauronan, a component of the proteoglycan
aggregate, discussed later.
Collagen IX assists in the process of cross linking fibrils.[10] It is attached to the fibrils in a
perpendicular manner. This offers a point of attachment for cross links (FIGURE 2). Also by
attaching to binding points on the fibrils it prevents lateral growth. Collagen IX is also
prominent in the pericellular region. It represents 2% of total collagen.[10]
Collagen X is found in growth plates and areas of calcified cartilage. It is produced by
hypertrophic chondrocytes and is normally found in the pericellular region. It is a short chain
that forms a hexagonal network, this assists in the mineralisation of the ECM.[11] It is used
as a marker for mineralisation.
5
PROTEOGLYCANS
Proteoglycans consist of glycosaminoglycan (GAG) chains bound to a protein core. They
account for up to 12.5% of the dry weight of cartilage.[7] The most common proteoglycan in
articular cartilage is aggrecan, making up 90% of the total proteoglycan mass. [8]
Aggrecans comprise 10% core protein and 90% GAG.[12] The protein backbone is made up
of 4 domains. The G1 domain (See Figure 2) is the proximal binding site. It can bind to
hyaluronan, with the assistance of a link protein, or other less common sites. G2 domain (See
Figure 2) is an area of structural importance. G3 domain (See Figure 2) is the termination of
the protein back bone. The 4th region between G2 and G3 is the GAG attachment domain.
The GAGs bind perpendicularly to the protein backbone. Around 150 GAG chains may be
attached to a single aggrecan protein backbone, [8] to form a ‘bottle brush’ structure.
FIGURE 2: MACROFIBIBILLAR COLLAGEN NETWORK & PROTEOGLYCAN AGGREGATE
Key: HA - Hylauronan CS - Chondroitin sulphate KS - Keratin sulphate
From “Composition and Structure of Articular Cartilage” [13]
6
GYLCOSAMINOGLYCANS (GAGS)
GAGs are repeating disaccharide units of uronic acid and a sugar. They are normally
sulphated which leads to the formation of a polyanion.[8] The three main GAGs in cartilage
are hyaluronan, chondroitin sulphate and keratan sulphate.
Hyaluronan is a repeating chain of hyaluronic acid which can reach up to 2 million repeats in
size.[14] Hyaluronan intertwines with the collagen fibrils, forming a “weave” that maintains
the hyaluronans position within the ECM (See Figure 2).
Hyaluronan offers the most
abundant source of binding sites for proteoglycans (PGs). Many PGs indirectly link to
hyaluronan which forms aggregates.
Hyaluronan also links the ECM to the chondrocytes via an interaction with the chondrocyte
cell surface receptor CD44.[15]
PROTEOGLYCANS (PGS)
PGs are formed by a protein backbone which binds to GAGs covalently attach. [8] The most
prolific PG in cartilage is aggrecan. [8]
Aggrecan contains two different forms of GAGs; chondroitin sulphate and keratin sulphate.
Both are repeating disaccharide units, chondonite sulphate is about 30 units long, while
keratan sulphate is shorter. Both types of chains contain carboxyl and sulphate groups that
when in contact with interstitial fluid become negatively charged and so attract cations. It is
this affinity for water that carries out aggrecans function. The water attracted by the negative
charge swells the aggregates, until their expansion is restricted by the collagen fibrils of the
ECM. Even after maximal swelling, aggregates still has further affinity for water. This
means that when cartilage is placed under compression, the compressive force is attempting
to force the water out of the cartilage, against frictional and osmotic forces which are
attempting to prevent water leaving. These forces are closely balanced in healthy cartilage,
causing resistance to compression.[16]
INTERSTITIAL FLUID
The role of the interstitial fluids is to provide nutrition to the chondrocytes and remove waste
products. As such it consists of; electrolytes, small molecules, glucose, metabolic wastes,
7
carbon dioxide and constituent parts of ECM all of which are suspended in water. Nutrients
and wastes have to be carried to and from the chondrocytes through the ECM, which acts as a
sieve. The ECM’s great affinity for water means that diffusion over any significant distance is
negligible. This accounts for the low oxygen tensions at the deep layers of cartilage.[4]
Articular cartilage has adaptations to these low oxygen tension which are discussed later.
8
ARTICULAR CARTILAGE
Articular cartilage is a form of hyaline cartilage, with particular structural variances to assist it
in its role. Articulating ends of involved joints are covered by this load bearing elastic
material. It serves as resistance to compressive forces and a low friction surface. Together
with the synovial fluid in the joint capsule they create an almost frictionless joint.[13]
ZONES
Articular cartilage is structurally divided into 4 layers that run parallel to the joint surface.
These 4 layers are; the superficial, transitional, deep and calcified zones. This organisation of
zones is of functional use and the composition of structural components differs between
zones.
SUPERFICIAL ZONE
The superficial zone runs on the free surface of articular cartilage. It is the thinnest of the
zones, but contains the highest cell density.[3] Cells in this layer are discoid or flattened as
opposed to their usual polygonal form.[4] Water concentrations are high as there is a low
proteoglycan content leaving room for the water.[17] The majority of the zone consists of
densely packed layers of collagen.
The thin collagen fibrils of this zone are uniformly packed and divided into 2 layers. The first
layer, known as the lamina slendens, is the linear fibres that form the articular surface. It is
formed by bundles of collagen running parallel to each other. This forms a smooth surface,
which is comparable to man-made metalwork.[18] The subsequent layers of collagen fibrils
are arranged perpendicular to the articular surface.
These layers produce a filtering effect on the fluid passing through to the deeper layers of the
cartilage. A molecule as small as an immunoglobulin may be prevented from entering the
cartilages interstial fluid, by this filtering effect.[8]
9
TRANSITIONAL ZONE
The transitional zone contains and is produced by rounded chondrocytes. Collagen of the
ECM is randomly arranged and proteoglycan content is greater than that of the superficial
zone.[14]
DEEP ZONE
The deep zone is characterised by chondrocytes arranged in columns. Chondrocytes are at
their lowest concentration in this zone, which is dominated by a proteoglycan rich ECM.[17]
An irregular line separates this zone from the calcified zone. Between the 2 zones bands of
collagen can be seen. These bands tether the deep zone to the calcified zone, to prevent
shearing forces separating the two zones.
CALCIFIED ZONE
Here, round hypertrophic chondrocytes remain in non-calcified area. They are surrounded by
an ECM which is almost absent in proteoglycan.[8] The collagen fibres are arranged
perpendicular to the surface and are anchored to the calcified matrix.[14]
The line of
communication between the calcified zone and underlying cortical bone is wave like in form.
ARTICULAR CARTILAGE FORMATION
Articular chondrocytes produce different quantities of ECM components due to variation in
the stimuli each zone of cells is subjected to. During joint loading, forces on the ECM cause
stimulation of the chondrocytes via CD44 and other receptors.[14] These stimuli vary in type
and intensity dependent on cell location.
It is this which causes the variance in gene
expression leading to ECM production.[19]
The second method of stimulation of the chondrocytes is through hydrodynamic
pressures.[20] Articular cartilage is effectively a very stiff sponge. When joint loading
occurs it forces liquid / synovial fluid out of the ECM, so causing fluid flows within the ECM.
This fluid flow takes positive charges ions with it, meaning the membrane potential is
depolarised. In addition the intracellular fluid composition has not changed and is now more
heavily attracted to the extra-cellular negative charge causing an osmotic swelling of the cell.
Both of these processes add to the stimulation of the call and can cause ECM production.
10
This fluid movement caused by loading of the joint also carries nutrients to the chondrocytes
and metabolites away from them.
11
ARTICULAR CARTILAGE INJURY & TREATMENTS
ARTICULAR CARTILAGE INJURY
Cartilage injuries fall into two main patterns; full or partial thickness.
The two are
differentiated by the involvement of subchondral bone, which is the barrier between the
cartilage and bone marrow. Partial thickness injuries do not repair on their own,[21] due to
cell immobility in cartilage. Other factors that effect cartilages ability to heal include; the
location of the injury, the age of the patient and any associated infection.
Full thickness injuries do heal, due to their contact with the marrow space and its
mesenchymal stem cells and blood supply. This forms a blood clot in the defect. Lesions 12mm in diameter can heal but larger lesions may not totally fill with clot,[22] leading to poor
healing. The clot adheres well to the bony surface but poorly to the articular edges, which can
lead to necrosis at the periphery of the neocartilage.[23] After a few days, mesenchymal cells
begin to infiltrate the haematoma. These cells help produce a fibrocartilage that fills the void
created by the injury. Ossified material is deposited deeper to reseal the marrow space.
Fibrocartilage is mechanically inferior to articular cartilage as it gradually degenerates over
time.[23]
TREATMENTS OF ARTICULAR CARTILAGE INJURY
Treatments for articular cartilage injury currently fall into three main categories,
arthroscopy/levage, marrow stimulating techniques and implantation techniques.
ARTHROSCOPY / LAVAGE
This procedure is carried out in two sets of circumstances. The first is if physical and
radiological assessment deems it likely a patient has a free body in the joint. A free body is
typically a piece of cartilage that has broken off during the injury event and is now floating
within the joint capsule. The lavage removes this free body so preventing it from damaging
the articular surface in any other area. This involves placing an access port into the capsule
and washing out the free body.
12
The second indication for lavage is for pain relief. If a patient has pain that is effecting their
activities of daily living then lavage may be of some use.[24] Pain relief is achieved through
an unknown mechanism. It is postulated that by replacing the synovial fluid, pain signalling
agents are also removed. However it also gives the surgeon the opportunity to place a fibre
optic camera into the joint and visually inspect the injury. This gives the surgeon a definitive
assessment of the injury and allows a decision to be made if any further treatment would be
beneficial.
MARROW STIMULATING TECHNIQUES
These techniques attempt to stimulate a healing of the injury similar to that found in full
thickness injuries. This is achieved by a range of techniques from debridment to subchondral
drilling. They are all base around the principle of exposing the bone marrow space to the
injury site by mechanical measures.
The microfracturing technique is an adaptation of the subchondral drilling technique first
described by Pirdie in 1959.[25] Steadman et al.[26] created microfracturing which consists
of creating holes; 4mm deep, 1mm in diameter and 3-4mm apart, across the extent of the
defect. This creates a communication to the bone marrow and allows a clot to form in the
defect so leading on to fibrocartilage creation. The holes are created by an arthroscopic awl
which has a number of benefits over Pirdies’ drill. It is carried out by arthroscopy so causes
less tissue damage, the awl does not cause the heat necrosis to surrounding tissue that a drill
might and the small holes mean that less trauma is caused to the subchondral plate, which
help maintains its biomechanical properties.
In young people it has been reported to improve joint functionality and gives pain relief in up
to 75% of patients.[27] The fibrocartilage created will however be of inferior quality to
articular cartilage and it is expected that these patients will suffer problems in the future.
IMPLANTATION
AUTOGENOUS CHONDROCYTE
IMPLANTATION
The first reported use of a autogenous chondrocyte implantation was by Grande et al, in
rabbits[28] This technique involved the removal of a 3mm full thickness segment from a
13
rabbits patella. The defect was then covered with a periosteal patch. The chondrocytes where
then liberated from the extracted segment and cell number was expanded in vitro. The cells
were then injected back into the defect under the periosteal patch. At six weeks the cells had
contributed to the repair of the defect and results were considerably better than the controls.
Following this Brittberg et al.,[29] published the first human study in 1994. The results of the
first 100 patients were subsequently published and are very encouraging.[30] 92% of the
repairs carried out, were rated good to excellent. The only major complication associated
with successful grafts is periosteal patch over growth, which occurs in 10-15% of patients.
These are relatively easily debrided by an arthroscopic procedure.
While these early results are encouraging , the use of these techniques is highly specialised
and long term results are unknown. In a canine study, cartilage produced is known to be
vastly inferior to native cartilage,[31] however clinical results in human studies seem to
exceed these expectations.
CARTILAGE TRANSPLANTATION
The main difference cartilage transplantation is the location of their donor site. Grafts are
either autografts (from the same patient) or allografts (from cadaver donors). The allografts
are then subdivided into fresh refrigerated grafts or frozen cryopreserved grafts.
AUTOGRAFTS
The advantages of autografts are the smaller risk of disease transmission and less risk of graft
failure due to immuno incompatibility. However these benefits must be compared to donor
site morbidity.
Mosaicplasty is a technique used in the knee, which was developed by Hangody et al. in
1992.[32] Small osteochondral grafts are harvested from the supra chondylar ridges or the
intra-chondylar ridges and then pressed into the pre-prepared holes in the defect. The size of
the donor grafts, 2.7-4.5mm in diameter, helps to reduce morbidity associated with their
harvesting.[32, 33] The bone of the graft is then pressed into the recipient hole, this heals
well and the small amount of blood that escapes forms a uniform cartilage surface between
14
the grafts. The graft cartilage survives and has intervening fibrocartilage, formed from the
blood, to support it.[28] Handgody et al., reports 91% of patients achieve good to excellent
scores on the Hospital for Special Surgery knee scoring system at 3 years.[33] There are
however limits as to the size of defect which can be treated. Ideally it should be less than
2cm in diameter and have a discrete margin. This technique can also not be used on anyone
who has an active inflammatory or infective process in the knee and can not be used if there is
any degenerative change to the cartilage.
ALLOGRAFTS
The first was carried out in 1907 by Lexer.[34] He performed a “half joint transplantation”.
After his initial success he continued to perform allografts for partial and whole joint
transplantations, reporting a 50% success rate.[34] This early work found that infection was
the main reason for failure and as such Lexer favoured fresh donor material.
It was Mankin et al. who pioneered the use of cryopreserved allografts which are logistically
more practical than fresh grafts.[35] However later analysis showed that the cartilage of the
frozen allografts rarely survived after 8 years.[36] It was hypothesised that the freezing
process killed the chondrocytes, which were dead after thawing. Without the chondrocytes to
maintain it the ECM begins to degrade with wear and tear.
Today, because of the above problem, fresh allografts are almost exclusively used. They tend
to be harvested 12-24hrs after the donors death. The graft is then placed in physiological
solution and stored at 4ºC. Most surgeons advocate its use within 24hrs of harvest.[37, 38]
Although chodrocytes remain viable up to 7 days in this environment, other parts of the graft
are less well adapted to survive in low oxygen environments.[39] Most clinical trials have
procured their allografts from donors below the age of 30.
Garret,[38] describes probably the most widely advocated surgical technique. The defect is
normally converted into a cylindrical shape with a depth of 8-10mm. Then a similarly sized
plug is harvested from the corresponding place on the donor, this is then press fitted into the
defect.
15
In clinical trials fresh allografts are used. The first of these trials were carried out in 1972 by
Gross et al.[40] and McDermott et al.[41] A recent review with an average follow up time of
7.5years showed an 85% success rate. Success was defined as no need to re-operate and nonrecurrence of pre-operative symptoms.
All patients undergo a similar regime of rehabilitation post-transplant. Non-weight bearing
for the first few days, followed by protected weight bearing until the graft has incorporated.
This is followed by limited exercise and finally by full weight bearing, which may take up to
2 years to achieve if the graft is slow to incorporate.
16
TISSUE ENGINEERING
In recent years, in-vitro strategies for the repair of cartilage defects have reached levels of
development, so as to make them plausible alternatives to in-vivo therapies.
Tissue
engineering strategies “focus on the delivery or in situ mobilisation of cells, capable of
restoring pathologically altered architecture and function”.[42] There are 2 main focuses of
research; the implantation of cells in an artificial supportive matrix and the in-vitro
development of a complete transplant. The development of these two different approaches,
are based on much of the same research. Developments in this area have been rapid, with the
application of old and new knowledge in the search for a viable clinical therapy.
The ultimate aim of a complete transplant would be to harvest cells from the patient, sort
them, place them into a growth platform and produce a high quality transplant that could be
used to replace damaged areas. The use of “bioreactors” holds the promise of producing high
quality cartilage for use in such a transplant.
There does however remain a lot of
development to be under taken to scale them up to an appropriate size.[43]
Cell transplantation in scaffolds uses a similar concept as Brittbergs et al. Instead of being
held in place by a periosteal patch, scaffolds attempt to maintain chondrocytes in the defect
by using a porous material,[44] until cells produce enough ECM to maintain their own
position. This research is also being used to fix chondrocytes in position in bioreactors and as
such much research is shared with cell implantation, tissue engineering.
SCAFFOLDS
These scaffolds must meet a number of design criteria. The material and its degradation
products must be compatible with all human tissues.[44, 45] In addition the scaffold must
allow nutrient diffusion to the encapsulated cells, be porous enough to allow native cells to
infiltration and be able to maintain structural integrity[44] and position.[42] The porosity of
the scaffold is required “both to encourage retention of implanted cells and to favour
colonisation by native cells”.[46] Native cell infiltration is imperative as implants that do not
bond and integrate with adjacent tissues are “destined to fail”.[47]
17
Scaffold Materials fall into two main categories; synthetic and natural materials.
SYNTHETIC SCAFFOLD MATERIALS
Polymeric materials are used heavily in the research of tissue engineering cartilage. Widely
used are polylatic acid and polyglycolic acid.
PLOYLACTIC ACID (PLA)
Initial animal studies with this scaffold showed discouraging results,[48, 49] with poor
subchondral bone repair and lower levels of biochemical markers than intrinsic cartilage.
Since then the initial retention of cells has been improved.[50] Also work has been carried out
using PLA as a carrier of exogenous transforming growth factors and its effects on
mesenchymal stem cells.
POLYGLYCOLIC ACID (PGA)
PGA comes in the form of a foam or fibre mesh that has been used in vivo cartilage defect
repair. In porcine models autologous chondrocytes implanted in defects produce good results
at 24 weeks.[51] Both biochemical and tissue interface results were good.
Co-polymers between PLA and PGA have been extensively investigated. The ratio between
PLA, PGA and additional constituents has been used to manipulate degradation time. The
addition of other constituents can, among other things, encourage bone growth into the
scaffold.[52]
NATURAL SCAFFOLD MATERIALS
FIBRIN
As the major component of a blood clot that forms at the site of full thickness injuries, fibrin
seems a logical choice as a potential scaffold. It has two major problems; it exhibits no
significant intrinsic mechanical strength and exogenous fibrin can cause an immune
response.[53]
Despite this fibrin has been shown to be an effective scaffold. With both cartilage chip[54]
and mesenchymal stem cell[55] studies using it with good outcomes. Fibrin glue is also used
to fixate other perichondral scaffold grafts in place.[56]
18
AGAROSE & ALIGINATE
These polysaccharides form hydrogels that allow even distribution of culture cells throughout
the scaffold as it is cast.[57] Agarose is used extensively in vitro but doesn’t break down well
and can induce an immune response in vivo.[58]
In contrast seeded alginate scaffolds shows poor biochemical markers,[59] but good
histological integration with native cartilage.[60]
COLLAGEN
As the largest constituent of the ECM collagen it also seems a logical choice as a scaffold.
There have been decades of work with collagen in vitro as well as a number of animal
studies.[61, 62] It is an essential tool within tissue engineering, as it can easily be combined
with proteins[62] and even gene therapy techniques[63] to provide improvements in the
quality of cartilage produced.
Recently collagens from bovine sources has been identified as a possible poroduct that could
spread prion induced bovine spongiform encephlalopathy.[64] This may limit its use to
research as in-vitro work would come with an unacceptable risk.
HYALURONAN
As another major component of ECM hyaluronic acid also seems an excellent potential
scaffold. However it was discovered to cause chondrocyte chondrolysis, inducing almost
total loss of proteoglycan rich areas.[65] Despite this rabbit studies have shown good results,
with marginally thinner cartilage at graft sites being the only major problem.[66]
CHITOSAN
Chitosan is a polysaccharide which forms a gel when cross-linked by a second substance.
Initially chondronitin sulphate was used,[67] but cellulose was later found to be a more
practical cross-linker.[68] Chitosan is such an attractive scaffold due to its thermal properties.
It is liquid at room temperature (21ºC) but forms a gel at body temperature (37ºC).[68] This
sparked a flurry of research into the properties of chitosan and its possible use as a scaffold
for cell transplantation in a variety of fields.[69] Though little attention has been focussed on
19
its possible use as a bioreactor scaffold. Its biocompatibility has been investigated and
confirmed.[70] It also has the advantageous quality of adherence with bone and cartilage.[71]
20
METHODS
MATERIALS
Dulbecco’s Modified Eagle Medium (DMEM-F12), foetal calf serum, Fungizone™,
gentamycin and Picogreen™ Dye were sourced from Invitrogen, UK.
Pronase E™ was sourced from VWR Interbational, UK.
Protasan UP (product: G213) was sourced from Novamatrix, Norway.
Hydroxyethyl-cellulose (cellulose), β-Glycerol phosphate (βGP) and all other reagents were
sourced from Sigma-Aldrich, UK.
MEDIUM
DIGESTION MEDIUM
Digestion medium (DM) consisted of DMEM-F12 media supplemented with 50µg/mL
gentamycin, 2.5ng/mL Fungizone™ and 50 µg/ml ascorbic acid-2-phosphate.
COMPLETE CULTURE MEDIUM
Complete culture medium (CCM) consisted of DM supplemented with foetal calf serum at
10% by volume.
GEL COMPONENTS
PROTASAN SOLUTION
Protasan powder was weighed and sterilised under UV light for 2 hours. Then, under a
lamina flow hood, it was placed in a bottled of sterile lab grade water with a magnetic stirrer
that had been autoclaved. It was stirred until all the powder had dissolved. After stirring it
was stored at 4˚C until required.
Β-GLYCEROL PHOSPHATE SOLUTION
βGP was weighed and made up to the appropriate concentration with autoclaved lab grade
water and vortex mixed. It was stored at 4˚C till required. It was filter sterilised through a
22µm filter when added to the protasan solution.
CELLULOSE SOLUTION
Cellulose was weighed and made up to the appropriate concentration with DMEM-F12 and
vortex mixed. It was filter sterilised through a 22µm filter when used.
21
EXPERIMENTAL METHODS
ISOLATION OF CHONDROCYTES FROM ARTICULAR CARTILAGE
Bovine joints were obtained from an abbatoir shortly after the animals slaughter. All animals
were aged under 30 months. A thickness from superficial to deep zone of cartilage was
dissected from the joint surface of the trochleal humerous and radial head of the thoracic
limb. Disected cartilage was placed into DM to prevent drying. Finally dissected pieces were
diced into 1-3mm pieces.
Chondrocytes were then isolated using a published method.[72] Briefly, the diced tissue is
digested with 700U pronase E™ for 1 hour at 37˚C on a rotary mixer. Then 100U/mL
collagenase XI and 0.1mg/mL for Dnase I, were added and the cartilage digested for 16 hours
to release the chondrocytes from the ECM.[72] Chondrocytes from the supernatant were then
strained through a 70µm sieve. Then washed and pelleted three times in 10% CCM by
centrifugation at 750g.
Cell viability was then determined by Trypan Blue exclusion. Briefly, 10µL of the final
chondrocyte suspension were mixed with buffered Trypan Blue dye and cells that excluded
the dye were counted on a Neubald Hemacytometer.
CHITOSAN GEL PREPERATION & CELL ENCAPSULATION.
Gel formation and cell encapsulation was based upon a previously published method.[71] In
brief, isolated cells were resuspended in 1 of 2 cellulose solutions and mixed with a 2% (w/v)
protasan solution which is dissolved in 0.1M βGP solution.
The βGP solution was added to the protasan solution on ice over a magnetic stirring plate.
700µL was then placed in the wells of 24 well plates using wide bore pipette tips.
The two cellulose concentrations used were 12.5mg/ml and 25mg/ml. Cells were pelleted
down then resuspended in the filter steralised cellulose solutions. 175µL of each cellulose
solution was pippeted into the wells and the gels were manual mixed. Negative control gels
were also cast with plain cellulose solution. Gels consisted a total of 875µL of materials,
giving an approximate depth of 0.5cm in the well. Cell concentration was 250,000 cells per
well.
22
Gels were placed in a 37˚C, 5% CO2 incubator to set. They were rinsed thrice with 1ml of
DMEM-F12 for 20 minutes. 1ml CCM was then placed on the gels for the duration of the
experiment. Gels were kept at 37˚C in a humidified atmosphere containing 95% air and 5%
CO2.
Positive controls with the same cell number where kept in 1ml CCM and digested with the
day 0 samples.
SAMPLING
5 gels were cast for each cellulose concentration (with corresponding controls), for each time
point. These gels and medium were biochemically analysed separately. 2 more gels were
cast for each cellulose concentration for live/dead analysis, with medium from these gels used
to monitor pH.
SAMPLE PREPERATION FOR BIOCHEMICAL ANALYSIS
Samples were digested with 10 U/ml proteinase K in 100mM ammonium Acetate, pH 7.0 at
60˚C for 2 hours with agitation. The protinase K was then inactivated by heating to 95˚C for
10 minuites and samples were store at -20˚C for analysis.
ANALYSIS
LIVE/DEAD VIABILITY
Viability in the gels was evaluated using a fluorescence technique. The gels designated for
this purpose were washed twice with 1ml of PBS for 20 minutes, to remove residue CCM.
1µL of ethidium homodimer (red) and 2µL of calcein AM (green) dissolved in 1ml PBS, was
placed in with each gel and left for 1 hour. After a further 20 minute wash in 1ml PBS to
remove excess reagents, part of each gel was compressed between a microscope slides and
cover. These were then visualized using a Zeiss fluorescence microscope equipped with a
digital camera. Under these conditions live cells fluoresce green and dead cells red. No
formal analysis was undertaken, other than a visual approximation of % viability. Images
were captured by the camera for possible further software analysis.
23
GAG / DIMETHYLMETHYLENE BLUE DYE
The GAG content was analysed using a standard method.[73] Sulphated GAGs were detected
spectrophotometrically in the digestion by 1,9-dimethylmethylene blue (DMMB) dye method.
This method detects anions, which when in complex with DMMB alters its absorbance.[74]
50µL of samples or standard, in triplicate, were mixed with 200µL of 32mg/L DMMB dye in
40 mM glycine buffer at pH3.0, in a 96 well plate. The plate was then read immediately on a
spectrophometric plate reader. Absorbance values at 490nm were read against a standard
curve plotted from 0, 2, 10, 50, 100, 200 µg/ml bovine tracheal chondroitin sulphate
standards.
DNA/ PICOGREEN™
DNA was quantified spectrophometrically using the Picogreen™ dye, which binds to double
stranded DNA. When bound it forms a fluorescent complex through a proposed binding of
the DNA polymer.[75]
10µL volume of each sample and control were added to 215µL of 10mM Tris-EDTA pH 7.4,
in duplicate.
An equal amount of Picogreen™ dye, dissolved in DMSO, was added.
Fluoresence emission counts were read at 535nm against a standard curve of 0, 25, 250, 2500,
25000 pg/ml DNA. Cell number was calculated using the reported average number of 7.7pg
of DNA per chondrocyte.[76]
STATISTICAL ANAYLSIS
All data represents the mean and standard deviation values derived from 5 replicates at each
time point. An paired Student’s t test was applied where indicated using SPSS™ v14
software. A level of 5% was considered significant (p<0.05).
24
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31
TABLE OF CONTENTS
Cartilage .................................................................................................................................... 3
Components of cartilage ........................................................................................................ 3
Chondrocytes ..................................................................................................................... 3
Components of the extracellular matrix (ECM) ................................................................ 4
Interstitial fluid .................................................................................................................. 7
Articular cartilage ...................................................................................................................... 9
Zones ..................................................................................................................................... 9
Superficial zone ................................................................................................................. 9
Transitional Zone............................................................................................................. 10
Deep Zone ....................................................................................................................... 10
Calcified zone .................................................................................................................. 10
Articular cartilage formation ............................................................................................... 10
Articular cartilage injury & treatments.................................................................................... 12
Articular cartilage injury ..................................................................................................... 12
Treatments of articular cartilage injury ............................................................................... 12
Arthroscopy / lavage ........................................................................................................... 12
Marrow stimulating techniques ........................................................................................... 13
Implantation......................................................................................................................... 13
Autogenous chondrocyte implantation ........................................................................... 13
Cartilage transplantation.................................................................................................. 14
Tissue engineering ................................................................................................................... 17
Scaffolds .............................................................................................................................. 17
Synthetic scaffold materials ............................................................................................ 18
Natural scaffold materials................................................................................................ 18
Methods ................................................................................................................................... 21
Materials .............................................................................................................................. 21
32
Medium ........................................................................................................................... 21
Gel Components .............................................................................................................. 21
Experimental Methods ........................................................................................................ 22
Isolation of chondrocytes from articular cartilage ........................................................... 22
Chitosan gel preperation & cell encapsulation. ............................................................... 22
Sampling .......................................................................................................................... 23
Analysis ........................................................................................................................... 23
Statistical anaylsis ............................................................................................................... 24
References ............................................................................................................................... 25
Table of Contents .................................................................................................................... 32
33