EURODISC: Interaction of age, genetics and environment

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Major biochemical components of the
intervertebral disc, their turnover and some
functional properties Department of Biomedical
Engineering Technion, IIT, Haifa, Israel
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The Intervertebral disc

• A large cartilaginous structure, which lies
  between the vertebral bodies, anchoring them
  together
• Discs vary in size down the spine, and are the
  largest in the lumbar region.
• All discs together make up approximately 25 of
  the length of the spinal column.

                         
Intervertebral Disc (a vertebral body, b
cartilage end-plate, c nucleus pulposus and d
annulus fibrosus)
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Normal vs. Degenerate disc
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Schematic representation of disc
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Functions of the intervertebral disc

• Mechanical transmitting loads down the spine
• Flexibility allowing movements in all planes.

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Balloon Model of Broom representing PG
constrained within the collagen network
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Collagen structure
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collagen fibrils
(from Hay, 1982)
Collagen fibrils act like string - strong in
tension, weak in compression
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Collagen network of the disc
Annulus lamellae
nucleus
Annulus lamellae
(adapted from Inoue and Takeda,1975)
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5D4 (KS)
Outer annulus
nucleus
5D4(KS)
Differences in proteoglycan populations and
synthesis rates across the disc
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Aggrecan Structurewhole monomer
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Main types of GAG
Chondroitin 4- and 6-sulfates composed of
D-glucuronate GalNAc-4- or 6-sulfate linkage
is b(1, 3)(the figure contains GalNAc 4-sulfate)
Keratan sulfatescomposed of galactose
GlcNAc-6-sulfate linkage is b(1, 4)

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• Fixed Charge Density (FCD)
• FCD is the concentration of negatively-charged
  groups (carboxyl and sulfonate) present in the
  GAG.
• Chondroitin sulfate (CS) contains 2 negatively
  charged groups/disaccharide and keratan sulfate
  (KS) contains 1 groups.
• The osmotic pressure of the GAG is largely due to
  the presence of negatively-charged groups.

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Gradient of PGs and osmolality

• Concentration of fixed
• negative charges
• governs local ion concentrations
• governs osmolality

c.400mOsm (plasma c.300mOsm)
FCD
PG
AF NP
AF
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Biochemical composition of the disc
Urban and Roberts, 1996.
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Swelling Pressure of the Disc Matrix
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The swelling pressure of a slice of tissue of
stated composition may be defined as the pressure
at which it neither loses nor gains hydration.
i.e. the swelling pressure is equivalent to the
equilibrium applied pressure.
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• Balance of forces in the disc

Externally applied pressure
Osmotic pressure of proteoglycans
Collagen stress


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• Pswelling Papplied ?PG - PC
• Pswelling tissue swelling pressure
• Papplied externally applied pressure
• ?PG proteoglycans osmotic pressure
• PC tensile stresses of the collagen network

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LOAD
LOAD
LOAD
Porous piston
Disc plug
water
Water expressed FCD rises osmolality rises
Effect of applied load on cartilage thickness
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LOAD
LOAD
LOAD
Porous piston
Cartilage plug
water
0.15M NaCl
1.0M NaCl
0.015M NaCl
Effect of NaCl concentration on equilibrium
thickness of loaded plug
Adapted from Eisenberg and Grodzinsky, 1985
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Dependence of FCD on location in the annulus of
human thoracic discs
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Swelling pressure of bovine disc compared to
extracted PG
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Swelling pressure of human disc annulus (62 yrs.)
compared to extracted PG (A1 fraction)
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Disc matrix
PG
Extrafibrillar water
Intrafibrillar
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Intra-fibrillar Water
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• How does this work relate to understanding
  disc degeneration?
• We want to know if there is repair of the disc
  is turnover increased or decreased with
  age/pathological situations.
• A fast turnover implies a possibility of natural
  repair whilst a long turnover means that defects
  can accumulate in the tissue without possibility
  of tissue renewal.

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Turnover of the Disc Matrix
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Degradation of large monomer during turnover
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Some facts.

• Aggrecan and collagen are key proteins of the
  matrix during aging, many changes occur in their
  composition and structure these changes include
  
• Racemization of the aspartic acid (L-gtD isomer)
• Accumulation of non-enzymatic glycation
  endproducts (AGEs)
• Collagen and aggrecan have long-lifetime, which
  make them susceptible to the accumulation of AGE
  (i.e.pentosidine).

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Racemization

• Racemization is chemical process in which a
  change in the three-dimensional structure of an
  amino acid from one form to its mirror image
  takes place.
• All amino acids in natural proteins exist as the
  L-configurational optical isomers, which with
  time convert into the D isomer by a spontaneous
  process dependent on time, temperature and pH.
• Racemization is complete when equal amounts of
  the L- and D-forms are obtained.

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• Racemization (or inversion) of L-amino acid in
  proteins proceeds via a slow, non-enzymatic
  reaction or sequence of reactions.

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• Aspartic acid is one of the fastest recemizing
  amino acids, so that D-aspartate can be detected
  in living subjects (during human life span) in
  proteins which are not renewed or which have a
  slow turnover.
• Hence, racemization of aspartic acid can be used
  as a method for assessing turnover of human
  cartilage and disc.

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Racemization of several free amino acids at
various temperatures (pH 7.6)
Amino acid 00 (years) 370 (years) 1000 (days)
Serine --- 50 (?) 4 (?)
Aspartic acid 430,000 460 35
Lysine --- --- 40
Histidine --- --- 40
Alanine 1.4 x 106 1,500 120
Isoleucine 6 x 106 6,500 300
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Non-enzymatic glycation

• Non-enzymatic glycation is a post transcriptional
  modification of protein in vivo, which is
  initiated by the spontaneous reaction of sugars
  with lysine residues and results in the formation
  of advanced glycation end-products (i.e.
  N-(carboxymethyl)lysine (CML), N-(carboxyethyl)lys
  ine (CEL) and pentosidine).
• Because AGEs are irreversible chemical
  modifications, they accumulate with age in
  long-lived proteins.

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Pentosidine

• Pentosidine is an advanced glycation end product
  (AGE) formed by combined processes of glycation
  and oxidation (glycoxidation) between
  carbohydrate-derived carbonyl group and protein
  amino group

Pentosidine involves Lys and Arg combined in an
imidazo-(4,5b)-pyridinium ring.
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A1-A2 fractionation profile
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A1D1-A1D8 sub-fractionation profile
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Removal of PG from GuCl-extracted specimens using
enzyme treatment
In order that aspartic acid racemization is not
falsified by the presence of PG, the latter
were Minimized.
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• Aggregate/non-aggregate separation
• By sepharose CL-2B
• Preliminary results show that the percent of
  aggrecan monomer in disc is between 10-30 of the
  total proteoglycan, compared to 70 aggregate in
  articular cartilage.
• The aggregate content is lower in the NP than the
  AF.

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Summary We have purified the major constituents
of the disc, necessary for the matrix
measurements of biochemical and biomechanical
properties Thus far, we have separated A1 and
A1D1-A1D6 sub-fractions by associative and
dissociative gradient ultra-centrifugation. We
have started the separation of A1 fraction into
aggregating and non-aggregating sub-fractions
using Sepharose CL-2B gel permeation
chromatography.
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• Matrix Measurements
• Turnover rate
• ( D-Asp and Pentosidine)

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D/LAsp ratio in A1 from nucleus and annulus of
human intervertebral disc

• D-Asp from A1 of both nucleus and annulus
  show similar trend with aging, with slightly
  higher values for nucleus.
• Flattening suggests an equilibrium between D-Asp
  racemization and turnover of the degraded
  fragments

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D/LAsp in A1 from different sources

• Accumulation of D-Asp from A1 of nucleus and
  annulus (IVD) is similar to that of cartilage
  from femoral head and is higher and more rapid
  compared to A1 from femoral condyle.

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D/LAsp in collagen from different sources

• D-Asp increases linearly with age
• The accumulation rate of collagen is as
  follows
• Annulus(IVD)gtnucleus(IVD)gt
• Articular cartilagegt skin

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D/LAsp accumulation in normal and prolapsed
specimens
Prolapsed discs (both nucleus and annulus) show
lower values of D-Asp (down to 50 in N, and 70
in A) compared to normal tissue, implying a
higher turnover rate. The same was the case with
articular cartilage.
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D/L ASP ratio in different buoyant density
fraction of PG extracted from human IVD
66 y e a r s 66 y e a r s 22 y e a r s 22 y e a r s
Nucleus Annulus Nucleus Annulus
D1 1.79 1.92 2.32
D2 2.61 1.95 2.43 1.61
D3 3.06 2.84 1.8
D4 3.12 2.92 1.87
D5 3.56 3.41 2.01
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• Non-enzymatic glycation
• Pentosidine measurements

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Pentosidine accumulation in A1 from IVD compared
with articular cartilage
Pentosidine accumulates more rapidly in the
intervertebral disc than in articular cartilage,
which implies a longer molecular age of A1 from
IVD than from articular cartilage.
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Pentosidine accumulation in A1 from IVD compared
with articular cartilage
Pentosidine accumulates more rapidly in the
intervertebral disc than in articular cartilage,
which implies a longer molecular age of A1
(IVD) compared articular cartilage.
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Pentosidine accumulation in collagen from
different sources
Accumulation of pentosidine from collagen of
nucleus and annulus (IVD) is similar to that of
collagen from articular cartilage and is higher
and more rapid compared to collagen from skin.
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Pentosidine accumulation collagen Vs. A1
Rates of pentosidine accumulation Rates of pentosidine accumulation Rates of pentosidine accumulation Rates of pentosidine accumulation Rates of pentosidine accumulation Rates of pentosidine accumulation Rates of pentosidine accumulation
Intervertebral disc Intervertebral disc Intervertebral disc Intervertebral disc Intervertebral disc Articular cartilage (Verzijl, 2001) Articular cartilage (Verzijl, 2001)
Nucleus Nucleus Annulus Annulus Annulus Articular cartilage (Verzijl, 2001) Articular cartilage (Verzijl, 2001)
collagen A1 collagen collagen A1 collagen A1
0.42 0.06 0.65 0.07 0.07 0.32 0.11
x 6.8 x 6.8 x 8.6 x 8.6 x 8.6 x 3 x 3
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• Conclusions..
• According to D-Asp and pentosidine accumulation
  values, turnover time is as follows
• In all cases Collagen lt A1
• For A1 nucleus, annulus (IVD) and articular
  cartilage from femoral condyle lt articulr
  cartilage from femoral head.
• For collagen annulusltnucleus lt articular
  cartilage lt skin

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• Summary
• We have measured the degree of racemization (
  D-Asp) and pentosidine accumulation in both A1
  and collagen in some of the discs.
• We have obtained some of the data which will
  enable us to estimate the turnover time of
  collagen and aggrecan in the disc (normal and
  degenerate) by measuring D-Asp and pentosidine
  levels in purified preparations of collagen and
  aggrecan.
• Material from other discs (including A1, D1-D6,
  Collagen types I and II, aggregating and
  non-aggregating samples) is being analyzed at
  present.

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• What have we learnt so far from this project?
• Turnover is slower IVD compared to cartilage
• Accumulation of pentosidine is similar in
  collagen from IVD and articular cartilage and is
  higher in A1 preparations
• Mean turnover rate of A1 is between 10-15 years,
  and collagen is over 100 years.
• There is a lot of non-aggregated material (70)
  in IVD A1 compared to 30 in articular cartilage.

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• How does this work relate to other work in the
  project?
• Cells produce the matrix, and the matrix affect
  the function of the cells. The osmotic pressure
  will help us understand the compression
  properties of the disc.

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• What are the final goals?
• To obtain more data on non-aggregated PG
  monomers
• To estimate quantitatively the t1/2 of aggrecan
  in is different forms.
• To understand better the turnover of the
  different constituents of the matrix and their
  function.
• To make measurements of swelling pressure of disc
  of different ages and degree of degeneration, and
  of the isolated PG, and from this data to attempt
  to estimate the contribution of collagen to the
  compressive properties of normal and degenerate
  discs.

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Acknowledgements

• Eve Citron
• Julia Nudelmann (Department of Biomedical Eng.
  Technion)
• Peter Roughley (Shriners Children Hospital,
  Canada)
• Jeroen DeGroot,
• Frits Van der Ham,
• Ruud Bank (Gaubius laboratory, Holland)
• Shirley Ayad

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• Thanks you all !!