The Effects of Risedronate on Molecular Expression of Bone Cytokines in Periarticular Tissues of an

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The Effects of Risedronate on Molecular
Expression of Bone Cytokines in Periarticular
Tissues of an Osteoarthritic Model
Caeley Lorincz, Cathy Huculak, Dr.R. Zernicke,
McCaig Centre for Joint Injury and Arthritis
Research
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Osteoarthritis Degenerative Disease

• Related to joint wear and tear with age, abnormal
  loading
• Characterized by degradation of joint cartilage
• Affects most periarticular tissues such as
• Ligament
• Meniscus
• Synovium
• Subchondral bone

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RANK/RANKL/OPG Cytokine System

• Responsible for regulation of osteoclast cell
  activity

Receptor Activator of Nuclear Factor Kappa B
Ligand (RANKL)-

• Binds with receptor RANK to promote pre-cursor
  osteoclast proliferation and differentiation
• Inhibits apoptosis of mature osteoclasts
• RESULT Increased number and activity of
  osteoclast cells

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Osteoprotegerin (OPG)-

• Soluble decoy receptor for RANKL
• Neutralizes biological actions of RANKL
• RESULT Decreased osteoclast activation

Receptor Activator of Nuclear Factor Kappa B
(RANK)-

• Transmembrane receptor
• Located primarily on osteoclast precursor cells

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OPG/RANKL Ratio Excess Inhibitor
(Adapted from Dr. Susan Ott)
No Osteoclastogenesis
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OPG/RANKL Ratio Excess Ligand
(Adapted from Dr. Susan Ott)
Osteoclastogenesis
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Risedronate

• BisphosphonatesNon-hormonal antiresorptive
  agents
• High affinity for calcium and therefore adsorb to
  bone mineral in vivo
• Selectively delivers bisphosphonate to sites of
  active bone remodeling
• Are internalized by active osteoclasts, causing
  injury by suggested toxic effects
• Rogers et al., 2000
• Expected

Decreased osteoclast activity
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Purpose
To examine the effects of Risedronate
antiresorptive therapy on mRNA expression of OPG
in periarticular tissues after ACL trauma
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Hypothesis
Antiresorptive therapy will perturb osteoclast
function in periarticular bone after ACL-rupture,
and will alter OPG mRNA expression
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Materials and Methods
Research Animal Female New Zealand White Rabbits
3 cohorts 1- Control rabbits 2- ACL transected
(ACLX) untreated rabbits- 6 weeks post-surgery 3-
ACLX Risedronate-dosed rabbits- 6 weeks
post-surgery
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Materials and Methods
MCL
MFC
TIBIA
FEMUR
Lateral Meniscus
LCL
Samples harvested from 3 locations
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Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
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Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
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Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
Sample 3 ligament midsubstance
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Materials and Methods

• Semi-quantitative RT-PCR (reverse transcription-
  polymerase chain reaction) was performed to
  amplify extracted mRNA products

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Semi-quantitative RT-PCR
Tissues
snap freeze and powder
TRIspin RNA isolation

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mRNA
Total RNA
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RANDOM PRIMERS
Reverse transcription
cDNA
Polymerase chain reaction (PCR)
SPECIFIC PRIMERS
Agarose Gel Electrophoresis EtBr staining
Normalize to internal control b-actin
Modified from Biotechniques. 1997
Jun22(6)1082-6.
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Results
OPG Expression Levels Relative to b-actin
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Results
OPG Expression Levels Relative to b-actin
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Results
OPG Expression Levels Relative to b-actin
OPG/bactin Ratio
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Discussion

• Increased OPG levels observed in ACLX untreated
  bone core samples could be an acute cellular
  adaptation to protect bone following injury

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Discussion

• Increased OPG levels observed in ACLX untreated
  bone core samples could be an acute cellular
  adaptation to protect bone following injury
• MCL expressing inhibitor- may be involved in the
  regulation of adjacent bone

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Discussion

• Increased OPG levels observed in ACLX untreated
  bone core samples could be an acute cellular
  adaptation to protect bone following injury
• MCL expressing inhibitor- may be involved in the
  regulation of adjacent bone
• Decreased OPG levels in untreated ACLX
  LCL-insertion could predispose bone at insertions
  to increased remodeling

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Ongoing Work

• Increase sample number for each cohort
• Focus on localizing differential expression of
  OPG by developing an RNA DIG labeled probe for in
  situ hybridization

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Acknowledgements
I would like to acknowledge Mike Doschak,
Jeremy LaMothe, Carol Reno, the Hart Lab
Group, Dr. R. Bray, and The Markin-Flanagen
Studentship for their contributions to this
study