Title: The Effects of Risedronate on Molecular Expression of Bone Cytokines in Periarticular Tissues of an
1The 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
2Osteoarthritis 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
3RANK/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
4Osteoprotegerin (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
5OPG/RANKL Ratio Excess Inhibitor
(Adapted from Dr. Susan Ott)
No Osteoclastogenesis
6OPG/RANKL Ratio Excess Ligand
(Adapted from Dr. Susan Ott)
Osteoclastogenesis
7Risedronate
- 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
8Purpose
To examine the effects of Risedronate
antiresorptive therapy on mRNA expression of OPG
in periarticular tissues after ACL trauma
9Hypothesis
Antiresorptive therapy will perturb osteoclast
function in periarticular bone after ACL-rupture,
and will alter OPG mRNA expression
10Materials 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
11Materials and Methods
MCL
MFC
TIBIA
FEMUR
Lateral Meniscus
LCL
Samples harvested from 3 locations
12Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
13Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
14Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
Sample 3 ligament midsubstance
15Materials and Methods
- Semi-quantitative RT-PCR (reverse transcription-
polymerase chain reaction) was performed to
amplify extracted mRNA products
16Semi-quantitative RT-PCR
Tissues
snap freeze and powder
TRIspin RNA isolation
AAAAAAAAAAAAA
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.
17Results
OPG Expression Levels Relative to Bactin
18Results
OPG Expression Levels Relative to Bactin
19Results
OPG Expression Levels Relative to Bactin
20Discussion
- 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
21Limitations
- Small Sample Size
- Calf Thymus rRNA standard (Sigma) used in RNA
quantification was found to be approximately 5
fold less concentrated when compared to a second
control standard (Sigma) - Implication Only 1/5 of stated RNA actually
present
22Future Directions Whats Next?
- Focus on localizing differential expression of
OPG by developing a DIG labeled probe for in situ
hybridization
23Acknowledgements
I would like to acknowledge Mike Doschak,
Jeremy LaMothe, Carol Reno, the Hart Lab
Group, and Dr. R. Bray for their contributions.