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

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3- ACLX Risedronate-dosed rabbits- 6 weeks post-surgery. Research Animal: Female New Zealand White Rabbits. FEMUR. TIBIA. MFC. MCL. LCL. Lateral Meniscus ... – PowerPoint PPT presentation

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


1
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
2
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

3
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

4
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

5
OPG/RANKL Ratio Excess Inhibitor
(Adapted from Dr. Susan Ott)
No Osteoclastogenesis
6
OPG/RANKL Ratio Excess Ligand
(Adapted from Dr. Susan Ott)
Osteoclastogenesis
7
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
8
Purpose
To examine the effects of Risedronate
antiresorptive therapy on mRNA expression of OPG
in periarticular tissues after ACL trauma
9
Hypothesis
Antiresorptive therapy will perturb osteoclast
function in periarticular bone after ACL-rupture,
and will alter OPG mRNA expression
10
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
11
Materials and Methods
MCL
MFC
TIBIA
FEMUR
Lateral Meniscus
LCL
Samples harvested from 3 locations
12
Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
13
Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
14
Sample Harvest
TIBIA
FEMUR
Sample 1 cancellous bone core
Sample 2 ligament insertion (bone ligament)
Sample 3 ligament midsubstance
15
Materials and Methods
  • Semi-quantitative RT-PCR (reverse transcription-
    polymerase chain reaction) was performed to
    amplify extracted mRNA products

16
Semi-quantitative RT-PCR
Tissues
snap freeze and powder
TRIspin RNA isolation

AAAAAAAAAAAAA
mRNA
Total RNA
AAAAAAAAAAAAA
AAAAAAAAAAAAA
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.
17
Results
OPG Expression Levels Relative to Bactin
18
Results
OPG Expression Levels Relative to Bactin
19
Results
OPG Expression Levels Relative to Bactin
20
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

21
Limitations
  • 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

22
Future Directions Whats Next?
  • Focus on localizing differential expression of
    OPG by developing a DIG labeled probe for in situ
    hybridization

23
Acknowledgements
I would like to acknowledge Mike Doschak,
Jeremy LaMothe, Carol Reno, the Hart Lab
Group, and Dr. R. Bray for their contributions.
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