Niccole Germscheid Faculty of Science

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a-Smooth Muscle Actin Expression in Posterior
Joint Capsules in Elbow Contractures

• Niccole Germscheid Faculty of Science
• Faculty Mentor Dr. K. Hildebrand

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INTRODUCTION

• Joint injuries
• Post-traumatic elbow contractures
• Contracture a loss of joint motion secondary to
  a permanent shortening of muscle, ligament, or
  scar tissue.
• Limit the function (range of motion) of an
  extremity.

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INTRODUCTION

• Joint Injuries
• Treatment Results
• Early joint motion can lessen the severity of the
  contracture.
• 5 of all elbow injuries result in a complete
  loss of motion. (Cooney III, 1993)
• New treatment is needed.

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INTRODUCTION

• Joint Injuries
• Joint Capsule
• Key structure that limits joint motion.
• Changes occur in the extracellular matrix (ECM)
  and very little is known about these changes.

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INTRODUCTION
Joint Capsule
Column Morrey JBJS-A 1998
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INTRODUCTION

• Joint Injuries
• Joint Capsule
• Myofibroblasts have been associated with several
  human conditions characterized by contractures
  and fibrosis.
• Dupuytrens contracture of the hand and frozen
  shoulder have an association with myofibroblast
  proliferation. (Berndt, 1994 and Bunker, 1995)

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INTRODUCTION

• Joint Capsule
• Myofibroblasts
• Specialized contractile fibroblast-like cells.
• Play a key role in the synthesis of the ECM and
  force generation.
• Participate in the contraction of granulation
  tissue in wound healing.

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INTRODUCTION

• Joint Capsule
• ?-Smooth Muscle Actin (?-SMA)
• Marker for myofibroblasts
• Contractile protein
• Found in smooth muscle

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INTRODUCTION

• Previous Studies
• Dr. Hildebrands lab
• Analyzed the anterior joint capsules of human
  post-traumatic elbow contractures.
• CONCLUSIONS
• Increase in myofibroblast numbers
• Increase in ?-SMA expression

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PURPOSE

• To investigate ?-SMA mRNA expressions of
  posterior joint capsules in patients with
  post-traumatic joint contractures.

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HYPOTHESIS

• The ?-SMA mRNA expression will be elevated in
  posterior joint capsules obtained from patients
  with post-traumatic contractures when compared to
  similar tissue obtained from elbow joints free of
  contractures.

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METHODS

• Experimental Group
• Twelve joint capsules obtained from patients with
  post-traumatic joint contractures.
• Control Group
• Ten joint capsules obtained from organ donors
  that were free of joint contractures.

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METHODS
Mean Standard Deviation
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METHODS

• Semi-quantitative reverse transcription-polymeras
  e chain reaction (RT-PCR) was used to evaluate
  the mRNA expression.

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METHODS

• Semi-quantitative RT-PCR
• Tissues
• TRI-spin method was used to isolate RNA
• Freeze tissue and powder
• Reverse Transcription
• Total RNA
• Random Primers
• Polymerase Chain Reaction
• cDNA
• Specific Primers

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METHODS

• Analysis
• Gel Electrophoresis
• Agarose gel stained with EtBr
• Photographed
• Optical density measurements
• Densitometric scanning of negatives
• Ratio of primer/GAPDH
• Statistics
• t-test (plt0.05)

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METHODS
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RESULTS
(A)
591bp
Contracture
Control
(B)
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RESULTS
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RESULTS
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RESULTS
(A)
500bp
(B)
500bp
Contracture
Control
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DISCUSSION

• Significant increase in ?-SMA expression in
  contracture specimens compared to specimens free
  of contractures.
• ?-SMA is one of the most reliable markers for
  myofibroblasts.
• Myofibroblast proliferation may be occurring
  within the ECM.
• Blood vessel walls contain smooth muscle.

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DISCUSSION

• Collagen type I
• Synthesized and secreted by myofibroblasts
• TGF-?1
• Stimulates myofibroblast differentiation
• Induces the synthesis of ?-SMA and collagen type
  I

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DISCUSSION

• Fibronectin
• Non-significant difference in Fibronectin
  expression.
• Cellular metabolisms of the genes were not all
  up-regulated.

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CONCLUSION

• There is an increase in the expression of ?-SMA
  in the posterior joint capsule of human elbows
  with post-traumatic joint contractures compared
  to the joint capsules of organ donors which were
  free of contractures.

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ONGOING RESEARCH

• Immunohistochemical studies
• Determine whether ?-SMA expression is due to
  myofibroblasts or blood vessels.
• Determine myofibroblast numbers.
• Western Blot Analysis
• Determine the protein levels of ?-SMA
• Comparison study
• Compare the results obtained from the anterior
  joint capsules with those obtained from the
  posterior joint capsules.

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ACKNOWLEDGEMENTS

• Dr. K. Hildebrand
• Mei Zhang
• Markin-Flanagan Research Grant