Effects of Cell Shape and Position on their Mechanical Environment & Effects of Dynamic Compression on iNOS and IL-1

1. Effects of Cell Shape and Position on their Mechanical Environment& Effects of Dynamic Compression on iNOS and IL-1 gene expression

2. What is the Meniscus? • Two ‘C’ shaped pieces of fibrous articular cartilage • Anchored to the tibia but free to move • Wedge shaped cross section • Blood vessels in outer third • Injuries don’t heal

3. What is the Meniscus?

4. Functions of Meniscus • Distributes stresses in the knee evenly over the tibia • Stabilizes the joint • Absorbs shock

5. What is Mechanotransduction? • Cells adapt surrounding tissue to help the body cope with external forces • Adaptation occurs by biochemical responses of cells to mechanical stimuli • This is how muscles grow bigger and bones get stronger

6. Mechanotransduction in the Meniscus • What is the mechanical environment of the cell? • What is the response of cells to mechanical loading?

7. What is the meniscus made of? • 70% interstitial fluid • Porous meniscal tissue • Collagen fibers arranged circumferentially Collagen fiber orientation

8. Hypotheses • Fluid velocities in meniscal tissue are affected by material properties and strain rate • Cell shape and location affects stresses, strains and fluid velocities within and around cells

9. Aims • To model the stresses, strains and fluid velocities within a meniscal tissue explant • To model and compare the stresses and strains in and around cells of different shapes and at different locations

10. METHODS

11. Computational model • Meniscal tissue explant • Fiber reinforced (using spring elements) • Porous, elastic material

12. Boundary conditions • 5% unconfined compression • Axis of cylinder constrained in horizontal direction • Pore pressure at the free edge is zero

13. Where were the cells placed? • At 1mm from the axis • At 2.5mm from the axis

14. What shapes were tested? • elliptical • circular Elliptical cell showing the cell, cell membrane, a region of tissue around a cell called the pericellular matrix and the meniscus itself or the extracellular matrix

15. RESULTS

16. Fluid velocities change radially Red shows high fluid velocities and blue shows low fluid velocities Fluid velocities um/s

17. Peak values altered by material properties Rate of change of fluid velocities and pressures remained the same. But the maximum velocity and pressure changed.

18. Rate of change affected by strain rate Peak values of fluid velocities and pressures AND the rate of change of fluid velocities and pressures increased as strain rates increased.

19. Fluid velocities are proportional to:

20. The Mechanical Environment of the Cell contour plot of fluid velocity. The abruptly high velocities around the cell are contained within the PCM. um/s

21. Range of Fluid Velocities are Highest in Circular Cells

22. Fluid flow induced shear stress is greater around elliptical cells Circle shows angular measurement around cell. Shear stress is affected by cell shape.

23. Range of Principal Strains are Highest in Circular Cells and change with position

24. Range of Principal Stresses are Highest in Circular Cells. Cell has low stresses.

25. Discussion • Tissue strain accommodated by: • Compression of solid material • Movement of fluid out of the material • Stretch of the spring elements • Stiffer springs & lower permeability reduced radial expansion higher velocities • Stiffer material and higher Poisson’s ratio increased radial expansion  lower fluid velocities

26. Discussion • Fluid velocities and shear stresses were altered by cell shape • Oblate ellipse offers higher resistance to flow • Pronate ellipse offers lower resistance to flow • Position of a cell • Cells closer to a free edge experienced higher velocities around them. Less resistance to fluid flow.

27. Discussion • Strain in the matrix is highest at the axis • Strains in cells decrease at the axis • Cellular strains may be governed by fluid flow or pore pressure. • Stresses are higher in an elliptical cell • Stresses around an elliptical cell are lower. • Shielding effect is diminished in elliptical cells

28. NO and IL-1 • Nitric Oxide (NO) • Highly reactive and short lived • Autocrine or paracrine action • Relaxes smooth muscles, neurotransmitter • Interleukin -1 (IL-1) • Inflammatory catabolic cytokine • Protein • Causes NO production

29. Dynamic Compression & iNOS and IL-1 expression • Compression increases biosynthesis • But compression with IL-1 does not • But compression with IL-1 and without iNOS does increase biosynthesis

30. Hypothesis • Dynamic compression upregulates iNOS via IL-1 upregulation in the mensicus

31. Aim • To measure the change in the amount of iNOS and IL-1 produced by cells of different shapes

32. Method • Compression of 5mm x 6mm cylindrical explants at 0, 5, 10, 15 and 20% • RT PCR to measure the change in iNOS and IL-1 production

33. Results

34. Further Research • Use Real time RT PCR to detect and quantify IL-1 • Block the production of IL-1 and measure the production of iNOS • Correlate the biochemical activity of cells to biochemical output

35. Questions