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

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Presentation transcript:

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

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

What is the Meniscus?

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

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

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

What is the meniscus made of? 1. 70% interstitial fluid 2. Porous meniscal tissue 1. Collagen fibers arranged circumferentially Collagen fiber orientation

Hypotheses 1. Fluid velocities in meniscal tissue are affected by material properties and strain rate 1. Cell shape and location affects stresses, strains and fluid velocities within and around cells

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

METHODS

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

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

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

What shapes were tested? elliptical elliptical circular 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

RESULTS

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

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

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.

Fluid velocities are proportional to:

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

Range of Fluid Velocities are Highest in Circular Cells

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

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

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

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

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

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

NO and IL-1 Nitric Oxide (NO) Nitric Oxide (NO) Highly reactive and short livedHighly reactive and short lived Autocrine or paracrine actionAutocrine or paracrine action Relaxes smooth muscles, neurotransmitterRelaxes smooth muscles, neurotransmitter Interleukin -1 (IL-1) Interleukin -1 (IL-1) Inflammatory catabolic cytokineInflammatory catabolic cytokine ProteinProtein Causes NO productionCauses NO production

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

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

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

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

Results

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

Questions