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

Effects of Cell Shape and Effects of Cell Shape and Position on their Position on their

Mechanical EnvironmentMechanical Environment& Effects of Dynamic & Effects of Dynamic

Compression on iNOS and Compression on iNOS and IL-1 gene expressionIL-1 gene expression

What is the Meniscus?What is the Meniscus?

Two ‘C’ shaped pieces of fibrous Two ‘C’ shaped pieces of fibrous articular cartilagearticular cartilage

Anchored to the tibia but free to Anchored to the tibia but free to movemove

Wedge shaped cross sectionWedge shaped cross section Blood vessels in outer thirdBlood vessels in outer third Injuries don’t healInjuries don’t heal

What is the Meniscus?What is the Meniscus?

Functions of MeniscusFunctions of Meniscus

Distributes stresses in the knee Distributes stresses in the knee evenly over the tibiaevenly over the tibia

Stabilizes the jointStabilizes the joint

Absorbs shockAbsorbs shock

What is Mechanotransduction?What is Mechanotransduction?

Cells adapt surrounding tissue to Cells adapt surrounding tissue to help the body cope with external help the body cope with external forcesforces

Adaptation occurs by biochemical Adaptation occurs by biochemical responses of cells to mechanical responses of cells to mechanical stimulistimuli

This is how muscles grow bigger and This is how muscles grow bigger and bones get strongerbones get stronger

Mechanotransduction in the Mechanotransduction in the MeniscusMeniscus

What is the mechanical environment What is the mechanical environment of the cell?of the cell?

What is the response of cells to What is the response of cells to mechanical loading?mechanical loading?

What is the meniscus made of? What is the meniscus made of?

1.1. 70% interstitial 70% interstitial fluidfluid

2.2. Porous meniscal Porous meniscal tissuetissue

1.1. Collagen fibers Collagen fibers arranged arranged circumferentiallycircumferentially

Collagen fiber orientation

HypothesesHypotheses

1.1. Fluid velocities in meniscal tissue Fluid velocities in meniscal tissue are affected by material properties are affected by material properties and strain rateand strain rate

1.1. Cell shape and location affects Cell shape and location affects stresses, strains and fluid velocities stresses, strains and fluid velocities within and around cellswithin and around cells

AimsAims

To model the stresses, To model the stresses, strains and fluid strains and fluid velocities within a velocities within a meniscal tissue meniscal tissue explantexplant

To model and To model and compare the stresses compare the stresses and strains in and and strains in and around cells of around cells of different shapes and different shapes and at different locationsat different locations

METHODSMETHODS

Computational modelComputational model Meniscal tissue Meniscal tissue

explantexplant Fiber Fiber

reinforced reinforced (using spring (using spring elements)elements)

Porous, elastic Porous, elastic materialmaterial

Boundary conditionsBoundary conditions 5% unconfined 5% unconfined

compressioncompression

Axis of cylinder Axis of cylinder constrained in constrained in horizontal directionhorizontal direction

Pore pressure at Pore pressure at

the free edge is the free edge is zerozero

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

axisaxis

At 2.5mm from the At 2.5mm from the axisaxis

What shapes were tested?What shapes were tested?

ellipticalelliptical

circularcircular

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

RESULTSRESULTS

Fluid velocities change radiallyFluid velocities change radially

Fluid Fluid velocities velocities um/sum/s

Red shows high fluid velocities and blue shows low fluid velocities

Peak values Peak values altered by altered by material material

propertiesproperties

Rate of change of fluid velocities and pressures remained the same. But the maximum velocity and pressure changed.

Rate of Rate of change change

affected by affected by strain ratestrain 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:Fluid velocities are proportional to:

The Mechanical Environment of the The Mechanical Environment of the CellCell

contour plot of fluid velocity. The abruptly high velocities around the cell are contained within the PCM.

um/s

Range of Fluid Velocities are Range of Fluid Velocities are Highest in Circular CellsHighest in Circular Cells

Fluid velocity magnitude range

17.7

5

2.58

09E

-04

16.7

1

3.47

85E

-04

0.65

4.50

E-0

5

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

entire model cell

velo

city

(u

m/s

)

reference

elliptical

at1mm

Fluid flow induced shear stress is Fluid flow induced shear stress is greater around elliptical cellsgreater around elliptical cells

Circle shows angular measurement around cell.

Shear stress is affected by cell shape.

Range of Principal Strains are Range of Principal Strains are Highest in Circular Cells and Highest in Circular Cells and

change with positionchange with positionIn-Plane Strain range

0.81

8

0.80

2

0.81

5

0.55

0

0.89

7

0.80

1

0.82

8

0.82

5

0.43

4

0.32

5 0.42

9

0.43

4

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

entire model PCM MEM CELL

Str

ain

(e/

e) reference

elliptical

at1mm

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

low stresses.low stresses.In-Plane Stress range

0.26

92

0.09

25

0.09

17

0.00

13

0.17

08

0.02

50

0.03

39

0.00

20

0.25

61

0.08

04

0.07

84

0.00

11

0.0

0.1

0.1

0.2

0.2

0.3

0.3

whole model PCM MEM CELL

Str

ess

(MP

a)

reference

elliptical

at1mm

DiscussionDiscussion

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 Stiffer springs & lower permeability reduced radial expansionreduced radial expansion higher higher velocitiesvelocities

Stiffer material and higher Poisson’s ratio Stiffer material and higher Poisson’s ratio increased radial expansion increased radial expansion lower fluid lower fluid velocitiesvelocities

DiscussionDiscussion Fluid velocities and shear stresses Fluid velocities and shear stresses

were altered by cell shapewere altered by cell shape• Oblate ellipse offers higher resistance to Oblate ellipse offers higher resistance to

flowflow• Pronate ellipse offers lower resistance to Pronate ellipse offers lower resistance to

flowflow Position of a cellPosition of a cell

• Cells closer to a free edge experienced Cells closer to a free edge experienced higher velocities around them. Less higher velocities around them. Less resistance to fluid flow.resistance to fluid flow.

DiscussionDiscussion

Strain in the matrix is highest at the axisStrain in the matrix is highest at the axis Strains in cells decrease at the axisStrains in cells decrease at the axis

• Cellular strains may be governed by fluid flow Cellular strains may be governed by fluid flow or pore pressure.or pore pressure.

Stresses are higher in an elliptical cellStresses 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-1NO 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, Relaxes smooth muscles,

neurotransmitter neurotransmitter Interleukin -1 (IL-1)Interleukin -1 (IL-1)

• Inflammatory catabolic cytokineInflammatory catabolic cytokine• ProteinProtein• Causes NO production Causes NO production

Dynamic Compression & iNOS and Dynamic Compression & iNOS and IL-1 expressionIL-1 expression

Compression increases biosynthesisCompression increases biosynthesis But compression with IL-1 does notBut compression with IL-1 does not But compression with IL-1 and But compression with IL-1 and

without iNOS does increase without iNOS does increase biosynthesisbiosynthesis

HypothesisHypothesis

Dynamic compression upregulates Dynamic compression upregulates iNOS via IL-1 upregulation in the iNOS via IL-1 upregulation in the mensicusmensicus

AimAim

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

MethodMethod

Compression of 5mm x 6mm Compression of 5mm x 6mm cylindrical explants at 0, 5, 10, 15 cylindrical explants at 0, 5, 10, 15 and 20%and 20%

RT PCR to measure the change in RT PCR to measure the change in iNOS and IL-1 productioniNOS and IL-1 production

ResultsResults

mean iNOS (n=3)

y = 0.2975x - 0.0985R2 = 0.5208

y = 0.2251x - 0.1123R2 = 0.6186

-0.3

0.2

0.7

1.2

1.7

2.2

2.7

0 0.05 0.1 0.15 0.2strain

inte

nsi

ties

iNO

S/G

AP

DH

TOP BOTTOM Linear (TOP) Linear (BOTTOM)

Further ResearchFurther Research

Use Real time RT PCR to detect and Use Real time RT PCR to detect and quantify IL-1quantify IL-1

Block the production of IL-1 and Block the production of IL-1 and measure the production of iNOSmeasure the production of iNOS

Correlate the biochemical activity of Correlate the biochemical activity of cells to biochemical outputcells to biochemical output

QuestionsQuestions