D.A.Mirea, A-M Trunfio-Sfarghiu, C.I.Matei, B.Munteanu, A.Piednoir, J.P. Rieu, M.G. Blanchin, Y. Berthier

38th LEEDS-LYON SYMPOSIUM ON TRIBOLOGY Lyon, 2011

Synovial fluid’s physicochemical analysis:

Role of the interactions between lubricant’s

biological components

Remarkable tribological performances:

Life expectancy over 80 years! Hydrodynamic lubrication- continuous fluid film hypothesis -

low wear Dowson et all (1992)

1 µm

Watanabe M. et all.(2000)

Discontinuities highlighted

2/16

Hills et all.(2002)

Which theory of lubrication for healthy joint is valid?

years 2000

Context

The ‘50

Lubricant volume Lubricant interface

Physiologic serum

+

Glucides:Hyaluronic acid 3g/l

Proteines: Albumin 18 g/lGlobulin 2 g/l

Lipids 3 g/l

+

+

Molecular chain

L ~ 12 000 nm

Globular protein

8 nm3 nm

Glycoproteic gelAlbumin

Hyaluronic acid

2,5 nm

0,5 nm

Lipid bilayer

Origin of discontinuities:  - Miscibility between the gel glycoprotein and lipid bilayers?

Non miscible liquids

3/16

Oates K.M.N. et all, 2005

Context: origin of discontinuitiesLubricant volume : composition

4/16

Structure viscosity Structure viscosity

What is the in vivo reality in volume?

PASQUALI-RONCHETTI I. et al., 1997, Journal of structural biology, 1997, vol 120, p. 1–10

CRESCENZIA V., et al., 2004, Colloids and Surfaces, 2004, vol 245, p. 133–135.

Unilamellar liposomes

= tens of nm

Liposomes and multilamellar tubes

j = hundreds nm

L = few µm

Context: origin of discontinuitiesLubrifiant volume : structure

1 2

Ex-vivo

0,5µm 0,5µm

Synthetic dense brushes

μ~0.0005

But not with lubricin or non dense brushes

5/16

Swann (1972]

L ~ 200 nm

- - -- - - -- -

Raviv Klein [2002, 2003]

Israelachvili [2007]

50-100 nm

Hydrophilic headHydrophobic tails

Stacks of membranes

Reduce μ

Polyelectrolyte concentrations (200 µg/ml)

SAPL concentration (100 – 300 µg/ml)

Biological surface (121cm2 – knee articulation)

Lubricin (polyelectrolyte)

Surface-active phospholipid (SAPL)

[Hills A.B., 1998]

3 - 5 nm

Polyelectrolytes can not form the dense molecular brush!

The SAPLs can form 3 to 7 stacks of bilayers

What is their in vivo interaction?

Context: origin of discontinuitiesInterface : two types of molecular

layers1

2

3

Trunfio-Sfarghiu [2007]

Objectives

1. Identifying the origin of discontinuities in the in vivo synovial fluid volume

2. Analyzing the intermolecular interactions in order to understand what molecular component is

responsible for the discontinuities in volume and interface

3. Analyzing the effects of tribological identified discontinuities in order to propose a mechanism for

lubrication

6/16

1. Identifying the origin of discontinuities in lubricant

volume TEM analysis – negative staining

SEM - Wet STEM analysis

View in dry state on continuous carbon film

Sample+ (APT) colorant

High resolution technique which requires a dilution of 80%

Offers a lower resolution but it

eliminates the need to dilute the samples

7/17

Methodology

Rat Knee samples

Healthy synovial liquid

samples

1

2

Wet STEM analysis

Undiluted synovial fluid visualization during drying

100 nm

Multilamellar vesicular structures surrounded by

3 to 7 lipid bilayers.

The size differs depending on dilution bursting the

vesicles

Vesicles of a few hundred nanometers, which fusion during drying

TEM analysis

8/16

1. Identifying the origin of discontinuities in lubricant

volume Results1

2

2. Analyzing the intermolecular interactions in the synovial fluid

Methodology: Atomic force spectroscopy

AFM cantileverCMA – a “separator” in order to keep the

molecular configuration in

solution

1. Hyaluronic acid

2. Globular proteins (BSA, globulin )

8 nm3 nm

3. Lubricin

Substances of interest

5 nm

Lipid bilayer

Intermolecular affinity measures

9/16

Analyzing the intermolecular interactions in the synovial fluid

10/16Z Piezo Displacement (nm)

Force Measurement

Intermolecular adhesion

AFM Results

11/17

Substance of Interest

Adhesion Force Curve

Adhesion Histogram

CMA

Hyaluronic Acid

Lubricin

Albumin

γ-Globulin

0,002nN 9%

1.2 nN 88%

0,6 nN 82%

0,07nN 24%

0,05nN 25%

1.6 nN

11/16

Tribological Analysis

Normal pressure: 0,3 – 1 MPa (similar to knee)

Speed : 0,1 – 1 mm/s (no hydrodynamic phenomena)

x

Moving table (v = 0.6 mm/s)

Fluorescence Microscope

Foucault sensor

Measurement of T

Normal load(NL = 2.5N)

Flexible lames

Flurescent Lipid

Bilayers

Hydrogel ~ few nm RMS

Glass0.2 nm RMS

Friction coefficient (f) = T/N

12/16

1. Physiological serum salt

4. Lipid vesicles containing

glycoproteic gel

2. Lubricin solution 200 µg/ml

3. Glycoproteic gel: solution HA

3mg/ml + BSA 18mg/ml + Globulin 2mg/ml

1

2

Tribological resultsLubricant Fluorescence

microscopyFriction

coefficientVelocity

accommodation

13/16

0,008

0,035

0,1

0,008

80μm

Physiologic salt

solution

Lubricinsolution

Glycoproteicgel

Lipid vesicles

Conclusions & interpretation

LubricinCartilage

Hyaluronic acid + seric

proteins

Lipid layers

Hyaluronic acid (HA)High affinity for lipid

Seric proteins – low adhesion lipid and reticulation with HA

glycoproteic gelCOF non included glycoproteic

gel COF glycoproteic gel included

HA and seric proteins

remain inside the vesicles

14/16

lipid multilamellar

vesicles0.1µ

m

VOLUME

Presence of lipid

multilamellar layers

Hills A.B., Internal Medicine

Journal 2002

INTERFACE

Lubricin- adhesion and

COF on lipid- adhesion on cartilage (Rhee D.K.,

2005)

Lubricin fixes the

lipid layers on

the cartilage

Conclusions & interpretation

Hyaluronic acid + seric proteins

Lipid multilayers (3-7 bilayers)

LubricinCartilageLipid layers

2 µm

Discontinuous structure of synovial liquid

Sliding location betwwen lipid

bilayers

c.f = 0.008

15/16

Lipid vesicular network in lubricant

volume2 µm

Hyaluronic acid + seric

proteins

Lipid multilayers

VOLUME INTERFACE

Thank you for your attention!