A very complex study unifying three different approaches to study the synovial fluid.

1. Synovial fluid’s
physicochemical analysis:
Role of the interactions
between lubricant’s
biological components
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

2. Context
Remarkable tribological performances:
Life expectancy over 80 years!
Hydrodynamic lubrication
- continuous fluid film hypothesis -
low wear
Dowson et all (1992)
years 2000
Discontinuities highlighted
Lubricant volume Lubricant interface
1 µm
Watanabe M. et all.(2000) Hills et all.(2002)
Which theory of lubrication for
healthy joint is valid?
The ‘50
2/16

3. Context: origin of discontinuities
Lubricant volume : composition
Physiologic serum
+ Molecular chain
Hyaluronic
L ~ 12 000 nm acid
Glucides:
Hyaluronic acid 3g/l
+
Proteines: 3 nm
8 nm
Albumin 18 g/l Albumin
Globulin 2 g/l Glycoproteic gel Non miscible
Globular protein
Oates K.M.N. et all, 2005 liquids
+
0,5 nm
Lipids 3 g/l
2,5 nm
Lipid bilayer
Origin of discontinuities:
- Miscibility between the gel glycoprotein and lipid bilayers?
3/16

4. Context: origin of discontinuities
Lubrifiant volume : structure
PASQUALI-RONCHETTI I. et al., 1997, Journal of structural biology, 1997, vol 120, p. 1–10
Ex-vivo
CRESCENZIA V., et al., 2004, Colloids and Surfaces, 2004, vol 245, p. 133–135.
1 2
0,5µm 0,5µm
Liposomes and
multilamellar tubes
Unilamellar liposomes = hundreds nm
= tens of nm L = few µm
Structure viscosity Structure viscosity
What is the in vivo reality in volume?
4/16

5. Context: origin of discontinuities
Interface : two types of molecular layers
1 Lubricin (polyelectrolyte)
3
--
-
-
-
-
Swann (1972]
- Polyelectrolyte concentrations
-
-
Synthetic dense
L ~ 200 nm
brushes
(200 µg/ml)
μ~0.0005 SAPL concentration
Raviv Klein [2002, 2003]
(100 – 300 µg/ml)
50-100 nm
But not with lubricin or
non dense brushes Biological surface
Israelachvili [2007]
(121cm2 – knee articulation)
2 Surface-active phospholipid (SAPL) Polyelectrolytes can not form
the dense molecular brush!
[Hills A.B., 1998]
The SAPLs can form 3 to 7
stacks of bilayers
Hydrophobic tails Hydrophilic head
3 - 5 nm
Stacks of What is their in vivo
Reduce μ
membranes interaction?
Trunfio-Sfarghiu [2007]
5/16

6. 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

7. 1. Identifying the origin of
discontinuities in lubricant volume
Methodology
Healthy
1 TEM analysis – negative staining
synovial liquid
Sample+ (APT)
samples colorant
View in dry state on continuous
carbon film
High resolution technique which
Rat Knee samples requires a dilution of 80%
2 SEM - Wet STEM analysis
Offers a lower
resolution but it
eliminates the need to
dilute the samples
7/17

8. 1. Identifying the origin of
discontinuities in lubricant volume
Results
1 TEM analysis
Multilamellar vesicular
structures surrounded by
3 to 7 lipid bilayers.
The size differs depending
on dilution bursting the
vesicles
100 nm
2 Wet STEM analysis
Vesicles of a
few hundred
nanometers, w
hich fusion
during drying
Undiluted synovial fluid visualization during drying 8/16

9. 2. Analyzing the intermolecular
interactions in the synovial fluid
Methodology: Atomic force spectroscopy
Substances of interest
1. Hyaluronic acid AFM cantilever
CMA – a “separator”
in order to keep the
molecular
configuration in
solution
2. Globular proteins (BSA, globulin )
Intermolecular
affinity measures
3 nm
8 nm 5 nm
3. Lubricin
Lipid bilayer
9/16

10. Analyzing the intermolecular
interactions in the synovial fluid
Force
Measurement
Intermolecular adhesion
Z Piezo Displacement (nm)
10/16

11. AFM Results
Substance of Adhesion Adhesion
Interest Force Curve Histogram
CMA 0,002nN 9%
Hyaluronic Acid
1.2 nN 1.6 nN 88%
Lubricin
0,6 nN 82%
11/17
Albumin 24%
0,07nN
γ-Globulin 25%
0,05nN
11/16

12. Tribological Analysis
Normal load Fluorescence
(NL = 2.5N) Microscope 1
1. Physiological serum salt
Glass
0.2 nm RMS
2. Lubricin solution
Flurescent Lipid 200 µg/ml
Bilayers
3. Glycoproteic gel: solution
Foucault sensor Hydrogel ~ HA
few nm Flexible
Measurement of RMS 3mg/ml + BSA 18mg/ml +
lames
T
Globulin 2mg/ml
x
Moving table (v = 0.6 mm/s) 4. Lipid vesicles
containing
Friction coefficient (f) = T/N glycoproteic gel
2 Normal pressure: 0,3 – 1 MPa (similar to knee)
Speed : 0,1 – 1 mm/s (no hydrodynamic phenomena)
12/16

13. Tribological results
Lubricant Fluorescence Friction Velocity
microscopy coefficient accommodation
Physiologic
salt 0,008
solution
Lubricin
solution 0,035
80μm
Glycoproteic
gel 0,1
Lipid vesicles
0,008
13/
16

14. Conclusions & interpretation
VOLUME INTERFACE
lipid multilamellar Presence of lipid Hills
A.B., Internal
vesicles multilamellar Medicine
layers Journal 2002
0.1µm
Lubricin
fixes the Lubricin
Hyaluronic acid (HA) HA and seric lipid layers - adhesion and
High affinity for lipid proteins remain
Seric proteins – low adhesion inside the
on the COF on lipid
cartilage
lipid and reticulation with HA vesicles - adhesion on
glycoproteic gel cartilage (Rhee D.K., 2005)
COF non included glycoproteic gel
COF glycoproteic gel included
Hyaluronic acid +
seric proteins
Cartilage
Lipid layers Lubricin
14/16

15. Conclusions & interpretation
VOLUME INTERFACE
Lipid multilayers (3-7 bilayers)
Hyaluronic acid + seric
proteins
Lipid layers Cartilage
Lubricin
Discontinuous structure of
Lipid vesicular
synovial liquid
network in lubricant
volume
2 µm
Lipid multilayers
Sliding location
betwwen lipid
bilayers
c.f = 0.008
Hyaluronic acid
+ seric proteins 2 µm
15/16

16. Thank you for your
attention!