Mapping Articular Cartilage Biomechanical Properties of Normal & - - PowerPoint PPT Presentation

Mapping Articular Cartilage Biomechanical Properties of Normal & Osteoarthritic Mice Using Indentation

J-F. Lavoie1,2,3 S. Sim1,3 E. Quenneville3 M. Garon3 A. Moreau2,4 M.D. Buschmann1 C.-E. Aubin1,2

• 1. Polytechnique Montreal, Montreal, QC, Canada,

2.Sainte-Justine University Hospital Center, Montreal, QC, Canada, 3. Biomomentum Inc., Laval, QC, Canada 4 Faculty of Medicine, Dept of Biochemistry and Dept of Stomatology, University of Montreal, Montreal, Qc, Canada.

21st Congress of the European Society of Biomechanics 05-08 July 2015

• E. Quenneville and M. Garon are owners of Biomomentum Inc.

J-F. Lavoie works for Biomomentum Inc.

Disclosure Funding

Introduction

Mouse models have unique advantages to study articular cartilage :

• Genetic modification (ex. transgenic)
• Availability of many different strains (some developing OA spontaneously,
• ex. STR/ort)
• Can be used for preclinical studies

Technical limitations:

• Histological assessment is tedious and while it gives important information

(ex. tissue organization), it gives no information on mechanical properties of cartilage.

• The small size of the mouse articular surfaces poses significant challenges

to measure the mechanical properties (ex. knee)

Mechanical assessment

• Mechanical testing of articular cartilage is a useful outcome measure in studies
• f cartilage degeneration and cartilage repair.
• Mechanical testing can be done in different experimental configurations:

Indentation Compression Shear Torsion Tension Bending

Practical advantages of indentation

• Cartilage doesn’t need to be harvested from articular

surface

• Minimal disruption of the articular surface
• Maintains the mechanical environment of the cartilage

layer and its interaction with the subchondral bone

• Testing multiple sites… multiple times!

Indentation requires the compression axis aligned perpendicular to the articular surface.

Pictures from: http://www.kneeclinic.info/

However…

Automated indentation technique

Contact coordinates (x,y,z) of predefined positions and 4 surrounding positions Surface

• rientation (θz)

Normal force/displacement vs time

• Spherical indenter for a new automated indentation mapping
• Multiaxial load cell – uses Fx, Fy and Fz to calculate the perpendicular force
• 3-axis mechanical tester – uses 3 displacement components to provide a perpendicular

displacement based on the surface orientation

Mach-1 v500css

Automated indentation mapping

Right Femoral Condyles Right Tibial Plateau Medial Lateral Instantaneous Modulus (MPa) Human

1 cm 1 cm

Sheep

3.44 ± 1.55 MPa 3.09 ± 2.97 MPa

5 mm 5 mm

Rabbit

3.31 ± 3.38 MPa 3.12 ± 2.44 MPa

Right Left

6.87 ± 3.70 MPa 5.88 ± 3.51 MPa

0.5 1 10 20

Indentation mapping - human

Instantaneous modulus shows degradation patterns that often extend beyond the visual lesion boundaries (red circles).

• Large variation within the medial and

lateral compartment of femoral condyle and tibial plateau.

• The cartilage is stiffer in regions covered

by the meniscus while a softer cartilage is

• bserved on the rest of the surface.
• Therefore the spatial location on the

cartilage surface must be considered when comparing the effect of treatments.

8.9 ± 4.0 MPa 1.8 ± 0.3 MPa Sheep (plateau) Rabbit (Condyles)

Automated indentation mapping

Rationale

Considering:

• That it is important to consider the region when analysing the

mechanical properties of cartilage;

• That indentation does not require to remove the cartilage from the

joint surface;

• That automated indentation can be performed on curved surface

without damaging the cartilage. We hypothesized that by using this automated indentation technique and by reducing the size of the indenter, it will be possible to map the mechanical properties of mouse knee cartilage.

Objectives

• First objective of this study was to scale down this

indentation technique to map the mechanical properties

• f the articular surfaces in murine knees.
• Second objective is to identify early alterations of the

articular cartilage of a mouse strain (STR/ort) that spontaneously develops osteoarthritis (OA).

Mechanical tester

Mach-1 v500css

• 3-axis mechanical tester

(Mach-1 v500css from Biomomentum)

• Multiaxial load cell

(force resolution: Fz = 3.5 mN and Fx = Fy = 2.5 mN)

• Motorized stages

(resolution vertical 0.1 um and 0.5 um horizontal)

• Spherical indenter

(glass bead with r = 0.175 mm)

Spherical Indenter

Male Balb/c – Healthy control 12 weeks old: N = 3 15 weeks old: N = 2 Do not spontaneously develop OA Male STR/ort – OA mouse 12 weeks old: N = 3 15 weeks old: N = 2 Spontaneously develop OA in the medial compartment of their knee with higher prevalence in the left joint (Walton, M., J. Pathol. 1977)

Animal model

STR/ort – OA mouse Balb/c – Healthy control

Images from : http://www.crj.co.jp/ and http://jaxmice.jax.org/

STR/ort

• Spontaneously develops osteoarthritis (closely resemble human OA)
• Causes of the induced desease are still unclear:
• Decrease in strength of anterior cruciate ligament?
• Patella subluxation?
• Combination of genetic factors?
• It first occurs at the interface of the cruciate ligament and medial tibial

articular cartilage then damage evolved in the medial plateau. Medial femoral condyle is also affected.

• The lateral cartilage is usually spared or mildly affected.

Mason 2001, Osteoarthritis and Cartilage 9:85-91

http://www.crj.co.jp/

STR/ort

• Mild histopathological lesions are often present at 10 weeks of age
• By 20 weeks, most animal have lesions in one or both knees.
• Damage can range from mild erosion to complete loss of cartilage with

exposed bone.

• Higher incidence in male (85% of all male develop OA in medial tibial

plateau)

• Higher incidence in the left joint.

Mason 2001, Osteoarthritis and Cartilage 9:85-91

http://www.crj.co.jp/

Methods

Dissect Tibia & femurs (age of 12 and 15 weeks)

Mouse images from : http://www.crj.co.jp/ and http://jaxmice.jax.org/

Left Tibial Plateau Left Femoral Condyles

Automated Indentation Mapping Tissue harvesting

Methods

Automated Indentation Mapping

3 mm

Reported is the structural stiffness (N/mm): Load (N) @ 0.010 mm 0.010 (mm) Indentation velocity: 30 mm/s

SS = Automated Indentation Mapping

• Paraffin embedded
• Serially sectioned (5mm thick)
• Safranin-O staining

Histological assessment

Maps of average structural stiffness on control and OA mouse left knee cartilage surfaces

Control OA Femoral Condyles

Anterior

Medial

12 weeks (N = 3) 15 weeks (N = 2)

Posterior Anterior Posterior

Tibial Plateaus

Lateral

Structural Stiffness (N/mm)

14 12 10 8 6 4 2 1

Control OA

I II III IV Zones

Femoral Condyles

2 4 6 8 10 12 2 4 6 8 10 12

2 4 6 8 10 12 I II III IV

Tibial Plateau

12 weeks

N = 3 per group

15 weeks

N = 2 per group

2 4 6 8 10 12 Structural Stiffness (N/mm)

Healthy OA

* * * *

Medial Lateral

Medial Lateral

I II III IV

Reduced structural stiffness is measured on medial side of OA mice joint

Error bar = SE

I II III IV I II III IV I II III IV

Medial Lateral

* = p < 0.05

Comparison of the Structural Stiffness and histology

Structural stiffness map

Zone: I II

100 mm Pixels (x axis of the map) 5 10 15

Structural Stiffness (N/mm)

Pixels (x axis of the map) 100 mm

Zone: I II

5 10 15

OA (15 weeks)

Medial Lateral

I II Healthy (15 weeks)

Medial Lateral

I II

Structural Stiffness (N/mm)

Saf-O Saf-O

Zone: Zone:

Conclusions

• Mechanical properties can be mapped on entire articular surfaces
• f tiny mouse joints.
• Structural stiffness maps show similar distribution patterns to

those previously observed for the stifle joints of larger species, with stiffer cartilage in the region covered by the meniscus.

• Decrease of the average structural stiffness for the medial

compartment of the OA-developing mouse is in agreement with the literature (Walton M. J. Pathol. 1977)

• The decrease in structural stiffness of the medial compartment is

more obvious than the decrease in proteoglycan staining.

Significance

• Automated Indentation Mapping can generate high resolution

map to help identify the location of affected cartilage (ex. in OA).

• It is rapid (about 1 minute/ position)
• This characterization technique can reveal itself very useful in

studies on the effect of age, gene modifications (transgenic- models) and disease (OA models) by reliably measuring the biomechanical properties of entire articular surfaces.

Acknowledgement

• We acknowledge the technical

contributions of:

• Anik Chevrier
• Geneviève Picard
• Saddallah Bouhanik
• Funding provided by :