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

Mapping Articular Cartilage Biomechanical Properties of Normal & Osteoarthritic Mice Using Indentation J-F. Lavoie 1,2,3 S. Sim 1,3 E. Quenneville 3 M. Garon 3 A. Moreau 2,4 M.D. Buschmann 1 C.-E. Aubin 1,2 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. 21 st Congress of the European Society of Biomechanics 05-08 July 2015

Disclosure E. Quenneville and M. Garon are owners of Biomomentum Inc. J-F. Lavoie works for Biomomentum Inc. 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 of cartilage degeneration and cartilage repair. • Mechanical testing can be done in different experimental configurations: Indentation Compression Bending Shear Tension Torsion

Practical advantages of indentation o Cartilage doesn’t need to be harvested from articular surface o Minimal disruption of the articular surface o Maintains the mechanical environment of the cartilage layer and its interaction with the subchondral bone o Testing multiple sites… multiple times! However … Indentation requires the compression axis aligned perpendicular to the articular surface. Pictures from: http://www.kneeclinic.info/

Automated indentation technique  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 Contact coordinates Normal (x,y,z) of predefined Surface force/displacement Mach-1 v500css orientation ( θ z ) positions and 4 vs time surrounding positions

Automated indentation mapping Instantaneous Modulus (MPa) Human Rabbit Sheep 3.31 ± 3.38 MPa 3.44 ± 1.55 MPa Condyles Femoral Right Lateral Medial 5 mm 1 cm 3.12 ± 2.44 MPa 3.09 ± 2.97 MPa 6.87 ± 3.70 MPa 5.88 ± 3.51 MPa Plateau Right Left Tibial Right 20 0.5 1 10 5 mm 1 cm

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

Automated indentation mapping  Large variation within the medial and Sheep lateral compartment of femoral condyle (plateau) and tibial plateau.  The cartilage is stiffer in regions covered by the meniscus while a softer cartilage is observed on the rest of the surface. Rabbit  (Condyles) Therefore the spatial location on the cartilage surface must be considered 1.8 ± 0.3 MPa when comparing the effect of treatments. 8.9 ± 4.0 MPa

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 of 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 • 3-axis mechanical tester (Mach-1 v500css from Biomomentum) • Multiaxial load cell (force resolution: F z = 3.5 mN and F x = F y = 2.5 mN) Mach-1 v500css • Motorized stages (resolution vertical 0.1 um and 0.5 um horizontal) • Spherical indenter (glass bead with r = 0.175 mm) Spherical Indenter

Animal model 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) STR/ort – OA mouse Male Balb/c – Healthy control 12 weeks old: N = 3 15 weeks old: N = 2 Do not spontaneously develop OA Balb/c – Healthy control Images from : http://www.crj.co.jp/ and http://jaxmice.jax.org/

STR/ort http://www.crj.co.jp/ • Spontaneously develops osteoarthritis (closely resemble human OA) • Causes of the induced desease are still unclear: o Decrease in strength of anterior cruciate ligament? o Patella subluxation? o 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

STR/ort http://www.crj.co.jp/ • 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

Methods Tissue harvesting Automated Indentation Mapping Left Tibial Plateau Left Femoral Condyles Dissect Tibia & femurs (age of 12 and 15 weeks) Mouse images from : http://www.crj.co.jp/ and http://jaxmice.jax.org/

Methods Automated Indentation Mapping Histological assessment -Paraffin embedded Indentation velocity: 30 m m/s -Serially sectioned ( 5 m m thick ) -Safranin-O staining Reported is the structural stiffness (N/mm): Load (N) @ 0.010 mm SS = 3 mm 0.010 (mm) Automated Indentation Mapping

Maps of average structural stiffness on control and OA mouse left knee cartilage surfaces Femoral Condyles Tibial Plateaus Control OA Control OA Anterior Posterior 12 weeks (N = 3) 15 weeks (N = 2) Posterior Anterior Medial Structural Stiffness (N/mm) Lateral 1 2 4 6 8 10 12 14

Reduced structural stiffness is measured on medial side of OA mice joint 15 weeks 12 weeks N = 2 per group N = 3 per group 12 12 Zones Femoral Condyles 10 10 * Structural Stiffness (N/mm) 8 8 6 6 4 4 I II III IV 2 2 0 0 I II III IV Medial I II III IV Lateral Healthy 12 12 OA 10 10 Tibial Plateau Error bar = SE * = p < 0.05 8 8 * * 6 6 * I II III IV 4 4 2 2 0 0 I II III IV I II III IV Medial Lateral Medial Lateral

Comparison of the Structural Stiffness and histology Structural stiffness map Medial Medial Lateral Lateral OA Healthy (15 weeks) (15 weeks) I II Zone: I II Zone: Structural Stiffness Structural Stiffness 15 15 (N/mm) (N/mm) 10 10 5 5 0 0 Pixels (x axis of the map) Pixels (x axis of the map) Saf-O Saf-O 100 m m 100 m m Zone: I II Zone: I II

Conclusions  Mechanical properties can be mapped on entire articular surfaces of 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 :