title: Guidelines for an optimized indentation protocol for measurement of cartilage stiffness: The effects of spatial variation and indentation parameters.
abstract: Mechanical properties of articular cartilage that are vital to its function are often determined by indentation tests, which can be performed at different scales. Cartilage tissue exhibits various types of structural, geometrical, and spatial variations that pose strict demands on indentation protocols. This study aims to define a reproducible micro-indentation protocol for measuring the effective (average) stiffness of the cartilage surface in a region around 1mm2. We elucidated how different parameters such as indenter size, indenter depth, and the location of the indentation influence the effective elastic modulus measured in micrometer scale on rat knee cartilage. When an indentation was performed (50μm radial probe, ≈10μm indentation depth) at exactly the same location, the variability was less than 10%, even with a recovery period of 30s. However, there was a high spatial variation and a small change of around 60μm in location could change the modulus values up to as much as 10-20 fold. The effective elastic modulus of cartilage surface layer cannot therefore be reproducibly determined from a few indentations on a cartilage sample, and requires at least 144 (12×12) indentations for a soft spherical probe with a 50μm radius. With higher depths, the spatial variation is slightly lower, allowing slightly lower number of indentations (≈80 measurements or a 9×9 frame) to provide a representative elastic modulus. Using this protocol, we determined an elastic modulus of 2.6±1.9N/mm2at the medial side versus a higher modulus of 4.2±2.6N/mm2at the lateral side of the tibia of 12 weeks old Wistar rats. Optimized indentation protocols similar to the one presented here are required for revealing such variations in the mechanical properties of cartilage with anatomical location.
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands; Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands. Electronic address: P.Rahnamaymoshtagh@umcutrecht.nl.
authors.forename: Parisa R
authors.initials: PR
authors.lastname: Moshtagh
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands; Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands. Electronic address: B.Pouran@umcutrecht.nl.
authors.forename: Behdad
authors.initials: B
authors.lastname: Pouran
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Electronic address: N.M.Korthagen@uu.nl.
authors.forename: Nicoline M
authors.initials: NM
authors.lastname: Korthagen
authors.affiliation: Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands. Electronic address: A.A.Zadpoor@tudelft.nl.
authors.forename: Amir A
authors.initials: AA
authors.lastname: Zadpoor
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands; Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands; Department of Rheumatology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: H.H.Weinans@umcutrecht.nl.
authors.forename: Harrie
authors.initials: H
authors.lastname: Weinans
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000502:physiology*; D055119:Elastic Modulus; D004548:Elasticity; D006614:Hindlimb / Q000502:physiology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013977:Tibia / Q000502:physiology
keywords: Articular cartilage; Indentation; Micro-stiffness; Spatial distribution