High spatial resolution analysis using automated indentation mapping differentiates biomechanical properties of normal vs. degenerated articular cartilage in mice
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Curated by eLife
Evaluation Summary:
The work presented by Masson et al. highlights experimental approaches using spatial indentation and contrast-enhanced 3-D x-ray imaging to topographically map cartilage thickness in mouse knee joints. This methods described have the potential to impact the field of musculoskeletal biomechanics, especially for researchers using mouse models to study cartilage wear and disease, given the high resolution and sensitivity of the described approaches.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Characterizing the biomechanical properties of articular cartilage is crucial to understanding processes of tissue homeostasis vs. degeneration. In mouse models, however, limitations are imposed by their small joint size and thin cartilage surfaces. Here we present a three-dimensional (3D) automated surface mapping system and methodology that allows for mechanical characterization of mouse cartilage with high spatial resolution. We performed repeated indentation mappings, followed by cartilage thickness measurement via needle probing, at 31 predefined positions distributed over the medial and lateral femoral condyles of healthy mice. High-resolution 3D x-ray microscopy (XRM) imaging was used to validate tissue thickness measurements. The automated indentation mapping was reproducible, and needle probing yielded cartilage thicknesses comparable to XRM imaging. When comparing healthy vs. degenerated cartilage, topographical variations in biomechanics were identified, with altered thickness and stiffness (instantaneous modulus) across condyles and within anteroposterior sub-regions. This quantitative technique comprehensively characterized cartilage function in mice femoral condyle cartilage. Hence, it has the potential to improve our understanding of tissue structure-function interplay in mouse models of repair and disease.
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Author Response
Reviewer #1 (Public Review):
In this work, the authors were trying to develop an approach for microindentation-based spatial mapping of articular cartilage of mouse femur. Because mouse cartilage in articulating joints is incredibly thin and challenging to indent repeatably and reliably, a need exists to increase resolution of indentation spacing on very small surfaces, improve sensitivity of indentation (e.g., surface detection), and reduce error and improve accuracy of indentation measurements. Using a relatively new multiaxis material test stand with repositioning capabilities and multi axis load cells, the authors developed a spatial indentation test protocol as well as used this array-based approach to measure cartilage thickness via needle probing. They then validated thickness measurements generated using …
Author Response
Reviewer #1 (Public Review):
In this work, the authors were trying to develop an approach for microindentation-based spatial mapping of articular cartilage of mouse femur. Because mouse cartilage in articulating joints is incredibly thin and challenging to indent repeatably and reliably, a need exists to increase resolution of indentation spacing on very small surfaces, improve sensitivity of indentation (e.g., surface detection), and reduce error and improve accuracy of indentation measurements. Using a relatively new multiaxis material test stand with repositioning capabilities and multi axis load cells, the authors developed a spatial indentation test protocol as well as used this array-based approach to measure cartilage thickness via needle probing. They then validated thickness measurements generated using needle probing with high resolution 3D x-ray imaging using contrast enhancement with phosphotungstic acid (PTA). The authors then compared cartilage thickness and indentation mechanical properties between wild type (C57BL6J) and Prg4 mutant mice.
This work is rigorous and includes new techniques that are validated using orthogonal approaches. Some of the techniques used in this work, especially indentation-based mapping of cartilage stiffness in small mouse joints, have been challenging for the field to overcome. This is especially true with the exploding number of small animal studies investigating cartilage health in transgenic mouse strains and injury models. While innovative and important, there remain a few key experiments that would help with validation of the data acquired in these experiments.
Specifically, a general rule of thumb for indentation testing is to test no more than 1/10th the thickness of the indented material. Because the cartilage thickness of the medial condyles (~0.04mm) was only ~2x that of the indentation depth used for automated indentation mapping (0.02mm), it is possible that this thin region of cartilage will lead to substrate effects from the subchondral bone on the indentation data. It is unclear if the indentation measurements are characterizing cartilage or substrate properties. This may not be a major issue for healthy, intact cartilage (including in the mutant strains) but will likely have a major impact on interpretation of results following cartilage degeneration and loss.
It is unclear if damage was caused by the 0.02mm indentations because the XRM scanning occurred after needle probing tests. The "bands" observed in the 3D XRM imaging following both indentation and needle probing (Fig 2A2) suggests that the indentation probes and individual needle probings at each site are not perfectly overlapping. Surface congruency of the cartilage suggest valley formation at indentation sites.
We thank the reviewer for the enthusiastic comments on our work and its importance to the field. We agree this is the known rule of thumb; however, by employing a microindentation rather than a nanoindentation approach (such as AFM) to also obtain spatial resolution, we are unable to probe the cartilage within a 1µm amplitude range reliably. Also, we had to accommodate for thickness variations throughout the cartilage surface (thinner and thicker regions) during indentation testing, which are unknowable before needle probing thickness measurements. We completely agree that substrate effects can play a role on indentation data as this is well described within the field. Therefore, to mitigate such effects, the instantaneous modulus was calculated at 20% strain for all positions and presented alongside thickness mappings of same surfaces to avoid misinterpretations on cartilage loss. Demonstrated repeatability in indentation peak forces during test-retest suggests indentations did not damage the cartilage surfaces. This can be further corroborated by XRM imaging of femurs subjected to indentation testing only. Nevertheless, we would also like to clarify (and will do so in the methods) that the indentation and needle probing were not undertaken on the exact same position, exactly because of this possibility. We apologize for any misunderstandings and would be happy to further clarify that on the methods section.
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Evaluation Summary:
The work presented by Masson et al. highlights experimental approaches using spatial indentation and contrast-enhanced 3-D x-ray imaging to topographically map cartilage thickness in mouse knee joints. This methods described have the potential to impact the field of musculoskeletal biomechanics, especially for researchers using mouse models to study cartilage wear and disease, given the high resolution and sensitivity of the described approaches.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this work, the authors were trying to develop an approach for microindentation-based spatial mapping of articular cartilage of mouse femur. Because mouse cartilage in articulating joints is incredibly thin and challenging to indent repeatably and reliably, a need exists to increase resolution of indentation spacing on very small surfaces, improve sensitivity of indentation (e.g., surface detection), and reduce error and improve accuracy of indentation measurements. Using a relatively new multiaxis material test stand with repositioning capabilities and multi axis load cells, the authors developed a spatial indentation test protocol as well as used this array-based approach to measure cartilage thickness via needle probing. They then validated thickness measurements generated using needle probing with high …
Reviewer #1 (Public Review):
In this work, the authors were trying to develop an approach for microindentation-based spatial mapping of articular cartilage of mouse femur. Because mouse cartilage in articulating joints is incredibly thin and challenging to indent repeatably and reliably, a need exists to increase resolution of indentation spacing on very small surfaces, improve sensitivity of indentation (e.g., surface detection), and reduce error and improve accuracy of indentation measurements. Using a relatively new multiaxis material test stand with repositioning capabilities and multi axis load cells, the authors developed a spatial indentation test protocol as well as used this array-based approach to measure cartilage thickness via needle probing. They then validated thickness measurements generated using needle probing with high resolution 3D x-ray imaging using contrast enhancement with phosphotungstic acid (PTA). The authors then compared cartilage thickness and indentation mechanical properties between wild type (C57BL6J) and Prg4 mutant mice.
This work is rigorous and includes new techniques that are validated using orthogonal approaches. Some of the techniques used in this work, especially indentation-based mapping of cartilage stiffness in small mouse joints, have been challenging for the field to overcome. This is especially true with the exploding number of small animal studies investigating cartilage health in transgenic mouse strains and injury models. While innovative and important, there remain a few key experiments that would help with validation of the data acquired in these experiments.
Specifically, a general rule of thumb for indentation testing is to test no more than 1/10th the thickness of the indented material. Because the cartilage thickness of the medial condyles (~0.04mm) was only ~2x that of the indentation depth used for automated indentation mapping (0.02mm), it is possible that this thin region of cartilage will lead to substrate effects from the subchondral bone on the indentation data. It is unclear if the indentation measurements are characterizing cartilage or substrate properties. This may not be a major issue for healthy, intact cartilage (including in the mutant strains) but will likely have a major impact on interpretation of results following cartilage degeneration and loss.
It is unclear if damage was caused by the 0.02mm indentations because the XRM scanning occurred after needle probing tests. The "bands" observed in the 3D XRM imaging following both indentation and needle probing (Fig 2A2) suggests that the indentation probes and individual needle probings at each site are not perfectly overlapping. Surface congruency of the cartilage suggest valley formation at indentation sites.
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Reviewer #2 (Public Review):
The authors are applying the application of a commercial instrument to mapping the properties of cartilage in the mouse knee. The primary advance is in the use of a needle probe to retrieve cartilage thickness in a site specific manner. Overall, the methods used here have been reported previously, as as the mouse model which is used for testing. There are many claims of novelty that are not strongly supported. The major strength is in the use and validation of the needle probe. There are some major technical issues with the reporting of peak force as a variable and the instantaneous modulus, which cannot be used in predictive modeling. The lack of measure of other cartilage properties such as permeability or Poisson's ratio are a weakness.
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