Atomic Force Microscopy Measurements of Cartilage in Intact and Regenerating Axolotl Limbs

Summary

In this protocol, we show how to prepare axolotl tissue for atomic force microscopy (AFM) and perform indentation measurements in intact and regenerating limb cartilage.

Abstract

Mechanical forces provide important signals for normal cell function and pattern formation in developing tissues, and their role has been widely studied during embryogenesis and pathogenesis. Comparatively, little is known of these signals during animal regeneration.

The axolotl is an important model organism for the study of regeneration, given its ability to fully restore many organs and tissues after injury, including missing cartilage and bone. Due to its crucial role as the main supporting tissue in the vertebrate body, regaining skeletal function during regeneration requires both the restoration of the missing structures as well as their mechanical properties. This protocol describes a method for processing axolotl limb samples for atomic force microscopy (AFM), which is the gold standard for probing cell and tissue mechanical properties at high spatial resolution.

Taking advantage of the regenerative capabilities of the axolotl, this study measured the stiffness of limb cartilage during homeostasis and two stages of limb regeneration: tissue histolysis and cartilage condensation. We show that AFM is a valuable tool for gaining insights into dynamic tissue restructuring and the mechanical changes that occur during regeneration.

Introduction

The skeleton, especially cartilage and bones, provides the main mechanical support for soft tissues of the body in vertebrates. Therefore, any damage in the skeletal system is likely to greatly compromise functionality and even survival. In humans, bone fractures are one of the most common traumatic injuries¹, most of which repair in a matter of weeks, but 5%-10% of these will have delays in healing or never fully recover^2,3. Moreover, humans are not able to recuperate from extensive bone or cartilage loss^4,5. Some salamanders, ....

Protocol

Axolotls (Ambystoma mexicanum) were grown in the Axolotl facility of the Center for Regenerative Therapies Dresden (CRTD) of the Dresden University of Technology (TUD). A full description of the husbandry conditions can be found in²⁴. Briefly, rooms were kept at 20-22 °C with a 12/12 h day/night cycle. All handling and surgical procedures were carried out in accordance with local ethics committee guidelines and were approved by the Landesdirektion Sachsen, Germany.

Discussion

Here, we demonstrate a technique for the measurement of cartilage stiffness in axolotl limbs with AFM. However, this method may be expanded for probing other tissue types as well. A key step for successful AFM measurements is sample preparation, which proved to be particularly challenging with axolotl samples. We found that probing the tissue surface that was still embedded in the agarose block was the best way to preserve tissue integrity. This is because the axolotl skin secretes high levels of mucous onto the surface .......

Materials

Name	Company	Catalog Number	Comments
Affinity Designer	Affinity	version 1.10.4	For figure assembling
Agarose Low Melt	Roth	6351.1	For sample preparation
Alexa Fluor 488 Phalloidin	Invitrogen	A12379	To stain tissue
Axiozoom	Zeiss		To image samplea under the AFM
Benzocaine	Sigma-Aldrich	E1501	To anesthetize the animals
Butorphanol (+)-tartrate salt	Sigma-Aldrich	B9156	As analgesic
Cantilever	NanoWorld	Arrow TL1	For AFM indentation measurements
Cellhesion 200 setup equipped with a motorstage	JPK/Bruker		For AFM indentation measurements
CellSense Entry			For imaging in Stereoscope Olympus UC90
Dulbecco’s Phosphate Buffered Saline (DPBS, 1x)	Gibco	14190-144	To clean samples and section under vibratome
FIJI (ImageJ2)	https://imagej.net/software/fiji	version 2.9.0/1.53t	For image processing
GraphPad Prism	GraphPad Software	(version 8.4.3)	To graph and statistically analyze the data
Heat-inactivated FBS	Gibco	10270-106	For cell culture medium
Histoacryl glue (2-Butyl-Cyanoacrylate)	Braun		To glue sample to petri dishes
Hoechst 33258	Abcam	ab228550	To stain tissue
Insulin	Sigma-Aldrich	I5500	For cell culture medium
Inverted confocal microscope	Zeiss	780 LSM	To image tissue sections
Inverted confocal microscope	Zeiss	980 LSM	To image tissue sections
JPK/Bruker data processing software	JPK/Bruker	SPM 6.4	To analyze force-distance curves
L15 medium (Leibovitz)	Sigma	L1518	For cell culture medium
L-Glutamine	Gibco	25030-024	For cell culture medium
Penicillin/Streptomycin	Gibco	15140-122	For cell culture medium
polystyrene beads ( 20 µm diameter); )	microParticles		For AFM indentation measurements
Pyjibe	written by Paul Müller https://github.com/AFM-analysis/PyJibe	0.15.0	For viscoelastic analysis
Stereoscope Olympus SX10	Olympus	SX10	For limb amputations and tissue mounting
Stereoscope Olympus UC90	Olympus	UC90	For imaging
Vibratome Leica	Leica	VT 1200S	For tissue sectioning