We thank all members of the Sandoval-Guzm&#225;n lab for continuous support and companionship during the development of this work. We are also grateful to Anja Wagner, Beate Gruhl, and Judith Konantz for their dedication to axolotl care. We also thank Paul M&#252;ller for providing codes for AFM data analysis. This work was supported by the Light Microscopy Facility of the CMCB Technology Platform at TU Dresden.&#160;AT is a fellow of the Mildred Scheel Early Career Center Dresden P2 funded by the German Cancer Aid (Deutsche Krebshilfe). RA is funded by a Temporary PI position (Eigene Stelle) from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) &#8211; AI 214/1-1.

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 &#176;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.
<p class="jove...

Mechanical Characterization of Tissue Stiffness During Axolotl Limb Regeneration

<ol>
	<li>Mills, L. A., Aitken, S. A., Simpson, A. H. R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+non-union+per+fracture:+current+myths+and+revised+figures+from+a+population+of+over+4+million+adults.">The risk of non-union per fracture: current myths and revised figures from a population of over 4 million adults.</a> Acta Orthop. 88 (4), 434-439 (2017).</li><li>Calori, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-unions.">Non-unions.</a> Clin Cases Miner Bone Metab. 14 (2), 186-188 (2017).</li><li>Nandra, R., Grover, L., Porter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+non-union+epidemiology+and+treatment.">Fracture non-union epidemiology and treatment.</a> Trauma. 18 (1), 3-11 (2016).</li><li>Nauth, A., Schemitsch, E., Norris, B., Nollin, Z., Watson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical-size+bone+defects:+Is+there+a+consensus+for+diagnosis+and+treatment.">Critical-size bone defects: Is there a consensus for diagnosis and treatment.</a> J Orthop Trauma. 32 (3 Suppl), S7-S11 (2018).</li><li>Schemitsch, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+Defining+critical+in+bone+defect+size.">Size matters: Defining critical in bone defect size.</a> J Orthop Trauma. 31 (10), S20-S22 (2017).</li><li>Joven, A., Elewa, A., Simon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+regeneration:+Salamanders.">Model systems for regeneration: Salamanders.</a> Development (Cambridge). 146 (14), 0 (2019).</li><li>Aires, R., Keeley, S. D., Sandoval-Guzm&#225;n, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+Self-Regeneration.">Basics of Self-Regeneration.</a> Cell Engineering and Regeneration. , (2020).</li><li>Stocum, D. 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The authors declare no conflicts of interest

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 ...

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 recover2,3. Moreover, humans are not able to recuperate from extensive bone or cartilage loss4,5. Some salamanders, however, can regenerate a variety of body structures, including full limbs6, making them an ideal model for the study of skeletal regeneration.
The axolotl (Ambystoma mexicanum) is a type of salamander where limb regeneration has been extensively studied. This process occurs in four main sequential but overlapping phases: 1) wound healing, 2) inflammation/histolysis, 3) blastema formation, and 4) blastema outgrowth/differentiation (reviewed in7,8). Following amputation, keratinocytes bordering the injury site migrate rapidly, closing the wound and forming the wound epithelium (WE). During the ensuing inflammation and histolysis, pathogens are eliminated, debris and damaged cells are cleared, and the extracellular matrix (ECM) under the amputation surface is remodeled9. Tissue histolysis is essential for limb regeneration to take place10, where the secretion of proteolytic enzymes is crucial not only for overall ECM remodeling, but also to release the cells giving rise to the blastema and to free bioactive molecules sequestered in the ECM itself8. In fact, studies in many regenerative contexts and model organisms have shown that the unique material properties of the ECM during histolysis are capable of inducing dedifferentiation processes or directing the migration of cells toward the injury site (reviewed in11). Furthermore, resorption of calcified tissue during the late stages of histolysis has shown to be key for proper integration of newly-formed limb skeletal elements12. Following the histolysis stage, the blastema is formed under the wound epithelium (WE) as an accumulation of undifferentiated, multi-lineage progenitors resulting from de-differentiated mature tissue cells or resident stem cells. Blastema cells proliferate and differentiate into all missing cell types. Finally, limb morphogenesis takes place, where skeletal tissue is regenerated through the condensation of chondroprogenitors derived from periskeletal cells and transdifferentiated dermal fibroblasts13,14,15.
Although many of the biochemical cues regulating changes in cell identity and ECM composition have been identified10,13,14,16,17,18, tissue mechanical properties during the different phases of limb regeneration, as well as their influence in regeneration, have remained largely unexplored. Many studies have shown that cells sense and integrate mechanical cues that regulate their fate and behavior in several contexts (reviewed in19,20). Therefore, complementing our cellular and molecular knowledge of limb regeneration with tissue mechanical measurements will greatly improve our understanding of these processes.
Different techniques have been developed that allow for mechanical characterization and force manipulation of biological samples21. Among these techniques, atomic force microscopy (AFM) has become the gold standard in mechanobiology, in which the viscoelastic properties of biological samples are probed at high spatial resolution by indenting with an ultrasensitive force sensor, the AFM cantilever22. Since this technique requires direct contact with the sample, typically, tissue slices are generated, which can be challenging in some cases. Thus, preparation conditions need to be adapted and optimized for each particular sample so that it can remain as close to physiological conditions as possible and minimal artifacts are generated23. This&#160;protocol describes how to measure tissue stiffness in axolotl limbs using AFM, focusing on cartilaginous tissues in intact conditions, while undergoing histolysis, and in cartilage condensation stages (Figure 1&#160;and Figure 2). This method may be expanded for the measurement of other tissue types as well.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Affinity Designer</td>
			<td>Affinity</td>
			<td>&#160;version 1.10.4</td>
			<td>For figure assembling</td>
		</tr>
		<tr>
			<td>Agarose Low Melt</td>
			<td>Roth</td>
			<td>6351.1</td>
			<td>For sample preparation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td>To stain tissue</td>
		</tr>
		<tr>
			<td>Axiozoom</td>
			<td>Zeiss</td>
			<td></td>
			<td>To image samplea under the AFM</td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>To anesthetize the animals</td>
		</tr>
		<tr>
			<td>Butorphanol (+)-tartrate salt</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;B9156</td>
			<td>As analgesic</td>
		</tr>
		<tr>
			<td>Cantilever</td>
			<td>NanoWorld</td>
			<td>Arrow TL1</td>
			<td>For AFM indentation measurements</td>
		</tr>
		<tr>
			<td>Cellhesion 200 setup equipped with a motorstage</td>
			<td>JPK/Bruker</td>
			<td></td>
			<td>For AFM indentation measurements</td>
		</tr>
		<tr>
			<td>CellSense Entry</td>
			<td></td>
			<td></td>
			<td>For imaging in Stereoscope Olympus UC90</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS, 1x)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>To clean samples and section under vibratome</td>
		</tr>
		<tr>
			<td>FIJI (ImageJ2)</td>
			<td>https://imagej.net/software/fiji</td>
			<td>version 2.9.0/1.53t</td>
			<td>For image processing</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>(version 8.4.3)</td>
			<td>To graph and statistically analyze the data</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>For cell culture medium</td>
		</tr>
		<tr>
			<td>Histoacryl glue (2-Butyl-Cyanoacrylate)</td>
			<td>Braun</td>
			<td></td>
			<td>To glue sample to petri dishes</td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Abcam</td>
			<td>ab228550</td>
			<td>To stain tissue</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td>For cell culture medium</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Zeiss</td>
			<td>780 LSM</td>
			<td>To image tissue sections</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Zeiss</td>
			<td>980 LSM</td>
			<td>To image tissue sections</td>
		</tr>
		<tr>
			<td>JPK/Bruker data processing software</td>
			<td>JPK/Bruker</td>
			<td>SPM 6.4</td>
			<td>To analyze force-distance curves</td>
		</tr>
		<tr>
			<td>L15 medium (Leibovitz)</td>
			<td>Sigma</td>
			<td>L1518</td>
			<td>For cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td>For cell culture medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>For cell culture medium</td>
		</tr>
		<tr>
			<td>polystyrene beads ( 20 &#181;m diameter); )</td>
			<td>microParticles</td>
			<td></td>
			<td>For AFM indentation measurements</td>
		</tr>
		<tr>
			<td>Pyjibe</td>
			<td>written by Paul M&#252;ller https://github.com/AFM-analysis/PyJibe</td>
			<td>0.15.0</td>
			<td>For viscoelastic analysis</td>
		</tr>
		<tr>
			<td>Stereoscope Olympus SX10</td>
			<td>Olympus</td>
			<td>SX10</td>
			<td>For limb amputations and tissue mounting</td>
		</tr>
		<tr>
			<td>Stereoscope Olympus UC90</td>
			<td>Olympus</td>
			<td>UC90</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Vibratome Leica</td>
			<td>Leica</td>
			<td>VT 1200S</td>
			<td>For tissue sectioning</td>
		</tr>
	</tbody>
</table>

atomic force microscopy measurements of cartilage in intact and regenerating axolotl limbs

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.

Author Spotlight: Exploring the Role of Mechanical Signals in Tissue Regeneration Through Atomic Force Microscopy 

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

We're interested in understanding the role of mechanical signals on the regeneration process. For this, it is key to have reliable tools that allows the measurement of mechanical properties such as the AFM. There are multiple techniques for probing mechanical properties of tissues.For example, share geometry or elastography. However, the gold standard for small tissue samples or probing mechanical properties of tissues at a cellular scale is still the AFM. One of the most challenging steps is preparing the sample because we need to ensure the maximum structural integrity so that we can reliably measure mechanical properties of samples with the AFM.We believe that our findings will motivate more mechanistic studies on how mechanical cues affect tissue regeneration. In addition, the obtained results can instruct more realistic tissue engineered models to conduct such experiments.

Using the protocol described above, we measured the apparent Young&#39;s modulus of cartilaginous axolotl limb tissues in homeostatic (&#34;Intact&#34;) conditions, during early cartilage histolysis, and later cartilage condensation stages (Figure 1A). We also probed the mechanical properties of the skeletal elements in different regions, including their center and periphery, as shown in the images depicting the cantilever position (Figure 1B). To display tissue...

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.

Mechanical forces provide important signals for normal cell function and pattern formation in developing tissues, and their role has been widely studied ...

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329 Views.  Technische Universität Dresden (TUD). We're interested in understanding the role of mechanical signals on the regeneration process. For this, it is key to have reliable tools that allows the measurement of mechanical properties such as the AFM. There are multiple techniques for probing mechanical properties of tissues.For example, share geometry or elastography. However, the gold standard for small tissue samples or probing mechanical properties of tissues at a cellular scale is still the AFM. One of the most challenging s...