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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Biology

Axolotl Tissue Mounting and Processing for Atomic Force Microscopy Measurements in Regenerating and Intact Limb Tissues

To prepare one-centimeter long cylinders of one centimeter diameter, heat a cardboard cutter under a Bunsen burner flame, and cut a 15-milliliter tube with the heated blade. Cover one side of the cylinders with Parafilm pieces. After anesthethetizing the axolotl and making sure it is unresponsive to tactile stimuli, orient the limb perpendicular to the body axis.Amputate the limb immediately distal to the calcified area of the zeugopodial region. Return the axolotl to a tank containing fresh holding water with analgesics. After 24 hours, transfer the axolotl back to fresh holding water and allow the limb to regenerate until the desired stage of interest.After placing the anesthetized axolotl under a stereo microscope, measure the length of the limb at the desired stage of regeneration for histolysis and condensing cartilage stages. After collecting the limbs, place them in a Petri dish for dissection. Under a stereo microscope, dissect the limbs with a scalpel at the elbow level, and place them in a Petri dish containing APBS solution.Arrange the Pasteur pipettes and the thermoblock set at 37 degrees Celsius with agarose aliquots on the working platform. Remove the cold block from the minus 20 degrees Celsius freezer and place the cylinder with the Parafilm-covered end facing down on top of it. Place the limb on a clean plate and gently remove excess liquid from the dissected limb with tissue paper.Add the melted low melting point agarose on top, and briefly move the limb around in the agarose to displace any remaining APBS from the skin surface. Quickly place the limb inside the cylinder, ensuring it is oriented vertically with the area of interest facing upwards. Add low melting point agarose inside the cylinder until the tissue is fully covered.Next, remove the cylinder from the cold block and allow the agarose to solidify for 30 seconds. After solidification, remove the Parafilm from the bottom of the cylinder and attach the agarose-containing tissue to the vibratome stage with cyanoacrylate glue. Then, remove any excess surrounding agarose with a scalpel.Submerge the stage in APBS solution for sectioning. Begin sectioning the agarose in 100-micrometer steps until the tissue tip is reached. Then, section until the distal portion of tissue is removed.Using a razor blade, carefully remove the tissue-containing agarose block from the vibratome stage. Immediately, attach the block to a 35-millimeter plastic Petri dish with surgical tissue adhesive glue. Add approximately two milliliters of culture medium to cover the tissue.

Atomic Force Microscopy-Based Indentation Measurements for Tissue Stiffness Analysis in Axolotl Limb Sections

Prior to atomic force microscopy or AFM measurements, mount the cantilever onto a glass block. Insert a Petri dish filled with PBS into the holder of the motor stage and insert the glass block into the AFM head. Then position the AFM head over the Petri dish so that the cantilever is immersed into the buffer and align the laser manually.In the JPK software, press the approach and acquire button to advance the cantilever onto a hard surface. Once the single force-distance curve is acquired, open the calibration manager and under method, select contact-based. Then adjust the room temperature to 20 degrees Celsius.Now zoom in on the curve and select the linear part for the sensitivity fit. Click on the sensitivity check box and retract the cantilever 200 micrometers from the surface. Then select the infinity symbol checkbox and click Run Thermal Noise.Next, zoom in on the resulting frequency graph and define a range using the right-click button of the mouse. Select the Spring Constant check box and close the calibration manager. Insert the Petri dish containing the regenerated axolotl limb embedded in the tissue block and culture medium into the dish holder of the AFM.Acquire an overview image of the tissue block in bright-field mode. Zoom into the region of interest and adjust the exposure time and focus. Set up the grid parameters for indentation measurements and record an array of force-distance curves.For data processing in the PyJibe software, open the file containing the force displacement curve. Select the preprocess tab and under the contact point estimation, select piecewise fit with line and polynomial. Then select the fit tab and under model, select spherical indenter, Under method, select nelder.For viscoelastic analysis in the PyJibe software, open the file containing the force distance curve. Select the preprocess tab and under the contact point estimation, select piecewise fit with line and polynomial. Then select the fit tab and under model, select the Hertz model corrected for viscoelasticity using KVM model.Under method, select leastsq. Set the accurate indenter radius to 10 micrometers and approach speed to 7.5 micrometers per second. Press Apply Model and Fit All to obtain apparent and unrelaxed Young's moduli, apparent viscosity, and Maxwell element relaxation time.Apparent Young's modulus measurements of the radius and ulna in intact limbs showed no significant difference. During the histolysis phase, the apparent Young's moduli of the radius and ulna dramatically decreased to 0.03 and 0.13 kilopascals respectively. The apparent Young's moduli in the center of intact cartilage were higher than in the periphery.At the histolysis stage, apparent Young's modulus measurements showed no significant difference between the cartilage center and periphery. During cartilage condensation, apparent Young's moduli significantly increased to 0.77 kilopascals representing intermediate stiffness values. Unrelaxed moduli showed substantial differences across intact tissue during histolysis and in condensing cartilage.The apparent Young's moduli were highly similar to unrelaxed moduli, indicating a predominantly elastic response. Apparent viscosity was significantly lower during histolysis compared to intact tissue and condensing cartilage.

329 Views.  Technische Universität Dresden (TUD).  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 Characterization of Tissue Stiffness During Axolotl Limb Regeneration

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