This article was supported by NSERC Grant # 299280, IRSST as a scholarship to G. Parent and FRSQ as an undergraduate student research award to M. Cyr. We thank Yoan Lemieux-Laneville for performing the manipulations recorded on video.

Prior to any manipulation, you must identify the group of tendons to be used depending on the experiment you are conducting and the apparatus at your disposition. For our purposes, ventral tendons were chosen because they are smaller and thus easier to manipulate when measuring the cross-sectional area and fitting them into the bioreactor chamber.
Please note that all instruments are autoclaved or sterilized with 70% ethanol. Moreover, a spray bottle containing 70% ethanol is placed beside each work station to sterilize the experimenter&#8217;s gloves before each operation.
Part 1: Extraction
After resection, the tail is carefully manipulated by its extremities to avoid damaging the tissues. Also, to conserve cell viability, all manipulations are carried out in cold saline solution.
1A) Materials:
<ul>
 <li>Cold saline solution (D-PBS)</li>
 <li>Crushed ice</li>
 <li>Surface protector</li>
 <li>Cutting board</li>
 <li>Individual manipulation plates</li>
 <li>2 500 ml dishes</li>
 <li>2 2L glass dishes</li>
 <li>Adhesive tape</li>
 <li>1 Tweezers</li>
 <li>1 Forceps</li>
 <li>1 Tweezers stand</li>
 <li>1 Pair of surgical shears</li>
 <li>1 Scalpel</li>
 <li>1 Pair of surgical scissors 
 
 <img src="/files/ftp_upload/2176/2176fig1.jpg" alt="Figure 1" /> 
 Figure 1. Individual manipulation with orientation identification ("P" for "proximal")</li>
</ul>
1B) Work station:
<ol>
 <li>Spread out the surface protector and place the cutting board on top.</li>
 <li>Fill each two larger glass dishes to halfway with crushed ice and place them on the paper.</li>
 <li>Stick a piece of tape on a corner of the manipulation plate and identify with a letter to indicate the proximal end. </li>
 <li>Fill the two small glass dishes to halfway and all grooves of the manipulation plates with cold saline solution. Place them in the large glass dishes containing ice.</li>
 <li>Organize all the sterile instruments on the surface protector.</li>
 <li>Transfer the tail into one of the glass dish containing saline solution.</li>
</ol>
1C) Manipulations:
<ol>
 <li>Observe the anatomy at the proximal end of the tail to distinguish the ventral side from the dorsal side. The ventral side has a larger number of tendons and a blood vessel that can be verified by gently squeezing the tail to produce tiny drops of blood.</li>
 <li>Using the surgical scissors, cut the skin along the dorsal side of the tail if you want to extract the ventral tendons (or along the ventral side if you need the dorsal tendons). </li>
 <li>Open the incision at the proximal end and remove the skin using forceps or your fingers and by carefully manipulating the tail ends.</li>
 <li>Rinse off the blood and hair in the saline solution and transfer the specimen into the remaining glass dish containing fresh solution. Change gloves. 
 
 A transversal section viewed under light microscopy shows the six tendon groups. One tendon from each group is attached to the caudal end of each vertebra, so we cut at the intervertebral disc to expose new tendons along the cut. 
 
 <img src="/files/ftp_upload/2176/2176fig2.jpg" alt="Figure 2" /> 
 Figure 2. Transversal section of the rat tail viewed under light microscopy.</li>
 <li>Cut the distal end through the cartilage 2-3 vertebrae shorter with surgical shears. Place the tail on the cutting board to cut the soft tissues using a scalpel and then immediately replace it in the solution. </li>
 <li>Using tweezers, pull out one tendon from the distal tail end.</li>
 <li>With a gentle hold at both ends, place the tendon into the individual manipulation plate. Repeat the last two steps until no more tendons can be teased out and cut one vertebra shorter whenever new tissues need to be exposed.</li>
</ol>
Part 2: Cross-sectional area measurement7
When the tissue undergoes mechanical characterization or stimulation, its mechanical properties are described by normalizing the force inside the tendon to stress. This is why we evaluate cross-sectional area.
2A) Materials:
<ul>
 <li>Optic Micrometer</li>
 <li>Stereomicroscope</li>
 <li>Digital camera</li>
 <li>Edge recognition and Profile reconstruction algorithms 5,6</li>
 <li>Cold saline solution (D-PBS)</li>
 <li>1 20-200&mu;L Micro volume pipette</li>
 <li>1 Hex key for optic micrometer adjustments</li>
 <li>2 Tweezers 
 
 <img src="/files/ftp_upload/2176/2176fig3.jpg" alt="Figure 3" /> 
 Figure 3. Optic Micrometer</li>
</ul>
2B) Work station:
<ol>
 <li>Open the software (Digital camera and algorithms).</li>
 <li>Place the optic micrometer so that there is enough room to load the tendon in the apparatus.</li>
 <li>Organize the instruments and glass dish containing the extracted tendons close at hand.</li>
</ol>
2C) Manipulations:
<ol>
	<li>Fill the measuring compartment with cold saline solution until both anchor systems are submerged.</li>
	<li>Transfer the tendon into the compartment by holding the ends with tweezers.</li>
	<li>Using suction via the micro-volume pipette, pull one extremity at a time into the silicon tube. Aspirate until the end extends beyond the shaft collars.</li>
	<li>Tighten the shaft collars to compress the tubes and install the apparatus under the stereomicroscope.</li>
	<li>At 105X magnification, stretch the tendons while observing the tissue until the creases are no longer perceptible. Stretch by additional 0.40%. 
 <img src="/files/ftp_upload/2176/2176fig4.jpg" alt="Figure 4" /> 
 Figure 4. Tendon projection at 105x magnification before and after stretching.</li>
	<li>Adjust position and focus to maintain a clear image of the tendon over a 180&deg; rotation.</li>
	<li>Take a photo at every 10&deg; rotation without modifying tension, focus or position. Use only the rotary shaft to carry out the rotation.
 Due to its cone shape, these steps may be repeated at different points along the tendon.</li>
 <li>Activate the edge recognition and profile reconstruction algorithms in order to analyse the database you have registered.</li>
 <li>Free the tendon by loosening the shaft collars and blowing air into the silicone tubes using the micro volume pipette.</li>
 <li>Reinsert the tendon onto the manipulation plate, again handling it by the ends.</li>
 <li>Verify the shape of the profile reconstruction obtained and the value of the cross-sectional area.</li>
</ol>
Part 3: Rinsing
To remove contamination that may have occurred during the previous manipulations, the tissues are rinsed under biosafety cabinet.
3A) Materials:
<ul>
 <li>Cold saline solution (D-PBS)</li>
 <li>Multiple-groove manipulation plate</li>
 <li>Individual Manipulation plates</li>
 <li>Adhesive tape</li>
 <li>2 Tweezers</li>
 <li>1 Tweezers stand</li>
</ul>
3B) Work station:
<ol>
 <li>Turn on the fan in the biosafety cabinet 15 minutes in advance and clean the inside surfaces with 70% ethanol.</li>
 <li>Bring all the instruments inside the biosafety cabinet.</li>
 <li>Stick pieces of tape onto a corner of each individual plate to identify the proximal end.</li>
 <li>Fill all grooves with sterile saline solution.</li>
</ol>
3C) Manipulations:
<ol>
 <li>Introduce the manipulation plate containing the extracted tendon in the cabinet.</li>
 <li>Using tweezers, remove the tendon from its plate with a gentle grasp on each end.</li>
 <li>Immerse and gently stir the tendon in the saline solution of each compartment of the multiple-groove manipulation plate, starting from the closest and proceeding to the furthest groove.</li>
 <li>Finally, submerge the tendon in an individual manipulation plate. </li>
</ol>
Part 4: Loading into bioreactor chamber
To avoid further contamination, the following manipulations are also conducted in the biosafety cabinet.
4A) Materials:
<ul>
 <li>Bioreactor chamber 4</li>
 <li>Sterile anchors</li>
 <li>1 Sterile Petri dish</li>
 <li>Cold cell cultured Medium (DMEM)</li>
 <li>Cyanoacrylate</li>
 <li>2 Tweezers</li>
 <li>Tweezers stand</li>
 <li>0.5-10&mu;l Micro-volume pipette</li>
 <li>1 Ruler 
 
 <img src="/files/ftp_upload/2176/2176fig5.jpg" alt="Figure 5" /> 
 Figure 5. Bioreactor chamber</li>
</ul>
4B) Work station:
<ol>
 <li>Since the previous step was also realized in biosafety cabinet, removal of unnecessary tools will give you more work space. </li>
 <li>Bring all other instruments inside the cabinet.</li>
</ol>
4C) Manipulations:
<ol>
 <li>Place an anchor on the manipulation plate with its spool centered over the tendon extremity and wound the tissue up and around the spool.</li>
 <li>Roll the anchor over and hold the loose extremity while ensuring that the tissue stays attached to the anchor. Stop after &frac34; turn. </li>
 <li>Carry out the same steps with the other end.</li>
 <li>Roll both anchors over equally until there is 6 cm between their centers1. </li>
 <li>Loosen the tendon to immerse it in the solution and allow the anchored sections to dry briefly to enhance its mechanical properties2-3.</li>
 <li>Pour cyanoacrylate into the Petri dish and draw 2.5&mu;L into the micro volume pipette.</li>
 <li>Apply a drop of cyanoacrylate to the wound-up tissue without dropping any glue in the saline solution. Repeat until 2 drops have been applied to each anchor.</li>
 <li>With the tendon still completely immersed, wait 5 minutes for the glue to dry completely.</li>
 <li>During this time, fill the chamber compartment with saline solution.</li>
 <li>Rehydrate all tissue with saline solution to prevent damage.</li>
 <li>By manipulating only the anchors, transfer the tendon into the bioreactor chamber and close it to prevent leakage and contamination during transfer.</li>
</ol>
Part 5: Representative Results:
The outcome of the protocol shows that when performed correctly, our tissue rigorous isolation and preparation procedure make it possible to maintain tissue sterility, viability and integrity.
First, using the simple and repetitive extraction method, we are able to extract tail tendons without damaging the collagen network as it can be observed by a microscopic analysis of H&amp;E tainted sections realized after the extraction.
<img src="/files/ftp_upload/2176/2176fig6.jpg" alt="Figure 6" /> 
 Figure 6. Tendon collagen network after extraction (5&mu;m longitudinal section stained with H&amp;E).
We then cultured tendons for up to ten days and conducted sterility tests. Each day, we plated used culture solution on agar and incubated it for 24 hours. Since no bacterial growth was observed, we concluded that our manipulations do not result in contamination.
With the profile reconstruction algorithm and optic micrometer, we are able to estimate the cross-sectional area within a 2% margin of error7.
<img src="/files/ftp_upload/2176/2176fig7.jpg" alt="Figure 7" /> 
 Figure 7. Profile reconstruction of a RTT.
Finally, we assessed viability using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells after the rinsing step and after a twelve-day culture period. Since a large majority of green fluorescent live cells were evident, we can confirm that our isolation procedures are successful in preserving live tissue. The same test was performed two hours after attaching the tendon into the bioreactor chamber. We verified that the dehydration and glue at the anchor had not spread in the tissue between both anchors.
<img src="/files/ftp_upload/2176/2176fig8.jpg" alt="Figure 8" /> 
 Figure 8. Tissue viability at the anchor (green = live cells, red = dead cells)

<ol>
	<li>Bennett, M. B., Ker, R., Dimery, N., Alexander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+various+mammalian+tendons.">Mechanical properties of various mammalian tendons.</a> J. Zool. Lond. A209, 537-548 (1986).</li><li>Dupuis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> D&#233;shydratation de matrices collag&#233;niques reconstruites in vitro: effets sur les propri&#233;t&#233;s m&#233;caniques et histologiques [dissertation]. , 114-114 (1981).</li><li>Haut, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+influence+of+strain+rate+on+the+tensile+failure+of+rat+tail+tendon.">Age-dependent influence of strain rate on the tensile failure of rat tail tendon.</a> Journal of Biomechanical Engineering. 105, 296-299 (1983).</li><li>Hupp&#233;, N., Lessard, J. -. L., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bioreactor+Design+for+the+Mechanobiological+Study+of+Soft+Tissue+Damage+and+Repair+in+Conditions+that+Provide+the+Best+Approximation+of+Actual+Use.">A Bioreactor Design for the Mechanobiological Study of Soft Tissue Damage and Repair in Conditions that Provide the Best Approximation of Actual Use.</a> , (1985).</li><li>Langelier, E., Dupuis, D., Guillot, M., Goulet, F. &. a. m. p. ;. a. m. p., Rancourt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+profiles+and+volume+reconstructions+of+soft+tissues+using+laser+beam+measurements.">Cross-sectional profiles and volume reconstructions of soft tissues using laser beam measurements.</a> J Biomech Eng. 126, 796-802 (2004).</li><li>Lee, T. Q., Woo, S. L. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+determining+cross-sectional+shape+and+area+of+soft+tissues.">A new method for determining cross-sectional shape and area of soft tissues.</a> J Biomech Eng. 110, 110-114 (1988).</li><li>Parent, G., Cyr, M., Cousineau-Pelletier, P., Desbiens-Blais, F. &. a. m. p. ;. a. m. p., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Techniques+for+the+Evaluation+of+Cross-Sectional+Area+in+Rat+Tail+Tendons+Generate+Significant+Errors.">Current Techniques for the Evaluation of Cross-Sectional Area in Rat Tail Tendons Generate Significant Errors.</a> , (1985).</li></ol>

No conflicts of interest declared.

By applying those experimental procedures, we can conduct a wide variety of in vitro studies on this kind of tissues. As for example, a study on tissue degeneration was carried out by the application of under-stimulation to RTTs for a period of ten days. Each day, we evaluated tissue mechanical properties in non-destructive stress relaxation tests. At the end, we were able to observe RTT stress variation and thus analyze the progression of mechanical properties.
<img src="/files/ftp_...

<table><tbody><tr><td>D-PBS </td><td>Reagent</td><td>Wisent Inc.</td><td>311-410-CL</td><td>Saline solution</td></tr><tr><td>Glucose </td><td>Reagent</td><td>Wisent Inc.</td><td>609-037-EL</td><td>Saline solution&lt;br/&gt;1g/L</td></tr><tr><td>Antibiotics-antimycotics </td><td>Reagent</td><td>Invitrogen</td><td>15240-062</td><td>Saline solution&lt;br/&gt;1%</td></tr><tr><td>DMEM</td><td>Reagent</td><td>Invitrogen</td><td>12800-017</td><td>Culture solution</td></tr><tr><td>Sodium Bicarbonate</td><td>Reagent</td><td>Wisent Inc.</td><td>600-105-CG</td><td>Culture solution&lt;br/&gt;3.7g/L</td></tr><tr><td>FBS</td><td>Reagent</td><td>Wisent Inc.</td><td>090150</td><td>Culture solution&lt;br/&gt;10%</td></tr><tr><td>Antibiotics-antimycotics</td><td>Reagent</td><td>Invitrogen</td><td>15240-062</td><td>Culture solution&lt;br/&gt;1%</td></tr><tr><td>Optic Micrometer</td><td>Tool</td><td>Custom Made</td><td></td><td></td></tr><tr><td>Manipulation plate</td><td>Tool</td><td>Custom Made</td><td></td><td></td></tr><tr><td>Bioreactor chamber</td><td>Tool</td><td>Custom Made</td><td></td><td></td></tr></tbody></table>

preparation of rat tail tendons for biomechanical and mechanobiological studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

Watch this Scientific Journal Video about Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies at JoVE.com

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

preparation-rat-tail-tendons-for-biomechanical-mechanobiological

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.8K Views.  Université de Sherbrooke. This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Video: Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Neuroscience

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Bioengineering

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Two-Photon Microscopy for the Study of Tendons

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence.
SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.

This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and development.

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in ...

Developmental Biology

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Medicine

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Summary

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Biomechanical Testing of Murine Tendons

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair