The protocol describes an efficient and reproducible method for biomechanical testing of mouse tendons through reusable custom-fit 3D printed fixtures. The new methods reduce the time required to test the sample from hours to minutes and eliminate a major gripping artifact that was a common problem in previous methods. This method is not limited to the supraspinatus and Achilles tendons.
It can easily be adapted to testing other mouse tendons and tendons from larger animals. To ensure reproducibility and efficiency of the approach, multiple cycles of design prototyping and validation may be needed for fixtures not described here. To begin, perform a microcomputed tomography scan of the entire bone in the agarose gel.
After reconstruction into cross-section images, select the command File Open to open the file dataset. Open the dialog file Preferences and select the Advanced tab. Use the adaptive rendering algorithm to construct the 3D models which provide smoother surface detail.
Use 10 as the locality parameter which defines the distance in pixels to the neighboring point used for finding the object border. Minimize tolerance to 0.1 to decrease the file size. To specify the volume of interest, manually select two images to set as the top and bottom of the selected VOI range, then move to the second page, Region of Interest.
Manually select the region of interest on a single cross-section image. Repeat selecting the ROI for every 10 to 15 cross-section images. Next, move to the third page, Binary Selection.
On the Histogram menu, click From Dataset to show the histogram distribution of brightness from all images of the dataset. Also on the Histogram menu, click the Create a 3D model file menu. Save a 3D model of the bone in STL file format.
In the Meshmixer software, import mesh and select All to edit. Choose Produce from the Tool Set edit, then select Triangle Budget from the tool set Reduce target. Reduce the tricount and accept changes.
Resave the newly-reduced file in STL format by choosing Export As.To design a supraspinatus tendon humeral bone, use a solid-modeling computer-aided design program to create a custom-fit model of humerus-gripping fixture. Open the STL format file of the humerus bone in a solid-modeling program and save as a part file in SLDPRT format. Then open the Part file to manually create three anatomically relevant planes, for example, sagittal, coronal, and transverse.
Click on File New to create the solid block component part. Click on File New to create an assembly model with two components, the solid block and either the right or left humerus bone. Define the orientation of the bone within the block to ensure the angle between the tendon and bone to be 180 degrees.
Ensure that the entire bone volume fits inside the block. In the Assembly window, select the block and click Edit Component from the Assembly toolbar. Click Insert, Features, Cavity.
Select Uniform Scaling and enter 0%as the value to scale in all directions. Suppress the bone part and save the assembly as a part. Open the cylinder with cavity part.
Create a sketch, ensure to only leave 0.5 millimeters above the humeral head. From Features, select Extruded Cut. Cut the assembly along the sagittal plane to create two symmetrical components that fit the bone anteriorly and posteriorly.
Cut the assembly part along the sagittal plane to create two symmetrical components that fit the bone anteriorly and posteriorly. It's critical to ensure the humeral head to prevent growth plate failure and to define a tight clearance that avoids disengaging of the humerus from the model during testing. Proceed as described in the manuscript to finish the posterior component and anterior component.
Save both components as separate part files. Now press Insert Pattern, Mirror, and select Mirror to create 3D mirror models for each component of the fixture for the opposite limb. Select the front face as a mirror face plain.
Select the part as the body to mirror. Select the mirror plane and create a sketch that includes all material. From Features, select Extruded Cut to remove the original part and to keep only mirrored part.
Click on the bottom of the part and use as a plane to make a sketch. Click on Sketch Text and add L or R.This adds an etch on the bottom of the fixtures to distinguish between the left and right sides. Save all fixture parts in STL standard file format in preparation for 3D printing.
To dissect the supraspinatus muscle-tendon, humerus bone specimen, first position the euthanized mouse in a prone position and make an incision in the skin from above the elbow of the forepaw towards the shoulder. Use forceps to carefully remove the skin with blunt dissection so that the musculature of the shoulder is visible. Remove the tissue surrounding the humerus until the bone is exposed.
Hold the humerus with forceps and carefully remove the deltoid and trapezius muscles to expose the coracoacromial arch. Identify the AC joint and carefully separate the clavicle from the acromion with a scalpel blade. Taking care not to damage the supraspinatus tendon and its bony attachment, remove the muscle from its scapular attachment using a scalpel blade.
Detach the humeral head from the glenoid and lacerate the joint capsule and the infraspinatus subscapularis and teres minor tendons. Disarticulate the elbow joint to separate the humerus from the ulna and radius. Isolate the humerus-supraspinatus tendon muscle specimen and clean off excess soft tissues on the humerus and humeral head.
To dissect the Achilles tendon, the calcaneus bone, position a euthanized mouse in a prone position. Taking care not to damage the Achilles tendon and its bony attachment, remove the skin with blunt dissection so that the musculature around the ankle and knee joints is exposed. Using a scalpel blade, starting at the Achilles tendon-calcaneus attachment, carefully detach the gastrocnemius muscle from its proximal attachments.
Carefully disarticulate the calcaneus from the various adjacent bones and isolate the Achilles tendon-calcaneus specimen and clean off excess soft tissues. To determine the cross-sectional area of the tendon, insert the bone upside down to suspend the specimen in the cryotube filled with agarose gel with the bone in the agarose gel and the tendon and muscle outside. After the scan using microcomputed tomography, gently remove the muscle from the tendon using a scalpel blade.
Insert the bone into the 3D printed fixture and attach them to the testing grids. Insert and glue the tendon between a folded thin tissue paper and clamp it using thin film grips. Insert the sample and the grips into a testing bath of PBS at 37 degrees Celsius and perform a mechanical test.
In this study, using reusable 3D printed fixtures to grip bone, specimen preparation time was reduced from hours to minutes. Representative load deformation curves for tensile testing of supraspinatus tendon using current methods eliminated major gripping artifact such as growth plate failure. The mechanical properties of supraspinatus and Achilles tendons demonstrate a significant effect of sex based on unpaired T tests with P value less than 0.05.
Micro CT successfully measured the cross-sectional area along the length of supraspinatus tendon along the length of Achilles tendon. It's important to remember that each anatomic site has specific design criteria necessary for effective gripping. Therefore, the design for each fixture should be adapted accordingly.
In addition to tensile testing to failure, these fixtures can be used for cyclic loading tests, which provide information about tendon fatigue and viscoeleastic properties. Characterizing the mechanical properties of murine tendons is uncommon in the literature due to a number of reasons, including the difficulty in gripping these small tissues, tedious and time-consuming specimen preparation methods, and growth plate fractures. This protocol presents a time-efficient and reproducible method for testing mouse tendons that has eliminated artifactual grip failures and has tripled the number of specimens that can be tested in a day.
