In Vivo Functional Assessment of Rat Masseter Muscle Following Surgical Creation of a Volumetric Muscle Loss (VML) Injury

Our mission is to leverage multi-institutional, multidisciplinary collaborations to develop novel and more efficacious tissue engineering and regenerative medicine technologies for unmet patient needs. Our major focus right now is on the musculoskeletal system, in particular, the development of technologies for the treatment of volumetric muscle loss injuries in civilians and wounded warriors. A variety of approaches have been tried in animal models ranging from application of biomaterials and biomaterial systems, with and without cells, through implantation of tissue engineered skeletal muscle.

However, none of these approaches have yet yielded full recovery of muscle form and function. Development of more high throughput, cost-effective, and biologically relevant animal models that address the full spectrum of volumetric muscle loss injuries, including standardized metrics for comparison of multi-scale, biomechanical and molecular mechanisms that drive functional deficits and mediate improved tissue repair and regeneration. Much of the research on volumetric muscle loss injuries and treatment is focused on limb and trunk muscles.

These muscles are developmentally different from muscles of the cranial-facial region, and therefore, may respond differently to injury and repair. It is imperative that we develop models in this region, which we accomplish here. The robust and consistent methods shown here permits the longitudinal assessment of jaw function in injured and healthy rats.

Researchers can track how animals respond to injury and treatment at multiple time points throughout the course of the study. This will provide another layer of insight into the mechanisms attending muscle regeneration after severe injury. To begin, weigh the anesthetized rat and place it onto a surgery board in the lateral position on its left side.

After shaving the right side of the face and sterilizing the surgical area, make an approximately two centimeter incision along the buccal region of the rat. Use blunt dissection to separate the skin and fascia. Make a similar incision along the fascia and gently dissect it away from the underlying masseter.

After locating the buccal and marginal nerves, use a sterile surgical marker and ruler to mark the area to be removed. With the help of micro scissors, carefully remove the tissue until the final injury reaches a depth of approximately four millimeters with 150 milligrams of excised tissue. Close the fascia using absorbable 6-O, and skin with non-absorbable 5-0 interrupted sutures.

To begin, open the Dynamic Muscle Control software, from the Setup menu, find InstantStim and change the parameters to the desired values. Under the Setup menu, create and select an auto save folder to store the data. Then, at the bottom of the screen, locate AutoSave Base, and change it to a title specific to the animal being tested.

Select Sequencer at the top of the screen. Then in the opened window, select Open Sequence at the bottom of the screen. Now from the file explorer, select the pre-made sequence.

Click on Load Sequence, followed by Close Window. To enable viewing of real time data acquisition, select File and Live Data Monitor. Then, in the Live Data Monitor window, set the timescale, Y-value minimum and Y-value Maximum Position the volumetric muscle loss injured, anesthetized rat in the supine position with its nose securely in the nose cone.

Place a securing loop over the upper half of the rat's jaw to hold its nose securely on the platform. Then, secure the upper half of the rat by placing and fastening a strap across its arms and chest. Adjust the bite lever position using the three rotating knobs located near the rig's platform to control the X, Y, and Z planes.

With the help of the top knob, bring the bite lever down towards the rat using the other two knobs, adjust the end of the lever just over the chin of the rat. Use tweezers to hook the hanging loop around the bottom teeth of the rat to secure the head and prevent it from moving. Adjust the height of the lever to increase the tension on the jaw.

Palpate the jaw, and locate the posterior corner of the mandible. Position electrodes subcutaneously around either side of the corner at approximately two to three millimeters apart. In the Live Data Monitor window, click on the Instant Stim button to activate it.

Then adjust electrode placement and lever arm position using the knobs as needed. Start turning the range knob to modulate the amperage. Then turn the adjust knob to modulate the percentage of the current range being used to stimulate the jaw.

After optimizing the twitch response, confirm that the electrodes are still securely in place and stop the Instant Stim. Then, from the Live Data Monitor window, click on the Start Sequence button and monitor the stimulation frequency responses. Injured rats produced significantly lower average maximum force at four, eight and 12 weeks post volumetric muscle loss compared to baseline.

The control cohort maintained consistent maximum force across all time points. No significant differences in animal body weights were observed between the control and no repair groups over time.