This research is supported by the UCSF Yearlong Research Fellowship Program and the C-Doctor Interdisciplinary Translational Project Program. Thanks to the members of the Pomerantz Lab and Craniofacial Biology Program at the University of California San Francisco for their contributions.

This study was conducted in accordance with all applicable regulations, including adherence to the recommendations outlined in The Guide for the Care and Use of Laboratory Animals. The UCSF Institutional Animal Care and Use Program approved all animal procedures and postoperative care (IACUC protocol #AN195944-01).
1. VML surgery
<ol>
	<li>Anesthetize male Sprague-Dawley rats at 12 weeks of age weighing 276-300 g in a sealed container using isofluorane general anesthesia at a rate of 1%-5%&#160;before being transferred to a sterile surgical field.
	<ol>
		<li>Position the rodents on their left side such that the neck is extended in a neutral position and the nose fitted into the anesthetic nosecone, allowing exposure to the right side of the face.</li>
		<li>Taper isofluorane flow rate from induction to maintenance flow rate (approximately 2%).</li>
	</ol>
	</li>
	<li>Apply ophthalmic ointment to the eyes of the rodents.</li>
	<li>Clear the operative site, bordered by a line between the right corner of the mouth, the base of the right ear superiorly, and the angle of the mandible inferiorly, of fur using depilatory cream.
	<ol>
		<li>Disinfect the operative site several times in a circular motion using both ethanol and betadine scrub. 
		NOTE: Sterile draping is not used in this protocol, given the size and location of access. The procedure is conducted using an aseptic technique in accordance with animal care guidelines.</li>
	</ol>
	</li>
	<li>Create a longitudinal incision 3-4 cm in length spanning from the inferior right whisker pad to the right ear.
	<ol>
		<li>Elevate a skin flap, allowing visualization of the right masseter as well as the buccal and mandibular branches of the facial nerve (Figure 1A).</li>
		<li>Clear the skin from the underlying fascia using blunt separation and hold the skin edges in a retracted position using surgical clamps for optimal visualization of the underlying muscle.</li>
	</ol>
	</li>
	<li>Make a transverse incision 1-2 cm in length in the fascia overlying the anterior portion of the masseter. Use blunt separation to expand the space between the fascia and the masseter with care to maintain the overall integrity of the remaining fascia.</li>
	<li>Using the fascial incision as a window to the superficial masseter, create a circular injury in the muscle measuring 5 mm across and 5 mm deep using a sterilized disposable punch biopsy. Ensure the defect is centered between the buccal and mandibular branches of the facial nerve, with care to avoid trauma to the nerves (Figure 1B).</li>
	<li>Add the biomaterial to the circular masseter injury (Figure 1C).</li>
	<li>Once the agent is suitably in place, suture the muscle-investing fascial window using a single 5-0 monofilament suture to avoid migration. 
	NOTE: Solid-form agents are directly placed in the wound, and the tissue can be perturbed to promote saturation with blood, whereas liquid agents are added and allowed to gelate if necessary or immediately closed in place using fascia to avoid unwanted movement. In the experiment demonstrated here, an acrylated hyaluronic acid-based cryogel is administered.</li>
	<li>Close the skin using either a simple interrupted or running subcuticular technique with 5-0 monofilament.
	<ol>
		<li>Apply skin glue over the sutures to help prevent postoperative incision dehiscence.</li>
	</ol>
	</li>
	<li>Allow the rats to recover in a cage with an external heating source (heating pad beneath the cage or sterile hand warmers in the cage) and observe for 30 min postoperatively.</li>
	<li>Dilute trimethoprim-sulfamethoxazole antibiotic (200 mg of trimethoprim and 40 mg of sulfamethoxazole in 5mL) in drinking water (5 mL/200 mL water) and administer it in rodents drinking water for 7 days postoperatively. Ensure perioperative analgesia is conducted according to institutional protocols. 
	NOTE: In this study, 0.25% SC bupivacaine was given prior to surgical incision, 0.05 mg/kg SC buprenorphine was administered prior to recovery from anesthesia, and 2 mg/kg IP meloxicam was administered prior to recovery from anesthesia and again the following morning.</li>
</ol>
2. Masseter harvesting, freezing, and analysis
<ol>
	<li>At predetermined time points, sacrifice the rats by inducing an overdose of a chemical agent (e.g., CO2 or anesthetic) in the anesthetic induction chamber.</li>
	<li>Confirm rodent sacrifice by conducting a bilateral thoracotomy using a single scalpel incision through 4th rib ventrally to puncture the lungs (bilateral thoracotomy).</li>
	<li>Reopen the surgical incision to allow visualization of the masseter, and remove the skin overlying the masseter for ease of access.</li>
	<li>Dissect and remove the masseter from the mandible, beginning with separation at the mandibular angle. Ensure the dissection occurs along the bone surface to capture the entirety of the masseter thickness.
	<ol>
		<li>Dissect anteriorly along the mandible, severing the attachment point of the tendon. Then, dissect along the zygomatic arch posteriorly (Figure 1D). At last, dissect the posterior attachment as there is an increased risk of bleeding.</li>
	</ol>
	</li>
	<li>Rinse the excised masseter in 1x PBS at room temperature (RT) and remove excessive moisture by thoroughly blotting the specimen with a paper towel.</li>
	<li>Place the muscle in a cryomold and submerge it in optimal cutting temperature (OCT) embedding media, with the anterior end facing downwards and the posterior end facing upwards.</li>
	<li>Add isopentane to a metal cup and cool it by submerging it in liquid nitrogen with care to prevent liquid nitrogen from entering it.
	<ol>
		<li>Once the isopentane has reached optimal temperature (-140 to -149 &#176;C) and thickened slightly, hold the cryomold containing OCT and the masseter partially submerged in the isopentane until the OCT has frozen through. Keep the frozen cryomold on dry ice until transferred to a freezer at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Cryosection frozen masseter samples with the orientation such that the anterior pole is sectioned first, moving posteriorly through the sample. Set the tissue block temperature to -20 &#176;C and cryosection blade temperature to -15 &#176;C. For every 200 &#181;m, cut 4 slides (2 with 10 &#181;m sections, 2 with 6 &#181;m sections) before moving 200 &#181;m further, cutting 4 additional slides and repeating throughout the sample.</li>
	<li>Use the 10 &#181;m sections for histology analysis (Masson&#39;s Trichrome, Hematoxylin and Eosin, Picrosirius Red, etc.) and the 6 &#181;m sections for immunohistochemistry. 
	NOTE: For histology, 6 &#181;m sections may also be used.</li>
	<li>Capture images using light or fluorescence microscopy.</li>
	<li>Measure the cross-sectional areas of ~200 muscle fibers of masseter muscle using Image J software.</li>
</ol>
3. Immunohistochemical analysis
<ol>
	<li>For immunohistochemical analysis of embryonic myosin heavy chain, fix slides in 4% PFA for 1 h at RT.</li>
	<li>Wash the slides in PBS 3 times at RT, allowing each wash to sit for 10 min.</li>
	<li>Permeabilize tissue by incubating the slides with 0.5% Triton X-100 at RT for 15 min.</li>
	<li>Wash the slides in PBS 3 times at RT, allowing each wash to sit for 10 min.</li>
	<li>Incubate with primary monoclonal antibody from host mice (1:200) in 2% normal goat serum (NGS) at 4 &#176;C overnight.</li>
	<li>Wash the slides in PBS-Tween 3 times at RT, allowing each wash to sit for 10 min.</li>
	<li>Incubate with fluorophore-labeled anti-mouse secondary antibody from rabbit host (1:500) in 2% NGS at RT for 2 h.</li>
	<li>Wash the slides in PBS-Tween 3 times at RT, allowing each wash to sit for 10 min.</li>
	<li>Mount the slides using 3 drops of DAPI fluorescent mount before placing a coverslip.</li>
</ol>

Evaluating Biomaterials in Craniofacial VML Injury Using a Rat Model

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+volumetric+muscle+loss+injury.">Pathophysiology of volumetric muscle loss injury.</a> Cells Tissues Organs. 202 (3-4), 180-188 (2016).</li><li>Kiran, S., Dwivedi, P., Kumar, V., Price, R., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulation+and+biomaterials:+Key+players+to+repair+volumetric+muscle+loss.">Immunomodulation and biomaterials: Key players to repair volumetric muscle loss.</a> Cells. 10 (8), 2016 (2021).</li><li>Greising, S. M., Corona, B. T., McGann, C., Frankum, J. K., Warren, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+approaches+for+volumetric+muscle+loss+injury:+A+systematic+review+and+meta-analysis.">Therapeutic approaches for volumetric muscle loss injury: A systematic review and meta-analysis.</a> Tissue Eng Part B: Rev. 25 (6), 510-525 (2019).</li><li>Bosse, M. 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The authors have nothing to disclose.

There are several critical steps in the protocol where special attention is needed to achieve an optimal result. Step 1.4 describes the initial incision and blunt separation of the skin from the superficial masseter fascia. Blunt dissection should be done directly along the skin with scissors pointing away from the underlying muscle and fascia to prevent nicking and unintentionally creating a window through the fascia. The caudal aspect of the superficial masseter should be avoided to prevent unintentional injury to the submandibular vein or external carotid artery. Step 1.5 and 1.6 describe the creation of the fascial window and VML defect, respectively. Care should be taken to ensure the fascial incision is the minimal size required in order to fit the punch biopsy through, as the fascia tends to contract away from the incision, and it can be difficult to locate the edges in step 1.8 if too large an incision is made. Again, blunt dissection beneath the fascia should be done only enough to allow the punch biopsy to fit within the window and angled appropriately to be perpendicular to the mandible. When creating the VML injury, it is important to identify the largest area between the mandibular and buccal branches of the facial nerve. The distal mandibular nerve divides into an upper and lower division, and there is variation in the location where the upper division branches away from the lower division towards the buccal branch of the facial nerve. Because this division courses superiorly, some animals may have the upper division of the mandibular nerve directly in the belly of the masseter where the defect should be created. In these cases, the animal should not be used for analysis, as there will likely be a peripheral nerve injury confounding the data. When creating the punch biopsy, gentle twisting may be required to penetrate the full 5 mm depth. Step 2.4 describes the blotting of the tissue sample after washing it to remove excessive moisture. This is done to prevent ice crystals from forming during the freezing of the tissue and subsequent reduction in freezing artifacts on histology20.
One of the advantages of this method is the ease of access to the superficial masseter muscle and the relative lack of high-risk structures that may be encountered during the surgery. Because the vasculature enters the caudal end of the muscle, significant bleeding can be avoided in the majority of cases. This is important as it not only decreases the risk of morbidity but also increases standardization and decreases the chance that ischemia has a confounding effect on muscle regeneration. Because of the availability of a fascial enclosure, this protocol may be used to evaluate biomaterials in liquid or solid form factors. While an acellular hydrogel was used as an example for this protocol, this method can be replicated to test biomaterials with human stem cell incorporation using immunodeficient rats as has been done in previous studies of extremity VML21.
This study does not formally define the critical size injury in VML, the precise point where endogenous repair mechanisms can no longer regenerate the muscle remains unclear. While it stands to reason that the definition may involve the removal of a certain percentage of the muscle&#39;s volume, and indeed 20% is often cited, there is conflicting evidence, and data suggest that other factors, including the location and geometry of injury, age of the animal, among others may also contribute16,21,22,23. Despite this, the observation of fibrous scar and visible contraction of the muscle border observed at 12 weeks following injury (Figure 4A) demonstrates that pathological remodeling has occurred, strongly suggesting that the injuries used here are of critical size with respect to the standard definition.
Because craniofacial muscles in humans, like intrinsic hand muscles, are small compared to proximal limb muscles and carry out more precise movements, they are more prone to having their function impacted by VML. The masseter was selected in this protocol because of adequate bulk for testing and ease of access. However, the geometry and location of injury are important factors in evaluating VML, and conclusions generated using this model may not directly translate to other craniofacial muscles. The masseter is a muscle of mastication, and while it contributes to facial expression in children24,&#160;its role in facial expression decreases in adulthood24. Injuries to other muscles of the face, such as the zygomaticus major, may result in greater psychological affliction following VML due to their more direct role in facial expression throughout life relative to the masseter25,26. In addition, this study did not evaluate in vivo force production, a limitation that is an area of need in the field. To date, no reproducible method for craniofacial muscle functional testing with sufficient sensitivity to compare treatment groups has been reported, and this is complicated by the extensive compensatory muscles within the face. Future developments of this protocol will aim to introduce approaches to directly measure force in individual facial muscles.
Alternative methods of histologic analysis of masseter VML in a rodent model are very limited. Kim et al. created a wearable EMG system to assess the functionality of VML-injured masseter muscles in mice. However, their terminal timepoint was 4 weeks, they did not demonstrate the visualization of biomaterial in histology images, did not describe a fascial closure, and focused primarily on validation of their EMG system versus analysis of muscle regeneration and biocompatibility of biomaterials-based treatments17. New treatments are needed for craniofacial volumetric muscle loss, and this masseter VML model is sufficiently reproducible to assess muscle response to VML injury and to evaluate the effects of treatments on regeneration, fibrosis, and contracture, especially in the first 3 months after injury.

Severe trauma, surgical excision, inflammation, and other acquired conditions can result in a degree of tissue loss that overwhelms the endogenous skeletal muscle repair mechanisms. Loss of resident cells and structures that promote the primary regenerative process can result in pathological remodeling and tissue fibrosis, resulting in long-term deficits of function and sensation and are referred to as volumetric muscle loss (VML)1,2,3. The inflammatory response to VML injuries involves a well-documented and complex mechanism involving macrophages, cytokines, and myogenic cells that presents many theoretical targets in regenerative medicine4. While many in vitro studies have utilized these targets in animal models of extremity VML treatment, there is a lack of research on skeletal muscle regeneration in animal models of craniofacial VML5,6,7.
Craniofacial tissue loss can result from conditions as described previously, and deficient craniofacial tissue can also occur in congenital conditions such as clefting, which in some cases involves a true volumetric deficiency of muscle tissue8,9. Because muscles of the craniofacial region are important for function as well as aesthetic appearance, long-term effects of VML may have significant psychological affliction. Several aspects of craniofacial skeletal muscle are different from the somite-derived skeletal muscle found in extremities, including variations in gene expression, embryonic origin, satellite cell phenotype, satellite cell quantity, fiber composition, and architecture10,11,12,13. These variations may result in VML injuries affecting craniofacial muscle differently than somite-derived muscle14,15. To date, tissue engineering approaches shown to increase regeneration in animal models of extremity VML have not translated equivalently to craniofacial VML animal models16. This underscores the need to optimize in vivo approaches to animal craniofacial VML models.
While several in vivo studies of craniofacial VML have been conducted, the studies are small and the creation of a robust craniofacial muscle defect in animal models is challenging8,13. Kim et al. reported the development of a mouse masseter VML model. However, this study only evaluated histology until 28 days following injury and had unclear power to detect differences in histologic outcomes between time points17. Rodriguez et al. reported the development of a sheep craniofacial VML model. However, they reported high variability within experimental groups, suggesting heterogeneity in the severity of the initial surgical injury16. Here, we report the protocol of our rat masseter VML model and demonstrate its utility in evaluating tissue-engineering approaches.

<table><tbody><tr><td>F1.652 Myosin heavy chain (embryonic) monoclonal antibody</td><td>DSHB</td><td>F1.652</td><td></td></tr><tr><td>Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td><td>Invitrogen</td><td>A-21242</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11034</td><td></td></tr><tr><td>Integra Standard Biopsy Punches, Disposable Standard biopsy punch; 5 mm, Diameter: 0.19 in., 0.5 cm</td><td>Integra</td><td>12460411</td><td></td></tr><tr><td>Mounting Medium with DAPI - Aqueous, Fluoroshield</td><td>Abcam</td><td>ab104139</td><td></td></tr><tr><td>Rabbit Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 647) preadsorbed</td><td>Abcam</td><td>ab150127</td><td></td></tr><tr><td>Sulfamethoxazole/Trimethoprim Oral Suspension, Cherry Flavored, 473 mL</td><td>Med-Vet International</td><td>SKU: RXBAC-SUSP</td><td></td></tr></tbody></table>

rodent model of masseter volumetric muscle loss for studying bioengineering materials

Craniofacial volumetric muscle loss (VML) injuries can occur as a result of severe trauma, surgical excision, inflammation, and congenital or other acquired conditions. Treatment of craniofacial VML involves surgical, functional muscle transfer. However, these procedures are unable to restore normal function, sensation, or expression, and more commonly, these conditions go untreated. Very little research has been conducted on skeletal muscle regeneration in animal models of craniofacial VML. This manuscript describes a rat model for the study of craniofacial VML injury and a protocol for the histological evaluation of biomaterials in the treatment of these injuries. Liquid hydrogel and freeze-dried scaffolds are applied at the time of surgical VML creation, and masseters are excised at terminal time points up to 12 weeks with high retention rates and negligible complications. Hematoxylin and eosin (HE), Masson's Trichrome, and immunohistochemical analysis are used to evaluate parameters of skeletal muscle regeneration as well as biocompatibility and immunomodulation. While we demonstrate the study of a hyaluronic-acid-based hydrogel, this model provides a means for evaluating subsequent iterations of materials in VML injuries.

Outcomes for the evaluation of craniofacial VML and tissue regeneration using biomaterials include both quantitative and qualitative outcomes.
Figure 2 depicts an example of qualitative evaluation using the previously described model. The observation of de novo muscle fiber growth within our hydrogel is a qualitative positive outcome (Figure 2A) and suggests a biomaterial can provide sufficient architectural support and growth factor or nutrient delivery. Retention of hydrogel within the wound bed (Figure 2B) is visible on HE stain. Qualitative assessment of other tissues within a biomaterial may be done, such as the identification of blood vessels using HE (Figure 2C) and Masson&#39;s trichrome (Figure 2D). The mandibular and buccal branches of the facial nerve are visible on the upper left and upper right corners of the image in Figure 2B, confirming the correct placement of hydrogel in the VML injury bed and providing orientation. A layer of fascia is seen above the hydrogel, confirming the fascial closure was successful and remained intact during the postoperative period. A negative result seen in this stage would include full loss of hydrogel (Figure 2E) or loss of fascial closure and extrusion from the wound bed (Figure 2F), with HE showing evidence of a wound bed or fibrotic scar depending on the elapsed time postoperatively.
Immunohistochemical analysis is critical in the evaluation of biomaterials for the treatment of craniofacial VML in order to identify cell types and molecular interactions at the cellular level. Because VML results in the loss of resident cells and architecture capable of regenerating the skeletal muscle, de novo muscle regeneration at the injured site is an important outcome in treatment4 and the evaluation of efficacy in biomaterials-based therapies for VML. Developmental isoforms of myosin, including embryonic (MYH3) and neonatal myosin (MYH8), are re-expressed in regenerating skeletal muscle fibers and provide a specific marker for regenerating fibers18,19. Figure 3 shows an example of how immunohistochemistry can be used to identify cellular types of interest, in this case, the embryonic myosin heavy chain.
Finally, ImageJ can be utilized to quantify features of histology slides, including immunohistochemistry and specialized stains such as Masson&#39;s trichrome, to measure the fibrotic response (Figure 4). Removal of a portion of muscle in VML injuries changes the muscle architecture and composition, leading to increased collagen I deposition and gross compartmental tissue fibrosis2,3. Many studies evaluating biomaterials-based treatments for VML compare fibrosis between their intervention group and control groups4. Using the method described here, the wound beds in animals that underwent VML injury can be identified on Masson&#39;s trichrome as fibrotic scars (Figure 4A). Color deconvolution through ImageJ can isolate and quantify the blue channel corresponding to connective tissue (Figure 4B), then set to grayscale for measurement (Figure 4C). The area of the scar can be isolated and measured using Image J (Figure 4D) and calibrated to a scale bar on the image for accurate size and reproducible area. Using this approach, the connective tissue deposition can be measured (Figure 4E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66450/66450fig01.jpg" /> 
Figure 1: Surgical schematic demonstrating the steps. (A,B) Surgical masseter VML injury. (C) Biomaterial application. (D) Masseter harvesting. (E) Muscle morphology following harvesting. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66450/66450fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66450/66450fig02.jpg" /> 
Figure 2: HE and Masson&#39;s trichrome staining. (A) HE-stained rat masseter sections from hydrogel-muscle interface with arrows indicating structures morphologically identified as skeletal muscle fibers. (B) HE-stained rat masseter section demonstrating the abundance of skeletal muscle fibers throughout hydrogel. (C) HE-stained and (D) Masson&#39;s trichrome-stained tissue with arrows indicating structures morphologically identified as blood vessels. (E,F) Negative results showing full loss of hydrogel with gel adhered to the superficial aspect of the masseter rather than the wound bed and incomplete loss of hydrogel with loss of fascial closure, respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66450/66450fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66450/66450fig03.jpg" /> 
Figure 3: Immunohistologic staining for embryonic myosin heavy chain (eMHC) and laminin on rat masseter cross sections. (A) 2 weeks post-surgery. (B) 12 weeks post-surgery. An increased concentration of eMHC is seen in the 12-week sample relative to the 2-week sample. Images captured at 20x magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66450/66450fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66450/66450fig04.jpg" /> 
Figure 4: Quantitative fibrosis analysis. (A) Masson&#39;s trichrome-stained rat masseter section for quantification of connective tissue deposition as a proxy for wound fibrosis after VML injury at 12 weeks. (B) Image J was used to deconvolute the blue channel corresponding to connective tissue. (C) The image was converted to grayscale for analysis. (D) The area corresponding to the fibrotic scar was isolated, and (E) the cross-sectional area and percentages of the field corresponding to white (background) and black (connective tissue) were calculated. Images captured using light microscopy at 10x magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66450/66450fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Rodent Model of Masseter Volumetric Muscle Loss for Studying Bioengineering Materials

Here, we describe a protocol for the surgical creation of a volumetric muscle loss (VML) injury in the rat masseter, providing a reproducible and accessible model for the study of craniofacial muscle injuries and their treatment using biomaterials such as the novel hydrogel.

Craniofacial volumetric muscle loss (VML) injuries can occur as a result of severe trauma, surgical excision, inflammation, and congenital or other ...

rodent-model-masseter-volumetric-muscle-loss-for-studying

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

337 Views.  University of California San Francisco (UCSF). Here, we describe a protocol for the surgical creation of a volumetric muscle loss (VML) injury in the rat masseter, providing a reproducible and accessible model for the study of craniofacial muscle injuries and their treatment using biomaterials such as the novel hydrogel.

Video: Rodent Model of Masseter Volumetric Muscle Loss for Studying Bioengineering Materials

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is essential to determine the effect of high ground impact forces on lower-body bone deformation to better understand the mechanisms of bone stress injuries. Conventional strain gauge measurement has been used to study in vivo tibia deformation. This method is associated with limitations including invasiveness of the procedure, involvement of few human subjects, and limited strain data from small bone surface areas. The current study intends to introduce a novel approach to study tibia bone strain under high impact loading conditions. A subject-specific musculoskeletal model was created to represent a healthy male (19 years, 80 kg, 1,800 mm). A flexible finite element tibia model was created based on a computed tomography (CT) scan of the subject&#39;s right tibia. Laboratory motion capture was performed to obtain kinematics and ground reaction forces of drop-landings from different heights (26, 39, 52 cm). Multibody dynamic computer simulations combined with a modal analysis of the flexible tibia were performed to quantify tibia strain during drop-landings. Calculated tibia strain data were in good agreement with previous in vivo studies. It is evident that this non-invasive approach can be applied to study tibia bone strain during high impact activities for a large cohort, which will lead to a better understanding of injury mechanism of tibia stress fractures.

During landing, lower-body bones experience large mechanical loads and are deformed. It is essential to measure bone deformation to better understand the mechanisms of bone stress injuries associated with impacts. A novel approach integrating subject-specific musculoskeletal modeling and finite element analysis is used to measure tibial strain during dynamic movements.

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Force Assessment of Rat Masseter Muscle After VML Injury

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

Volumetric Muscle Loss (VML) is prevalent in civilian and military populations and represents a debilitating skeletal muscle injury surpassing the body&#39;s natural regenerative capacity. These injuries disrupt not only muscle fibers but also nerves, blood vessels, and the extracellular matrix, overwhelming the regenerative capacity of skeletal muscle and leading to severe fibrosis and permanent deficiencies in muscle structure and function. Current clinical management has many limitations, and thus, research is ongoing to develop more effective therapeutic approaches. Notably, however, much of the preclinical emphasis on VML injuries has focused on limb and trunk muscles, with limited investigation into craniofacial muscles. Differences in developmental biology and regenerative capacity between craniofacial and limb/trunk muscles may provide crucial insights that drive more injury-specific VML treatment options. Moreover, evaluation of functional recovery is critical to establishing therapeutic efficacy. In this regard, in vivo testing of muscle contraction with percutaneous nerve stimulation is a minimally invasive method that allows for repeated functional assessment over the course of a study - in the same animal. In light of these considerations, this paper describes a method for the in vivo assessment of muscle function in the rat masseter muscle before and after a VML injury. This protocol is the first published instance to detail the creation and functional evaluation of a biologically relevant craniofacial VML injury in the rat.

Volumetric muscle loss (VML) injuries exceed endogenous regenerative ability, resulting in permanent functional deficits. Current VML research primarily focuses on limb and trunk muscles. To extend mechanistic studies of VML to craniofacial muscles, this article describes an in vivo method for longitudinal assessment of masseter muscle function pre- and post-VML injury.

Volumetric Muscle Loss (VML) is prevalent in civilian and military populations and represents a debilitating skeletal muscle injury surpassing the ...

Establishing an Orofacial Muscle Fibrosis Model in Mice via a Freezing Injury

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.

Author Spotlight: Exploring Orofacial Muscle Regeneration &#8211; Insights and Innovations

The goal of this protocol is to establish an orofacial muscle fibrosis model. Comparison of the histology between mice masseter and tibialis anterior muscle after freezing injury confirmed masseter muscle fibrosis. This model will facilitate further investigation into the mechanism underlying orofacial muscle fibrosis.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived ...

Rodent Model of Masseter Volumetric Muscle Loss for Studying Bioengineering Materials

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Przeglądaj więcej filmów

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Microfluidic Mixers for Studying Protein Folding

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

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

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model