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. 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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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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 growt...

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 ...

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281 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 ...

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. ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...