Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.

Diagnosis of subglottic stenosis in a rabbit model using long-range optical coherence tomography.

OBJECTIVES/HYPOTHESIS: Current imaging modalities lack the necessary resolution to diagnose subglottic stenosis. The aim of this study was to use optical coherence tomography (OCT) to evaluate nascent subglottic mucosal injury and characterize mucosal thickness and structural changes using texture analysis in a simulated intubation rabbit model. STUDY DESIGN: Prospective animal study in rabbits. METHODS: Three-centimeter-long sections of endotracheal tubes (ETT) were endoscopically placed in the subglottis and proximal trachea of New Zealand White rabbits (n = 10) and secured via suture. OCT imaging and conventional endoscopic video was performed just prior to ETT segment placement (day 0), immediately after tube removal (day 7), and 1 week later (day 14). OCT images were analyzed for airway wall thickness and textural properties. RESULTS: Endoscopy and histology of intubated rabbits showed a range of normal to edematous tissue, which correlated with OCT images. The mean airway mucosal wall thickness measured using OCT was 336.4 µm (day 0), 391.3 µm (day 7), and 420.4 µm (day 14), with significant differences between day 0 and day 14 (P = .002). Significance was found for correlation and homogeneity texture features across all time points (P < .05). CONCLUSIONS: OCT is a minimally invasive endoscopic imaging modality capable of monitoring progression of subglottic mucosal injury. This study is the first to evaluate mucosal injury during simulated intubation using serial OCT imaging and texture analysis. OCT and texture analysis have the potential for early detection of subglottic mucosal injury, which could lead to better management of the neonatal airway and limit the progression to stenosis. LEVEL OF EVIDENCE: NA Laryngoscope, 127:64-69, 2017.

Model to Estimate Threshold Mechanical Stability of Lower Lateral Cartilage.

IMPORTANCE: In rhinoplasty, techniques used to alter the shape of the nasal tip often compromise the structural stability of the cartilage framework in the nose. Determining the minimum threshold level of cartilage stiffness required to maintain long-term structural stability is a critical aspect in performing these surgical maneuvers. OBJECTIVE: To quantify the minimum threshold mechanical stability (elastic modulus) of lower lateral cartilage (LLC) according to expert opinion. METHODS: Five anatomically correct LLC phantoms were made from urethane via a 3-dimensional computer modeling and injection molding process. All 5 had identical geometry but varied in stiffness along the intermediate crural region (0.63-30.6 MPa). DESIGN, SETTING, AND PARTICIPANTS: A focus group of experienced rhinoplasty surgeons (n = 33) was surveyed at a regional professional meeting on October 25, 2013. Each survey participant was presented the 5 phantoms in a random order and asked to arrange the phantoms in order of increasing stiffness based on their sense of touch. Then, they were asked to select a single phantom out of the set that they believed to have the minimum acceptable mechanical stability for LLC to maintain proper form and function. MAIN OUTCOMES AND MEASURES: A binary logistic regression was performed to calculate the probability of mechanical acceptability as a function of the elastic modulus of the LLC based on survey data. A Hosmer-Lemeshow test was performed to measure the goodness of fit between the logistic regression and survey data. The minimum threshold mechanical stability for LLC was taken at a 50% acceptability rating. RESULTS: Phantom 4 was selected most frequently by the participants as having the minimum acceptable stiffness for LLC intermediate care. The minimum threshold mechanical stability for LLC was determined to be 3.65 MPa. The Hosmer-Lemeshow test revealed good fit between the logistic regression and survey data (χ23 = 0.92, P = .82). CONCLUSIONS AND RELEVANCE: This study presents a novel method of modeling anatomical structures and quantifying the mechanical properties of nasal cartilage. Quantifying these parameters is an important step in guiding surgical maneuvers performed in rhinoplasty. LEVEL OF EVIDENCE: 5.

In-depth analysis of pH-dependent mechanisms of electromechanical reshaping of rabbit nasal septal cartilage.

OBJECTIVES/HYPOTHESIS: Electromechanical reshaping (EMR) involves reshaping cartilage by mechanical deformation and delivering electric current to the area around the bend axis, causing local stress relaxation and permanent shape change. The mechanism of EMR is currently unclear, although preliminary studies suggest that voltage and application time are directly related to the concentration and diffusion of acid-base products within the treated tissue with little heat generation. This study aims to characterize local tissue pH changes following EMR and to demonstrate that local tissue pH changes are correlated with tissue damage and shape change. STUDY DESIGN: Ex vivo animal study involving EMR of rabbit nasal septal cartilage and biochemical estimation of tissue pH changes. METHODS: The magnitude and diffusion of acid-base chemical products in control (0V, 2 minutes), shape change (4V, 4 minutes; 6V, 1, 2, 4 minutes; 8V, 1, 2 minutes), and tissue damage (8V, 4, 5 minutes; 10V, 4, 5 minutes) parameters following EMR are approximated by analyzing local pH changes after pH indicator application. RESULTS: There is a direct relationship between total charge transfer and extent of acid-base product diffusion (P <0.05). A "pH transition zone" is seen surrounding the bend apex above 8V, 2 minutes. Colorimetric analysis suggests that small local pH changes (10(-8) hydrogen ions) are at least partly implicated in clinically efficacious EMR. CONCLUSIONS: These results provide additional insight into the translational applications of EMR, particularly the relationship among pH changes, shape change, and tissue injury, and are integral in optimizing this promising technology for clinical use.

The rabbit costal cartilage reconstructive surgical model.

Rib grafts in facial plastic surgery are becoming more frequently used. Small animal models, although not ideal may be used to emulate costal cartilage-based procedures. A surgical characterization of this tissue will assist future research in the selection of appropriate costal segments, based on quantitative and qualitative properties. The objective of this study is to assess the surgical anatomy of the rabbit costal margin and evaluate costal cartilage for use in either in vivo or ex vivo studies and to examine reconstructive procedures. Detailed thoracic dissections of 21 New Zealand white rabbits were performed post-mortem. Costal cartilage of true, false, and floating ribs were harvested. The length, thickness, and width at proximal, medial, and distal locations of the cartilage, with perichondrium intact were measured. Further qualitative observation and digital images of curvature, flexibility, and segmental cross-sectional shape were recorded. The main outcome measure(s) is to characterize, describe, and assess the consistency of dimensions, location, and shape of costal cartilage. In this study, 12 to 13 ribs encase the thoracic cavity. Cartilage from true ribs has an average length, width, and depth of 23.75 ± 0.662, 3.02 ± 0.025, and 2.18 ± 0.018 mm, respectively. The cartilage from false ribs has an average length, width, and depth of 41.97 ± 1.48, 2.00 ± 0.07, 1.19 ± 0.03 mm, and that of floating ribs are 7.66 ± 0.29, 1.98 ± 0.04, and 0.96 ± 0.03 mm. Rib 8 is found to be the longest costal cartilage (49.10 ± 0.64 mm), with the widest and thickest at ribs 1 (3.91 ± 0.08 mm) and 6 (2.41 ± 0.11 mm), respectively. Cross-sectional segments reveal the distal cartilage to maintain an hourglass shape that broadens to become circular and eventually ovoid at the costochondral junction. The New Zealand white rabbit is a practical source of costal cartilage that is of sufficient size and reproducibility to use in surgical research where the long-term effects of operations, therapies, devices, and pharmacologic on cartilage can be studied in vivo.

Precise and rapid costal cartilage graft sectioning using a novel device: clinical application.

IMPORTANCE The use of costal cartilage as a graft in facial reconstructive surgery requires sectioning the cartilage into a suitable shape. OBJECTIVE To evaluate the accuracy of a novel mechanical device for producing uniform slices of costal cartilage and to illustrate the use of the device during nasal surgery. DESIGN Basic and clinical study using 100 porcine ex vivo costal cartilage slices and 9 operative cases. METHODS This instrument departs from antecedent devices in that it uses compression to secure and stabilize the specimen during sectioning. A total of 75 porcine costal cartilage ribs were clamped with minimal compression just sufficient to secure and stabilize the specimen while cutting. Slices having a length of 4 cm and width of 1 cm were obtained using the cartilage cutter at 3 thicknesses: 1 mm (n = 25), 2 mm (n = 25), and 3 mm (n = 25). The procedure was repeated for the 2-mm thick samples; however, the ribs in this group (n = 25) were clamped using the maximum amount of compression attainable by the device. Thickness was measured using a digital micrometer. Case presentations illustrate the use of the device in secondary and reconstructive rhinoplasty surgery. RESULTS All specimens were highly uniform in thickness on visual inspection and appeared to be adequate for clinical application. Sectioning was completed in several seconds without complication. In the porcine specimens sectioned using minimal compression, the percentage difference in thickness for each individual sample averaged 18%, 10%, and 11% for the 1-mm-, 2-mm-, and 3-mm-thick slices, respectively. Within the specimens sectioned using maximum compression, the percentage difference in thickness for each individual sample averaged 35% for the 2-mm-thick slices. In the setting of nasal reconstructive surgery, slices having a thickness from 1 to 2 mm were found to be well suited for all necessary graft types. CONCLUSIONS AND RELEVANCE The simple mechanical device described produces costal cartilage graft slices with highly uniform thickness. Securing the rib by clamping during cutting reduces uniformity of the slices; however, the imperfections are minimal, and all sectioned grafts are adequate for clinical application. The device can be adjusted to produce slices of appropriate thickness for all nasal cartilage grafts. This device is valuable for reconstructive procedures owing to its ease of use, rapid operation, and reproducible results.

In vivo laser cartilage reshaping with carbon dioxide spray cooling in a rabbit ear model: a pilot study.

BACKGROUND/OBJECTIVES: Similar to conventional cryogen spray cooling, carbon dioxide (CO2) spray may be used in combination with laser cartilage reshaping (LCR) to produce cartilage shape change while minimizing cutaneous thermal injury. Recent ex vivo evaluation of LCR with CO2 cooling in a rabbit model has identified a promising initial parameter space for in vivo safety and efficacy evaluation. This pilot study aimed to evaluate shape change and cutaneous injury following LCR with CO2 cooling in 5 live rabbits. STUDY DESIGN/MATERIALS AND METHODS: The midportion of live rabbit ears were irradiated with a 1.45 µm wavelength diode laser (12 J/cm(2)) with simultaneous CO2 spray cooling (85 millisecond duration, 4 alternating heating/cooling cycles per site, 5 to 6 irradiation sites per row for 3 rows per ear). Experimental and control ears (no LCR) were splinted in the flexed position for 30 days following exposure. A total of 5 ears each were allocated to the experimental and control groups. RESULTS: Shape change was observed in all irradiated ears (mean 70 ± 3°), which was statistically different from control (mean 37 ± 11°, P = 0.009). No significant thermal cutaneous injury was observed, with preservation of the full thickness of skin, microvasculature, and adnexal structures. Confocal microscopy and histology demonstrated an intact and viable chondrocyte population surrounding irradiated sites. CONCLUSIONS: LCR with CO2 spray cooling can produce clinically significant shape change in the rabbit auricle while minimizing thermal cutaneous and cartilaginous injury and frostbite. This pilot study lends support for the potential use of CO2 spray as an adjunct to existing thermal-based cartilage reshaping modalities. An in vivo systematic evaluation of optimal laser dosimetry and cooling parameters is required.

Real-time subglottic stenosis imaging using optical coherence tomography in the rabbit.

IMPORTANCE: Subglottic stenosis (SGS) is a severe, acquired, potentially life-threatening disease that can be caused by endotracheal tube intubation. Newborns and neonates are particularly susceptible to SGS owing to the small caliber of their airway. OBJECTIVE: To demonstrate optical coherence tomography (OCT) capabilities in detecting injury and scar formation using a rabbit model. Optical coherence tomography may provide a noninvasive, bedside or intensive care unit modality for the identification of early airway trauma with the intention of preventing progression to SGS and can image the upper airway through an existing endotracheal tube coupled with a small fiber-optic probe. DESIGN: Rabbits underwent suspension laryngoscopy with induction of of SGS via epithelial injury. This model was used to test and develop our advanced, high-speed, high-resolution OCT imaging system using a 3-dimensional microelectromechanical systems-based scanning device integrated with a fiber-optic probe to acquire high-resolution anatomic images of the subglottic epithelium and lamina propria. SETTING: All experiments were performed at the Beckman Laser Institute animal operating room. INTERVENTION OR EXPOSURE: Optical coherence tomography and endoscopy was performed with suspension laryngoscopy at 6 different time intervals and compared with conventional digital endoscopic images and histologic sections. Fifteen rabbits were killed at 3, 7, 14, 21, and 42 days after the induction of SGS. The laryngotracheal complexes were serially sectioned for histologic analysis. MAIN OUTCOME AND MEASURE: Histologic sections, endoscopic images, and OCT images were compared with one another to determine if OCT could accurately delineate the degree of SGS achieved. RESULTS: The rabbit model was able to reliably and reproducibly achieve grade I SGS. The real-time OCT imaging system was able to (1) identify multiple structures in the airway; (2) delineate different tissue planes, such as the epithelium, basement membrane, lamina propria, and cartilage; and (3) detect changes in each tissue plane produced by trauma. Optical coherence tomography was also able demonstrate a clear picture of airway injury that correlated with the endoscopic and histologic images. With subjective review, 3 patients had high correlation between OCT and histologic images, 10 demonstrated some correlation with histologic images, and 2 showed little to no correlation with histologic images. CONCLUSIONS AND RELEVANCE: Optical coherence tomography, coupled with a fiber-optic probe, identifies subglottic scarring and can detect tissue changes in the rabbit airway to a depth of 1 mm. This technology brings us 1 step closer to minimally invasive subglottic airway monitoring in the intubated neonate, with the ultimate goal of preventing SGS and better managing the airway.

Identifying ideal brow vector position: empirical analysis of three brow archetypes.

BACKGROUND: Surgical browlifts counteract the effects of aging, correct ptosis, and optimize forehead aesthetics. While surgeons have control over brow shape, the metrics defining ideal brow shape are subjective. OBJECTIVES: This study aims to empirically determine whether three expert brow design strategies are aesthetically equivalent by using expert focus group analysis and relating these findings to brow surgery. METHODS: Comprehensive literature search identified three dominant brow design methods (Westmore, Lamas and Anastasia) that are heavily cited, referenced or internationally recognized in either medical literature or by the lay media. Using their respective guidelines, brow shape was modified for 10 synthetic female faces, yielding 30 images. A focus group of 50 professional makeup artists ranked the three images for each of the 10 faces to generate ordinal attractiveness scores. RESULTS: The contemporary methods employed by Anastasia and Lamas produce a brow arch more lateral than Westmore's classic method. Although the more laterally located brow arch is considered the current trend in facial aesthetics, this style was not empirically supported. No single method was consistently rated most or least attractive by the focus group, and no significant difference in attractiveness score for the different methods was observed (p = 0.2454). CONCLUSION: Although each method of brow placement has been promoted as the "best" approach, no single brow design method achieved statistical significance in optimizing attractiveness. Each can be used effectively as a guide in designing eyebrow shape during browlift procedures, making it possible to use the three methods interchangeably.

In vivo electromechanical reshaping of ear cartilage in a rabbit model: a minimally invasive approach for otoplasty.

OBJECTIVE: To report the first successful study to date of in vivo electromechanical reshaping of ear cartilage in a rabbit model. METHODS: Ears of New Zealand white rabbits were reshaped using percutaneous needle electrode electromechanical reshaping (5 V for 4 minutes) and were then bolstered for 4 weeks. Ten ears were treated, with 2 undergoing sham procedures and serving as controls. The treatment was performed using a platinum array of electrodes consisting of 4 parallel rows of needles inserted across the region of flexures in the ear. After 4 weeks, the animals were killed, and the ears were photographed and sectioned for conventional light microscopy and confocal microscopy (live-dead fluorescent assays). RESULTS: Significant shape change was noted in all the treated ears (mean, 102.4°; range, 87°-122°). Control ears showed minimal shape retention (mean, 14.5°; range, 4°-25°). Epidermis and adnexal structures were preserved in reshaped ears, and neochondrogenesis was noted in all the specimens. Confocal microscopy demonstrated a localized zone of nonviable chondrocytes (<2.0 mm in diameter) surrounding needle sites in all the treated ears. CONCLUSIONS: Electromechanical reshaping can alter the shape of the rabbit auricle, providing good creation and retention of shape, with limited skin and cartilage injury. Needle electrode electromechanical reshaping is a viable technique for minimally invasive tissue reshaping, with potential applications in otoplasty, septoplasty, and rhinoplasty. Further studies to refine dosimetry parameters will be required before clinical trials.

Mechanical analysis of the effects of cephalic trim on lower lateral cartilage stability.

OBJECTIVE: To determine how mechanical stability changes in the lower lateral cartilage (LLC) after varying degrees of cephalic resection in a porcine cartilage nasal tip model. METHODS: Alar cartilage was harvested from fresh porcine crania (n = 14) and sectioned to precisely emulate a human LLC in size and dimension. Flexural mechanical analysis was performed both before and after cephalic trims of 0 (control), 4, and 6 mm. Cantilever deformation tests were performed on the LLC models at 3 locations (4, 6, and 8 mm from the midline), and the integrated reaction force was measured. An equivalent elastic modulus of the crura was calculated assuming that the geometry of the LLC model approximated a modified single cantilever beam. A 3-dimensional finite element model was used to model the stress distribution of the prescribed loading conditions for each of the 3 types of LLC widths. RESULTS: A statistically significant decrease (P = .02) in the equivalent elastic modulus of the LLC model was noted at the most lateral point at 8 mm and only when 4 mm of the strut remained (P = .05). The finite element model revealed that the greatest internal stresses was at the tip of the nose when tissue was flexed 8 mm from the midline. CONCLUSION: Our results provide the mechanical basis for suggested clinical guidelines stating that a residual strut of less than 6 mm can lead to suboptimal cosmetic results owing to poor structural support of the overlying skin soft-tissue envelope by an overly resected LLC.

Electromechanical reshaping of costal cartilage grafts: a new surgical treatment modality.

OBJECTIVES/HYPOTHESIS: Needle electrode-based electromechanical reshaping (EMR) is a novel, ultra-low-cost nascent surgical technology to reshape cartilage with low morbidity. EMR uses direct current to induce mechanical relaxation in cartilage that is first deformed into a required geometry, which in turn leads to permanent shape change. The objective of this study was to determine the effect of EMR voltage and time on the shape change of costal cartilage grafts. STUDY DESIGN: EMR of ex vivo porcine costal cartilage. METHODS: Graft specimens obtained from the central core of porcine costal cartilage were bent at a 90-degree angle with a custom jig and then reshaped via EMR. The effects of voltage (3-7 V) and application time (1-5 minutes) on the amount of shape change were systematically examined. Bend angles were analyzed using analysis of variance and paired t tests to determine significant reshaping times at each voltage setting. RESULTS: There is a threshold for voltage and time above which the retention of bend angle is statistically significant in treated specimens compared to the control (P < .05). Above the threshold of 3 V, shape retention initially increased with application time for all voltages tested and was then observed to reach a plateau. Shape retention was noted to be greatest at 6 V without a rise in temperature. CONCLUSIONS: EMR provides a novel method to bend and shape costal cartilage grafts for use in facial plastic surgery. A low voltage can reshape cartilage grafts within several minutes and without the heat generation. This study demonstrates the feasibility of EMR and brings this minimally invasive procedure closer to clinical implementation.