Tissue-Engineered Implant for Hemilaryngectomy Reconstruction with Recurrent Laryngeal Nerve Injury.

OBJECTIVE: Laryngeal cancer resections often require excision of portions of the larynx along with sacrifice of the ipsilateral recurrent laryngeal nerve (RLN). In such cases, there are no reconstructive options that reliably restore laryngeal function, rendering patients with severe functional impairment. To address this unmet clinical need, we extend our evaluation of a 3-implant mucosal, muscle, cartilage reconstruction approach aimed at promoting functional laryngeal restoration in a porcine hemilaryngectomy model with ipsilateral RLN transection. METHODS: Six Yucatan mini-pigs underwent full-thickness hemilaryngectomies with RLN transection followed by transmural reconstruction using fabricated collagen polymeric mucosal, muscle, and cartilage replacements. To determine the effect of adding therapeutic cell populations, subsets of animals received collagen muscle implants containing motor-endplate-expressing muscle progenitor cells (MEEs) and/or collagen cartilage implants containing adipose stem cell (ASC)-derived chondrocyte-like cells. Acoustic vocalization and laryngeal electromyography (L-EMG) provided functional assessments and histopathological analysis with immunostaining was used to characterize the tissue response. RESULTS: Five of six animals survived the 4-week postoperative period with weight gain, airway maintenance, and audible phonation. No tracheostomy or feeding tube was required. Gross and histological assessments of all animals revealed implant integration and regenerative remodeling of airway mucosa epithelium, muscle, and cartilage in the absence of a material-mediated foreign body reaction or biodegradation. Early voice and L-EMG data were suggestive of positive functional outcomes. CONCLUSION: Laryngeal reconstruction with collagen polymeric mucosa, muscle, and cartilage replacements may provide effective restoration of function after hemilaryngectomy with RLN transection. Future preclinical studies should focus on long-term functional outcomes. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Early Changes in Porcine Larynges Following Injection of Motor-Endplate Expressing Muscle Cells for the Treatment of Unilateral Vocal Fold Paralysis.

OBJECTIVES: No curative injectable therapy exists for unilateral vocal fold paralysis. Herein, we explore the early implications of muscle-derived motor-endplate expressing cells (MEEs) for injectable vocal fold medialization after recurrent laryngeal nerve (RLN) injury. METHODS: Yucatan minipigs underwent right RLN transection (without repair) and muscle biopsies. Autologous muscle progenitor cells were isolated, cultured, differentiated, and induced to form MEEs. Three weeks after the injury, MEEs or saline were injected into the paralyzed right vocal fold. Outcomes including evoked laryngeal electromyography (LEMG), laryngeal adductor pressure, and acoustic vocalization data were analyzed up to 7 weeks post-injury. Harvested porcine larynges were examined for volume, gene expression, and histology. RESULTS: MEE injections were tolerated well, with all pigs demonstrating continued weight gain. Blinded analysis of videolaryngoscopy post-injection revealed infraglottic fullness, and no inflammatory changes. Four weeks after injection, LEMG revealed on average higher right distal RLN activity retention in MEE pigs. MEE-injected pigs on average had vocalization durations, frequencies, and intensities higher than saline pigs. Post-mortem, the MEE-injected larynges revealed statistically greater volume on quantitative 3D ultrasound, and statistically increased expression of neurotrophic factors (BDNF, NGF, NTF3, NTF4, NTN1) on quantitative PCR. CONCLUSIONS: Minimally invasive MEE injection appears to establish an early molecular and microenvironmental framework to encourage innate RLN regeneration. Longer follow-up is needed to determine if early findings will translate into functional contraction. LEVEL OF EVIDENCE: NA Laryngoscope, 134:272-282, 2024.

Enhanced Abductor Function in Bilateral Vocal Fold Paralysis with Muscle Stem Cells.

INTRODUCTION: Patients with bilateral vocal fold paralysis (BVFP) experience airway obstruction because of loss of abductor function of posterior cricoarytenoid (PCA) muscles. We previously reported that implantation of autologous muscle progenitor (stem) cells into thyroarytenoid muscles during reinnervation resulted in improved adductor function. In this study, that same approach was applied to treating PCA muscles in a canine model of BVFP. DESIGN: Animal study. METHODS: Two canines underwent baseline measures of glottal resistance (GR), then complete transection and suture repair of both recurrent laryngeal nerves. Muscle stem cells were isolated from skeletal muscle and cultured. Two months later, GR was measured, and then 107 stem cells were implanted into one PCA muscle of each animal. After four more months, GR and glottal opening force (GOF) were measured and the muscles were harvested for histologic study. One control dog underwent the same procedures without stem cell implantation, for comparison. RESULTS: GR increased by 21%-25% over baseline at 2 months, but after stem cell implantation, improved to 10%-14% over baseline at 6 months. PCA muscle strength, as determined by GOF, was 61%-65% on control sides (no stem cells), and 78%-83% on treated sides (with stem cells). Histology confirmed survival of stem cells and a 50% higher rate of innervation of motor endplates in the stem cell treated sides. CONCLUSION: Autologous muscle progenitor (stem) cells show promise as a potential new therapy for patients with bilateral vocal fold paralysis. Additional studies are needed to determine the optimal number of cells, timing of implantation, and other variables before launching a clinical trial. LEVEL OF EVIDENCE: NA (animal study) Laryngoscope, 134:324-328, 2024.

Impact of Needle Selection on Survival of Muscle-Derived Cells When Used for Laryngeal Injections.

Objective: To describe how differing injector needles and delivery vehicles impact Autologous Muscle-Derived Cell (AMDC) viability when used for laryngeal injection. Methods: In this study, adult porcine muscle tissue was harvested and used to create AMDC populations. While controlling cell concentration (1 × 107 cells/ml), AMDCs including Muscle Progenitor Cells (MPCs) or Motor Endplate Expressing Cells (MEEs) were suspended in either phosphate-buffered saline or polymerizable (in-situ scaffold forming) type I oligomeric collagen solution. Cell suspensions were then injected through 23- and 27-gauge needles of different lengths at the same rate (2 ml/min) using a syringe pump. Cell viability was measured immediately after injection and 24- and 48-hours post-injection, and then compared to baseline cell viability prior to injection. Results: The viability of cells post-injection was not impacted by needle length or needle gauge but was significantly impacted by the delivery vehicle. Overall, injection of cells using collagen as a delivery vehicle maintained the highest cell viability. Conclusion: Needle gauge, needle length, and delivery vehicle are important factors that can affect the viability of injected cell populations. These factors should be considered and adapted to improve injectable MDC therapy outcomes when used for laryngeal applications.

Engineered collagen polymeric materials create noninflammatory regenerative microenvironments that avoid classical foreign body responses.

The efficacy and longevity of medical implants and devices is largely determined by the host immune response, which extends along a continuum from pro-inflammatory/pro-fibrotic to anti-inflammatory/pro-regenerative. Using a rat subcutaneous implantation model, along with histological and transcriptomics analyses, we characterized the tissue response to a collagen polymeric scaffold fabricated from polymerizable type I oligomeric collagen (Oligomer) in comparison to commercial synthetic and collagen-based products. In contrast to commercial biomaterials, no evidence of an immune-mediated foreign body reaction, fibrosis, or bioresorption was observed with Oligomer scaffolds for beyond 60 days. Oligomer scaffolds were noninflammatory, eliciting minimal innate inflammation and immune cell accumulation similar to sham surgical controls. Genes associated with Th2 and regulatory T cells were instead upregulated, implying a novel pathway to immune tolerance and regenerative remodeling for biomaterials.

Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly.

BACKGROUND/OBJECTIVES: While voice-related therapeutic interventions are often researched preclinically in the porcine model, there are no well-established methods to induce porcine glottic phonation. Described approaches, such as training animals to phonate for positive reinforcement are time-consuming and plagued by inherent variability in the type of phonation produced and contamination of background noise. Thus, a reliable method of assessing glottic phonation in the porcine model is needed. METHODS: In this study, we have created a novel pulley-based apparatus with harness for "pig-lifting" with surrounding acoustic insulation and high-directional microphone with digital recorder for recording phonation. Praat and Matlab were used to analyze all porcine vocalizations for fundamental frequency (F0), intensity, duration of phonation and cepstral peak prominence (CPP). Glottic phonation was detected using F0 (≥2000 hz), duration (≥3 seconds) and researcher perceptual judgment. Partial-glottic phonations were also analyzed. Reliability between researcher judgment and acoustic measures for glottic phonation detection was high. RESULTS: Acoustic analysis demonstrated that glottic and partial-glottic phonation was consistently elicited, with no formal training of the minipigs required. Glottic vocalizations increased with multiple lifts. Glottic phonation continued to be elicited after multiple days but became less frequent. Glottic and partial-glottic phonations had similar CPP values over the 6 experimental days. CONCLUSION: Our cost-effective, reliable method of inducing and recording glottic phonation in the porcine model may provide a cost effective, preclinical tool in voice research.

Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study.

OBJECTIVE/HYPOTHESIS: There are currently no treatments available that restore dynamic laryngeal function after hemilaryngectomy. We have shown that dynamic function can be restored post hemilaryngectomy in a rat model. Here, we report in a first of its kind, proof of concept study that this previously published technique is scalable to a porcine model. STUDY DESIGN: Animal study. METHODS: Muscle and fat biopsies were taken from three Yucatan minipigs. Muscle progenitor cells (MPCs) and adipose stem cells (ASCs) were isolated and cultured for 3 weeks. The minipigs underwent a left laterovertical partial laryngectomy sparing the left arytenoid cartilage and transecting the recurrent laryngeal nerve. Each layer was replaced with a tissue-engineered implant: 1) an acellular mucosal layer composed of densified Type I oligomeric collagen, 2) a skeletal muscle layer composed of autologous MPCs and aligned oligomeric collagen differentiated and induced to express motor endplates (MEE), and 3) a cartilage layer composed of autologous ASCs and densified oligomeric collagen differentiated to cartilage. Healing was monitored at 2 and 4 weeks post-op, and at the 8 week study endpoint. RESULTS: Animals demonstrated appropriate weight gain, no aspiration events, and audible phonation. Video laryngoscopy showed progressive healing with vascularization and re-epithelialization present at 4 weeks. On histology, there was no immune reaction to the implants and there was complete integration into host tissue with nerve and vascular ingrowth. CONCLUSIONS: This pilot study represents a first in which a transmural vertical partial laryngectomy was performed and successfully repaired with a customized, autologous stem cell-derived multi-layered tissue-engineered implant. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2277-2284, 2021.

Motor endplate-expressing cartilage-muscle implants for reconstruction of a denervated hemilarynx.

OBJECTIVE: Tissue engineering of the larynx requires a complex, multiple tissue layer design. Additionally, spontaneous reinnervation of the larynx after recurrent laryngeal nerve (RLN) injury is often disorganized, resulting in subpar function. This study investigates use of tissue-engineered cartilage and motor endplate-expressing (MEE) tissue-engineered skeletal muscle implants for laryngeal reconstruction and the promotion of organized reinnervation after RLN injury. METHODS: F344 rat primary muscle progenitor cells (MPCs) were isolated. Three-dimensional muscle constructs were created by encapsulating MPCs in type I oligomeric collagen under passive tension. Constructs were then cultured in differentiation medium (MPC control constructs) or induced to form motor endplates (MEE constructs) with neurotrophic agents. Three-dimensional cartilage constructs were created with adipose stem cells differentiated in chondrocyte medium. The muscle and cartilage constructs were implanted into surgically created myochondral defects in the F344 rat larynx with injured or intact (control) RLN. At 1-, 3-, and 6-month timepoints, videolaryngoscopy, electromyography (EMG), histology, and immunohistochemistry were used to assess outcomes. RESULTS: At all timepoints, cartilage-muscle implants were well integrated into host tissue. Functionally, there was increased vocal fold adduction and EMG activity in nerve-injured rats treated with the MEE constructs when compared to those treated with the MPC control constructs. Motor endplate-expressing constructs had increased myofiber cross-sectional area compared to MPC control constructs. CONCLUSION: Although our laboratory previously demonstrated that muscle and cartilage constructs could be used separately for hemilaryngeal reconstruction, this study suggests combining them with the modification of MEEs rather than MPCs, resulting in improved muscle recovery after recurrent laryngeal nerve injury. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1293-1300, 2019.

Three-dimensional tissue-engineered skeletal muscle for laryngeal reconstruction.

OBJECTIVE: There is an unmet need for tissue-engineered three-dimensional (3D) muscle constructs for laryngeal reconstruction. Functional engineered muscle could be used to repair postoncologic or traumatic defects or to medialize the vocal fold in cases of paresis/paralysis. Autologous, organized, engineered muscle that has adequate bulk integrates into host tissue and restores function currently does not exist. METHODS: Primary skeletal muscle progenitor cells (MPCs) were isolated from F344 rats. Three-dimensional muscle constructs were created by encapsulating MPCs via flow alignment in a customized collagen formulation and cultured under passive tension. Muscle-specific immunohistochemistry and confocal microscopy were used to evaluate muscle tissue differentiation. After 2 weeks of culture, muscle constructs were implanted into surgically created defects in the rat larynx. Postmortem function testing and histology was performed at 1 and 3 months. RESULTS: Immunohistochemistry with confocal microscopy demonstrated well-differentiated myotubes, which were well aligned and distributed throughout the engineered construct in vitro. There was evidence of restoration of normal laryngeal function at 1 month postoperative, as indicated by safe swallow (no aspiration events), weight gain, and excellent animal survival. Postmortem specimens demonstrated functional muscle contraction on ex vivo testing, and histology confirmed integration into host tissue. CONCLUSION: This is the first study to demonstrate that functional, 3D tissue-engineered skeletal muscle can be developed from primary MPCs and standardized oligomeric collagen. Collectively, these findings may have tremendous clinical implications for autologous laryngeal muscle repair and reconstruction. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:603-609, 2018.

Improved adductor function after canine recurrent laryngeal nerve injury and repair using muscle progenitor cells.

OBJECTIVE: Muscle progenitor cells (MPCs) can be isolated from muscle samples and grown to a critical mass in culture. They have been shown to survive and integrate when implanted into rat laryngeal muscles. In this study, the ability of MPC implants to enhance adductor function of reinnervated thyroarytenoid muscles was tested in a canine model. STUDY DESIGN: Animal study. METHODS: Sternocleidomastoid muscle samples were harvested from three canines. Muscle progenitor cells were isolated and cultured to 107 cells over 4 to 5 weeks, then implanted into right thyroarytenoid muscles after ipsilateral recurrent laryngeal nerve transection and repair. The left sides underwent the same nerve injury, but no cells were implanted. Laryngeal adductor force was measured pretreatment and again 6 months later, and the muscles were harvested for histology. RESULTS: Muscle progenitor cells were successfully cultured from all dogs. Laryngeal adductor force measurements averaged 60% of their baseline pretreatment values in nonimplanted controls, 98% after implantation with MPCs, and 128% after implantation with motor endplate-enhanced MPCs. Histology confirmed that the implanted MPCs survived, became integrated into thyroarytenoid muscle fibers, and were in close contact with nerve endings, suggesting functional innervation. CONCLUSION: Muscle progenitor cells were shown to significantly enhance adductor function in this pilot canine study. Patient-specific MPC implantation could potentially be used to improve laryngeal function in patients with vocal fold paresis/paralysis, atrophy, and other conditions. Further experiments are planned. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage.

OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E123-E129, 2018.

Frequency of behavior witnessed and conformity in an everyday social context.

Conformity is thought to be an important force in human evolution because it has the potential to stabilize cultural homogeneity within groups and cultural diversity between groups. However, the effects of such conformity on cultural and biological evolution will depend much on the particular way in which individuals are influenced by the frequency of alternative behavioral options they witness. In a previous study we found that in a natural situation people displayed a tendency to be 'linear-conformist'. When visitors to a Zoo exhibit were invited to write or draw answers to questions on cards to win a small prize and we manipulated the proportion of text versus drawings on display, we found a strong and significant effect of the proportion of text displayed on the proportion of text in the answers, a conformist effect that was largely linear with a small non-linear component. However, although this overall effect is important to understand cultural evolution, it might mask a greater diversity of behavioral responses shaped by variables such as age, sex, social environment and attention of the participants. Accordingly we performed a further study explicitly to analyze the effects of these variables, together with the quality of the information participants' responses made available to further visitors. Results again showed a largely linear conformity effect that varied little with the variables analyzed.

Response theory for confined systems.

In this work, we use the transient time correlation function (TTCF) method to evaluate the response of a fluid confined in a nanopore and subjected to shear. The shear is induced by the movement of the boundaries in opposite directions and is made of moving atoms. The viscous heat generated inside the pore is removed by a thermostat applied exclusively to the atomic walls, so as to leave the dynamics of the fluid purely Newtonian. To establish a link with nonlinear response theory and apply the TTCF formalism, dissipation has to be generated inside the system. This dissipation is then time correlated with a phase variable of interest (e.g., pressure) to obtain its response. Until recently, TTCF has been applied to homogeneous fluids whose equations of motion were coupled to a mechanical field and a thermostat. In our system dissipation is generated by a boundary condition rather than a mechanical field, and we show how to apply TTCF to these realistic confined systems, comparing the shear stress response so obtained with that of homogeneous systems at equivalent state points.

Entertainment-education in the context of humor: effects on safer sex intentions and risk perceptions.

Past research has examined the effects of entertainment narratives on story-related behaviors, but most has focused primarily on dramatic genres rather than comedy. The present study examines how the presence or absence of pregnancy-related humor influences viewers' counterarguing, perceived severity, and intentions to engage in unprotected sexual behavior. Results were consistent with expectations in that related humor reduced counterarguing while also trivializing the severity of the consequences of sexual behavior. When the pregnancy storyline was presented in its original humorous context, viewers reported greater intentions to engage in unprotected sex than when pregnancy was presented in a more serious tone. Model testing clarified this finding by revealing the underlying mechanisms. Practical and theoretical implications are discussed.

Potassium channel blocker, 4-aminopyridine-3-methanol, restores axonal conduction in spinal cord of an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a severely debilitating neurodegenerative diseases marked by progressive demyelination and axonal degeneration in the CNS. Although inflammation is the major pathology of MS, the mechanism by which it occurs is not completely clear. The primary symptoms of MS are movement difficulties caused by conduction block resulting from the demyelination of axons. The possible mechanism of functional loss is believed to be the exposure of potassium channels and increase of outward current leading to conduction failure. 4-Aminopyridine (4-AP), a well-known potassium channel blocker, has been shown to enhance conduction in injured and demyelinated axons. However, 4-AP has a narrow therapeutic range in clinical application. Recently, we developed a new fast potassium channel blocker, 4-aminopyridine-3-methanol (4-AP-3-MeOH). This novel 4-AP derivative is capable of restoring impulse conduction in ex vivo injured spinal cord without compromising the ability of axons to follow multiple stimuli. In the current study, we investigated whether 4-AP-3-MeOH can enhance impulse conduction in an animal model of MS. Our results showed that 4-AP-3-MeOH can significantly increase axonal conduction in ex vivo experimental autoimmune encephalomyelitis mouse spinal cord.