Browsing by Author "Voytik-Harbin, Sherry"
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Item Computational Mechanobiology Model Evaluating Healing of Postoperative Cavities Following Breast-Conserving Surgery(bioRxiv, 2023-04-28) Harbin, Zachary; Sohutskay, David; Vanderlaan, Emma; Fontaine, Muira; Mendenhall, Carly; Fisher, Carla; Voytik-Harbin, Sherry; Buganza Tepole, Adrian; Surgery, School of MedicineBreast cancer is the most commonly diagnosed cancer type worldwide. Given high survivorship, increased focus has been placed on long-term treatment outcomes and patient quality of life. While breast-conserving surgery (BCS) is the preferred treatment strategy for early-stage breast cancer, anticipated healing and breast deformation (cosmetic) outcomes weigh heavily on surgeon and patient selection between BCS and more aggressive mastectomy procedures. Unfortunately, surgical outcomes following BCS are difficult to predict, owing to the complexity of the tissue repair process and significant patient-to-patient variability. To overcome this challenge, we developed a predictive computational mechanobiological model that simulates breast healing and deformation following BCS. The coupled biochemical-biomechanical model incorporates multi-scale cell and tissue mechanics, including collagen deposition and remodeling, collagen-dependent cell migration and contractility, and tissue plastic deformation. Available human clinical data evaluating cavity contraction and histopathological data from an experimental porcine lumpectomy study were used for model calibration. The computational model was successfully fit to data by optimizing biochemical and mechanobiological parameters through the Gaussian Process. The calibrated model was then applied to define key mechanobiological parameters and relationships influencing healing and breast deformation outcomes. Variability in patient characteristics including cavity-to-breast volume percentage and breast composition were further evaluated to determine effects on cavity contraction and breast cosmetic outcomes, with simulation outcomes aligning well with previously reported human studies. The proposed model has the potential to assist surgeons and their patients in developing and discussing individualized treatment plans that lead to more satisfying post-surgical outcomes and improved quality of life.Item Computational Mechanobiology Model Evaluating Healing of Postoperative Cavities Following Breast-Conserving Surgery(Elsevier, 2023) Harbin, Zachary; Sohutskay, David; Vanderlaan, Emma; Fontaine, Muira; Mendenhall, Carly; Fisher, Carla; Voytik-Harbin, Sherry; Buganza Tepole, Adrian; Surgery, School of MedicineBreast cancer is the most commonly diagnosed cancer type worldwide. Given high survivorship, increased focus has been placed on long-term treatment outcomes and patient quality of life. While breast-conserving surgery (BCS) is the preferred treatment strategy for early-stage breast cancer, anticipated healing and breast deformation (cosmetic) outcomes weigh heavily on surgeon and patient selection between BCS and more aggressive mastectomy procedures. Unfortunately, surgical outcomes following BCS are difficult to predict, owing to the complexity of the tissue repair process and significant patient-to-patient variability. To overcome this challenge, we developed a predictive computational mechanobiological model that simulates breast healing and deformation following BCS. The coupled biochemical-biomechanical model incorporates multi-scale cell and tissue mechanics, including collagen deposition and remodeling, collagen-dependent cell migration and contractility, and tissue plastic deformation. Available human clinical data evaluating cavity contraction and histopathological data from an experimental porcine lumpectomy study were used for model calibration. The computational model was successfully fit to data by optimizing biochemical and mechanobiological parameters through Gaussian process surrogates. The calibrated model was then applied to define key mechanobiological parameters and relationships influencing healing and breast deformation outcomes. Variability in patient characteristics including cavity-to-breast volume percentage and breast composition were further evaluated to determine effects on cavity contraction and breast cosmetic outcomes, with simulation outcomes aligning well with previously reported human studies. The proposed model has the potential to assist surgeons and their patients in developing and discussing individualized treatment plans that lead to more satisfying post-surgical outcomes and improved quality of life.Item Early Changes in Porcine Larynges Following Injection of Motor-Endplate Expressing Muscle Cells for the Treatment of Unilateral Vocal Fold Paralysis(Wiley, 2024) Kaefer, Samuel L.; Zhang, Lujuan; Morrison, Rachel A.; Brookes, Sarah; Awonusi, Oluwaseyi; Shay, Elizabeth; Hoilett, Orlando S.; Anderson, Jennifer L.; Goergen, Craig J.; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjectives: 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.Item Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly(Elsevier, 2022-04-30) Zhang, Lujuan; Fujiki, Robert Brinton; Brookes, Sarah; Calcagno, Haley; Awonusi, Oluwaseyi; Kluender, Keith; Berry, Kevin; Venkatraman, Anumitha; Maulden, Amanda; Sivasankar, M. Preeti; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineBackground/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.Item Impact of Needle Selection on Survival of Muscle-Derived Cells When Used for Laryngeal Injections(Longdom Publishing, 2023) Awonusi, Oluwaseyi; Harbin, Zachary J.; Brookes, Sarah; Zhang, Lujuan; Kaefer, Samuel; Morrison, Rachel A.; Newman, Sharlé; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjective: 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.Item Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study(Wiley, 2021-10) Brookes, Sarah; Zhang, Lujuan; Puls, Theodore J.; Kincaid, John; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjective/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.Item Multi-Layered Implant Approach for Hemilaryngectomy Reconstruction in a Porcine Model(Wiley, 2025) Wesson, Troy; Morrison, Rachel A.; Zhang, Lujuan; Brookes, Sarah; Kaefer, Sam; Finnegan, Patrick R.; Calcagno, Haley; Campiti, Vincent J.; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjective: Partial laryngectomies result in voice, swallowing, and airway impairment for thousands of patients in the United States each year. Treatment options for dynamic restoration of laryngeal function are limited. Thus, there is a need for new reconstructive approaches. Here, we evaluated early (4 week) outcomes of multi-layered mucosal-myochondral (MMC) implants when used to restore laryngeal form and function after hemilaryngectomy in a porcine model. Methods: Six Yucatan minipigs underwent transmural hemilaryngectomies followed by reconstruction with customized MMC implants aiming to provide site-appropriate localization of regenerated laryngeal tissues, while supporting laryngeal function. All implants were fabricated from polymeric collagen, with a subset of muscle and cartilage implants containing motor endplate-expressing muscle progenitor cells or cartilage-like cells differentiated from adipose stem cells, respectively. Vocalization and laryngeal electromyography (L-EMG) measurements with nerve conduction studies were performed post-operatively and compared with baseline along with gross and histological analyses of the healing response. Results: All animals (n = 6) survived and maintained airway patency, safe swallowing, and phonation, without the use of tracheostomy and/or gastrostomy tubes. Histological evaluation indicated no adverse tissue reaction or implant degradation, showing progressive regenerative remodeling with mucosa reformation and ingrowth of new muscle and cartilage. Preliminary L-EMG suggested weak but detectable motor unit action potentials. Although vocalization duration, frequency, and intensity decreased post-operatively, all animals retained vocal capacity and parameter recovery was evident over the study duration. Conclusion: Engineered collagen polymeric implants in the presence or absence of autologous cell populations may serve as a feasible reconstructive option to restore dynamic function after hemilaryngectomy. Long-term follow-up is needed to further assess functional outcomes.Item Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis(Wiley, 2025-01-07) Kaefer, Samuel L.; Shay, Elizabeth O.; Morrison, Rachel A.; Zhang, Lujuan; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjectives: The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha-bungarotoxin (α-BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf-life. However, a parallel approach to identify NMJs in paraffin-embedded tissue sections has not been previously described. Methods: Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5-μm thick paraffin-embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons. Results: Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti-nicotinic acetylcholine receptor alpha-1 subunit (CHRNα1, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×). Conclusions: Herein, we describe a new methodology to visualize NMJs within paraffin-embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin-embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin-embedded protocol serves to enhance histological analysis of NMJs.Item Oligomeric collagen as an encapsulation material for islet/β-cell replacement: effect of islet source, dose, implant site, and administration format(American Physiological Society, 2020-08) Hernandez Stephens, Clarissa; Morrison, Rachel A.; McLaughlin, Madeline; Orr, Kara; Tersey, Sarah A.; Scott-Moncrieff, J. Catharine; Mirmira, Raghavendra G.; Considine, Robert V.; Voytik-Harbin, Sherry; Pediatrics, School of MedicineReplacement of islets/β-cells that provide long-lasting glucose-sensing and insulin-releasing functions has the potential to restore extended glycemic control in individuals with type 1 diabetes. Unfortunately, persistent challenges preclude such therapies from widespread clinical use, including cumbersome administration via portal vein infusion, significant loss of functional islet mass upon administration, limited functional longevity, and requirement for systemic immunosuppression. Previously, fibril-forming type I collagen (oligomer) was shown to support subcutaneous injection and in situ encapsulation of syngeneic islets within diabetic mice, with rapid (<24 h) reversal of hyperglycemia and maintenance of euglycemia for beyond 90 days. Here, we further evaluated this macroencapsulation strategy, defining effects of islet source (allogeneic and xenogeneic) and dose (500 and 800 islets), injection microenvironment (subcutaneous and intraperitoneal), and macrocapsule format (injectable and preformed implantable) on islet functional longevity and recipient immune response. We found that xenogeneic rat islets functioned similarly to or better than allogeneic mouse islets, with only modest improvements in longevity noted with dosage. Additionally, subcutaneous injection led to more consistent encapsulation outcomes along with improved islet health and longevity, compared with intraperitoneal administration, whereas no significant differences were observed between subcutaneous injectable and preformed implantable formats. Collectively, these results document the benefits of incorporating natural collagen for islet/β-cell replacement therapies.Item Optimizing transport methods to preserve function of self‐innervating muscle cells for laryngeal injection(Wiley, 2024-12-08) Kaefer, Samuel L.; Zhang, Lujuan; Brookes, Sarah; Morrison, Rachel A.; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjectives: Recently, our laboratory has discovered a self-innervating population of muscle cells, called motor endplate-expressing cells (MEEs). The cells innately release a wide variety of neurotrophic factors into the microenvironment promoting innervation when used as an injectable treatment. Unlike other stem cells, the therapeutic potential of MEEs is dependent on the cells' ability to maintain phenotypical cell surface proteins in particular motor endplates (MEPs). The goal of this study is to identify transport conditions that preserve MEE viability and self-innervating function. Methods: Muscle progenitor cells (MPCs) of adult Yucatan pigs were cultured and induced to generate MEEs. Effects of short-term cryopreservation methods were studied on MPC and MEE stages. A minimally supplemented medium was investigated for suspension-mediated transport, and MEEs were loaded at a constant concentration (1 × 107 cells/mL) into plastic syringes. Samples were subjected to varying temperatures (4, 22, and 37°C) and durations (6, 18, 24, and 48 h), which was followed by statistical analysis of viability. Transport conditions maintaining viability acceptable for cellular therapy were examined for apoptosis rates and expression of desired myogenic, neurotrophic, neuromuscular junction, and angiogenic genes. Results: Cryopreservation proved detrimental to our cell population. However, a minimally supplemented medium, theoretically safe for injection, was identified. Transport temperature and duration impacted cell viability, with warmer temperatures leading to faster death rates prior to the end of the study. Transport conditions did not appear to affect apoptotic rate. Any expression change of desirable genes fell within the acceptable range. Conclusions: Transport state, medium, duration, and temperature must be considered during the transport of injectable muscle cells as they can affect cell viability and expression of integral genes. These described factors are integral in the planning of general cell transport and may prove equally important when the cell population utilized for laryngeal injection is derived from a patient's own initial muscle biopsy.