Keynote abstract

PARAMETRIC SENSITIVITY ANALYSIS OF DISC MECHANICS USING A NONLINEAR BIPHASIC AND FIBER-REINFORCED FINITE ELEMENT ANALYSIS

Debabrata Auddya, Harrah Newman, John Peloquin, Dawn M Elliott

University of Delaware

Finite element analysis (FEA) is widely used to assess motion segment mechanics under disc degeneration or surgical interventions. Despite numerous spine FEA studies, the selection of material models and properties is often unjustified, and the impact of tissue-level material properties on overall motion segment behavior remains unclear. This study aimed to conduct a parametric sensitivity analysis of annulus fibrosus (AF) material properties to identify key parameters influencing motion segment mechanics. Using FEBio, we simulated torsion and axial creep loading on a non-degenerated intervertebral disc model with AF-nucleus pulposus (NP) transition regions based on Newman et al. 2021. The inner AF, outer AF, and NP were modeled as a nonlinear biphasic material incorporating a Holmes-Mow elastic solid with strain-dependent permeability, Donnan equilibrium osmotic swelling, and, in the AF, nonlinear fibers. A one-parameter-at-a-time sensitivity analysis was performed on selected AF and NP parameters. The results identified material parameters that significantly affect motion segment response, essential for modeling different degeneration stages. These findings help refine FEA models, enabling more accurate simulations and guiding the development of patient-specific disc models based on a reduced set of key material properties.

Lab: Elliott

IN VIVO ASSESSMENT OF PATELLAR TENDON LOADING DURING BASAS SPANISH SQUATS WITH NEUROMUSCULAR ELECTRICAL STIMULATION

Jack Felipe, Claudia Kacmarcik, João Luiz Quaglioti Durigan (Universidade de Brasília, Laboratory of Muscle and Tendon Plasticity, Graduate Program in Rehabilitation Sciences, DF, Brazil), Karin Grävare Silbernagel, Stephanie Cone

University of Delaware

Patellar tendinopathy causes load-related pain in the patellar tendon that can be managed through progressive tendon loading. In this study, we employ shear wave tensiometry in healthy participants to measure loading in the patellar tendon during Basas Spanish squats (squat) and Basas Spanish Squats with superimposed neuromuscular electrical stimulation (squat+NMES). We hypothesized that patellar tendon loading would be greater during squat+NMES. Participants performed Basas Spanish squats with NMES (squat+NMES) and without NMES (squat) while patellar tendon load was tracked via shear wave tensiometry. Using statistical parametric mapping (SPM), we found no significant differences in shear wave speed patterns between squat and squat+NMES trials (SPM{t} > 3.30). While there were no significant differences in loading patterns, there was an increase in average patellar tendon loading during the squat+NMES condition with a high degree of variance between participants. These results suggest that Basas Spanish squats with superimposed NMES could increase patellar tendon strain compared to standard Basas Spanish squats. However, these results remain inconclusive due to the high degree of variability caused by individual tissue properties of each participant.

Lab: Cone

TRIPLY PERIODIC MINIMAL SURFACE ARCHITECTURES ENHANCE SHORT-TERM BONE GROWTH IN PCL SCAFFOLDS

Hisham Omar, Paulina Bargallo Gonzalez Lugo, Rachel Bonfini, Matthew Fainor, Sarah Gullbrand, Michael W. Hast

University of Delaware

Polycaprolactone (PCL) scaffolds are widely used for bone reconstruction due to their biocompatibility and tunable properties. Recent advancements in additive manufacturing provide new opportunities to design PCL scaffolds with intricate lattice architectures. Notably, honeycomb designs are known for their superior mechanical strength. Conversely, triply periodic minimal surface (TPMS) architectures, which feature interconnected pores and high surface areas, are known to improve cell proliferation. The relationships between scaffold lattice architecture and bone regeneration remain unclear. This study used PCL test coupons to evaluate the impact of scaffold architecture (Honeycomb vs. TPMS Gyroid) and porosity (small and large pores) on bone regeneration. We hypothesized that TPMS Gyroid designs would enhance bone regeneration with only minimal losses in mechanical strength. Four unique scaffold designs were 3D-printed, seeded with juvenile bovine bone marrow, and cultured for 4 or 8 weeks. Metabolic activity, calcium deposition, bone volume, and compressive mechanics were analyzed. At week 4, Gyroid scaffolds showed significantly greater metabolic activity, calcium deposition, and bone volume than Honeycomb samples. Honeycomb scaffolds exhibited superior mechanical strength. There were minimal differences between groups at week 8. These findings demonstrate a trade-off between bone formation and mechanical strength, highlighting the need for further optimization of scaffold designs.

Lab: Hast

SPONTANEOUS CALCIUM SIGNALING AND CELL-CELL COMMUNICATION IN HUMAN ARTICULAR CARTILAGE

Ying Peng, Annie Porter, Michael Axe, X. Lucas Lu

University of Delaware

Intracellular calcium ([Ca²⁺]i) signaling plays an essential role in chondrocyte mechanotransduction and serves as a key regulator of cartilage metabolism. We previously revealed spontaneous [Ca²⁺]i signaling, a phenomenon commonly associated with excitable cells such as neurons and cardiomyocytes, in bovine chondrocytes. However, spontaneous [Ca²⁺]i signaling in human cartilage, particularly in healthy joints, remains understudied. In addition, chondrocytes are considered isolated from each other, with little evidence supporting the existence of direct cell-cell communication. Therefore, this study aimed to (1) characterize spontaneous [Ca²⁺]i signaling in healthy human cartilage and compare it with that in late-stage osteoarthritis (OA) cartilage, (2) quantify the matrix synthesis capability of chondrocytes in healthy and OA cartilage and correlate it with [Ca²⁺]i signaling, and (3) investigate potential cell-cell communication through spontaneous [Ca²⁺]i signaling among chondrocytes. Our results demonstrate spontaneous [Ca²⁺]i signaling in cartilage from both healthy donors and OA patients. Notably, OA chondrocytes exhibited a higher proportion of active cells and more frequent [Ca²⁺]i peaks compared to healthy chondrocytes. Finger-print like [Ca²⁺]i peak patterns in [Ca²⁺]i transient and cell-cell communication among in situ chondrocytes were observed in both healthy and OA cartilage. Additionally, OA chondrocytes exhibited increased synthesis of glycosaminoglycans, and collagen compared to healthy chondrocytes.

Lab: Lu

EFFECTS OF ENGINEERED SURFACE ROUGHNESS ON ADDITIVELY MANUFACTURED ZINC FOR ORTHOPAEDIC IMPLANTS

Rachel Bonfini, Jacklyn Griffis, Stephen Ching, Kazi Shahed, Guha Manogharan, Michael Hast

University of Delaware

Laser Powder Bed Fusion (LPBF) is an additive manufacturing technique that offers precise design capabilities for fabricating complex scaffold geometries, which may improve orthopaedic implant designs. Zinc is a promising material for such applications due to its essential role in bone metabolism, moderate degradation rate, and biocompatibility. Because additive manufacturing of zinc is in its nascency, little is known about the material properties of LPBF zinc parts and how we can optimize surface properties to enhance cell adhesion and proliferation. Pure zinc test coupons with various geometries and surface textures were created using LPBF. We performed tensile tests, scanning electron microscopy (SEM), optical profilometry, and energy dispersive X-ray spectroscopy (EDS) to quantify mechanical properties, microstructure, and chemical composition. We also performed a cell culture study with polished 316L stainless steel parts as a control. Mean tensile strength of parts was 72.88 MPa, 6.4% oxygen was found in EDS analysis, and changes in surface texture were significant. All zinc samples expressed higher metabolic activity compared to 316L stainless steel (p<0.0001), but changes in surface textures had minimal impact. Overall, this study provides key insights into how LPBF zinc implants may provide new avenues for fracture repair.

Research Area: Bone

CHONDROTOXICITY OF LIDOCAINE AND BUPIVACAINE ON CARTILAGE

Annie Porter, Naod Asres, Chloe Kappen, Ying Peng, Michael Axe, X. Lucas Lu

University of Delaware

Lidocaine and bupivacaine are the most commonly used anesthetics in orthopedics for procedures and post-operative pain management. However, there are clinical concerns that local anesthetics may be chondrotoxic and lead to cartilage degeneration. The evidence is conflicting, varying with the in vitro model used and the type and dosage of anesthetic. In this study, we submerged young healthy bovine cartilage in lidocaine and bupivacaine according to their physiological doses and half-lives. We then quantified chondrocyte viability and cartilage metabolic activity using click chemistry methods. Both lidocaine and bupivacaine significantly reduced chondrocyte viability in the top zone after 24 hours but had no significant effect in the middle zone. Lidocaine also reduced chondrocyte viability in the top zone after 7 days, but the effect was not significant. Lidocaine significantly reduced GAG and collagen synthesis, whereas bupivacaine had no significant effect on either. Neither lidocaine nor bupivacaine had any significant effect on GAG loss. Cell death is not necessarily sufficient to induce GAG loss in vitro, but the same may not be true in vivo. In summary, caution must be taken when selecting local anesthetics, particularly when injecting in conjunction with stem cell therapies.

Research Area: Cartilage

SINGLE-CELL SPATIAL PROTEOMIC MAPPING OF TMJ INNERVATING NEURONS IN PRECLINICAL MODELS OF TMJ-OA

Robert Ndzeidze, Neda K. Dezfuli, Skyler Brand, Daria Chefan, Joohyun Lim, Austin Keeler

University of Delaware

Temporomandibular disorders (TMDs) are a collective group of diseases that are associated with severe pain and joint dysfunction, which affects 11-12 individuals in the United States alone. Temporomandibular joint osteoarthritis (TMJ-OA) is frequently associated with synovial inflammation in TMD patients and cartilage infiltration by inflammatory mediators originating from the synovium that may play a role in cartilage degeneration and sensitization of joint innervating neurons in TMJ. Patients with TMDs can develop chronic neuropathic pain that does not effectively respond to current modes of therapy, which may negatively impact their quality of life. However, pathological mechanisms that cause chronic pain associated with TMDs are not well understood. Previous studies suggest that abnormal infiltration of immune cells such as macrophages in the trigeminal ganglia (TG) can activate trophic signaling pathways in neuronal cells leading to increased sensitivity of pain neurons.
In this study, we aim to characterize immune infiltration in the TMJ and TG in preclinical mouse models of TMJ-OA to determine trophic signaling pathways that are activated in the TG neuronal subtypes and to test their role in the persistence of chronic pain. To this end, we have used a multiplexed protein-based single cell platform, known as Imaging Mass Cytometry (IMC), which can analyze up to 50 protein markers simultaneously in single cells through rare earth metal-conjugated antibodies that allow for multiplexing. To develop a single-cell proteomic panel of immune, trophic signaling, neuronal subtype, as well as markers for mandibular condyle cartilage and bone, we completed metal conjugation and validation of our antibody panel for IMC.
Overall, results from this study will allow us to gain more insights into the mechanisms of TMJ-OA and chronic pain. In future studies, we will investigate the impact of TMJ-OA on immune and neuronal cell subtypes and their role in pathophysiology.

Research Area: Cartilage

HIGH-THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL OSTEOARTHRITIS THERAPEUTICS

Julie Nguyen, Annie Porter, X. Lucas Lu

University of Delaware

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and extracellular matrix dysregulation due to chronic inflammation. Current clinical treatments primarily focus on symptom relief, with limited options to prevent cartilage damage. In this study, we developed a high-throughput drug screening (HTS) protocol to identify FDA-approved compounds that mitigate glycosaminoglycan (GAG) loss, an early marker of OA progression. Using juvenile bovine cartilage explants, we screened 80 compounds in duplicate and measured GAG loss under IL-1β-induced inflammatory conditions. Resveratrol, a known anti-inflammatory compound, was included as a positive control. HTS hits were defined as compounds that reduced GAG loss by at least two standard deviations below the IL-1β group. Sixteen hits were identified, including Zafirlukast, Dioscin, and Telmisartan, which target OA-relevant pathways like GPCR signaling, autophagy regulation, and cytokine/chemokine inhibition. Additional hits, such as Revefenacin and Iptacopan, act on less explored pathways like cholinergic signaling and complement system inhibition, providing novel therapeutic potential. Data mining revealed interconnected relationships between the hits and key biological mechanisms involved in cartilage maintenance. In summary, our HTS protocol identified multiple chondroprotective candidates, providing opportunities for drug repurposing and further investigation into their therapeutic potential for OA treatment.

Research Area: Cartilage

TRIAMCINOLONE ACETONIDE’S EFFECTS ON HUMAN CARTILAGE METABOLISM

Annie Porter, Michael Axe, X. Lucas Lu

Corticosteroid injections are often used to control joint inflammation, but there is a widely held fear among clinicians that steroid injections could cause degeneration of otherwise healthy cartilage. Previously, we found that in contrast to concerns, triamcinolone acetonide (TA, a corticosteroid) protected against inflammation-induced degradation in juvenile bovine cartilage. In this study, we evaluated the effects of TA on healthy and OA human cartilage. TA had no effect on chondrocyte viability in either human cartilage. TA did have some effects on GAG and collagen synthesis, although the effects were dose, tissue, and donor dependent. A 200 μM dose of TA exposed for 14 days reduced GAG synthesis by ~25% in OA cartilage, but a 1 nM dose had no effect. In healthy cartilage, a worst-case 14 day exposure to TA reduced GAG or collagen synthesis by <25% in some donors, but all donors were not affected following a two day TA exposure and 14 day recovery period. No dose of TA induced GAG loss from healthy or OA cartilage. In summary, the effects of TA on healthy and OA human cartilage were minimal and recoverable, indicating TA may be a safe and effective option for managing joint inflammation clinically.

Research Area: Cartilage

ESTABLISHING METHODS FOR MEASURING MENISCUS DISPLACEMENTS IN-VIVO

Jamie Benson, Isabelle Larche, Dawn M. Elliott

Nemours Children’s Health

The meniscus plays a critical role in transmitting axially loaded forces while mitigating excessive stress and strain on the articular cartilage of the knee. Meniscus tears disrupt its mechanical abilities, leading to altered joint loading, joint instability and increased risk of developing osteoarthritis. Current standard treatment is surgical repair. While most meniscus repair surgeries are highly successful, some studies have shown repair failure rates upwards of 20% —which may be attributed to the lack of consensus on post-operative rehabilitation protocols. Studies have shown that early weight-bearing and mobilization leads to better patient outcomes. However, studies have also shown that it can compromise repair integrity and potentially lead to repair failure. While there have been several mechanical studies in cadaveric samples, meniscus mechanics under load in-vivo has remained largely unstudied. In this study, we addressed this gap by fabricating an in-vivo magnetic resonance imaging compatible knee loading device to quantify meniscus mechanics under physiologic loading conditions. The objective of this study was to establish methods for measuring meniscus displacements in healthy adults. Establishing methods in healthy adults will provide a baseline of typical meniscus mechanics and lay the groundwork for future studies evaluating the mechanics of repaired menisci in surgical populations.

Research Area: Meniscus

CALIBRATION OF WEARABLE KNEE SLEEVE SENSORS FOR KNEE FLEXION ANGLE MEASUREMENT

Rosalind C. Bendell, Sagar M. Doshi, Amit Chaudhari, Erik T. Thostenson, Jill S. Higginson

University of Delaware

Wearable sensors have the potential to help prevent injury, monitor rehabilitation efforts and disease progression, and quantify activity levels regardless of the environment. Our collaborators have developed a novel wearable sensor integrated into a knee sleeve to provide feedback during exercise or rehabilitation. The integrated sensors measure a change in resistance when elongated. In this preliminary study, a single participant performed seated leg extensions – with knee flexion angles from 0 to 100 degrees – while wearing the sensing knee sleeve. An increase in knee flexion angle produced an increase in the stretch sensor resistance. Peak values, measured at a knee flexion angle of 90 degrees, were 874 ± 8.5 kOhms. An increase in knee flexion angle produced an increase in the stretch sensor resistance. Preliminary work suggests a consistent relationship between sensor resistance and knee flexion angles. Future work will improve repeatability and remove sensor noise. Then, a linear regression will be used to calibrate the change in sensor resistance with knee flexion angle, reported in degrees. The calibration of the sensing knee sleeve will provide opportunities for accurate joint angle measurements of human movements beyond a motion capture lab for applications in sports performance and knee injury rehabilitation.

Research Area: Design & Innovation, Rehabilitation & Treatment

ADAPTIVE SPLIT-BELT TREADMILL TO REDUCE PROPULSIVE ASYMMETRY IN PEOPLE WITH CHRONIC STROKE: A PRELIMINARY STUDY

Rucha Kulkarni, Jill S. Higginson

University of Delaware

Stroke is a leading cause of disability, with over 100 million stroke survivors worldwide. Post-stroke rehabilitation often focuses on improving walking speed to enhance community participation. Propulsion (push-off) during walking is highly correlated with higher walking speeds, and targeting propulsion could improve walking outcomes. Although treadmills are commonly used in gait rehabilitation, they do not target the paretic (affected) side and show mixed effectiveness in improving walking function. Split-belt treadmills can target the paretic side and adaptive treadmills (ATMs) can promote healthy gait variability, but show mixed efficacy in stroke survivors. We developed an adaptive split-belt treadmill (sATM) that can be preferentially weighted to encourage increased paretic propulsion during walking. As a preliminary study, we had N=2 participants with chronic stroke walk on the sATM for a series of trials with low, medium and high preferential propulsive weighting. We hypothesized that as propulsive weighting increased, peak propulsive force asymmetry would decrease while walking speed is maintained. Both participants showed a lower asymmetry as paretic propulsion was preferentially weighted, as compared to equal weighting between paretic and non-paretic sides. This suggests that an adaptive split-belt treadmill could improve propulsive symmetry and thereby act as a beneficial tool for post-stroke gait rehabilitation.

Research Area: Gait Analysis, Rehabilitation & Treatment

ACHILLES TENDINOPATHY AND ACHILLES TENDON RUPTURE DIFFERENTIALLY AFFECT ACHILLES TENDON LOADING DURING GAIT

Madeleine L. Krotine, Morgan N. Potter, Karin Grävare Silbernagel, Stephanie G. Cone

University of Delaware

Achilles tendinopathy (AT) and Achilles tendon rupture (ATR) can take months or years to rehabilitate and cause lasting changes in tendon structure and function. This study used shear wave tensiometry to assess long term changes in Achilles tendon loading during walking following Achilles tendon injury. Shear wave tensiometry data was collected bilaterally in 15 participants: 5 healthy control, 5 AT, and 5 ATR. The tensiometer, consisting of a mechanical tapper and two mini accelerometers, was secured to the Achilles tendon. Data was collected while participants walked on a treadmill at a self-selected speed and then at a 20% faster speed. A modified Chi-squared method showed shear wave speed varied significantly between sides, with greater tension in the involved limb than the uninvolved limb for both injuries, with no side-to-side difference found in the healthy population (p=0.953). Loading also varied between healthy, AT, and ATR groups. Walking speed did not significantly affect shear wave speed, suggesting greater changes may be needed to induce changes in loading. These results indicate asymmetries in walking mechanics of both injured groups for comparable portions of the gait cycle, with AT displaying the greatest magnitude of asymmetry.

Research Area: Ligament & Tendon

MORPHOMETRIC AND FUNCTIONAL ASSESSMENT OF ANTERIOR CRUCIATE LIGAMENT INJURY IN A RAT MODEL

Olivia Dyer, Stephanie Cone

University of Delaware

Rat models are used to understand the effects of injury, yet current techniques are limited in their ability to measure 3D morphometry and multiaxial function. This work presents a novel approach that leverages magnetic resonance imaging (MRI) and a multiaxial robotic testing system to understand the effects of anterior cruciate ligament (ACL) injury on knee stability. Joints were dissected from Long-Evans rats for 9.4T MRI imaging and robotic testing. MRI images were segmented and 3D models of the tissues of interest were generated for morphometric analysis. For robotic testing, the knee joint was set to 40° of flexion and moved through anterior-posterior (AP; peak load: 2.5N) and varus-valgus (VV; peak torque: 0.025Nm) paths in intact and ACL deficient states. There was a tissue dependent relationship in ligament CSA and an anatomical location dependent relationship in meniscus width. In the ACL deficient knees, increases in AP translations by 2.02mm ± 0.12mm and VV rotations by 13.87° ± 7.21° were observed, highlighting the importance of the ACL in knee stability. Future work will focus on understanding age- and sex-based differences in knee joint structure and function in rodents.

Research Area: Ligament & Tendon

ESTABLISHING METHODS FOR ANALYZING 3D RECONSTRUCTION, GEOMETRY AND STRUCTURE OF TENDON FIBRILS

Jamie M. Benson, Ellen T. Bloom, Chandran Sabanayagam, Lily M. Lin, Jeffrey Caplan, Dawn M. Elliott

University of Delaware

Tendon function relies on the hierarchical organization of aligned collagen fibrils, which transmit forces between muscle and bone. Damage to tendons disrupts fibril alignment, altering mechanical properties and increasing susceptibility to further injury. Traditional electron microscopy provides two-dimensional (2D) assessments of fibril structure but fails to capture their three-dimensional (3D) organization. To overcome this limitation, we previously utilized serial block face scanning electron microscopy (SBF-SEM) and a U-Net machine learning algorithm to segment and reconstruct tendon fibrils in 3D. However, this method failed to quantify their 3D structural geometric properties. This study aimed to develop methods to quantify 3D fibril metrics and compare them across three tendon types: healthy rat tail tendon, healthy rat plantaris tendon, and degenerated rat plantaris tendon. Using custom MATLAB scripts, we calculated relative fibril length, tortuosity, and angular deviations from the overall bulk fibril alignment. Results showed that healthy rat tail tendons had the most uniform fibril organization, while degenerated plantaris tendons exhibited increased variability and misalignment. These findings suggest that fibril disorganization may contribute to impaired tendon mechanics. Future work will refine segmentation methods to analyze more complex fiber-aligned tissues.

Research Area: Ligament & Tendon

DEFINING A CYTOPLASMIC ROLE FOR A HISTONE METHYLTRANSFERASE IN C. ELEGANS MUSCLE

Jessica Tanis

University of Delaware

The histone lysine methyltransferase SETDB1 trimethylates lysine 9 on histone 3 (H3K9me3), which leads to the formation of heterochromatin, silences gene expression, and impacts three-dimensional chromatin structure. We are using the C. elegans model system to define a new physiological role for MET-2, the worm homolog of SETDB1. We imaged adult C. elegans that express functional GFP-tagged MET-2 from the endogenous locus and observed that MET-2::GFP localized to the cytoplasm of the body-wall muscles (BWMs) as well as nuclei of germ cells and embryos. Knockout of MET-2 or sequestration of MET-2 in the nucleus caused hypersensitivity to levamisole, an agonist of acetylcholine receptors on the BWMs. Since levamisole hypersensitivity is observed in animals with reduced ATP, we reasoned that MET-2 could impact the mitochondria. Using red fluorescent protein tagged TOMM-20 to visualize mitochondria, we discovered that loss of MET-2 caused extensive fragmentation, swelling, and disorganization of muscle mitochondria. These results demonstrate a profound and previously undescribed effect of a histone methyltransferase on mitochondria morphology.

Research Area: Muscle

INVESTIGATING THE PHENOTYPIC CONSEQUENCES OF R384C MUTATION IN GALNS GENE IN A MOUSE MODEL: TRANSLATIONAL IMPLICATIONS IN MOUSE MODELS

Dione A. Holder, Shunji Tomatsu, Betul Celik

University of Delaware

Mucopolysaccharidosis IVA (MPS IVA) is a rare lysosomal storage disorder (LSD) caused by mutations in the GALNS encoding gene, leading to the accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. While significant progress has been made in understanding MPS IVA, a major gap persists in modeling its most prevalent subtype, bearing the most frequent and severe human missense mutation c.1156C>T, hindering complete insight into its skeletal and muscular pathology. Preliminary multi-modal investigations integrating micro‐CT imaging, histopathological examination, and biochemical assays of key lysosomal and cartilage-specific biomarkers have begun to reveal early signs of disease pathology. Early histopathological assessments suggest the presence of cellular degeneration and inflammatory infiltrates in joint tissues, while enzyme assays have shown trends toward low or absent GALNS activity. Although not all analyses are complete, these preliminary findings provide promising evidence of MPS IVA pathology in its most prevalent form. This study underscores the critical need to develop a representative experimental model to further elucidate the interplay between skeletal dysplasia and cartilage/connective tissue degeneration to inform targeted therapeutic strategies.

Research Area: Musculoskeletal Development

IDENTIFICATION OF SURROGATE BIOMARKERS FOR MUCOPOLYSACCHARIDOSIS TYPE IVA

Yasuhiko Ago, Shaukat Khan, Kimberly Klipner, Andrea Klenotiz, Allison Bradford, Shunji Tomatsu

Nemours Children’s Health

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disorder caused by N-acetylgalactosamine-6-sulfatase (GALNS) deficiency, leading to glycosaminoglycan (GAG) accumulation and severe skeletal dysplasia. Reliable biomarkers for monitoring disease progression and treatment efficacy remain limited. Given the role of C-type natriuretic peptide (CNP) in skeletal development, its inactive cleavage product, NT-proCNP, may serve as a potential biomarker for skeletal growth failure in MPS IVA.
This study evaluated NT-proCNP, collagen type I, and collagen type II as biomarkers in 60 MPS IVA patients. Plasma NT-proCNP and collagen levels were measured using ELISA, while GAGs were quantified via LC-MS/MS. Age-controlled correlations were analyzed to assess relationships between biomarkers, height z-scores, and enzyme replacement therapy (ERT) response.
NT-proCNP was significantly elevated in MPS IVA patients and strongly correlated with height z-score, especially after eight years of age. Collagen type I was increased in adults. Urinary GAGs correlated with growth failure in pediatric patients and decreased with ERT in adults. NT-proCNP and collagen levels remained unchanged with or without ERT in adult patients.
These findings suggest NT-proCNP as a promising biomarker for skeletal growth failure in MPS IVA, warranting further research into its role in disease pathology and therapeutic monitoring.

Research Area: Musculoskeletal Development

NOVEL INTERACTION AT THE PROTEIN LEVEL BETWEEN HEY FACTORS AND SIX1

Matthew E. Smith, Eleanor Helm, Shuo Wei, and Andre L. P. Tavares

University of Delaware

Birth defects from neural crest cell (NCC) and placode mispatterning require the transcription factor Six1 for proper craniofacial development. Six1 function requires co-factor binding, including the co-activator Eya1. six1 and eya1 mutations account for ~45% of Branchio-oto-renal (BOR) syndrome cases, characterized by craniofacial anomalies, hearing loss, and kidney defects. This suggests there are other causative genes for BOR. Yeast-2-Hybrid analysis in Xenopus suggests Hey factors as potential Six1 binding partners. In mice, Six1 interacts with Notch signaling during mandibular arch NCC patterning. These data support the hypothesis that Hey1/Hey2 act as Six1 co-factors during craniofacial development and could be responsible for BOR-related defects. In situ hybridization shows hey1 and six1 co-expression in the otic vesicle and branchial arches at larval stages. Co-immunoprecipitations suggest Hey1 and Six1 bind. Luciferase assays show ~20-fold increase in Six1 transcriptional activity when both Eya1 and Hey1 are present. Loss- and gain-of-function experiments are being performed in Xenopus using CRISPR/Cas9 and morpholino knockdown to verify if Hey1/Hey2, along with Six1, regulate patterning of NCCs within the branchial arches and otic vesicle. These results will establish whether Hey1/Hey2 act as Six1 co-factors, possibly accounting for unresolved BOR cases. Funding: University of Delaware College of Arts & Sciences

Research Area: Musculoskeletal Development

ADAPTIVE VS DEGENERATIVE TENDON RESPONSE TO OVERLOAD IS DURATION AND AGE DEPENDENT

Lily M. Lin, Rita C. Marqueti, Hailey Bonelli, Justin Parreno, Karin G. Silbernagel, Dawn M. Elliott

University of Delaware

Tendon homeostasis is regulated by mechanical loading, where moderate increases promote adaptation, but excessive or prolonged overload can lead to degeneration. Aging further increases susceptibility to tendon degeneration, yet the threshold between adaptation and degeneration remains unclear. This study examined tendon mechanical, structural, and cellular responses to overload over time and aging using a rat model. Female Long-Evans rats, aged 3 months (Juvenile) or 12 months (Adult), underwent bilateral synergistic ablation (SynAb) surgery, Sham surgery, or served as Intact controls. Juvenile rats were euthanized at 3 days, 8 weeks, or 16 weeks, while Adults were euthanized at 8 weeks. Tendon mechanics, histology, MRI, and gene expression were performed.
Juvenile tendons initially adapted to overload by increasing cross-sectional area (CSA) and ultimate load without compromising material properties. However, after 16 weeks, a decline in linear modulus and ultimate stress indicated a shift toward degeneration, with increased inflammatory and matrix degradation mRNA expression. Adult tendons exhibited similar adaptation at 8 weeks, suggesting that aging may not drastically alter initial overload responses. These findings, suggests a critical threshold exists between 8- and 16-weeks when adaptation transitions to degeneration, emphasizing the importance of load management in tendon health across different ages.

Research Area: Ligament & Tendon

COMBINED SENOLYTICS AND 11-WEEK TREADMILL RUNNING REVERSED THE DECLINING OF BOBE TISSUE MINERAL DENSITY IN AGED MICE WITH PARKINSON DISEASE

Tiankuo Chu, Rosa Guerra, Maya Collado, Samantha Izquierdo, Ella Cooper, Krish Shajpaul, Crystal P James, Sharee McGriff, Darice Wheeler, Kwadwo Ofori, Ho Ming Chow, Hwan Y Kim, Liyun Wang

University of Delaware

INTRODUCTION: Aging led to skeletal and neurodegenerative disorders like osteoporosis and Parkinson’s disease (PD).1 Senolytics and exercise can improve both brain and bone functions and delay aging-associated diseases.2,3,4 However, the interactions of these two treatments have not been investigated in the context of skeletal health and PD, which was the objective of this study.
METHODS: Under IACUC approval, fifty-eight 11-month-old C57BL/6J mice, including normal controls or PD (induced by α-synuclein preformed fibrils), received either senolytics (ABT-263), treadmill exercise, or both. All mice underwent microCT scans at baseline Week 7, and Week 11.5,6
RESULTS: As expected, the normal mice (PBS+Veh) showed aging-induced decreases in Ct.TMD at the 3 sites and an average loss of -4.1% (Week 11 vs. Week 0), while non-treated PD mice (PFF+Veh) showed similar loss (-3.9 %), which was reduced (but not significantly) with senolytics by 79% or exercise by 51% (Fig. 1A). Amazingly, combining the two treatments almost completely reversed the Ct.TMD loss (-0.5%) and the improvements on TMD (significance or trend) were detected on all sites (Fig. 1A). Further, the % of PD mice showing positive response was increased 3.6 fold by senolytics with or without exercise (vs. non-treated controls, p=0.005, Fig. 1A). Combined interventions also improved Ct.pMOI, reduced body weight, indicative of better structural integrity (Fig. 1B-E). Gains were slightly reduced in Week 7 and in female mice (not shown due to space).
DISCUSSION: Although exercise and senolytics are promoted to treat PD patients2 and osteoporosis4, their combined effects on aged mice with PD remained unclear until now. For the first time, this large preclinical study revealed that combining a 5-dose senolytic treatment and 11-week treadmill running effectively reversed the declining of bone material properties and mitigated the impaired structural integrity due to aging and the motor deficits from Parkinson disease.
REFERENCES: [1] Reeve 2014/PMID: 24503004; [2] Goodwin 2011/21856692; [3] Baker 2018/29457783; [4] Farr 2017/28825716; [5] Verma 2021/34359864; [6] Wang 2021/34246808

Research Area: Bone

MECHANICAL OVERLOAD REDUCES F-ACTIN LEADING DYSREGULATED CHONDROCYTE HOMEOSTASIS

Marin Herrick , Mark Arranguez , Justin Parreno

University of Delaware

Post-traumatic Osteoarthritis (PTOA) is a debilitating disease caused, in part, by mechanical overload onto cartilage. The mechanotransduction mechanisms by which overload influences cartilage degeneration are unclear. A surgical PTOA model previously revealed that induced injury reduced native chondrocyte cortical filamentous (F-)actin, which we predict is pivotal in promoting degeneration. In this study, we test the hypothesis that: Mechanical overload will destabilize cortical F-actin to dysregulate chondrocyte homeostasis.
Applied mechanical overload by either ex vivo compression of osteochondral tissues or in vitro stretch of isolated chondrocytes revealed that high strains >25% reduces cortical F-actin. To investigate the effects of F-actin loss, we treated chondrocytes with TR100, an inhibitor of F-actin stabilization. This significantly reduced F-actin in chondrocytes correlating decreases in chondrogenic genes: Collagen Type 2, Aggrecan, and Proteoglycan 4 and increases in degradative gene Matrix Metalloprotease 3.
Our results support our hypothesis that cortical F-actin reduces during overloading and that F-actin stability is critical for maintaining chondrocyte homeostasis. This suggesting F-actin loss seen during overload may be contributing to catabolism seen during PTOA. Future studies aim to characterize these changes in vivo. Maintaining cortical F-actin during overload by targeting actin stabilizing molecules may lead to novel therapeutic insights against Osteoarthritis.

Research Area: Cartilage

MIMICKING DEVELOPMENTAL BIOMECHANICAL CUES TO ENHANCE BIOENGINEERED CARTILAGE FORMATION BY PASSAGED CHONDROCYTES WITHIN TUNABLE HYALURONIC ACID BASED HYDROGELS

Thomas J. Manzoni, Aditya Natu, Anh Ho, Lilly Smull, Yinzhi Fang, Joseph M. Fox, Alvin W. Su, Xinqiao Jia, Justin Parreno

University of Delaware

Cell-based bioengineering to repair focal defects in articular cartilage remains to be a challenge stemming from the inability to form tissues that recapitulate native matrix composition and organization. Recently, matrix stiffening has been shown to be a critical developmental cue that guides chondroprogenitors toward cartilage matrix expression. Therefore, we aimed to improve bioengineering outcomes by examining the hypothesis that dynamic tuning of matrix stiffness will enhance cartilage matrix expression by cells in bioengineered constructs. To test our hypothesis, we evaluated the redifferentiation of passaged chondrocytes, which resemble interzonal chondroprogenitors, within hyaluronic acid-based hydrogels functionalized with Tetrazine (HA-Tz) that can be dynamically stiffened in culture. We found that passaged chondrocytes within unstiffened HA-Tz are highly viable (88%), exhibit cortical F-actin organization, and decrease expression of fibroblastic matrix mRNA levels. When supplemented with TGFβ3, passaged chondrocytes re-express cartilage-specific matrix. To examine the effects of dynamic stiffening, a rapid Tz/trans-cyclooctene reaction is used to fully crosslink remaining Tz groups, stiffening the hydrogel matrix. Rapid dynamic stiffening elevated expression of Collagen-2 and Aggrecan by passaged cells. Therefore, HA-Tz is chondro-supportive and can be used to mimic the dynamic mechanical cues during cartilage development to enhance the production of cartilage matrix by passaged chondrocytes.

Research Area: Cartilage

METFORMIN’S EFFECTS ON CHONDROCYTE METABOLIC ACTIVITY

Annie Porter, Ying Peng, Rachel Perry, Chloe Kappen, Julie Nguyen, Naod Asres, Rebecca Wang, Michael Axe, X. Lucas Lu

University of Delaware

Metformin is commonly used for managing type 2 diabetes, but it has also exhibited anti-inflammatory properties, attracting attention as a potential preventative treatment for osteoarthritis (OA). This study explores metformin’s effects on cartilage, treating cartilage samples with a range of metformin doses (10 μΜ-10 mM) and evaluating chondrocyte viability and extracellular matrix metabolic activity. In juvenile bovine cartilage, a high 10 mM dose reduced chondrocyte viability, but a physiological 10 μM dose did not. In OA cartilage, no dose of metformin had any significant effect. No tested dose of metformin affected GAG synthesis, but a high 10 mM dose reduced collagen synthesis in juvenile bovine, healthy human, and OA human cartilage. A low 10 μM dose reduced collagen synthesis in juvenile bovine and healthy human cartilage but had no effect on OA cartilage. In inflamed cartilage, metformin could not recover reduced GAG or collagen synthesis, but at all tested doses metformin did reduce the loss of GAG. In this study, metformin exhibited anti-inflammatory properties, but at high doses had negative effects on chondrocyte viability and collagen synthesis and was more harmful to young, healthy cartilage. Careful consideration should be given to using metformin on young patients.

Research Area: Cartilage

CHONDROCYTE VOLUME AND PROTEOGLYCAN REMODELING

Ying Peng, Lam Vien Che (University of Rostock, Germany), Julie Nguyen, Ursula van Rienen (University of Rostock, Germany), X. Lucas Lu

University of Delaware

Joint inflammation can initiate osteoarthritis (OA) by altering the chondrocyte phenotype, often associated with changes in cell morphology and metabolism. One of the early biomarkers of OA progressions is altered glycosaminoglycan (GAG) turnover in cartilage extracellular matrix. To assess GAG turnover, we developed a click chemistry-based technique for the visualization, quantification, and spatial mapping of newly synthesized GAG. However, the relationship between chondrocyte morphology and new GAG synthesis under inflammatory challenge remains poorly understood. This study aimed to (1) develop three-dimensional imaging methods to quantify the volumes of chondrocytes, their nuclei, and newly synthesized GAGs in cartilage; (2) examine the effects of inflammatory challenge on chondrocyte morphology and new GAG synthesis. Our results showed that, following 2, 7, and 14 days of inflammatory challenge, chondrocytes in young articular cartilage exhibited reduced cell and nucleus volumes but increased cell density. Under inflammatory conditions, GAG synthesis of chondrocytes was reduced with newly synthesized GAG accumulating near the cell membrane in a denser yet more spatially condensed distribution. Moreover, the GAG synthesis rate was positively correlated with both cell and nuclear volumes under inflammatory stress. These findings provide new insight into the interplay between chondrocyte morphology and matrix metabolism in inflammation-induced OA progression.

Research Area: Cartilage

THE EFFECT OF UNLOADED REST AND TIME OF DAY ON MRI MEASUREMENTS OF DISC DEGENERATION

Mackenzie N. Conner, Harrah R. Newman, Dawn M. Elliott

University of Delaware

MRI is often used to quantify disc degeneration from T2 relaxation time and disc height. Disc hydration and height loss occur throughout the day, thus studies scan after an unloaded rest or in the morning to mitigate these effects. However, the effectiveness of these efforts is unknown. Therefore, participants (23-37 years old, n=8) underwent MRI before and after 45-minutes of unloaded rest a day and throughout the day.

T2 following rest significantly decreased (-4 ± 18 ms), but ~half of the discs’ T2 increased and was highly variable. Therefore, it is unnecessary to scan after unloaded rest. The T2 across the day was variable and not significantly different, besides 11:00 am to 2:00 pm (-10 ± 17 ms). Therefore it is not necessary to control for time of day when measuring T2. Disc height significantly decreased ~1.8% between each timepoint, decreasing ~5.4% in total, which is consistent with previous studies. Therefore, in contrast to T2, it is necessary to control for time of day when measuring disc height. However, since height loss was linear, it may be possible to account for time of day using a correction factor. These results should be considered to improve scanning consistency across studies.

Research Area: Spine

VALIDATION OF A SENSOR FOR MONITORING LOAD IN LOWER LIMB PROSTHESIS

Theophilus Annan, Amit Chaudhari, Sagar Doshi, Erik Thostenson, Jill Higginson

University of Delaware

Patients with unilateral lower limb amputation often exhibit asymmetric interlimb gait patterns. Over time, the asymmetries between the intact and prosthetic limbs and excessive loading on the intact limb increase the risk of developing osteoarthritis (OA) in the knee or hip of the intact limb than non-amputees. Clinicians continue to use qualitative assessments in part due to high-cost equipment, complex protocols, diverse training, and varying biomechanics experience and knowledge. This study aims to validate the repeatability of a wearable sensor to monitor prosthetic limb load. We have developed a portable novel fabric-based pressure sensor that can potentially monitor limb loads during activities both in and outside laboratory settings. We compared the sensor’s repeatability within and between tests for a selected force range and observed the sensor’s ability to detect distinct force range values with a 1% change in resistance. This is observed for both within and between tests. This could be used to detect 25%, 50%, 75%, and 100% of total body weight during weight monitoring training for amputees. Additionally, it can be used to monitor limb loads during activities of daily living, thereby preventing the risk of developing OA in the intact limb.

Research Area: Design & Innovation, Rehabilitation & Treatment

WALKING THROUGH PAIN: GAIT VARIATIONS AMONG ADULTS WITH LOWER LIMB AMPUTATION AND CHRONIC LOW BACK PAIN

Vallery C, Witt JM, Stauffer, SJ, Sahi A, Sions JM

University of Delaware

Chronic low back pain (LBP) is the leading cause of secondary disability among individuals with lower-limb amputation (LLA). LBP is linked to gait deviations after transfemoral-level amputation (TFA), but little is known about LBP and gait after transtibial-level amputation (TTA). This study evaluated relationships between LBP presence or absence, amputation level (TFA versus TTA), and spatiotemporal gait metrics.
Adults >1-year post-unilateral LLA (n=41, 56.0±17.2 years-old, 78% male, 54% TFA) reported on LBP and underwent instrumented gait analysis (Mobility Lab, APDM) during the 6-Minute Walk Test (6MWT). Level-specific (TFA and TTA) between-group (LBP versus no LBP) and group-specific between-level difference in gait parameters were identified using independent sample t-tests and Mann-Whitney U tests, as appropriate (p≤.050).
Adults with TTA reported LBP more frequently than TFA (84% versus 45% prevalence). Among individuals with TTA, those with LBP displayed decreased 6MWT distance and decreased cadence, single-limb support, gait cycle duration, and stride length (p≤.001-.018). With TFA, adults with LBP exhibited greater hip circumduction (p=.043).
LBP presence correlates with more gait deviations among adults with TTA when compared to adults with TFA. Further level-specific research is necessary to identify if abnormal gait results in or is a result of LBP after LLA.

Research Area: Gait Analysis, Rehabilitation & Treatment

MOTION CAPTURE AND SHEAR WAVE TENSIOMETRY ASSESSMENTS OF ACHILLES TENDON LOADING ARE SIGNIFICANTLY CORRELATED

Zahra McKee, Stephanie G. Cone, Karin Grävere Silbernagel, Elisa S. Arch

University of Delaware

The Achilles tendon is prone to injuries like tendinopathy and tendon ruptures. For these injuries, managing the load experienced by the tendon is important for optimal recovery. Currently, estimates of tendon loading have primarily used motion capture (MC) but this method has some limitations. Shear wave tensiometry (SWT), an emerging technique, allows for direct measurement of the Achilles tendon. SWT centers around the idea that tendon shear wave speed (SWS) varies in proportion to the square root of tendon axial stress and therefore SWS can be used as a proxy for tendon load. This study aimed to compare Achilles tendon loading assessed with MC and SWT. 5 healthy adults (2 M/3 F; 28.0± 2.92 yr) visited the lab. MC, force plate and SWT data were collected while participants performed several iterations each of nine dynamic exercise activities. MC load estimate and SWS peaks (p<0.001, ⍴=0.55), impulses (p<0.001, ⍴=0.62), and peak rates of change (p<0.001, ⍴=0.83) were significantly correlated (Figure 1), indicating these methods similarly represent tendon loading. Trends in peaks, impulses, and peak rates of change can be compared between these two measurement methods.

Research Area: Ligament & Tendon

TRANSGELIN’S ROLE IN REGULATING MYOFIBROBLAST DIFFERENTIATION DURING FIBROSIS

Rylee King, Christian Le, Justin Parreno

University of Delaware

Tendon fibrosis results in biomechanically inferior scar tissue prone to failure. This arises from the persistent differentiation of tendon cells into myofibroblasts, which deposit and contract fibrotic extracellular matrix, causing tissue thickening. During myofibroblast differentiation, structural changes to the cells occur. During myofibroblast differentiation, actin reorganizes from globular (G-) actin to filamentous (F-) actin stress fibers. These fibers enable contraction, partly due to increased alpha-smooth muscle actin (αSMA), which incorporates into stress fibers, rendering cells hypercontractile. Repressing stress fiber formation has been shown to reduce both matrix deposition and contraction in other cell types.

Transgelin (Tagln), an F-actin cross-linker, is upregulated during fibrosis. We hypothesized that Tagln is necessary for maintaining stress fibers during myofibroblast differentiation. Treating tenocytes with TGFβ1 increased Tagln1 mRNA, correlating to enhanced stress fiber formation. However, Tagln1-/- knockout tenocytes still formed αSMA positive stress fibers, suggesting stress fiber formation does not solely depend on Tagln1 in tenocytes. Other actin-binding proteins, such as Tagln2, may compensate. Thus, we performed siRNA knockdown of Tagln1 and Tagln2, finding that stress fibers still form. Understanding the role of actin-binding proteins in myofibroblast differentiation could provide insights into targeting persistent myofibroblast activation in fibrosis progression.

Research Area: Ligament & Tendon

MULTIFIDI MUSCLE DYSFUNCTION IS PRESENT IN ADULTS WITH LOWER-LIMB AMPUTATION AND LOW BACK PAIN

Stauffer SJ; Shahi AK; Roseberry RL; Vallery CE; Sions JM

University of Delaware

Low back pain (LBP) affects up to 89% of adults with lower-limb amputation. Dysfunction of the lumbar multifidi muscles, which are important spinal stabilizers, is strongly linked to LBP in the general population. However, after lower-limb amputation, associations between lumbar multifidi muscle activity and LBP remain unknown. This study evaluated differences in multifidi muscle activity after amputation between adults with and without LBP. Fifty-three adults (n=35 with LBP; n=18 without LBP) who underwent unilateral amputation >1-year prior (median age: 62 years; 73.1% male; 51.9% above-knee amputation; 34.6% traumatic etiology) received ultrasound imaging to assess lumbar multifidi muscle activity, which was calculated as the percent change in muscle thickness from a resting to a contracted state facilitated with a contralateral lower-limb lift. Multifidi activity across 3 trials was recorded for lumbar levels L3/L4 to L5/S1, and average multifidi muscle activity across levels was calculated. Adults with LBP were heavier (p=0.007), more likely to have a below-knee amputation (p=0.012), and had reduced amputated-side (14.7% versus 22.7%; p=0.042) and contralateral-side (17.6% versus 22.4%; p=0.025) multifidi muscle activity when compared to peers without LBP. Further research is needed to determine if interventions enhancing multifidi muscle activity mitigate LBP among adults with lower-limb amputation.

Research Area: Muscle

SAND TIGER SHARK TEETH MORPHOLOGY THROUGHOUT LIFE STAGES ANALYZED VIA MICRO-CT

Caitin Bailey, Dr. Jennifer Wyffels (Ripley’s Aquariums and Center for Bioinformatics & Computational Biology), Dr. Kady Lyons (Georgia Aquarium)

University of Delaware

Several species of shark exhibit tooth morphology changes throughout their lifespan likely reflecting changes in diet as they grow. Sand tiger shark (Carcharias taurus) tooth morphology has not been examined throughout its lifespan despite a rare requirement for teeth during embryonic development. Sand tiger sharks use a reproductive mode termed adelphophagy. In this reproductive mode, developing embryos break out of their egg cases and seek out and kill other developing embryos. Embryos have a precocious dentition that allows them to tear free of their egg case effectively and lethally wound other developing embryos. This reproductive strategy results in robust, high-quality young.
Sand tiger sharks must hunt for prey on their own after parturition because there is no parental care for sharks. Changes in prey require changes in tooth form and function to be effective. To better understand how tooth shape relates to prey for sand tiger sharks, teeth from various life stages were imaged using microtomography (micro-CT) and reconstructed in 3D using NRecon, DataViewer, and Amira. Once reconstructed, a suite of morphology measurements were collected for each tooth and compared across life stage.These findings contribute to a deeper understanding of tooth development and reproductive adaptations of sand tiger sharks.

Research Area: Musculoskeletal Development

TRANSFERRIN RECEPTOR-ASSOCIATED UPTAKE OF HGALNS ENZYMES AMELIORATES THE DISEASE PROGRESSION IN MUCOPOLYSACCHARIDOSIS IVA MICE

Georgina Neema Baya, Betul Celik, Shaukat Khan, Shunji Tomatsu

University of Delaware

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a mutation in the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) gene, which results in GALNS enzyme deficiency and accumulation of glycosaminoglycans (GAGs) in bone and cartilage, leading to progressive systemic skeletal dysplasia. Currently, no effective treatment is available for this skeletal condition. However, lentiviral gene therapy (LVGT) promises a one-time, permanent solution to produce defective enzymes by integrating a functional copy. We hypothesize that the transferrin receptor (Tfr) domain tagged-hGALNS enzymes produced by LVs elevate the hGALNS activity via Tfr-mediated endocytosis while reducing GAG accumulation within the chondrocytes. Our LVs were designed under the CBh promoter driving Tfr sequence-inserted-hGALNS gene. We transduced MPS IVA patients’ fibroblasts and HEK293T cells via LVs with multiplication of infection (MOI 20) and collected media and cells to evaluate the therapeutic efficacy. MPS IVA patients’ fibroblasts and HEK293T cells had increased intracellular and extracellular GALNS enzyme activity via mannose-6-phosphate (M6P) and Tfr receptor endocytosis. While GAG levels were reduced, cross-correction mechanisms indicated an efficient uptake of Tfr-tagged hGALNS enzymes by neighboring cells. Our findings revealed that the Tfr-tagged hGALNS via LVGT could potentially cure MPS IVA in vitro. Ex vivo studies are still underway to investigate therapeutic efficacy.

Research Area: Musculoskeletal Development

ROLE OF THE SIX1 TRANSCRIPTIONAL COMPLEX IN BONE AND CARTILAGE DEVELOPMENT DURING CRANIOFACIAL MORPHOGENESIS

Kelechi Chukwuocha, Gabriel DaSilva, Visnu Chowdhury, Andre L P. Tavares

University of Delaware

The transcription factor SIX1, belonging to the SIX family of homeobox genes, is essential for craniofacial development. Notably, Six1 function relies on the interaction with co-factors that either induce (EYA) or repress (TLE4, SOBP) its transcriptional activity. Published and preliminary studies show that the Six1 transcriptional complex (Six1+co-factors) is expressed in the precursors for bone and cartilage in the developing face. This study investigates the mechanistic role of the SIX1 transcriptional complex in directing the differentiation of craniofacial skeletal tissues. We hypothesize that SIX1 transcriptional activity modulates the differentiation of neural crest cells into osteoblasts and chondrocytes, driving the formation and maturation of craniofacial structures. Using genetic, molecular, and histological analyses, we aim to delineate the role of SIX1 and its co-factors in neural crest cell patterning and differentiation within the developing mouse embryo head. Additionally, we will characterize the impact of disease-associated mutations in SIX1 in craniofacial development. Elucidating the role of the SIX1 transcriptional complex in craniofacial development may reveal underlying mechanisms of craniofacial malformations, pointing toward potential therapeutic targets for regenerative interventions.

Research Area: Musculoskeletal Development

ROLE OF SIX1 IN CALVARIAL BONE AND SUTURE DEVELOPMENT

Visnu P. Chowdhury, Andre L. Pasqua Tavares

University of Delaware

One third of all congenital anomalies involve craniofacial defects. Branchio-oto-renal syndrome (BOR) is such a defect where patients show hearing loss (>90%), branchial cyst/fistula, and occasional kidney defect. Research shows mutation in transcription factor Six1 correlates with increased incidence of craniosynostosis, while to exert the function Six1 relies on its cofactors like Eya1 or Sobp. Thus, we hypothesize Six1 modulates cranial bone and suture development, in a cofactor dependent manner. We are using Six1KO and Six1p.R110W mouse lines for in vivo, and mesodermal and preosteoblast cell lines for in vitro experiments, to examine how different Six1 dosage or functionality affects the calvarial bone development. Micro-CT of E18.5 embryos show that the absence or lack of functional Six1 impairs frontal and parietal bone development. We also observed bridging between frontal and parietal bones in E18.5 embryo and partial sagittal suture fusion at 2 months in presence of Six1-haploinsufficiency, probably reflecting craniosynostosis observed in some BOR patients. Mutant embryos at E15.5 also show defects in the calvarium, suggesting Six1 is impacting the patterning of frontal and parietal bone primordia. Together, our data is generating new knowledge on underlying mechanisms of Six1 in cranial bone and suture development to find novel therapeutic targets.

Research Area: Musculoskeletal Development

A NOVEL PROTEIN-LEVEL INTERACTION BETWEEN Ebf3 AND Six1 DURING DEVELOPMENT IN Xenopus

Asif Ahmed, Matthew E. Smith, Andre L. P. Tavares

University of Delaware

Each year, around 7.9 million infants are born with major birth defects, with approximately 35% exhibiting craniofacial abnormalities. These defects often arise from developmental disruptions in neural crest cells and cranial sensory placodes. The transcription factor Six1 is essential for the normal development of these cells and requires co-factors, such as Eya1, for proper function. Mutations in the SIX1 and EYA1 genes account for roughly 45% of Branchio-oto-renal (BOR) syndrome cases, which are characterized by craniofacial deformities, hearing loss, and kidney anomalies; however, a significant number of cases remain genetically unexplained, suggesting other genetic factors may play a role. Recent yeast two-hybrid studies by Dr. Sally Moody’s team have identified Ebf3 as a potential co-factor for Six1 in Xenopus larvae. Evidence shows that Ebf3 is co-expressed with Six1 in critical developmental structures, including the otic vesicle, branchial arches, and somites. Together, these data support the hypothesis that Ebf3 functions as a co-factor for Six1 in craniofacial development and may contribute to unresolved BOR cases. To test this hypothesis, we are performing in situ hybridization for ebf3 and six1 to confirm colocalization in neural crest cells and sensory placodes, co-immunoprecipitation (IP) to confirm binding between ebf3 and six1, luciferase assays to determine if ebf3 regulates six1 transcriptional activity, and both morpholino (MO) knockdown and mRNA overexpression injections in Xenopus embryos to characterize function during embryonic development. Preliminary results from in situ hybridization experiments validate overlapping expression patterns of ebf3 and six1 in otic vesicles, branchial arches, and somties, and from luciferase assays suggest that Ebf3, in combination with Eya1, reduces Six1 transcriptional activity.

Lab: Tavares

KNOCKOUT OF PLS3/FIMBRIN RESULTED IN THE LOSS OF F-ACTIN IN OSTEOCYTE DENDRITES IN MATURE BUT NOT YOUNG MICE

Rosa Guerra, Megan Coffin, Velia Fowler, Liyun Wang

University of Delaware

Osteocytes, master orchestrators of bone remodeling, form extensive dendrite networks for mechanosensing and communication. However, little is known about the structure, composition, and regulating factors involved in the formation and maintenance of their dendrites. We aim to investigate the role of PLS3/fimbrin, a crosslinking protein, in the osteocyte dendrite cytoskeleton’s initiation, formation, and maintenance, using a global fimbrin knockout mouse model and high-resolution imaging. Full body Pls3 knockout mice (termed PLSKO) were generated in the Fowler Lab. Long bones and calvarias from 2 and 6-month-old male PLSKO and WT littermates were processed (fixed, decalcified, OCT embedded) before stained to reveal the fimbrin and actin structure with high resolution airyscan confocal imaging. It appears that Pls3/fimbrin is not required for the osteocyte dendrite formation, given the normal F-actin staining in 2-month PLS3-KO dendrites, suggesting that other factors may drive the dendrite’s actin cytoskeleton initiation and elongation. However, for the first time, we demonstrated Pls3/fimbrin is crucial for stabilization, without which F-actin staining was nearly completely lost. Further research is needed to understand the time course of the changes of the F-actin cytoskeleton induced by Pls3/fimbrin KO, and the roles of fimbrin on cell viability and mechanosensing.

Lab: Wang

INVESTIGATING THE IN VIVO IMPACTS OF HIGH BMP2 CONCENTRATION ON THE FEMORAL BONE MICRO-ENVIRONMENT OF AGING MICE

Md Tamzid Hossain Tanim, Jordan Groves, Dr. Anja Nohe

University of Delaware

Dysregulation in the adipo-osteogenic fate determination of bone marrow mesenchymal stem cells (BMSCs) is a critical factor contributing to bone diseases like osteoporosis. While BMP2 is traditionally recognized as an osteogenic driver, accumulating evidence suggests that its effects are dose-dependent, with high concentrations paradoxically inducing adipogenesis. Previous in vitro studies have demonstrated that BMP2 can activate adipogenic transcription factors, including PPARγ and C/EBPα, through both SMAD-dependent and p38 MAPK pathways. Moreover, excessive BMP2 signaling has been shown to suppress Wnt/β-catenin activity, a key pathway in osteogenesis, shifting the balance toward adipogenesis. However, in vivo validation of these mechanisms remains limited.
This study investigates the effects of systemic administration of high-dose BMP2 in a murine osteoporosis model, revealing a significant increase in marrow adiposity. Histological analysis confirmed an increased presence of adipocytes, while immunostaining demonstrated enhanced nuclear co-localization of BMPR1A C-terminal with PPARγ, consistent with findings that BMP2 can drive adipogenesis in BMSCs. These results align with prior studies in which high BMP2 doses in animal models led to marrow fat accumulation indicating that BMP2’s osteogenic effects may be counteracted under supraphysiological conditions.

Lab: Nohe

IL1β-MEDIATED DIRUPTION OF THE ACTIN CYTOSKELETON AND ITS ROLE IN CARTILAGE DEGRADATION

Stephanie Richardson-Solorzano and Justin Parreno

University of Delaware

In Osteoarthritis, inflammatory mediators disrupts chondrocyte function leading to matrix degradation. Prior in vitro studies showed IL1β reorganizes chondrocyte cortical F-actin into stress fibers. Preventing this reorganization suppresses degenerative gene changes, suggesting a relationship between stress fibers and cartilage breakdown. However, in vivo OA models reveal that stress fibers do not form, rather cortical F-actin is reduced in native cartilage. Earlier in vitro studies used extensively cultured chondrocytes; thus the response to inflammatory mediators may be context dependent. Here, we investigate IL1β on freshly isolated chondrocytes with intact cortical F-actin. We hypothesize IL1β reduces cortical F-actin to dysregulate chondrocyte homeostasis.

We found that chondrocyte morphology, cortical F-actin, and chondrogenic gene expression is preserved in chondrocytes cultured in serum-reduced media for 3 days. We found exposure of chondrocytes to IL1β on day 2 does not lead to stress fibers but rather, reduces F actin, upregulates matrix metalloproteases (MMPs) and downregulates chondrogenic genes (COL2) mRNA levels. To investigate the specific effect of chondrocyte F-actin loss, we exposed chondrocytes to actin depolymerizer, Latrunculin. Latrunculin downregulates COL2 and upregulates MMPs. Thus, inflammation may dysregulate chondrocyte homeostasis by cortical F-actin loss.

Lab: Parreno