2023 CBER/DCMR Research Symposium

Keynote Speaker Session 1: Dr. Hani A. Awad

Title: Peritendinous Adhesions: Decoding the Roles of TGF-β and Myofibroblasts

Abstract:

Injuries to flexor tendons in zone II of the hand remain among the most difficult injuries for surgeons to repair. Restoration of function during healing is often impaired due to the formation of debilitating peritendinous adhesions, and the incidence of repair rupture typically necessitates additional surgery. Our research focuses on the mechanobiology regulating tendon healing with the goal of manipulating this biology to develop clinically translatable biological therapies to improve outcomes of healing. Our approach uses genetic mouse models of zone II flexor tendon injury to uncover the molecular mechanisms of fibrotic healing.   In this talk, I will present emerging paradigms about how inflammatory pathways alter TGF-β1 regulation of myofibroblasts in peritendinous adhesions. I will also present our efforts to translate our discoveries in the mouse model to human microphysiological systems to recreate vascular inflammatory fibrosis on a chip to enable studies of pathogenic mechanisms and discovery or validation of disease-modifying drug candidates.

Podium Session 1

1 Kayla Pariser

Computational Treatment Design Of Adaptive Treadmill Controllers

Kayla Pariser, Jill Higginson

University of Delaware

Treadmill gait training is frequently prescribed for individuals poststroke to promote improved walking function. However, with commonly used fixed-speed treadmill paradigms only 50% of stroke survivors improve gait mechanics, possibly because fixed-speed treadmills limit natural stride-to-stride variability essential for motor learning. To address this concern, we developed an adaptive treadmill (ATM) that adjusts belt speed in real-time via changes in user propulsion and step length, promoting healthy dynamic variability. While healthy young adults improve gait mechanics with the ATM versus fixed-speed treadmills, the response of stroke survivors to the ATM is mixed, perhaps due to lack of controller customization. Computational treatment design using predictive simulations may allow for more efficient selection of optimal rehabilitation compared to fatiguing trial-and-error experiments. The purpose of this study was to develop and evaluate a predictive simulation framework to estimate changes in gait with various ATM controllers. With musculoskeletal modeling and optimal control methods we simulated different ATM controllers we previously tested experimentally. The ATM simulation framework successfully captured changes in gait mechanics that we observed experimentally. This is the first study to show how computational modeling can inform design of ATM controllers and test hypotheses regarding how individuals will respond to novel ATM controllers.

Track: Skeletal Disorders, Treatments & Rehabilitation
Research Area: Neuromuscular Modeling & Control

2 Daniel Halloran

Age-Related Low Bone Mineral Density In C57Bl/6 Mice Is Reflective Of Aberrant Bone Morphogenetic Protein-2 Signaling Observed In Human Patients Diagnosed With Osteoporosis

Daniel Halloran, Presenter, Venu Pandit, Connor MacMurray, Victoria Stone, Kailey DeGeorge, Mark Eskander, Delaware Orthopaedic Specialists; Denise Root, Orthopedic Surgery, ChristianaCare Hospital; Sean McTague, Orthopedic Surgery, ChristianaCare Hospital; Heather Pelkey, Orthopedic Surgery, ChristianaCare Hospital; Anja Nohe, Principal Investigator

University of Delaware, ChristianaCare Hospital

Osteoporosis (OP) is a bone disorder characterized by decreased bone mineral density (BMD). Bone Morphogenetic Protein-2 (BMP-2) injections are used to promote bone formation in OP patients. However, patients are unresponsive to BMP-2 while displaying an upregulation of BMP Receptor Type 1a (BMPRIa) and protein kinase CK2? (CK2?). A synthetically produced peptide named casein kinase 2.3 (CK2.3) utilizes the BMP-signaling pathway as it enhances osteogenesis of primary osteoblasts isolated from OP patients, whereas BMP-2 does not. Although shown in OP patients, there is currently no reliable mouse model to study BMP-2 and CK2.3 signaling. In this publication, we show that BMPRIa was required for CK2.3-mediated osteogenesis in C2C12 cells with a CRISPR-Cas9-mediated gene knockout for BMPRIa. We utilized the C57BL/6 (B6) mouse strain as an aging-model to study aberrant BMP-2 signaling, demonstrating that, like OP patients, in 15 and 20-month mice, BMP-2 did not increase bone growth and displayed upregulated BMPRIa and CK2? protein expression. Furthermore, CK2.3 enhanced osteogenesis and decreased osteoclastogenesis in all age groups, whereas BMP-2 only increased mineralization in 6-month mice while increasing osteoclast formation in all age groups. These data demonstrated that aging B6 mice were a reliable model and mimicked data obtained from OP patients.

Track: Skeletal Disorders, Treatments & Rehabilitation
Research Area: Bone

3 Murtaza Wasi

Yoda1 Augmented Loading Rescued Bone After Chemotherapy

Murtaza Wasi, Tiankuo Chu, Rosa Guerra, Lidan You (U Toronto), Liyun Wang

University of Delaware

Chemotherapeutic agents such as anthracyclines directly affect bone cells, marrow and induce bone loss. Rats receiving anthracycline showed reduction in the densities of osteocytes and bone lining cells. Using a piezo1 channel agonist, Yoda1, with loading improved bone properties and bone formation in mice without and with breast cancer. Hence the aim of our study was to understand the efficacy of Yoda1 augmented exercise in mitigating bone loss after chemotherapy. Healthy aged mice were injected with Doxorubicin at two different doses: 2.5 and 5 mg/kg in 6 cycles. After one week of recovery time, two weeks of Yoda1 intraperitoneal injection and tibial loading (4.5 N peak load, 4 Hz, 300 cycles for 5 days/week) was performed. Cortical bone properties were analyzed from the micro CT data at weeks 0, 2 and 5 at two locations above Tibia-Femur Junction. Both doses of doxorubicin declined bone properties examined in our study with the higher dose causing drastic decline at both cortical regions, due to elevated bone resorption. Loading alone did not have any beneficial effect on the cortex. Yoda1 treatment dampened the decline in the cortical polar moment of inertia (Ct.pMOI). The Yoda1 augmented loading group resulted in significant improvement in Ct.pMOI.

Track: Skeletal Disorders, Treatments & Rehabilitation
Research Area: Bone

4 Cody Helm

Subthreshold Tms For Cortical Inhibition Of Long-Latency Responses In One Forearm Muscle

Cody A Helm, Kyle Grossman, Sheldon-Anthony Amofah, Fabrizio Sergi

University of Delaware

In-depth knowledge of the function of the reticulospinal tract (RST) would improve our understanding of its role in stroke recovery. Function of the RST can be evaluated using long-latency responses (LLR). Transcranial magnetic stimulation (TMS) can modulate corticospinal output to independently quantify function of the RST. Thus, methods that combine TMS and stretch reflexes can study RST function associated with LLRs. We combined single subthreshold TMS pulses, wrist perturbations to evoke forearm LLR, and surface electromyography to determine the effect of subthreshold TMS stimulus intensity and timing on the long-latency response amplitude (LLRa). We divided 24 participants into two groups (N=12) to test two TMS intensities (90% or 95% Active Motor Threshold). TMS pulses were timed to arrive at the muscle at three different timings (0ms, 20ms, 50ms) prior to perturbation onset. Perturbation only and background only conditions were included as controls. TMS timing had a significant effect on LLRa (p<0.0001*) with no significant effect between TMS intensities (p=0.6719). Furthermore, TMS pulse timing of 50 ms resulted in the greatest reduction in LLRa (p=0.0001*). In conclusion, subthreshold TMS can significantly reduce the LLRa and the RST may contribute to motor commands when corticospinal output is reduced.

Track: Skeletal Disorders, Treatments & Rehabilitation
Research Area: Neuromuscular Modeling & Control

5 Betul Celik

Direct Infused Lentiviral Gene Therapy For Morquio Syndrome In A Histo-Pathological Perspective

Betul Celik, Estera Rintz, Nidhi FNU, Andres Felipe Leal, Shaukat Khan, Shunji Tomatsu

Nemours Children’s Health System

Morquio syndrome is an autosomal recessive disease caused by a mutation in the N-acetylgalactosamine-6-sulfate sulfatase gene. No effective treatment for this skeletal disease is present. Therefore, a novel therapy is an unmet challenge to reverse or ameliorate the disease progression. We hypothesized that the proposed novel lentiviral vectors (LV) could permanently produce the active enzyme by transduced cells into the circulation and would significantly impact bone and cartilage abnormalities in mice with Morquio. LVs carrying the native GALNS gene were produced under three different promoters (ubiquitous-CBh, collagen type II-COL2A1, hematopoietic stem cells-CD11b). Then, we treated GALNS knockout (KO) mice intravenously at newborns and 4 weeks old with low/high doses of LVs under three different promoters. Blood samples were collected biweekly following direct in vivo infusion, and mice were autopsied at 16 weeks old to collect tissues. We aimed to investigate vector copy number, enzyme activity levels and the GAG concentrations in blood and tissue samples, plasma AST/ALT levels, pathology, and bone morphology. In vivo experiment data demonstrated that LVs under ubiquitous CBh promoter with high dose in KO newborn mice had the highest enzyme activity. Bone pathology showed little impact of the LV treatment in bone.

Track: Skeletal Disorders, Treatments & Rehabilitation
Research Area: Bone

Poster Session 1

1 Tiankuo Chu

Effect Of Yoda1-Augmented Whole-Body Vibration On Bone Integrity Of Adult Mice Undergoing Radiotherapy

Tiankuo Chu, Jason Jiang, Wiley Gong, Murtaza Wasi, Rosa Guerra, Shubo Wang, Lidan You (University of Toronto), Liyun Wang*

University of Delaware

Physical exercise, as an adjuvant therapeutic, improves bone quality, but routine aerobic or strength exercise can be challenging for some elderly cancer patients with unintended injuries.1?2 Even though whole-body vibration (WBV) is safer and easier-to-perform and shows overall wellness benefits for metastatic patients including bone protection, but the bone remodeling respond is limited due to the aging effect.3 What’s more, radiotherapy is a common treatment of cancer inhibition and pain management, but radiation was found to increase the apoptosis of osteocytes (the major mechanosensing cells in bone).4 Yoda1, an agonist to the mechanosensitive Piezo1 channels highly expressed in osteocytes, promotes bone growth in young mice,5 but its effects on mature bone in the presence of WBV and radiotherapy remain to be determined. In our study, the 8-month-old mature Balb/c and C57BL/6J female mice received WBV (12 Hz, 0.25 g, 30 min with 7.5 min rest in between) 1h after Yoda1 injection, 5 days/week for 4 weeks after 8Gy radiation twice before intervention. Under the challenges of radiation, we did observe smaller pMOI decline (a trend) in the Yoda1-augmented WBV group and a significant increase in bone formation at week 2 for the combined treatment. Besides, relative to non-treated groups, the Yoda1-augmented WBV significantly inhibited the osteoclast’s TRAP activity regardless of radiation. Our results demonstrated the benefits of Yoda1-augmented WBV are retained in mature mice after radiotherapy.
REFERENCES: [1]. Chen+ 2017. [2]. Alfano+ 2012, [3]. Margaret+ 2021, [4]. Abhishek+ 2017, [5]. Li+ 2019

Track: Cell and Tissue Studies
Research Area: Bone

3 Rosa Guerra

F-Actin Bundles And Cross-Linkers In Osteocyte Dendrites

Rosa Guerra, Megan Coffin, Shannon Modla, Velia Fowler, Liyun Wang

University of Delaware

Osteocytes are the master orchestrator of bone remodeling, and their dendrites enable them to communicate with other cells. With aging, osteocyte dendrites deteriorate leading to declined bone quality. Similar to epithelial microvilli and inner ear stereocilia, osteocyte dendrite integrity is thought to depend on tightly packed cross-linked F-actin bundles. However, the dendrite cytoskeleton details remain unclear due to technical challenges. In this study, we screened three RNA sequencing databases and identified four candidate F-actin cross-linkers. Our approach is to detect if they are present in osteocytes using immunohistochemistry and then dissect their role in dendrite structure via gene deletion. The protocols of cell culture, immunohistology, confocal and high-resolution STED imaging, and transmission electron microscopy (TEM) are being optimized. The objectives are to form long dendrites, preserve F-actin filaments and cross-linkers, and image F-actin and cross-linkers in normal and gene-deleted osteocytes. Our pilot data demonstrated multiple F-actin filaments within the dendrite and fimbrin (a known F-actin cross-linker) expressed in both cytoplasm and dendrites. Our long-term goal is to uncover the molecular mechanisms underlying the osteocyte dendrite formation, maintenance, and eventual decline with aging.

Track: Cell and Tissue Studies
Research Area: Bone

5 Thomas Manzoni

Passaged Zonal Chondrocytes Can Regain Cartilage Zone-Specific Properties After Redifferentiation

Thomas Manzoni, Justin Parreno

University of Delaware

Mechanical competence of bioengineered cartilage depends on its matrix composition, which must contain collagen-2 (COL2) and aggrecan (ACAN). The biomechanical properties in native cartilage depend on the zonal architecture consisting of the superficial zone (SZ), middle zone (MZ), and deep zone (DZ). The SZ cells (SZC) produce proteoglycan-4 (PRG4) for surface lubrication, while DZ cells (DZC) produce collagen-10 (COLX) for compressive strength. Monolayer expansion to increase cell number causes loss of chondrogenic expression. We test the hypothesis that both SZC and DZC can form bioengineered cartilage rich in COL2 and ACAN, while only SZC will express PRG4, and only DZC will express COLX. SZC and DZC were isolated from bovine joints, and separately expanded. Monolayer expansion decreased chondrogenic and zonal expression in expanded SZC and DZC, while dedifferentiated molecule collagen-1 expression increased. Passaged cells were then seeded in 3D culture to allow for redifferentiation. After redifferentiation both subpopulations increased expression of COL2 and ACAN, while only DZC expressed COLX, and only SZC expressed and secreted PRG4. Zonal properties are regained by the original zonal cell subpopulation after redifferentiation. Therefore, bioengineering of cartilage rich in COL2 and ACAN, with expression of PRG4 and COLX must contain both SZC and DZC.

Track: Cell and Tissue Studies
Research Area: Cartilage

7 Emily Lambeth

The Role Of Hyaluronic Acid In The Synergistic Lubrication Of Articular Cartilage

Emily P. Lambeth; David L. Burris; Christopher Price

University of Delaware

Articular cartilage facilitates remarkably low friction coefficients in our joints1. Only recently has this lubricity been replicated on the benchtop, using a configuration that promotes tribological rehydration and synovial fluid2,3. Synovial fluid is composed of numerous molecules that may influence cartilage lubrication, including hyaluronic acid (HA)3,4. Here, our objective was to explore the effects of hydrodynamics and interstitial lubrication (ISL) and HA presence on cartilage friction. Osteochondral explants (19mm) were extracted from mature bovine femoral condyles2,3. All explants underwent loading and sliding preconditioning before undergoing “speed sweep” tests where sliding speed and lubricant were altered. In study 1, four increasing concentrations of HA (0 – 5mg/mL, 1.4MDa) were assessed, while in study 2, four increasing molecular weights of HA (0-1.4MDa, 3mg/mL) were utilized. As expected, the lowest observed equilibrium frictions occurred at sliding speeds >40mm/s, while peak frictions occurred at <5mm/s regardless of lubricant. Interestingly, peak and minimum frictions decreased with increasing HA concentration and molecular weight. These findings suggest that upon the sliding-dependent recovery of fluid load support and ISL, HA becomes a power synergistic lubrication partner, and that these benefits are HA concentration and molecular weight dependent.
References: [1] Linn J Biomech 1968; [2] Moore OA&C 2017; [3] Farnham Tribol Let 2021; [4] Fam Biorheol 2007.

Track: Cell and Tissue Studies
Research Area: Cartilage

9 Venu Pandit

Investigating Chondrogenic Mechanism Induced By Bmpr1A Mimetic Peptide Ck2.1

Venu Pandit, Kailey DeGeorge, Daniel Halloran, Xinqiao Jia, Anja Nohe

University of Delware

Osteoarthritis (OA), called ‘wear and tear arthritis’ has affected more than 50% of older adults over the age of 60. It involves the deterioration of articular cartilage in weight-bearing joints accompanied by inflammation and pain. A synthetic peptide CK2.1 restored articular cartilage in an OA mouse model. It is a BMP receptor (BMPR) 1A mimicking peptide. In our study, we intend to understand the underlying mechanism of the repair. Knee tissue sections of mice operated to induce artificial OA followed by intra-articular injections of CK2.1 conjugated with hydrogel particles were obtained. Tissue sections from mice injected with only hydrogel particles and otherwise operated similarly served as control. From immunofluorescence, localization of CK2.1 majorly in the proximity of superficial and middle zone chondrocytes of articular cartilage was seen. It co-localized with the expression of sox9 and collagen type IX. There was an increase in expression of sox9 and collagen type IX as compared to the control mice. Our results demonstrate that CK2.1 majorly accumulates in chondrocytes from the superficial and middle zone. An increase in the expression of markers for superficial and middle zone chondrocytes can be investigated further to study the molecular mechanism of cartilage repair activated by CK2.1.

Track: Cell and Tissue Studies
Research Area: Cartilage

11 Rafael Castro

Design And Synthesis Of Multifunctional Collagen Mimetic Peptides For Biomedical Applications

Rafael Castro

University of Delaware

Peptides, functionally encoded by their amino acid sequence, are increasingly being designed within the biomaterials community for creating mimics of the extracellular matrix found within human tissues. Collagen-like peptides have been designed to mimic parts of the structure and bioactivity of collagen I, the most prevalent protein in the human body and important in the structure and properties of many tissues. Recently, synthesis methods have been developed for self-assembling multifunctional collagen mimetic peptides (mfCMP) for the formation of synthetic matrices with robust and tunable properties. There remains a need for self-assembling peptides with tunable properties to better mimic and potentially replace natural collagen I, including matrix stiffening, stability, and bioactivity. I plan to design mfCMP sequences with reactive handles for triggering intra-fibrillar crosslinking and inducing matrix stiffening with light, building from an established polymer-peptide materials system. Next, I will design mfCMP sequences to assemble, stabilize, and stiffen purely peptide-based materials using light-activated inter- and intra-fibrillar crosslinking. Finally, I will design mfCMPs coassembled with integrin binding sequences and with tunable melting temperature for controlling material structure and biochemical content, creating a modular system of building blocks that has the potential to be a fully synthetic surrogate for natural collagen I.

Track: Cell and Tissue Studies
Research Area: Design & Innovation

13 Lily Lin

Tendon Overload Using A Rodent Model Of Synergist Ablation Leads To Mechanical Degeneration

Lily M. Lin, Ellen T. Bloom, John M. Peloquin, Michael H. Santare, Justin Parreno, Karin G. Silbernagel, Dawn M. Elliott

University of Delaware

Introduction: Many tendon disorders are related to overloading, not overuse, thus, we recently established a rodent overload model of synergist ablation (SynAb). We showed multi-scale structural changes that suggest tendon degeneration, however, it was unclear whether the degeneration was induced exclusively from structural changes. Therefore, the objective of this study was to investigate the mechanical changes induced by tendon overload.

Methods: Bilateral plantaris tendons from 7-month-old female Long Evans rats were randomly assigned to two groups: SynAb and Control. At 8 weeks post-surgery, rats were sacrificed and the plantaris tendons were dissected. Each tendon underwent stress relaxation at 25% then a ramp to failure. All loading and unloading rates were 1%/s. The stress-strain curves were analyzed for transition point, yield point, failure point, and linear region modulus.

Results: The modulus and transition stress in SynAb was lower than Control. Yield and failure strain were not statistically different between the treatment groups, however, the SynAb yield stress was 52% lower than Control, and the SynAb failure stress was 65% lower than Control.

Conclusion: This study showed degenerative mechanical changes, specifically, we observed a decrease in modulus in the SynAb group indicating that increasing mechanical load from SynAb impairs mechanical function.

Track: Cell and Tissue Studies
Research Area: Ligament & Tendon

15 Ying Peng

Resveratrol Protects Meniscus From Inflammatory Damage By Strengthening Collagen Fibers And Inhibiting Matrix Metalloproteinases Activity

Ying Peng, Asma Arshad, Shuo Wei, X. Lucas Lu

University of Delaware

Resveratrol (trans-3,5,4’-trihydroxystilbene) is a natural polyphenol present in human diet such as red grapes, blueberries, and peanuts. Over 200 clinical trials in the US are studying resveratrol’s health benefits for patients with cardiovascular disease, cancer and other diseases. Animal studies have shown that resveratrol can ameliorate inflammatory damage on joint tissues and prevent the initiation of post-traumatic osteoarthritis. Inflammation also impacts meniscus metabolism, and the subsequent meniscus degeneration contributes to the onset and the progression of osteoarthritis. The aim of this study is to test if resveratrol can protect meniscus from inflammatory challenge and investigate the potential protective mechanisms. We use meniscus explants harvested from bovine knee joint for this study. Resveratrol was applied to the explants to study its effects on anabolic activity, and catabolic activity under IL-1? treatment, of menisci cells. The interacts of resveratrol with meniscus extracellular matrix and catabolic enzyme were investigated for mechanism studies. Our results shown that resveratrol has protective effects on meniscus by (1) improving the anabolic activities of menisci cells; (2) interacting directly with collagen fibers and protect them from enzymatic digestion, and (3) inhibiting the activity of catabolic enzyme, such as matrix metalloproteinases (MMP) 13.

Track: Cell and Tissue Studies
Research Area: Meniscus

17 Sampurna Saikia

Oral Administration Of Peptides For Galns (N-Acetylgalactosamine-6-Sulfate Sulfatase) Into Oral Administration Of Peptides For Galns (N-Acetylgalactosamine-6-Sulfate Sulfatase) Into Mucopolysaccharidosis Iva Mice To Produce Immune Tolerance For Efficacy Of Aav Gene Therapy

Sampurna Saikia, Yasuhiko Ago, Shunji Tomatsu

University of Delaware

Background: Mucopolysaccharidosis IVA or MPS IVA is one of the most important genetic disorders grouped in Lysosomal storage diseases, because of accumulation of the undegraded substrate in absence of the enzyme N-acetylegalactyosamine-6-sulphate sulphates. Different phenotypic symptoms like skeletal abnormalities, short stature, knock knees, cloudy eyes, vision loss, course facial features are the outcome of the disease onset. Although gene therapy has been established for promising outcome for one-time permanent treatment, as the continual enzyme is expected to produce after injection with transduced cells, high immune responses against the gene therapy treatment are an overriding phenomenon to consider, which eventually decrease the treatment efficacy. Therefore, a new strategy is required to induce immune tolerance to specific proteins and viral vectors using mouse models. In this study we hypothesized that oral delivery of peptides for GALNS will induce the tolerance in mice for MPS IVA, which will eventually increase the efficiency of gene therapy treatments by improving bone and cartilage lesions. Methods: Neonatal male mice will be treated with three epitope peptides and GALNS enzyme within 48 hours of birth for every alternate day continuing for 20 days in two doses at the rate of 2.5µg/g body weight and 5µg/g body weight followed by tail vein injection with 5×1013 GC/kg CAG-GALNS AA9 vectors to the peptide and GALNS experimental treatment group at the age of 30 days. Collection of plasma samples from treated mice bi-weekly after each gene therapy treatments for GALNS activity was carried out until euthanization of the mice at age of 24 weeks to collect various tissues. We target to evaluate the vector biodistribution, antibody and enzymatic activity along with GAG levels in plasma and various tissues followed by pathology and bone morphology. Results: ELISA for anti-GALNS antibody by using purified GALNS as primary antibody for standards, showed a various range of anybody concentrations in different groups of treated animals including the control group. Similarly, enzymatic activities among the treated mice groups varied widely with the highest enzymatic activity shown for the group treated with GALNS 2.5µg/g body weight. The therapeutic efficacy of the gene therapy treatment will be evaluated by conducting GAG assay, KS levels in plasma and tissues, bone pathology and vector copy number analysis.

Track: Cell and Tissue Studies
Research Area: Rehabilitation & Treatment

19 Apoorva Metkari

Rock Inhibition Promotes The Development Of Polarized Acini Structures With Secretory Functions In Hyaluronic Acid- Based Hydrogel Matrices

Apoorva Metkari, Mugdha Pol, Robert Witt (Helen F. Graham Cancer Center), and Xinqiao Jia

University of Delaware

Xerostomia is a salivary gland hypofunction caused by radiation therapy for head and neck cancers. Xerostomia severely affects patients’ quality of life and salivary gland tissue engineering offers a regenerative solution. Previously, we cultured primary human salivary stem/progenitor cells (hS/PCs) in hyaluronic acid (HA)-based matrices with varying proteolytic degradability and demonstrated that cell-mediated matrix degradation is necessary to maintain the pro-acinar stem/progenitor phenotype. However, the multicellular structures developed in these HA gels did not contain correctly polarized lumens. In the current study, we investigate the effects of ROCK inhibition on the phenotypic changes and polarization of hS/PCs. We also study the effects of matrix degradation and ROCK inhibition on stiffness of the cells via mitotracking microrheology. We demonstrate that ROCK inhibition led to the establishment of multicellular structures that were correctly polarized, as evidenced by apical localization of GM130, and basal localization of basement membrane proteins. We also saw an increased expression of acinar markers (AQP3, SLC12A2, AMY1A) at the gene and the protein level in constructs treated with an ROCK inhibitor. In summary, we show that ROCK inhibition promotes the formation of polarized acini structures in HA-based synthetic matrices. This finding is an important step forward in salivary gland tissue engineering.

Track: Cell and Tissue Studies
Research Area: Tissue Engineering

21 Hannah Cohen

A Novel Controller for Belt Accelerations During Late Stance to Modulate Propulsion Mechanics at Multiple Speeds

Hannah Cohen and Fabrizio Sergi

University of Delaware

Walking is critical for completing activities of daily living. Propulsion, a major subtask of walking, has two components: propulsive force, and pushoff angle. Our lab previously developed a training protocol based on belt accelerations applied at push-off to modulate propulsion mechanics but we have not assessed its efficacy for use for gait training of participants with asymmetric motor impairment. In this study, we recruited healthy adults to a two-session experiment where we measured the effect of belt accelerations on propulsion via anterior-posterior ground reaction forces, propulsive impulse (PI), TLA, and electromyography in three plantarflexor muscles. While walking on a dual-belt treadmill, participants experienced an increase in belt speed as they pushed off the treadmill. In the unilateral condition, only one leg was accelerated, while both legs were accelerated during their respective push-off in the bilateral condition. The electromyography results showed 5-10% increase in plantarflexor activation during training compared to baseline in the targeted legs. PI increased by 3-4% in the left leg during late aftereffects compared to baseline in the bilateral condition, and in the unilateral condition increased by 7% ± 0.38 in the targeted leg and 5% ± 0.20 in the non-targeted leg.

Track: Joint and Clinical Studies
Research Area: Gait Analysis

23 Rebecca Nikonowicz

Functional Imaging Of The Brainstem During Stretch-Evoked Responses Under Different Task Instructions

Rebecca Nikonowicz, Fabrizio Sergi

University of Delaware

Despite the importance of the reticulospinal tract (RST) in motor recovery after lesions of the corticospinal tract, in-vivo measurement of RST function is currently not possible. Because the RST may contribute to the increase in long latency response amplitude (LLRa) associated when participants are asked to “resist” a perturbation, functional magnetic resonance imaging (fMRI) during LLRs under different instructions may be a method to stimulate the RST and decouple the contribution of the corticospinal tract and RST to LLRs. We built an MR-compatible robotic perturbator for experiments with surface electromyography (sEMG) and fMRI. We conducted a preliminary study outside the MRI scanner on five participants using sEMG to measure wrist flexor muscle activity. Participants were given a “Yield” or “Resist” instruction prior to each trial. In a second pilot study, during whole-brain fMRI sequencing, ten participants completed sessions of blocked perturbations under i) Yield, ii) Resist, and iii) Yield 2 (slow perturbation, control) conditions. Analysis of sEMG data shows significantly greater LLRa in “Resist” relative to “Yield” (Resist: 4.483+/-0.966n.u., Yield: 1.205+/-0.964n.u., p=0.001). Analysis of functional images shows increased activation in the brainstem primarily localized in the bilateral medulla and midbrain, contralateral pons, and primary motor cortex in the resist condition.

Track: Joint and Clinical Studies
Research Area: Neuromuscular Modeling & Control

25 Margo Donlin

Development And Validation Of Adaptive Functional Elctrical Stimulation

Margo C. Donlin; Jill S. Higginson, PhD

University of Delaware

Stroke is one of the leading causes of disability in the United States, often resulting in physical impairments like hemiparesis. Hemiparesis to the lower leg commonly causes foot drop and decreased forward propulsion, leading to reduced mobility and poor overall health. Rehabilitation protocols often use functional electrical stimulation (FES) to augment muscle function in the lower leg and restore mobility, but only some participants experience meaningful improvements in foot drop and forward propulsion. To address these limitations, we developed a novel adaptive FES (AFES) system that adjusts the stimulation amplitude at every stride to target subject-specific impairments and deliver optimal stimulation. At each stride, the AFES system measures real-time ankle dorsiflexion angle and forward propulsion, then compares these variables to a healthy reference value. If the error between the real-time and reference values is large, the stimulation amplitude to that muscle group will increase to promote healthier gait. Upcoming experiments with individuals post-stroke will validate that the stimulation amplitude changes as prescribed and compare the effects of the AFES system to the existing FES system. The goal of these experiments is to develop and validate a novel post-stroke gait rehabilitation tool that addresses individual-specific impairments and improves functional walking outcomes.

Track: Joint and Clinical Studies
Research Area: Rehabilitation & Treatment, s

27 Zahra McKee

Ankle Mechanics Of Pd-Afo Walking Can Improve After Physical Therapy Training: A Case Study

Zahra McKee, Jacob Skigen, Darcy Reisman, Elisa Arch

University of Delaware

To supplement post-stroke muscle weakness, passive dynamic ankle-foot orthoses (PD-AFOs) provide dynamic levels of rotational spring-like assistance about the ankle joint. This case study aimed to investigate if physical therapy training could improve a patient’s loading of the PD-AFO and if so, if enhanced walking function followed. The participant was a 72-year-old male 8 years post-stroke. They participated in 5 physical therapy sessions, focused on increasing midstance dorsiflexion on the PD-AFO (paretic) side to engage the rotational spring. Instrumented gait analysis data was collected pre- and post-training at the participant’s pre-training PD-AFO self-selected walking speed. Paretic peak dorsiflexion angle increased 181.7% (Pre: 2.52°, Post: 7.11°), paretic peak plantar flexion moment increased 21.33% (Pre: -0.72 Nm/kg, Post: -0.88 Nm/kg ), and paretic peak positive ankle power increased 51.81% (Pre: 0.19 W/kg, Post: 0.30 W/kg ). Self-selected walking speed was virtually unchanged (Pre: 0.32 m/s, Post: 0.35 m/s) and total mechanical cost of transport increased by 10% (Pre: 1.88 J/kg/m, Post: 2.07 J/kg/m). Training resulted in meaningful improvements in ankle biomechanics, but these did not translate to enhanced walking function.

Track: Joint and Clinical Studies
Research Area: Rehabilitation & Treatment

Poster Session 2

2 Nidhi Nidhi

Idlv-Based Crispr-Cas9 System Mediates Highly Efficient Gene Editing In The In Vitro Model Of Morquio Syndrome A

FNU Nidhi 1,2, Shaukat A Khan 1, Shunji Tomatsu 1,3,4,5*

1 Department of Biomedical Research, Nemours/Alfred I. DuPont Hospital for Children; 2 Department of Biological Sciences, University of Delaware; 3 Department of Pediatrics, Shimane University; 4 Department of Pediatrics, Graduate School of Medicine, Gifu University; 5 Department of Pediatrics, Thomas Jefferson University

Background: Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive, lysosomal storage disorder (LSD) caused by the deficiency of the N-acetylgalactosamine-6-
sulfatase (GALNS). Deficiency of GALNS causes glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), to which accumulate in cartilage and its extracellular matrix as well as cornea and heart valves, leading to short stature and neck, pectus carinatum, laxity of joints, kyphoscoliosis, knock-knee, tracheal obstruction, corneal clouding, and heart valvular disease. No effective treatment for this skeletal disease is available; therefore, an unmet challenge to develop an effective therapy. The most revolutionary genome editing platform, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated System (Cas), provided consistent gene expression and successive positive findings in various LSDs. Even though lentiviral vectors is are the first choice to accommodate a large CRISPR-Cas9 system, problems still exist with undesirable off-target effects. The Integrase-deficient lentiviral vector (IDLV) presents an attractive alternate means to deliver CRISPR-Cas9 system due to its superior packaging and low integration capacity, transient expression, and capability to transduce different cells and tissues. Here, we aimed to evaluate IDLV-based CRISPR/Cas9 systems. Materials; In vitro expression levels of GALNS and GAG levels in transduced mouse MPS IVA fibroblasts were assessed for IDLV-based CRISPR/Cas9 systems using two synthetic guide RNAs (sgRNA) and human GALNS cDNA. The target site was before ATG of exon1 in mouse galns gene.
Results and Discussion: . The results demonstrated successful homologous recombination and stable, long-term GALNS expression up to a 6.4-fold change as compared to WT. In addition, we observed marked amelioration in MPS IVA fibroblasts, evident in the normalization of lysosomal mass and total GAGs. These findings suggested IDLV-based CRISPR-Cas9 system as a highly efficient and specific platform for a novel therapy for MPS IVA.

Track: Cell and Tissue Studies
Research Area: Bone

4 Kelechi Chukwuocha

Caspase1 Cleavage Of Bmpr1A Leads To Aberrant Signaling Of The Bmp2 Pathway And Drives Adipogenesis In Mice Myoblasts Cells

Kelechi Chukwuocha, Anja Nohe (PI)

University of Delaware

The skeletal system is an essential part of developmental process that provides structural support and protection for other tissues and organs. For bone homeostasis to be maintained, there must be a balance between bone mineralization and bone resorption through the activities of osteoblasts and osteoclasts. An imbalance between the two activities can degenerate to diseases such as osteoporosis. About 20% of American women suffer from osteoporosis which results in 1.5 million fractures annually, and about thirty five percent of the adults are obese. These conditions have recently been found to be related with evidence showing that increased abdominal fat is associated with increased risk of hip fracture, reduced bone mineral density and increased marrow adiposity. Bone morphogenic proteins especially bone morphogenic protein 2 (BMP2), are potent growth factors that play crucial roles in osteogenesis, adipogenesis and/or adipocyte apoptosis based on concentration. However, the molecular mechanism directing adipogenesis within marrow is not well understood. In this study, we examine the implication of a possible proteolytic cleavage of the BMP type 1 receptor (BMPR1A) by the Interleukin-1 converting enzyme (Caspase1). Defining key pathways to specifically direct adipogenesis or osteogenesis may lead to new therapeutics regulating osteoblast and adipocyte numbers and functions.

Track: Cell and Tissue Studies
Research Area: Bone

6 Annie Porter

Comparison Of Mechanical Response Of Tmj And Knee Cartilage Under Dynamic Loading

Annie Porter, Jonathan Kim, Lin Han, Drexel University; John Peloquin, Michael Santare, X. Lucas Lu

University of Delaware, Drexel University

Disorders of the temporomandibular joint (TMJ), the only moving joint in the human head, affect over 10 million Americans. TMJ condylar cartilage however remains an understudied tissue. It has a unique, bi-layered structure in which the top layer is fibrocartilage and the bottom layer is primarily hyaline cartilage. The layers are integrated by collagen bundles which extend from the top surface and root into the subchondral bone. In this study we hypothesized that the unique bilayer structure endows the TMJ condylar cartilage with a low dynamic stiffness, especially under small stress and high frequency loads, to minimize the impact on the brain from TMJ daily functions, i.e., eating and talking. At low stresses, the dynamic modulus of TMJ cartilage was lower than that of the knee. This trend implies that during daily activities, such as talking, TMJ cartilage provides better shock absorption than knee cartilage, potentially reducing impact on the brain. It is critical to note that the dynamic moduli here were obtained when the knee and TMJ tissues were under the same initial load, but different initial strains. Thus, the dynamic moduli of TMJ could be even lower than the knee with similar initial strain.

Track: Cell and Tissue Studies
Research Area: Cartilage

8 Shamimur Akanda

Effect Of Bath Osmolarity On Tribological Rehydration And Cartilage Lubrication

Shamimur Akanda, David Burris, Christopher Price

University of Delaware

Articular cartilage, a triphasic tissue in our joint, maintains vanishingly low friction coefficient over decades. The presence of negatively charged proteoglycans and counter ions in the tissue causes Donnan osmotic swelling pressure influencing cartilage material properties. Although cartilage is compromised by its lubricity under habitual static loading, our recent discovery of ‘Tribological Rehydration’ fosters active fluid recovery into cartilage. However, how external bathing solution osmolarity influences cartilage tribological rehydration is yet to be identified. The objective of this study is to determine the influence of bathing solution osmolarity and composition on tribological rehydration and cartilage lubrication under the biofidelic sliding conditions of the cSCA configuration. For all approaches,19 mm diameter explants from femoral condyles were extracted. In the first two approaches approach, the effect of bath osmolarity and composition on tribological rehydration was assessed from the initiation of sliding (Both high and low fluid load support) and no significant effect of osmolarity was observed on cartilage lubrication. However, in the third approach, explants were stiffened or softened by bath osmolarity before sliding. Interestingly, it resulted in significantly compromised lubrication for the stiffer explants. This study concludes that stiffening the tissue through bath osmolarity compromises the lubricating ability of cartilage.

Track: Cell and Tissue Studies
Research Area: Cartilage

10 Stephanie Richardson

Transdifferentiation Of Fibroblasts To Chondrocytes Using Decellularized Matrices

Stephanie Richardson-Solorzano (1), Travis Block (2), and Justin Parreno (1)

1 Department of Biology, University of Delaware; 2 StemBioSys, San Antonio

Articular cartilage is incapable of self-repair. Even small defects to cartilage results in Osteoarthritis progression where eventually total joint replacements would be necessary. Therefore, repairing small focal defects to prevent Osteoarthritis is critical, especially for young patients that injure their cartilage. Autologous Chondrocyte Implantation (ACI) is a gold standard therapy to heal small focal defects. In ACI, a small portion of cartilage is removed from a healthy cartilage site and primary chondrocytes are isolated from donor cartilage. The chondrocytes are then expanded and then reimplanted into the damage joint. Two major issues assoicated with ACI are donor site morbidity and the loss of phenotype during monolayer expansion of chondrocytes. In this study, we attempt to address these issues by investigating the use of another autologous cell source, skin fibroblasts, for ACI. We test the hypothesis that skin fibroblasts can be stimulated to express chondrogenic matrix.
To stimulate chondrogenic expression, we culture embryonic fibroblasts (NIH3T3) on decellularized extracellular matrix. I have determined that short-term exposure stimulates chondrogenic gene expression. Transdifferentiation of fibroblasts into chondrocytes would result in a potential cell source for cartilage repair.

Track: Cell and Tissue Studies
Research Area: Cartilage

12 Breanna Huntington

To The Rescue! – Altering The Fate Of Pulmonary Fibroblasts Using Cellularly And Externally Responsive Hydrogels

Breanna M. Huntington, Qi Zhang, Samantha E. Cassel, Eric M. Furst, April M. Kloxin

University of Delaware

Cells rely on detection of biophysical and biochemical cues from their microenvironment to drive a plethora of events, including proliferation and migration. In fibrotic diseases, the cellular microenvironment stiffens, and cells respond by commencing a feedback loop to uncontrollably and maladaptively remodel the extra cellular matrix (ECM). To probe and understand underlying cell-matrix interactions in this complex process, in-vitro model systems that integrate synthetic ECMs can be used for recapitulating cell-matrix interactions in three dimensions with well-defined and tunable biophysical and biochemical cues. We use a system of cellularly and externally responsive hydrogels as synthetic ECMs to mimic healthy and fibrotic cellular microenvironments. To further understand cell-matrix interactions, we use material characterizations, notably bulk rheometry and atomic force microscopy (AFM), and cellular assays, including immunostaining and a dynamic ?SMA lentiviral fibroblast reporter cell line, to assess the dynamic reciprocity and its role in cell activation in our cell culture systems. We further apply dynamic material softening events using an externally applied enzyme to alter microenvironmental mechanics and probe changes in fibroblast activation, which is a typical hallmark of fibrotic disease progression. These studies aim to provide insights into important biomechanical regulators of cellular responses and their temporal dynamics.

Track: Cell and Tissue Studies
Research Area: Disease models

14 Valerie West

Actin Regulates Tenocyte Gene Expression Via Mrtf

Valerie West, Kameron Inguito, Karl Matthew Ebron, Justin Parreno

University of Delaware

Tendinosis as a result of tissue overload counterintuitively causes under stimulation of tenocytes. We previously demonstrated that cellular under stimulation alters cellular phenotype through a decrease in tenogenic expression levels and an increase in both chondrogenic and protease expression levels. Therefore, we hypothesize that this regulation of genes in response to cellular stress-deprivation is the result of actin depolymerization. Specifically, that actin depolymerization regulates genes through monomeric (globular; G-) actin-binding myocardin-related transcription factor (MRTF).

We exposed isolated primary tenocytes to Latrunculin A, sequestering G-actin, preventing actin polymerization, and causing a nuclear export of MRTF from the nucleus of cells. Coinciding with nuclear export of MRTF are decreases to tenogenic genes (collagen-1, scleraxis, and a-smooth muscle actin) and increases to the expression of chondrogenic (Sox9) and proteases (Mmp-3 and Mmp-13). To determine if genes were regulated directly by MRTF, we exposed tenocytes to MRTF inhibitor, CCG1423, resulting in significant decreases to tenogenic genes with minimal effects on chondrogenic or protease genes. In conclusion, actin depolymerization is a regulator of gene expression in tendon cells, partially through regulation of MRTF. Ultimately, further understanding the regulation of gene expression during tendinosis by actin may lead to new therapeutic opportunities against disease progression.

Track: Cell and Tissue Studies
Research Area: Ligament & Tendon

16 Ziqing Wang

Spatial Transcriptomics Study Revealed Alterations In Lipid Metabolism Of Lipid Laden Macrophages In Broiler Chickens With Wooden Breast

Ziqing Wang, Paul Khondowe, Erin Brannick, Behnam Abasht

University of Delaware

The goal of this study is to use spatial transcriptomics to characterize expression profile particular to different cell types and their potential interactions at early stage of Wooden Breast myopathy (WB). Four randomly sampled broiler chickens were euthanized at 23 days post-hatch, and a 1 cm3 sample was dissected from the cranial part of the right pectoralis major muscle. These samples were subsequently processed with Visium Spatial Gene Expression kits (10X Genomics) followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified by an ACVP certified veterinary anatomic pathologist. Obtained sequence reads were aligned to the chicken reference genome (Galgal6), mapped to histological images and analyzed for clustering as well as differential expression. Unsupervised K-means clustering differentiated histological features and their expression pattern including lipid laden macrophages (LLM), myositis and vasculature. Particularly, LLM exhibited reprogramming of lipid metabolism with increased expression of lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through CD36 molecule mediated signaling. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositis regions, increased cathepsins expression in lysosome may play a role in muscle homeostasis and repair.

Track: Cell and Tissue Studies
Research Area: Muscle

18 Joohyun Lim

Ectopic Ossification Of The Tmj In A Murine Model Of Osteogenesis Imperfecta

Joohyun Lim

University of Delaware

Temporomandibular disorders (TMDs) derive from abnormalities in the temporomandibular joint (TMJ) and adjacent connective tissues that cause severe orofacial pain and reduced range of motion. Here, we report a murine model of osteogenesis imperfecta (OI) which displays aberrant bone formation in the tendon-bone interface (TBI) of TMJ due to defects in the posttranslational modification and cross-linking of collagen I. Loss-of-function mutations in FKBP10 causes OI with joint contracture. In addition, we recently reported that tendon-specific deletion of Fkbp10 causes postnatal joint deformities and impaired locomotor function which corroborates the phenotypic spectrum observed in patients. Interestingly, conditional deletion of Fkbp10 also induced abnormal ossification in TMJ that increases mandibular condyle length and width at 6 and 18 months-of-age, despite normal mandibular development. The ectopic bone formation in TMJ of Fkbp10-deficient mice was triggered by ectopic bone growth in the TBI at 1.5 months-of-age. Interestingly, abnormal bone growth in Fkbp10 mutants coincided with a significant increase in ?SMA-expressing cell populations in the TBI. Thus, Fkbp10 is likely required for preventing abnormal differentiation of this cell population. Collectively, the data suggests that posttranslational modification of the collagen matrix is critical for postnatal tissue homeostasis of the TMJ, in part through regulating a unique population of cells in the TBI.

Track: Cell and Tissue Studies
Research Area: Skeletal Disorder & Treatments

20 Shaukat Khan

Small Activating Rna Therapy For Mucopolysaccharidosis Type I

Shaukat Khan

Nemours

BACKGROUND AND SIGNIFICANCE
Mucopolysaccharidosis type I (MPS I) is a rare disorder caused deficiency of the lysosomal enzyme ?-L-iduronidase (IDUA). MPS I leads to the accumulation of glycosaminoglycans (GAGs), heparan and dermatan sulfates, in the cells of various tissues, resulting in multi-system dysfunction, including CNS and musculoskeletal systems, which if untreated results in death in the first decade of life. Enzyme replacement therapy and hematopoietic stem cell transplantation are available. However, neither of the treatments completely restore CNS and skeletal system. We propose developing a small activating RNA (saRNA) therapeutic approach to test our hypothesis that targeted overexpression of gene products corrects abnormal systems biology and ameliorates brain and bone lesions. saRNA is chemically synthesized double-strand RNA oligonucleotides, which are highly selective and activate specific genes, leading to increased production of target proteins.
HYPOTHESIS
We will use gentamycin to read through stop codon mutations (W402X and Q70X) to restore low level (~3%) of IDUA activity followed by saRNA therapy that will boost the IDUA supraphysiological level and reduction in GAGs to normal level.
SIGNIFICANCE
There is no treatment for MPS I to completely restore CNS and bone lesions and removal of storage materials in the tissues. The current proposal with saRNA allows to activate the endogenous gene product of IDUA to deliver sufficient enzymes to hard-to-reach tissues; brain and bone.

Track: Cell and Tissue Studies
Research Area: Skeletal Disorder & Treatments

22 Diego Caban-Rivera

Quantification Of Muscle Stiffness In Spastic Hemiplegic Cerebral Palsy Using Magnetic Resonance Elastography

Diego A. Caban-Rivera BS, Curtis L. Johnson PhD, Daniel R. Smith PhD (Emory University), M. Wade Shrader MD (Nemours A.I. duPont Hospital for Children), Stephanie Lee MS (Nemours Children’s Health-Delaware), Ellie Wright BA (Nemours Children’s Health-Delaware), Chris Church MPT (Nemours Children’s Health-Delaware), Faaiza Kazmi MD (Nemours Children’s Health-Delaware), Jason J. Howard MD (Nemours A.I. duPont Hospital for Children)

University of Delaware

Increased muscle stiffness is common in cerebral palsy (CP), but a reliable method of quantification is lacking. Magnetic resonance elastography (MRE) enables simultaneous stiffness measurements across muscles within an anatomic space. The study objective was to measure muscle stiffness in the gastrocnemius-soleus (G-S) complex from children with spastic hemiplegic CP, comparing more affected (MA) to less affected (LA) sides.
Participants underwent an MRE sequence with 80Hz external vibration delivered to each leg by a pneumatic transducer. A nonlinear inversion algorithm produced shear stiffness outputs for the entire G-S complex.
MRE/MRI scans were performed for 6 patients (4F), mean age 10.7 (range 5-17) years. Mean shear stiffness between sides (LA/MA) for gastrocnemius and soleus were not different (3.337 vs 3.4178 kPA, p=0.31; 4.224 vs 4.365 kPA, p=0.26, respectively), while significant differences between volumes were found (67.6 vs 47.1 cm3, p=0.046; 100.2 vs 71.7 cm3, p=0.005). Shear stiffness (LA/MA) was correlated with age for soleus [r=0.86 (p=0.03)/r=0.89 (p=0.02)].
These results suggest that G-S complex muscle belly stiffness is not different between sides in hemiplegic CP, yet soleus for both sides stiffened with age. Future studies are required to determine contributions of the entire muscle-tendon unit to overall stiffness of the gastrocnemius-soleus complex.

Track: Joint and Clinical Studies
Research Area: Muscle

24 Kristin Schmidt

Feasibility Of Using Task-Induced Changes In Resting State Functional Connectivity To Predict Motor Recovery In Post-Stroke Individuals

Kristin Schmidt, Tamara Wright, Andria J. Farrens, Henry Wright, Susanne M. Morton, Fabrizio Sergi

University of Delaware

As stroke is a disease with high inter-subject variability, there is growing interest to establish individualized predictive biomarkers of motor recovery. Recent work shows that changes in functional connectivity between brain networks measured immediately before and after exposure to a motor task reflect the motor memory consolidation process and may predict responsiveness to training programs based on a similar paradigm. To investigate the relationship between changes in resting state functional connectivity (rsFC) and motor recovery, we acquired resting state fMRI scans in fourteen post-stroke individuals immediately before and after performing an upper extremity motor task and compared changes in rsFC to motor performance evaluated via Upper Extremity Fugl-Meyer (UE-FM) before and after four weeks of physical therapy training. We found that a linear regression model based on the connectivity between the contralateral sensory cortex (S1) and the contralateral premotor cortex (PM) explained 66% of the variance in motor recovery. These results suggest that relevant task-induced changes in functional connectivity may serve as a predictive assessment of an individual’s responsiveness to training.

Track: Joint and Clinical Studies
Research Area: Neuromuscular Modeling & Control

26 Khushboo Verma

Improvements In Somatosensation And Dynamic Balance During Postural Transitions With The Use Of Stochastic Resonance Stimulation In An Individual With Cerebral Palsy – A Case Study

Khushboo Verma, Ashwini Sansare, Eman Alsaqabi, Hendrik Reimann, Samuel C.K. Lee

University of Delaware

People with Cerebral Palsy (CP), a group of disorders affecting balance, have a high risk of falls, leading to serious injuries. Falls can occur either due to inability to sense how the body moves through space or failure to generate an appropriate motor response. Multiple interventions focus on improving motor responses, however, sensory processing rehabilitation approaches that may enhance proprioception are currently underexplored. Stochastic Resonance (SR) stimulation is a promising method to overcome sensory information deficits. The overall hypothesis of this proposed research is that SR stimulation may enhance sensory detection and processing to improve balance control in people with CP.
One individual with CP and one typical developing peer (TD) performed somatosensation tests, sit-to-stand and gait initiation while SR stimulation was applied. SR improved vibration and joint position sense for the individual with CP, whereas the TD participant showed slightly better performance in vibration and worse performance in joint position sense. SR stimulation reduced the total time and anteroposterior center-of-pressure excursions during balance activities in CP while TD showed little change indicating better locomotor planning and balance control.
Knowledge gained from this study will give greater insights into the balance control strategies and provide a new sensory-centric rehabilitation approach.

Track: Joint and Clinical Studies
Research Area: Rehabilitation & Treatment

28 Samantha Stauffer

Is Gritti-Stokes Amputation Functional For Active Adults? A Case Series

Samantha J Stauffer, MSOP, CPO; J. Megan Sions, PT, DPT, PhD; John R Horne, Cped, CPO (Independence Prosthetics-Orthotics, Inc)

University of Delaware

The Gritti-Stokes amputation (GSA), in which the femur is resected at the level of the epicondyle and the patella is sutured to the distal end, is traditionally performed on older adults who would not be prosthetic candidates in order to give them a limb functional for transfers. However, the procedure may also yield a highly functional limb for a prosthesis due to provision of a long lever arm with distal weightbearing capacity without the problematic bulbous end of a traditional knee disarticulation procedure. The purpose of this study is to report on functional outcomes of the surgery among 3 young adults who are < 1 year post-GSA. All individuals underwent standardized clinical assessment involving self-report and performance-based outcome measures. A review of their prosthetic medical charts yielded timeline to prosthetic fitting and current componentry. Two of the three participants were fit with their preparatory prosthesis within 90 days of initial amputation. Self-report mobility indicated functional mobility at the 70th percentile or above for adults with lower-limb loss. During physical performance testing, all participants performed at the K3 or K4 functional level. Results indicate GSA may be an appropriate surgical technique for young adults with traumatic amputations.

Track: Joint and Clinical Studies
Research Area: Rehabilitation & Treatment

Keynote Speaker Session 2: Dr. Carla Scanzello

Title: Osteoarthritis as a Chronic Wound:  The Role of Toll-like Receptors.

Abstract:

Osteoarthritis (OA) is a leading source of disability in adults, causing chronic progressive joint pain and degeneration of the entire joint structure. No current medical treatments prevent the progressive joint deterioration, pain and disability characteristic of OA. Synovial inflammation is a common pathologic feature of OA and is strongly associated with disease progression and pain. Moreover, inflammatory transcriptional pathways play critical roles in the phenotypic changes to chondrocytes that promote catabolic activity and result in disruption of the functional integrity of articular cartilage. Therefore, targeting inflammation in the OA joint holds promise for preventing chronic pain while also slowing structural progression.  But it is not yet clear which pathways are most targetable to ameliorate chronic inflammation in the setting of OA.   Our laboratory has been investigating the role of Toll-like receptors in OA-associated inflammation. TLRs are pattern-recognition receptors (PRRs) of the innate immune response, and are important in initiating inflammation in response to both infectious and non-infectious tissue damage. Several observations support a role for TLR signaling in OA, both in promoting pathology and pain. We have recently demonstrated a critical role for the TLR co-receptor CD14 in an injury-induced OA model.  This work will be discussed in the context of the broader literature implicating TLRs and other PRRs of innate immunity in OA. Taken together, evidence implicating TLRs as central mediators of inflammation in OA suggests a hypothetical model of OA as a chronic wound of the joint, in which TLRs sustain chronic inflammation through interaction with molecular “danger-signals” produced by ongoing tissue damage and cellular stress.

Podium Session 2

1 Annie Porter

Effects Of Triamcinolone Acetonide On Metabolic Activities Of Young Cartilage

Annie Porter, Emily Newcomb, The College of William & Mary; Jacob Poplawski, Michael Axe, X. Lucas Lu

University of Delaware

Triamcinolone acetonide (TA) is a corticosteroid commonly used to reduce synovial inflammation following traumatic joint injury. Whether TA is harmful to chondrocytes remains unclear, depending on the injury models tested and TA dosage. A fear of clinicians is whether TA injections following injury in joints with otherwise healthy cartilage (lack prior osteoarthritis) will lead to cartilage degeneration and an early onset of osteoarthritis. We used click chemistry techniques to evaluate the metabolic effects of TA on in situ chondrocytes to evaluate its safety in clinical applications. We found chondrocyte viability, proliferation, and anabolic extra-cellular matrix (ECM) activities were not harmed by short-term TA treatment. In contrast to concerns, TA was able to reduce the ECM loss caused by both low-level long-term and high-level short-term inflammation. Previous studies which showed harmful effects of TA on chondrocytes primarily used monolayer cells with much higher doses of TA than we expect actually reaches the chondrocytes in vivo. Our study shows when chondrocytes remain in the natural solid matrix and are treated with more clinically relevant doses of TA, the harmful effects are mediated. This study supports the clinical use of intra-articular TA injections to reduce synovial inflammation in otherwise healthy cartilage.

Track: Osteoarthritis, Treatments & Mechanisms
Research Area: Cartilage

2 Sofia Gonzalez-Nolde

The Regulation Of Proteoglycan 4 Via F-Actin Reorganization In Native Superficial Zone Chondrocytes

Sofia Gonzalez-Nolde, Zsanaia Badiang, Cameron Schweiger, Justin Parreno

University of Delaware

The initial stage of Osteoarthritis (OA) includes a decrease in surface lubricant proteoglycan 4 (Prg4), secreted by superficial zone chondrocytes (SZCs). Prg4 has been shown to be regulated via the actin cytoskeleton in vitro however, it remains unclear if F-actin reorganizes and regulates Prg4 in native cartilage. We develop imaging methodology of an ex vivo whole organ culture of mouse femoral head cartilage to test the hypothesis that Prg4 is regulated by F-actin in native SZCs. Culturing native cartilage in serum free DMEM results in decreased Prg4 mRNA levels. We assessed actin reorganization by the F/G-actin ratio in SZCs captured via en face images of F- and globular (G-) actin. We determined that the reduction of Prg4 via culturing correlated with a decrease in the F/G-actin ratio. Treatment with transforming growth factor beta (TGF-?) sustains the SZC F/G-actin ratio and increases Prg4 expression. We found Tropomyosin3.1 (Tpm3.1), an F-actin stabilizer, regulates Prg4; with Tpm3.1 inhibition and in Tpm3.1 KO cartilage, induction of Prg4 via TGF-? is repressed. Our findings support that Prg4 is regulated by F-actin reorganization and that Tpm3.1 is critical for growth factor induced Prg4 expression.

Track: Osteoarthritis, Treatments & Mechanisms
Research Area: Cartilage

3 Meghan Kupratis

Detrimental Effects Of Collagen Crosslinking On The Frictional Response Of Bovine Articular Cartilage

Meghan E. Kupratis, Uriel Gonzalez, Atia Rahman, Elise A. Corbin, David L. Burris, Christopher Price

University of Delaware

During osteoarthritis (OA), decreased stiffness is thought to expose cartilage to elevated compressive and shear strains. Increasing cartilage stiffness via collagen crosslinking (CXL) has seen interest as a means to stabilize cartilage and prevent degeneration. However, recent studies from our group reveal that mechanical properties are poor predictors of healthy cartilage tribomechanics during biofidelic sliding. We can replicate in vivo friction behaviors in the convergent stationary contact area (cSCA) via a novel mechanism known as tribological rehydration. To determine whether CXL alters cartilage lubricity, bovine osteochondral explants underwent sequential microindentation and cSCA characterization in their naïve state and after CXL via glutaraldehyde or genipin exposure. As expected, CXL significantly increased cartilage tensile and compressive moduli. During cSCA tests, strain during ramp loading to 7N decreased following CXL, resulting in decreased in situ contact areas. Consequently, CXL cartilage experienced 66% higher contact stresses. Naïve cartilage sustained near-physiological friction in PBS-lubricated contacts (µ=0.010?0.003) and in vivo-like friction (µ=0.003?0.001) in synovial fluid (SF). CXL led to elevated friction in both PBS- and SF-lubricated contacts (µ=0.16?0.007 and µ=0.038?0.017, respectively). Collectively, this work demonstrates that increased cartilage stiffness does not necessarily equate to improved, or even adequate, tribomechanical function under biofidelic sliding conditions.

Track: Osteoarthritis, Treatments & Mechanisms
Research Area: Cartilage

4 Shu-jin Kust

In Vivo Strain Recovery Of Articular Cartilage

Shu-Jin Kust, Dana Voinier, Kyle D. Meadows, Dawn M. Elliott, Daniel K. White, and Axel C. Moore

University of Delaware

Articular cartilage lines the ends of long bones in synovial joints and functions to provide load-bearing and low-friction articulation. These biomechanical functions are largely driven by the poroelastic mechanics (fluid pressurization) of the articular cartilage. Unfortunately, the same poroelastic pressure that leads to load-bearing and lubrication also drives fluid exudation and concurrent tissue strain. In vivo static loading (e.g., standing) produces as much as -30% strain in 60 min. Interestingly, in vivo active loading (e.g., walking, knee bends, cycling, running) produces an initial period of fluid exudation and strain (~ -5%); after which, active loading arrests further fluid loss and tissue strain. While static and active loading drive fluid exudation and cartilage strain, static unloading (e.g., lying down) provides recovery, and to date, is the only in vivo mechanism that has been shown to restore hydration and strain. We hypothesize that active loading is actually a recovery mechanism that has yet to be observed.

In this study, we use magnetic resonance imaging to evaluate the in vivo strain of articular cartilage in human knees following: (1) standing, (2) lying down, and (3) walking. The results demonstrate that active loading (walking) is indeed an in vivo recovery mechanism for articular cartilage.

Track: Osteoarthritis, Treatments & Mechanisms
Research Area: Cartilage

5 Shalini Sundar

Optics-Free, In Situ Swelling Monitoring Of Articular Cartilage With Graphene Strain Sensors

Shalini Sundar, University of Delaware; Renata Linardi, University of Pennsylvania School of Veterinary Medicine; Angela Gaesser, University of Pennsylvania School of Veterinary Medicine; Tianzheng Guo, University of Delaware; Kyla Ortved, University of Pennsylvania School of Veterinary Medicine; Julie Engiles, University of Pennsylvania School of Veterinary Medicine; Justin Parreno, University of Delaware; Charles Dhong, University of Delaware

University of Delaware, University of Pennsylvania

Swelling has presented itself as a key mechanical biomarker of cartilage functionality and integrity during osteoarthritis (OA) disease progression. In healthy unloaded cartilage, the collagen network elastically restrains against internal hydrostatic and proteoglycan-associated electrostatic forces, providing the tissue with a unique swelling capacity. However, molecular reorganization events occurring during OA progression damages the extracellular matrix and thus disrupts normal swelling processes. Traditional measurement techniques used to explore these changes, such as wet weight measurements, often lack resolution and throughput. To address these limitations, we have developed a novel minimally-invasive and high throughput platform, that uses ultrasensitive thin-film graphene strain sensors, to measure the interfacial swelling mechanics of cartilage explants in situ. Through continuous temporal measurements, we demonstrate how we can specifically measure changes in explants during enzymatic and osmotic treatments. Characterization of these interfacial swelling mechanics can provide a more complete mechanical picture of OA disease progression when combined with traditionally used mechanical properties such as stiffness.

Track: Osteoarthritis, Treatments & Mechanisms
Research Area: Cartilage

 


 

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