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.
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.
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.
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.
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.
Research Area: Bone
Podium 2: Osteoarthritis, Treatments & Mechanisms
1
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.
Research Area: Cartilage
Podium 2: Osteoarthritis, Treatments & Mechanisms
2
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.
Research Area: Cartilage
Podium 2: Osteoarthritis, Treatments & Mechanisms
3
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.
Research Area: Cartilage
Podium 2: Osteoarthritis, Treatments & Mechanisms
4
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.
Research Area: Cartilage
Podium 2: Osteoarthritis, Treatments & Mechanisms
5
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.
Research Area: Cartilage
Poster 1: Cell and Tissue Studies
1
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
Research Area: Bone
Poster 2: Cell and Tissue Studies
2
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.
Research Area: Bone
Poster 1: Cell and Tissue Studies
3
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.
Research Area: Bone
Poster 2: Cell and Tissue Studies
4
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.
Research Area: Bone
Poster 1: Cell and Tissue Studies
5
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.
Research Area: Cartilage
Poster 2: Cell and Tissue Studies
6
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.
Research Area: Cartilage
Poster 1: Cell and Tissue Studies
7
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.
Research Area: Cartilage
Poster 2: Cell and Tissue Studies
8
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.
Research Area: Cartilage
Poster 1: Cell and Tissue Studies
9
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.
Research Area: Cartilage
Poster 2: Cell and Tissue Studies
10
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.
Research Area: Cartilage
Poster 1: Cell and Tissue Studies
11
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.
Research Area: Design & Innovation
Poster 2: Cell and Tissue Studies
12
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.
Research Area:
Poster 1: Cell and Tissue Studies
13
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.
Research Area: Ligament & Tendon
Poster 2: Cell and Tissue Studies
14
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.
Research Area: Ligament & Tendon
Poster 1: Cell and Tissue Studies
15
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.
Research Area: Meniscus
Poster 2: Cell and Tissue Studies
16
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.
Research Area: Muscle
Poster 1: Cell and Tissue Studies
17
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.
Research Area: Rehabilitation & Treatment
Poster 2: Cell and Tissue Studies
18
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.
Research Area: Skeletal Disorder & Treatments
Poster 1: Cell and Tissue Studies
19
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.
Research Area: Tissue Engineering
Poster 2: Cell and Tissue Studies
20
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.
Research Area: Skeletal Disorder & Treatments
Poster 1: Joint and Clinical Studies
21
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.
Research Area: Gait Analysis
Poster 2: Joint and Clinical Studies
22
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.
Research Area: Muscle
Poster 1: Joint and Clinical Studies
23
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.
Research Area: Neuromuscular Modeling & Control
Poster 2: Joint and Clinical Studies
24
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.
Research Area: Neuromuscular Modeling & Control
Poster 1: Joint and Clinical Studies
25
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.
Research Area: Rehabilitation & Treatment
Poster 2: Joint and Clinical Studies
26
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.
Research Area: Rehabilitation & Treatment
Poster 1: Joint and Clinical Studies
27
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.
Research Area: Rehabilitation & Treatment
Poster 2: Joint and Clinical Studies
28
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.
Research Area: Rehabilitation & Treatment
Poster 1:
1
Using Image Analysis to Determine the Effectiveness of ck2.1 on Subchrondral Bone Damage in Mice With Destabalized Meniscus.
Masoud Salim, Venu Pandit and Anja Nohe
Osteoarthritis is a devastating disease affecting millions of people. Treatment options are limited and usually lead to knee replacement. Our lab developed CK2.1 as a treatment option. To validate CK2.1 as a treatment DMM mice were injected with CK2.1 and processed the knee by histology, measured with Image J, and analyzed the subchondral bone region. Ck2.1 injected mice showed values similar to the Sham-operated mice. Ck2.1 injection led to a decrease in the ratio of bone volume to total volume (BV/TV) and growth plate thickness compared to PBS-injected mice. HGP-injected mice showed a reduction in growth plate thickness but not the BV/TV. CK2.1 was effective in restoring subchondral bone damage and may be a valuable option for the treatment of OA.
Research Area:
Poster 2:
2
Role of SIX1 in the Development of Cranial Bone and Suture in the Mouse
Visnu P. Chowdhury, Karyn Jourdeuil*, Sally Moody*, Andre L. P. Tavares (*Department of Anatomy & Cell Biology, The George Washington University, Washington, DC 20037)
Craniofacial anomalies affecting cranial bone and suture development constitute 1/3rd of all congenital birth defects. Branchio-oto-renal (BOR) syndrome is such a disorder that shows hearing loss, craniofacial and kidney defects. SIX1 or EYA1 mutations are found in 50% of BOR patients. SIX1 is a transcription factor, while EYA1 is its co-activator. SIX1 mutations were recently linked to craniosynostosis, causing abnormal cranial bone fusion. Previous studies showed that SIX1 function is co-factor dependent and required for craniofacial osteogenesis. Thus, we hypothesize that SIX1 regulates cranial bone development that is co-factor dependent. To test this, we are characterizing novel co-factors of SIX1, SOBP, MCRS1, and PA2G4 in mice, identified in Xenopus. Our findings indicate, while SOBP, MCRS1, and PA2G4 are expressed with SIX1 in the developing mice face, only SOBP and MCRS1 are bona fide SIX1 co-factors. We are using the Six1-KO mouse, neural crest, and pre-osteoblast cell lines to characterize Six1’s role in cranial bone development, particularly frontal and parietal bones. Mutant mouse embryos present defects in these bones and associated sutures demonstrating that SIX1 is required for proper development of these structures. Altogether, our work is generating new knowledge on SIX1’s mechanism of action in cranial bone and suture development.
Research Area:
Poster 1:
3
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, Liyun Wang
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
Research Area:
Poster 2:
4
In Situ F-Actin Bundles and Cross-Linkers in Osteocyte Dendrites
Rosa Guerra, Megan Coffin, Shannon Modla, Velia Fowler, Liyun Wang
Osteocytes, master orchestrators of bone remodeling, have extensive dendrite networks to sense external mechanical signals and communicate with other cells. With aging, the dendrites deteriorate with bone quality declined. Similar to epithelial microvilli and inner ear stereocilia, osteocyte dendrites are thought to contain rigid tightly packed cross-linked F-actin bundles as in previous mechanosensing models. However, the dendrite cytoskeleton details have not been revealed experimentally due to their mineralized bone surroundings and the high resolution needed to resolve the F-actin fibers (5-7 nm diameter). The goal of this study is to optimize protocols for high-resolution Confocal Airyscan and STED imaging of the osteocyte dendrite cytoskeleton in long bones. Decalcified neonatal mice tibia and femur cryosections (5-10 micron thickness) were stained for F-actin and selected cross-linkers with phalloidin dye and fimbrin and actinin antibodies, followed with 2-3 color STED (pixel ~16.5 nm) and confocal imaging (pixel ~100-200 nm). The cytoskeleton is more complex than earlier models predict. Fimbrin and actinin were found, but not fully colocalized, along the F-actin fibers as previously suggested. Overall, the F-actin cytoskeleton is arranged differently than the current linear actin core model. To understand osteocyte dendrites in vivo, further investigation is required.
Research Area:
Poster 1:
5
Bioluminescent Imaging and Ultrasound Technology to Develop an Orthotopic Model of Nsclc in Vivo.
Nicole Haas, MS*, Emily Geilda, MS*, Kristen Pisarcik*, Kelly Banas, Ph.D.*, Katelynn Owens, Ph.D.* *CCHS
Genome Editing is a promising tool for the correction of genetic mutation-driven human cancers. Some tumor-driving mutations present themselves as viable targets for Gene Editing with CRISPR/Cas technology. In our in vivo model of subcutaneous and orthotopic Non-Small Cell Lung Cancer (NSCLC), we used Lipid Nanoparticles (LNP) to deliver Cas9 mRNA and gRNA directly to the tumor. Bioluminescent Imaging was utilized for the screening of various LNP formulations using Firefly Luciferase mRNA. Top performing (steady and sustained signal, low biodistribution, and moderate retention) LNP’s were then formulated with Cas9 mRNA and gRNA for efficacy studies. The Visualsonic (Fujifilm) Vevo 3100 was used to precisely implant FLuc-positive NSCLC cells directly into the right lung of our mouse model. After inoculation, mice were imaged once per week for four weeks to observe growth and development of the orthotopic lung tumor. Equipment provided by the DCMR Core at the University of Delaware was vital to the collection of our data.
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6
Pss-Based Polymer for Long-Term Treatment of Osteoarthritis
Ai Nin Yang
Cartilage is identified by its fibrous collagens within a heavily hydrated extracellular matrix (ECM), typically comprising 60–80% water. Within this ECM, highly anionic glycosaminoglycan (GAG) molecules play a crucial role, providing cartilage with its ability to withstand compressive loads by retaining and organizing water. The early stages of osteoarthritis (OA) involve the loss of GAG chains, leading to cartilage breakdown. Chondroitin sulfate, widely used as a treatment for OA today, is a topic of ongoing research and discussion, with its effectiveness being somewhat controversial. Therefore, here we propose to use negatively charged synthetic polymers (polyelectrolytes), that are derived from FDA-approved poly(sodium styrene sulfonate) (PSS) to restore the fixed charge density in degraded cartilage. In this poster, I will present the synthesis of PSS-based polymer for long-term treatment and avoiding leaching out from the tissue. N-isopropylacrylamide (NIPAM) was added or grafted onto PSS backbone (PSS-block-PNIPAm or PSS-graft-PNIPAM to form polyelectrolytes that undergo a phase transition around body temperature to either form large micelles or hydrogels. The ultimate goal of this molecular design is to ensure long-term retention of this polymeric treatment for osteoarthritis.
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7
Tropomyosin 3.1 Promotes F-Actin Stability in In-Vitro Chrondrocytes
Marin Herrick, Mark Arranguez, Sofia Gonzalez-Nolde, Justin Parreno
Osteoarthritis is a debilitating disease caused, partially, by mechanical overload onto cartilage. We suspect that the mechanical overload transmission onto chondrocytes has major implications in the etiology of Osteoarthritis via activation of chondrocyte death. Recently, using a surgical mouse Osteoarthritis model that leads to cartilage overload, we revealed a reduction in chondrocyte cortical filamentous (F-) actin. We predict loss of F-actin is instrumental in promoting apoptosis, a degenerative feature of Osteoarthritis. We sought to determine the role of Tropomyosins, which stabilize F-actin, in chondrocytes during overload. We aim to test the hypothesis: Tpm3.1 promotes F-actin stability and prevents chondrocyte death. First we determined that Tpm3.1 knockout reduces F-actin in chondrocytes. Then, using isolated bovine chondrocytes, we found TPM3.1 inhibition substantially reduces F-actin in chondrocytes. Finally, we determined that mechanical overloading of chondrocytes by 30% uniaxial stretch reduces F-actin, mimicking TPM3.1 inhibition. Our results support our hypothesis that Tpm3.1 promotes F-actin stability. Further studies aim to investigate if overexpression of Tpm3.1 will prevent F-actin depolymerization and offer chondroprotection. Maintaining cortical F-actin may be critical for maintaining chondrocyte/cartilage health, and understanding the mechanisms regulating F-actin stability may lead to new therapeutic insights against Osteoarthritis.
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8
Multiscale in Situ Imaging of Collagen-Targeting Biomimetic Nanovesicles for Drug Delivery to Post-traumatic Osteoarthritic Joints
Ann Thomas, Sanjna Rao, Kristi Kiick, Christopher Price
Post-traumatic osteoarthritis (PTOA) is characterized by progressive degradation of articular cartilage. The efficacy of therapeutics proposed to modify PTOA progression are limited by poor delivery and joint retention. Work from our team has shown that biomimetic elastin-collagen nanovesicles (ECnVs) can encapsulate PTOA-modifying drugs while targeting degraded cartilage. However, the retention, distribution, and targeting of ECnVs (and their cargo) in situ has yet to be established. Leveraging multiscale visualization approaches (e.g., in vivo, epi-fluorescent, and confocal imaging) we have characterized the intra- & peri-articular localization of injected ECnVs. Far-red fluorescently-labeled ECnVs were introduced into intact tissues (ex vivo) and multiscale imaging performed to optimize injection protocols. After confirming in situ stability to tissue processing and optical clearing, labeled ECnVs were intra-articularly injected into murine joints (post-mortem). In situ and ex vivo imaging was performed on processed joints to establish ECnV robustness to optical clearing, which enabled visualization of particles deep into tissues (>500µm). Overall, these results confirm our ability to directly characterize ECnVs in situ in murine musculoskeletal tissues. Future work involving intra-articular ECnV administration to healthy and injured murine joints, coupled with longitudinal and multiscale imaging, will further establish ECnVs as an effective delivery system for targeted PTOA therapeutics.
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9
Establishment of a Surrogate Biomarker Specific to Skeletal Dysplasia
Yasuhiko Ago, Allison Bradford, Andrea Klenotiz, Shunji Tomatsu
Early and precise determination of the skeletal phenotype is essential in congenital skeletal dysplasia, including mucopolysaccharidoses (MPS). However, accurate biomarkers to predict prognosis, staging, and therapeutic efficacy of skeletal dysplasia are lacking. Recently, C-type natriuretic peptide (CNP), a growth regulatory factor, has been identified as a critical molecule in several skeletal dysplasia including achondroplasia. The analogue of CNP has been approved for this disorder. Hence, we hypothesized that the levels of CNP in patients with skeletal dysplasia are elevated in response to impaired bone growth. Through our natural history program (NIH: 5R01HD102545-02), we collected plasma samples of MPS IVA patients with systemic progressive skeletal dysplasia and measured N-terminal proCNP (NT-proCNP) levels. We found a significant elevation in patients under 16 years of age (Welch’s t-test, p = 1.9 x 10^-12). This biomarker also positively correlated with glycosaminoglycan levels (r=0.670 vs. plasma keratan sulfate (KS), 0.626 vs. urine KS, 0.636 vs. plasma di-sulfated KS). On the other hand, CNP levels in the MPS IVA mouse model with less skeletal phenotype did not show significant elevation. These preliminary data suggest that CNP is a potential surrogate biomarker in the pathophysiology of skeletal dysplasia in MPS IVA.
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10
Effects of Spontaneous Calcium Signaling on Cartilage Anabolic Activities
Ying Peng, Annie Porter, Steven DiStefano, X. Lucas Lu
Intracellular calcium, [Ca2+]i, signaling is a crucial mechano-transduction response in chondrocytes and regulates cartilage metabolism. We discovered that chondrocytes in cartilage exhibit spontaneous [Ca2+]i signaling, similar to active cells such as neurons and cardiomyocytes. A spontaneous [Ca2+]i peak is initiated by the influx of extracellular Ca2+ and can be amplified by the release of Ca2+ stored in the endoplasmic reticulum (ER). Additional Ca2+ influx may occur through the gap junctions connecting adjacent cells. The roles of spontaneous [Ca2+]i signaling in chondrocyte metabolism are not well understood. In this study, we investigate: (1) the roles of extracellular Ca2+ source, PLC-IP3 pathway, ER Ca2+ store, and gap junctions in spontaneous [Ca2+]i signaling in human cartilage, (2) the effects of these pathways on the synthesis rates of proteoglycan and collagen using a novel click chemistry technique, and 3) sex difference in spontaneous [Ca2+]i signaling, proteoglycan synthesis, and collagen synthesis in cartilage samples.
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11
Triamcinolone Acetonide Has Minimal Effect on Short and Long-Term Metbolic Activities of Cartilage
Annie Porter, Emily Newcomb, Steven DiStefano, Michael Axe, X. Lucas Lu
Corticosteroid injections are often used to control synovial joint inflammation. There is a widely held fear among clinicians, however, that steroid injections could cause degeneration of otherwise healthy cartilage and lead to premature osteoarthritis. Whether steroids are harmful to cartilage remains unclear, with controversial evidence reported in literature. This study used a click chemistry-based technique to evaluate the metabolic effects of Triamcinolone Acetonide (TA, a corticosteroid) on chondrocytes in cartilage. In juvenile bovine cartilage, a short-term (2-day) or long-term (14-day) treatment with TA did not affect chondrocyte viability, proliferation or GAG and collagen synthesis. TA at all doses reduced GAG loss from short and long-term inflammatory challenged cartilage and recovered control tissue mechanical properties. In senior human cartilage, a 14-day treatment with TA alone did not affect chondrocyte viability or collagen synthesis but did reduce GAG synthesis by ~25%. TA treatment with simultaneous inflammatory challenge also had minimal effect. In summary, when chondrocytes remain in their native matrix and are treated with physiologically relevant doses of TA, TA has minimal harmful effect on their anabolic activities in juvenile bovine or senior human cartilage. In contrast to concerns, TA protects against inflammation-induced cartilage degradation.
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12
Measuring Limb Loads Using a Novel Prosthetic Pylon Force Sensor
Hanna Armstrong
An estimated 1.4 million Americans currently live with lower extremity amputations. Despite high rates of prosthesis usage, a significant majority of amputees express dissatisfaction and experience pain while using conventional prosthetic devices. Given the considerable variability in outcomes following amputation, there’s a pressing need for interventions to enhance functional performance for prosthesis users. To address this gap, we developed a new pressure sensor system (PSS) secured at the top of the prosthetic pylon and the base of the residual limb socket. The objective of our study was to validate sensor measurements against gold standard force plate data. With 1 individual with lower limb amputation, we recorded synchronous sensor and force plate data during quiet standing, sit-to-stand, and normal treadmill walking at self-selected speed. Raw sensor data was detrended, rectified, and filtered then normalized to body weight. Results show no significant difference in peak forces in normal walking(NW) (n=12, p=0.497), difference for sit-to-stand (SS) peaks (p=0.016, 4% difference), and a moderate to strong correlation for loading curves of both activities (NW: R2 = 0.403, SS: R2 = 0.667). This preliminary data shows promise in accurate monitoring of pylon loads during walking which may have important implications for prosthetic usage.
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13
Kinetics and Enhanced Retention of Polystyrene Sulfonate Over Chondroitin Sulfate for Gag Replacement in Cartilage
Shalini Sundar, Allison Koopman, Thomas J. Manzoni, Weiran Xie, Qurat-Ul-Ain Bhatti, Chun-Yuan Lo, Vidhika S. Damani, Ai Nin Yang, Darrin Pochan, Justin Parreno, Julie B. Engiles, Laure V. Kayser, Charles Dhong
Tissue hydration provides articular cartilage with crucial viscoelastic properties. During early stages of osteoarthritis (OA), there is a loss of proteoglycans and glycosaminoglycans (GAG) from the tissue matrix, impairing tissue mechanical function. Currently, there are limited options for GAG replacements as traditionally-used chondroitin sulfate has failed to show effectiveness in pre-clinical and clinical studies, either as an oral supplement or via intra-articular injections. Here, we investigated a synthetic polyelectrolyte, poly(styrene sulfonate) (PSS), as a potential candidate to restore fixed charge density in cartilage with GAG loss. Through cartilage explant studies and histological analysis, we determined zonal-based effective diffusion coefficients for three PSS (11 kDa, 20 kDa, 65 kDa) and compared performance against that of chondroitin sulfate A (CS-A). We found that PSS was retained longer in GAG-depleted cartilage than CS-A, in static and compression-based desorption experiments. This enhanced solute performance could be explained by the improved compactness and charge density of PSS compared to CS-A, as measured by small-angle x-ray scattering, which permits better diffusion through and electrostatic binding with the cartilage matrix. This work may lead to better design and functionalization of GAG mimetic molecules for repairing osmotic function in OA cartilage or endowing osmotic pressure in synthetic matrices.
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14
Patient-Reported Surveys Are Not Sensitive to Post-stroke Changes in Biomechanics or Walking Function With Ankle Foot Orthoses
Zahra McKee, Jacob Skigen, Darcy Reisman, Elisa Arch
Passive dynamic ankle foot orthoses (PD-AFO) are used post-stroke to improve walking function and ankle-foot mechanics. Methods to evaluate AFO effectiveness require expensive equipment that cannot be implemented in a clinical setting. Instead, clinicians often use subjective observations and standardized patient surveys. We aimed to investigate whether a relationship exists between commonly used surveys, and biomechanical measures. 20 individuals with chronic stroke walked in a previously prescribed standard of care (SOC) AFO and a stiffness-customized PD-AFO for a 10-meter walk test to collect self-selected walking speed and an instrumented gait analysis to calculate peak paretic plantarflexion moment and mechanical cost of transport. Responses to the Activity-specific Balance Confidence Scale, Orthotics and Prosthetics User Survey, and Quebec User Evaluation of Satisfaction with assistive Technology 2.0 surveys were also recorded. Linear regressions were performed comparing changes between SOC and PD-AFO results for the 3 surveys and the 3 biomechanical outcomes (9 total regressions). No significant relationships were found (p=0.16-0.65), indicating that improvements in self-selected walking speed, cost of transport, or plantarflexion moment were not reflected in the survey scores. Clinicians using these surveys should not equate improvements in patient preference or experience with better walking function or biomechanics.
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15
A Matching Game: Prosthetic Knee Center Height & Gait After Above-Knee Amputation
Stauffer SJ, Sions JM
After above-knee amputation, anatomic and cosmetic concerns may necessitate differences between non-prosthetic and prosthetic knee-center height (KCH); how KCH mismatch affects gait remains unknown. This study evaluates the relationship between prosthetic KCH and gait parameters. Twenty-one individuals who underwent above-knee amputation >1 year prior [median age: 55 years, 71.4% male, 47.6% traumatic etiology,] underwent instrumented gait analysis (Mobility Lab, APDM) during the 10-Meter Walk Test at self-selected and fast walking speeds. We calculated Pearson correlations (r2) and 90% confidence intervals (CI) between KCH difference and change in gait parameters between self-selected and fast walking speed. With further distal shifts in KCH, there were trends toward greater increases in prosthetic-side cadence (r2=0.440; 90% CI: 0.073, 0.702) and sagittal lumbar range of motion (r2=0.699; 90% CI: 0.435, 0.852), and shorter prosthetic gait cycles (r2=-0.385; 90% CI: -0.667, -0.007). Results suggest individuals with lower relative prosthetic KCH increase gait speed by increasing prosthetic-side cadence, shortening the gait cycle. Increased lumbar motion may be leveraged to drive forward momentum. Further research is needed to determine if differences in gait are driven by the biomechanical advantage of a longer residual limb or by the impact of shorter knee-to-foot distance on swing phase kinematics.
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16
The Effect of Time of Day and Activity on T2 Time and Disc Height From in Vivo Disc Magnetic Resonance Imaging
Mackenzie N. Conner, Harrah R. Newman, Dawn M. Elliott
The intervertebral disc supports loads and allows spinal motion; disc degeneration is associated with low back pain. MRI is used to quantify disc degeneration through measures of disc height and T2 time [1], however, it is unknown how variable these measures are with time of day, with activity during the day, and over several months. Therefore, subjects (25-30 y.o., n=3) underwent repeated MRI 1) at multiple timepoints throughout a day, 2) before and after a 45-minute rest to unload the disc, and 3) longitudinally for 0.5- 4.5 years.
T2 relaxation time trended down across the day but this was not significantly different (118.1 ± 31.4 ms). The disc height was significantly lower at the end of the day compared to mid-day (at timepoint 2 (11am) and 4 (5pm)) There was no significant difference in the T2 relaxation before/after unloaded rest (117.6 ± 30.8 ms) or longitudinally over 0.5 to 5 years (122 ± 27.2 ms). Based on these findings, future studies do not need unloaded rest time before scanning. For future studies concerning disc geometry, scanning before noon is recommended but for studies concerning T2 time the time of day does not need to be controlled.
[1] Meadows+ JOR Spine 2023
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17
Morphometric Analysis of Menisci and Ligaments in a Rat Model
Olivia L. Dyer, Mackenzie N. Conner, Stephanie G. Cone
Identification and quantification of 3D morphometric properties in healthy rat knees is important to be able to assess and quantify changes due to injury and disease. Briefly, rat knees (n = 11) were imaged in full extension in a 9.4T Bruker Biospec 94/20 AV Neo MRI machine with a T1 FLASH 3D sequence. Images were segmented and 3D models of the four ligaments (ACL, PCL, MCL, LCL) and the medial and lateral meniscus were created. The cross-sectional area (CSA) for the ligaments, height and width along 3 locations (central, and anterior and posterior horns) and volume of the menisci were determined. The PCL had the largest average cross sectional area, while the MCL had the smallest. The widths of the posterior horn on the menisci were larger than the widths at the other two locations, while heights were consistent across all three locations. This study established a methodology to quantify 3D rat knee morphometry using high field MRI and will enable future work to better understand how anatomy changes during growth and due to musculoskeletal injuries, such as osteoarthritis.
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18
Shear Wave Tensiometry for the Assessment of Loading in the Healthy Iliotibial Band
Jack T. Felipe, Adam M. Burk, Stephanie G. Cone
Understanding force transmission in muscle-tendon-units (MTUs) is essential in studying the biomechanics of human motion. Inverse dynamics and electromyography (EMG) based models are common practice for quantifying forces generated during movement. However, inverse dynamics models operate under specific assumptions that can give inaccurate readings of force in individual MTUs, while accurate EMG readings could require the use of invasive methods. Forces in individual MTUs can be quantified through the use of shear wave tensiometry. A shear wave tensiometer is a wearable device that consists of a mechanical tapper, which taps on the skin to induce a shear wave through underlying tissues such as tendons, and two mini uniaxial accelerometers, which detect movement of the tissue caused by the shear wave. The goal of this study was to utilize a non-invasive wearable tensiometer to quantify loading on the IT band during walking, jogging, and various functional movements. In this study, we used shear wave tensiometry to measure IT band mechanics during these movements in 30 subjects. Across subjects we found similar trends in wave speed variations when looking at walking, running, and squatting. Moving forward we can use this technique to compare healthy versus injured IT band populations.
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19
Mechanosensitivity of Tendon Using a Novel in Vitro Microtissue Platform
Connor Virgile, Joohyun Lim, Elise Corbin
The elucidation of isolated cellular pathways is a complex matrix of local and systemic signals. To isolate specific pathways, there exists a need to create accurate models of specific organs and tissues. In tendon, current models are optimal for quantifying protein expression and use for tensile testing but require high cell numbers and long periods of time to contraction. Here, we hypothesize that using previously established load-bearing models, we can create a high throughput, translatable model for tendon using a modular chip platform. We find that primary tendon cells isolated from ScxGFP transgenic mice can spontaneously form 3D “tendon tissue” and undergo contraction within 3 hours and increase ScxGFP reporter activity. Furthermore, we show after just 24 hours, tendon tissues formed on relatively stiff cantilevers form highly aligned collagen fibers, in conjunction with increased amounts of ScxGFP reporter activity. Lastly, we show that modulating the stiffness of the modular chip alters biomechanical properties of the in vitro tissue, with the ability to introduce chemical dosing and genetic knockdowns to further determine in vivo translatability.
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20
Transgelin as a Regulator of Myofibroblast Differentiation in Fibrosis
Rylee King, Gracie Emin, Christian Le, Justin Parreno
Tendon fibrosis following injury leads to the formation of scar tissue that is functionally deficient and failure-prone. Fibrosis is the result of sustained activation of myofibroblasts. While myofibroblasts deposit matrix and contract to promote tendon matrix remodeling, sustained myofibroblast activation results in tissue thickening and scarring. Scaring is partly attributed to elevated alpha-smooth muscle actin, which incorporates into actin stress fibers to render cells hypercontractile.
Transgelin (Tagln), is a cross-linker of F-actin in stress fibers. During fibrosis, Tagln is upregulated, preceding the upregulation of αsma. Here, we hypothesized that Tagln is required to maintain stress fibers and incorporate αsma into stress fibers during myofibroblast differentiation.
We stimulated myofibroblast differentiation by treating tenocytes with TGFβ1 and determined that treatment increased expression of Tagln1mRNA levels. To examine the role of Tagln in differentiation, we next exposed tail tenocytes from Tagln1-/- to TGFβ1. Tagln1 knockout tenocytes still contained stress fibers with incorporated αSMA.
Thus, stress fiber formation is not entirely dependent on Tagln1. Other critical actin-binding proteins may be compensating for the loss of Tagln1. Delineating the role of actin-binding proteins during myofibroblast differentiation will provide new insight into targeting sustained myofibroblast activation during fibrosis progression.
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21
Enzymatic Activity and Structure of E. Coli’s Rna Cap Decapping Proteins: Nudc and Rpph
Yogeshwari Singh, Henry Anderson, Lingting Li, Imalka Mudiyanselage, Jared Schrader, Yu Zhang, Karl Schmitz, Jeremy G. Bird
RNA stability relies on a 5′ identity. Eukaryotes use m7G cap, while prokaryotes use metabolite caps like NAD(H). These caps, acting as non-canonical initiating nucleotides (NCINs), are incorporated during transcription initiation. NAD(H) is the most studied NCIN cap, impacting RNA stability in both prokaryotes and eukaryotes. The Nudix hydrolase family enzymes remove 5′ caps in both domains. E. coli possesses NudC, a decapping enzyme removing NAD(H) and other NCIN caps, leading to RNA degradation. With NudC showing evolutionary conservation, I hypothesize that NudC serves as a universal decapping enzyme in E. coli, exhibiting varied specificities for distinct cap moieties. In vitro transcription (IVT) assays, which I helped develop in Bird Lab, were conducted, generating RNAs with diverse NCIN caps. Radiolabeled nucleotides labeled the RNA, followed by NudC incubation are run on TBE-Urea gel which separates RNA based on length. Radiolabeled RNA was visualized and analyzed using a Saphire Phosphorous screen reader. Structural studies on E. coli NudC were carried out through X-ray Crystallography to comprehend the molecular mechanism of NudC-dependent bacterial RNA-decapping reaction. NudC displays differing activities for specific capped RNA, exhibiting superior decapping activity for NADH-capped RNAs compared to NAD+ capped RNAs, and for longer RNA over short-length RNA. Structural data shows that NudC active site can accommodate more than just the cap moiety.
The addition of NCIN caps by RNAP on specific RNAs and the existence of enzymes responsible for removing these caps suggests that NCIN-capping is a highly regulated process that affects the transcriptome of the cell under different growth phases or conditions. This research and its result can help us understand RNA regulation and gene expression, which are fundamental processes used by all branches of life.
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22
Motor Deficiencies and Altered Social Behavior in Mice With Deletion of Nak-Atpase B1-Subunit in Cerebellar Granule Cells
Mikail Rasheed, Mohamed Khalife, Aysegul Cort Donmez, Karen Sperle, Zhiqin Li, Elmira Tokhtaeva, Nils W. Lambrecht, Xuyi Yue, Amanda E. Hernan, John Peloquin, Olga Vagin, and Sigrid A. Langhans
Dysregulated function of Na,K-ATPase α-subunit has been implicated in cerebellum-associated neurological disorders. Besides pumping, it has pump-independent roles, acting as signaling scaffolds, particularly its α- and β-subunits. β-subunits, (β1 and β2 isoforms), also serve as cell adhesion molecules. Recent research has focused on unraveling the physiological significance of the pump-independent functions of Na,K-ATPase, particularly those attributed to its β-subunit. We aim to elucidate motor deficiencies and altered social behavior in mice with deletion of Na,K-ATPase b1-subunit in cerebellar granule cells.
We selectively deleted the β1-subunit in cerebellar granule cells in mice. This led to complex neurological deficits, where Heterozygous KO mice showed learning/memory and social interaction deficiencies compared to littermates. Homozygous KO mice exhibited age-worsened cerebellar ataxia symptoms and a shorter lifespan. Interestingly, phenotypes in β1-subunit-deleted mice differed significantly from β2-subunit deletions, highlighting a unique role of β1-isoform in cerebellar granule cells and cerebellum development.
We further characterized morphological changes in adult mice lacking β1-subunit using various techniques. While histology and MRI analysis didn’t show major structural differences, ex vivo MRI revealed a reduction in cerebellar volume in homozygous KO mice compared to the littermates. Studies aimed at identifying molecular and electrophysiological changes underlying the deficiencies in mice with β1-subunit deletion in cerebellar granule cells are ongoing.
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23
Controlled Release of Amphiregulin From Alginate Hydrogels
Brennagh Shields, Lauren Mottel, Lindsay Gallagher, Brian Kwee
Ischemia, the loss of blood flow, as a result of injury or disease affects millions of people per year. Ischemia in skeletal muscle, in particular, can lead to muscle degeneration, muscle denervation, and tissue necrosis. Amphiregulin (AREG), a cytokine that can stimulate the growth of muscle stem cells, blood vessel cells, and motor neurons, may be a potent therapeutic for treating ischemic muscle injuries. Furthermore, AREG can activate Tregs, which are anti-inflammatory immune cells that can also promote muscle regeneration. As a treatment of ischemic muscle injuries, we engineered an injectable, oxidized (degradable by hydrolysis), calcium crosslinked alginate hydrogel that provides sustained release of AREG. Incorporation of AREG into these alginate hydrogels resulted in a moderate burst release followed by a sustained release over 14 days, which was enhanced by reducing the calcium crosslinking concentration of the hydrogel. The addition of charged laponite nanodiscs into the hydrogel reduced the burst release and rate of release of AREG over 10 days. Ongoing work focuses on evaluating the ability of the AREG-releasing hydrogels to promote vascularization, innervation and muscle repair in the ischemic muscles of mice with severe hindlimb ischemia.
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1
The Evaluation of Tissue Specific and Ubiquitous Promoters Under Lentiviral Vectors in Genetically Modified Hsc-Transplanted Mps Iva Mice
Betul Celik, Shaukat Khan, Shunji Tomatsu
Mucopolysaccharidosis IVA (MPS IVA) is caused by a mutation in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene. GALNS enzyme deficiency contributes to glycosaminoglycans (keratan sulfate, KS, and chondroitin-6-sulfate) accumulation in multiple tissues, including bone, cartilage, and heart valves, resulting in progressive skeletal dysplasia. There is no effective treatment for this skeletal disease. We hypothesized that the tissue specific promoter (COL2A1) driving GALNS gene under lentiviral vectors (LVs) produces the continuous supraphysiological level of GALNS enzyme through hematopoietic stem cells (HSCs) better than ubiquitous promoter (CBh), impacting bone abnormalities in MPS IVA. Busulfan conditioned newborn knockout recipients were transplanted with genetically modified bone marrow HSCs transduced with CBh- or COL2A1-hGALNS LVs. Transplanted mice were autopsied at 16 weeks and all tissues were processed for further analysis. The results indicated that COL2A1-hGALNS LVs reduced KS GAG accumulation in related tissues with a relatively low GALNS enzyme expression. Histo-pathological analysis demonstrated the GALNS expression in liver and tibia, and a clear normalization in chondrocyte size in the growth plate of tibia and femur. Furthermore, both vectors achieved complete correction of heart lesions. Based on the notable therapeutic efficacy, we conclude that ex vivo LV gene therapy is a premise for treating MPS IVA.
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2
Skeletal Effects of YODA1-Augmented Loading in Mice With Breast Cancer Bone Metastasis Treated With Doxorubicin
Murtaza Wasi, Rosa Guerra, Tiankuo Chu, Rory Kooker, Maya Collado, Samantha Izquierdo, Xuehua Li, Jinhu Xiong, Liyun Wang
Breast cancer and chemotherapeutic agents directly affect bone cells and marrow, inducing bone loss. Using a piezo1 channel agonist, Yoda1, we previously showed that bone structure was improved, and bone formation increased in mice when used alone or combined with tibial loading. The hypothesis for this study was that Yoda1-augmented loading mitigates bone loss in mice with breast cancer bone metastasis after chemotherapy. Middle aged mice (55 weeks) received 1000 Py8119 breast cancer cells followed by Doxorubicin (DOX, 5 mg/kg, 3 times) intraperitoneal injections. After one week of recovery, Yoda1 injection and unilateral tibial loading (4.5N peak load, 4 Hz, 600 cycles for 5 days/week) was performed for two weeks. Sequential in vivo microCT scans were performed to track bone changes. Additional assessments (i.e., mechanical testing, histology, and flow cytometry) were performed post-sacrifice. DOX’s toxic effect was the main driving force behind the bone loss found in our study. Loading alone did not have a beneficial effect on bone cortex, while Yoda1 alone slightly reduced the decline of the cortical polar moment of inertia (Ct.pMOI) in DOX treated mice. Yoda1-augmented loading, despite resulting in the most improvement in Ct.pMOI, could not counter the effects of DOX.
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2
Mechanical and Structural Response Following Increased Tendon Loading in Rodent Model
Lily M. Lin, Rita C. Marqueti, John M. Peloquin, Hailey Bonelli, Stephanie Cone, Justin Parreno, Karin G. Silbernagel, Dawn M. Elliott
Loading is necessary for maintaining tendon homeostasis: moderate load increases lead to adaptation, whereas overload leads to degeneration. The threshold from adaptation to degeneration is unknown, therefore, animal models are needed. The objective of this study was to evaluate tendon structure and function following overload in juvenile rats using synergistic ablation (SynAb), where the Achilles tendon is resected to overload plantaris tendon.
Methods: Juvenile female Long Evans rats were assigned to three treatment groups: SynAb, Sham, or Intact. Activity levels were evaluated pre- and post-surgery. 8-weeks post-surgery, plantaris tendons were evaluated for tensile failure and fatigue mechanics, 3D tissue structure with MRI, and cellularity and fiber structure with histology.
Results and Discussion: Activity levels were not affected by treatment. The SynAb group exhibited increases in cross-sectional area, stiffness, and ultimate load compared to Intact and Sham. Fatigue testing revealed increased resistance to failure in the SynAb, indicating an adaptive response to overload. Histology displayed elevated cell density in the SynAb, and MRI revealed scar tissue formation adjacent to the plantaris, impacting load distribution and remodeling.
This study demonstrates an adaptive mechanical response of tendons to increased loading in juvenile rats, contrasting previous findings of degenerative outcomes in older rats.
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4
A Click Chemistry Method to Measure Glycosaminoglycan Composition and Metabolics in Articular Cartilage
Annie Porter, Steven DiStefano, Emily Newcomb, Michael Axe, X. Lucas Lu
A primary component of articular cartilage is aggrecan, a proteoglycan composed of glycosaminoglycan (GAG) chains attached along a protein core. GAG chain composition influences cartilage mechanical properties and osteoarthritis development. The dominant GAG chains are chondroitin sulfate, keratan sulfate, and hyaluronan (CS, KS, HA). In this study, we used a novel technique to separately quantify the synthesis and degradation of new GAG chains in cartilage. Human and bovine cartilage samples were cultured in media containing azide-modified monosaccharide building blocks of GAG chains, either N-azidoacetyl-galactosamine-tetraacylated (GalNAz) or N-azidoacetylglucosamine-tetraacylated (GlcNAz). Chondrocytes incorporate GalNAz into CS chains and GlcNAz into KS&HA chains. A fluorescent dye was “clicked” onto the labeled GAGs in a copper-free click chemistry reaction. The total amount of GAG (CS+KS&HA) did not vary between anatomic regions of the bovine knee, but the ratio of GAG types was lower in the medial condyle compared to other regions. Inflammation decreased both CS and KS&HA synthesis in bovine cartilage, but only CS synthesis in human cartilage. In both tissues, inflammation induced loss of CS and KS&HA chains, however there was greater loss of CS. In summary, the click chemistry method can quantify the metabolism of the different GAG chains in cartilage.
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1
Chondrocyte-Derived Decellularized Matrices Support the Expansion of Superficial Zone Chondrocytes
Thomas Manzoni, Anh Ho, Lilly Smull, Anne Sheldrake, Travis Block, Alvin W. Su, Justin Parreno
The superficial zone and superficial zone chondrocytes (SZC) play a large role in establishing articular cartilage mechanical properties. SZC produces the lubricating molecule, Proteoglycan-4, which is critical in providing a frictionless environment. When damaged, cartilage is incapable of self-repair. Passaged chondrocytes, harvested from full-thickness cartilage, are an FDA approved cell source for cartilage repair. However, we determined that the cell expansion process on polystyrene (PS) limits the number SZC after passaging due to poor attachment and slow proliferation. Also, SZC in culture undergo dedifferentiation, causing a loss Proteoglycan-4 expression. Previous studies have shown that culturing full-thickness chondrocytes on a chondrocyte derived-decellularized extracellular matrix (CD-ECM) improves proliferation and reduces dedifferentiation. Here, we hypothesize that passaging SZC on CD-ECM will enhance cellular attachment, proliferation, and repress dedifferentiation. Primary SZC were seeded on PS or CD-ECM. We examined cell attachment, expansion rate, and gene expression throughout the passaging process. Seeding SZC on CD-ECM enhances cell attachment and expansion rate. Passaging SZC on CD-ECM reduces chondrogenic mRNA levels similar to PS and represses mRNA levels for dedifferentiated genes. Overall, passaging SZC on CD-ECM may help in achieving an adequate cell number of SZC for bioengineering purposes.
Research Area:
Podium 1:
3
Optimizing the Uptake of Chondrocyte Penetrating Peptide ck2.1 for the Treatment of Osteoarthrtis
Venu Pandit, Anja Nohe
Peptide drugs are promising candidates for targeted treatment without the danger of immunogenicity and toxicity associated with monoclonal antibodies and small-molecule drugs. Osteoarthritis is a debilitating condition with an ever-increasing incidence rate. A synthetic peptide drug developed at our lab, CK2.1, reversed the symptoms of surgically induced Osteoarthritis in a mouse model. The drug was delivered by local systemic injections of CK2.1 conjugated to hydrogel particles (CK.21-HGP) for sustained release. We investigate the mechanism of uptake of CK2.1 in the hyaline cartilage extracellular matrix and the chondrocytes. We used the paraffin-embedded tissue section of the knee cavity where the OA was induced, followed by local injection of CK2.1-HGP. With the immunofluorescence technique, we show that the peptide CK2.1 accumulates in the superficial and middle zones of the hyaline cartilage. There is an increase in the expression of chondrogenic markers SOX9 and Type IX collagen. Additionally, the chondrocyte hypertrophy markers were not upregulated compared to mice injected with HGP alone. Intracellular localization of the peptide is seen to be within the cytoplasm. Finally, with the blockage of endocytosis by clathrin-coated pits or caveolae or both, there is a reduction in the uptake of CK2.1 in C3H10T1/2 cells.
Research Area:
Podium 2:
3
Chondrocyte Volume Regulation and Proteoglycan Remodeling Under Inflammatory Challenge
Ying Peng, Julie Nguyen, Shuo Wei, X. Lucas Lu
Osteoarthritis (OA) is the most prevalent joint disease and a leading cause of chronic pain and disability in the United States. Elevated concentrations of pro-inflammatory cytokines, such as interleukin-1 (IL-1), in the joint cavity disrupt the metabolic balance of cartilage and play a significant role in OA pathogenesis. Chondrocytes are the exclusive cell type in healthy cartilage. The volume regulation of chondrocytes is one of the cellular responses to OA progression. Proteoglycans are the second most abundant component within the cartilage matrix, consisting of glycosaminoglycans (GAGs). The homeostasis of GAGs is important to maintain healthy cartilage. However, little is known about the volume regulation and GAG homeostasis of in situ chondrocytes under inflammatory challenge. The present study quantified chondrocyte volume regulation and nascent GAG turnover in response to inflammatory stimulation at days 2, 7, and 14 using custom-developed image processing software. Furthermore, the correlation between cell volume and GAG metabolism was analyzed. RNA sequencing analysis was conducted to investigate the transcriptional level changes in cells associated with cell volume regulation and GAG homeostasis.
Research Area:
Podium 2:
4
Synovial Fluid Constituents Are Not Unique in Their Ability to Drive Articular Cartilage Super-Lubricity
Emily P Lambeth, Brooklyn Tyndall, David L Burris, Christopher Price
Articular cartilage is a biological bearing material that easily sustains friction coefficients (μ)<0.004. This lubricity has historically been attributed to cartilage’s structure and/or its bathing (synovial) fluid composition. Our recent work indicates that hyaluronic acid (HA), in isolation, can sustain biofidelic cartilage μ (~0.004) under physiological sliding conditions on the benchtop (in the cSCA configuration); findings that present multiple firsts-of-their-kind. However, whether these biofidelic μ are due to unique biologic or generalizable lubricant properties remains unclear. Therefore, osteochondral explants underwent tribomechanical characterization in multiple putative lubricant formulations, including those of synovial fluid constituents (HA, phospholipids, gamma-globulins [IgG], & albumin) and non-physiological lubricants exhibiting comparable structural and/or rheologic properties to HA (polyethylene oxide [PEO], dextran, glycerol, & mucin). At biologic concentrations, the only synovial fluid constituent individually capable of replicating biofidelic cSCA μ was HA (μ = 0.004). Unexpectedly, PEO, mucin, dextran, glycerol, & IgGs drove concentration/molecular weight-dependent reductions in μ, culminating in μ approaching/exceeding super-lubricity (and >5-fold reductions compared to PBS). Intriguingly, this work suggests that cartilage’s remarkable ‘super lubricity’ may not be unique to its natural bathing solution but can be replicated using “non-natural” lubricants. These first-of-their kind observations provide new insights into possible mechanisms for cartilage’s phenomenal lubricity.
Research Area:
Poster 2:
24
Development of Noncontractile Bioengineered Cartilage Neo-Tissues Using Passaged Chondrocytes
Stephanie Richardson-Solorzano, Justin Parreno
Damage to articular cartilage leads to Osteoarthritis(OA) progression. In OA, inflammatory mediator signaling alters cartilage tissue homeostasis causing cartilage degradation. We aim to develop a new model that can mimic native cartilage to study the effect of inflammatory molecules on OA progression using monolayer expanded chondrocytes in three-dimensional(3D) culture. Unfortunately, monolayer expansion (passaging) results in dedifferentiation whereby chondrocytes lose cartilage matrix expression. We have developed methodology to redifferentiate passaged bovine chondrocytes and aim to adapt our methodology for redifferentiation of human passaged chondrocytes. We aim to test our hypothesis that: the treatment of human cartilage-like bioengineered tissue by passaged human chondrocytes with inflammatory mediators will mimic aspects of OA. We determined that the formation of stable human 3D bioengineered tissue requires repression of passaged chondrocyte contractility in combination with stimulation of matrix deposition using TGFβ3. After 20 days stable cartilage was formed which expressed cartilage matrix, Exposure of bioengineered tissue to IL-1β increased degrative enzymes and decreasing chondrogenic factors. Our findings suggest that the generation of cartilage-like bioengineered tissue will serve as a model to mimic key aspects of OA progression .
