An NIH-designated Center of Biomedical Research Excellence (COBRE)

Research Projects 2020-21

The Delaware Center for Musculoskeletal Research Projects Awards offer needed assistance to early-career investigators on the path to research independence.

Elise Corbin, PhD
Assistant Professor
Biomedical Engineering
Materials Science and Engineering
University of Delaware
Profile

Thematic area: Disease Modeling and Tissue & Regenerative Engineering

Project title: Competition between Resistance Training and Inflammation in an On-Chip Skeletal Muscle Microtissue Model of Sepsis

Elise Corbin

Summary: Chronic and acute inflammation are significant contributors to skeletal muscle pathology in multiple diseases. Severe inflammation associated with sepsis has profound short- and long-term effects on muscle. Sepsis is characterized by a dysregulated immune response to infection that can alter muscle force generation, wasting, and bioenergetics. Survivors of sepsis have increased risk for the development of persistent acquired weakness syndromes. The inflammatory response in sepsis is mediated by the release of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β). While we know that sepsis-induced changes in skeletal muscle are associated with inflammation, the mechanisms underlying muscle dysfunction in sepsis are not well understood, and there is a significant need to capture the evolution of these impairments to establish effective treatment strategies. Harnessing in vitro models of cytokine-induced myopathy in human skeletal muscle can inform and elucidate fundamental mechanisms of pathology in sepsis enabling development of effective treatments. Resistance training is a widely accepted prescriptive treatment for rebuilding muscle strength and mass. However, post-recovery resistance training has minimal long-term effects in many sepsis patients, and recent studies suggest that early (pre-recovery) physical therapy may preserve muscle fiber cross-sectional area though not strength, indicating a need for further analysis of the complex evolution of sepsis. This evidence formed the cornerstone of our hypothesis that inflammation limits the therapeutic effects of resistance training, which will be tested in a 3D in vitro organoid model.

Justin Parreno, PhD
Assistant Professor
Biological Sciences
Biomedical Engineering
University of Delaware
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Thematic area: Articular cartilage

Project title: Cytoskeletal Mechanisms that Regulate Chondrocyte Architecture and Phenotype

Justin Parreno

Summary: Osteoarthritis (OA) is an irreversible, debilitating, and chronic disease. Current OA treatments are either surgical or aimed at pain with poor long-term reparative outcomes. Thus, there is a need to develop new OA treatments. Targeting the actin cytoskeleton in chondrocytes may be a promising strategy for treatments against OA. Actin is an abundant, ubiquitously expressed protein in cells that exists as globular (G-) molecules which polymerize to form filamentous (F-) actin. Proper F-actin organization into diverse higher order structures is required for the maintenance of chondrocyte morphology which determines phenotype. Despite strong links between actin reorganization and the chondrocyte phenotype it remains unclear if targeting actin reorganization is a feasible strategy against OA. This is due in large part to two critical knowledge gaps: 1) It remains unclear how specific deregulated F-actin populations (i.e. stress fibers) can be abolished, while retaining other vital F-actin networks (i.e. cortical actin). To fill this knowledge gap, a greater understanding on the regulation of F-actin networks by actin binding proteins is needed. 2) It is unclear if F-actin reorganization occurs and plays a role in OA pathogenesis in native chondrocytes. Previous studies have determined that treatment of chondrocyte with inflammatory mediators results in reorganization of cortical F-actin networks into stress fibers. However, these studies were performed on in vitro cultured cells. It is unknown if F-actin occurs in vivo. To assess actin reorganization in cartilage, the development of new high-to-super resolution imaging methodology of chondrocytes within native cartilage is required. Our long-term goal is to enable actin-based interventions against OA.

Pilots 2020-21

The Delaware Center for Musculoskeletal Research Pilot Awards offer needed assistance to early-career investigators on the path to research independence.

Charles Dhong, PhD
Assistant Professor
Material Science & Engineering
Biomedical Engineering
University of Delaware
Profile

Thematic area: Osteoarthritis & Diagnosis

Project title: Resolving chondrocyte stiffening from matrix stiffening in osteoarthritis models

Charles Dhong

Summary: Although multiple mechanisms have been identified as a target for osteoarthritis, current therapies are ultimately not effective at reversing osteoarthritis and no treatment option exists. To provide new treatment strategies, our long-term goals are to provide a comprehensive timeline of the early stages of osteoarthritis, which has been difficult because osteoarthritis originates from closely associated changes between the collagenous extracellular matrix and chondrocytes. This pilot project will provide a reference of mechanical changes in cartilage undergoing simulated conditions which mimic early stages of osteoarthritis. Crucially, we will decouple key processes in the extracellular matrix from those originating within the chondrocytes in pre-osteoarthritis. Our approach uses in situ, highly sensitive strain sensors placed onto cartilage explants undergoing simulated osteoarthritis. In separate experiments, simulated osteoarthritis will target either extracellular matrix degradation or aberrant chondrocyte physiology. The mechanical stiffness of cartilage is a powerful biomarker of key stages of osteoarthritis because it synthesizes contributions from multiple pathways, but, by the same reason, it is historically challenging to attribute changes in stiffness to specific processes. Due to our time-resolved in situ measurements, high sensitivity, and matrix/filler analysis, we will be able to attribute mechanical stiffness primarily to the extracellular matrix or chondrocytes. Characteristic mechanical changes in the extracellular matrix will be separated from changes within chondrocytes in two aims. In the first aim, the extracellular matrix will be targeted by simulating stages of osteoarthritis which primarily degrade the extracellular matrix, including degradation from cellular processes or mechanical injury. In the second aim, characteristic mechanical changes attributable to the chondrocytes—proliferation, dedifferentiation, and inflammatory responses, will be simulated through soluble factors. While we expect crosstalk between the chondrocytes and extracellular matrix, our time-resolved measurements will help classify mechanical changes to a primary source. The significance of a more unified timeline of osteoarthritis progression may help redeploy or combine existing strategies to provide new treatments.

Christopher Price, PhD
Associate Professor
Biomedical Engineering
University of Delaware
Profile

Thematic area: Articular cartilage and bone

Project title: Chemogenetic Approaches for Promoting Chondrogenesis and Cartilage Tissue Engineering

Christopher Price

Summary: As a result of articular cartilage’s limited ability to spontaneously regenerate/heal in vivo, focus has been placed on the use of cell-based tissue-engineering/regeneration strategies to aid cartilage repair. However, ‘native-tissue-like’ repair of articular cartilage represents a major, and yet unrealized, clinical challenge, in part due to an incomplete understanding of the (mechano) biology of articular cartilage development and the presence of technical hurdles to scale-up. Nonetheless, one mechanism conclusively tied to chondrogenesis and chondrocyte physiology is intracellular calcium signaling ([Ca2+]i). Calcium is a ubiquitous second messenger crucial for regulating the proliferation, differentiation, homeostasis, and matrix metabolism of chondrocytes and their precursors (e.g., mesenchymal stem cells [MSCs]). Mature chondrocytes and MSCs exhibit robust spontaneous [Ca2+]i signaling, which have been linked to enhanced chondrogenic and increased matrix synthesis (anabolic) outcomes. [Ca2+]i activation is also the earliest, and an obligate response of chondrocytes and MSCs in their adaptation to the necessary mechanical stimulation required for articular cartilage development and health. However, controversy remains regarding the primary drivers of chondrocytes [Ca2+]i signaling. Nonetheless, mechanical loading is routinely utilized as an adjuvant to improve/accelerate cartilage tissue engineering; however, physically driving construct maturation through mechanical loading is not without limitations. Alternatively, recent studies have demonstrated the utility of non-physical manipulation of chondrocyte ‘mechanosensory’ pathways in promoting engineered cartilage development and maturation. Indeed, exogenous manipulation of [Ca2+]i through targeting native TRPV4 calcium channels enhances MSC chondrogenesis, and matrix deposition in and maturation of engineered cartilage constructs. Building upon such findings, the PI proposes the use of a novel chemogenetic strategy, the use of the engineered designer receptors exclusively activated by designer drugs [DREADDs] to: i) demonstrate the capacity for synthetic activation of [Ca2+]i-regulating Gaq- & Gas-coupled DREADD-GPCR cascades to control chondrogenesis, and chondrocyte (re)differentiation, physiology and matrix metabolism in both primary chondrocytes and their MSC precursors, and ii) establish the efficacy of synthetically driven DREADD-mediated Ga-protein signaling for enhancing the development of MSC and/or primary chondrocyte-derived engineered 3D cartilage tissues. Ultimately, this work will introduce a simple, yet transformative methodology for precisely studying the role of GPCR-driven [Ca2+]i signaling in chondrogenesis and cartilage physiology while providing a novel tool for enhancing engineered cartilage maturation, and improving cartilage tissue engineering for joint repair and regeneration.