Browsing by Author "McNulty, Amy L"
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Item Open Access CXCL10 is Upregulated in Synovium and Cartilage following Articular Fracture.(J Orthop Res, 2017-09-14) Furman, Bridgette D; Kent, Collin L; Huebner, Janet L; Kraus, Virginia B; McNulty, Amy L; Guilak, Farshid; Olson, Steven AThe objective of this study was to investigate the expression of the chemokine CXCL10 and its role in joint tissues following articular fracture. We hypothesized that CXCL10 is upregulated following articular fracture and contributes to cartilage degradation associated with post-traumatic arthritis (PTA). To evaluate CXCL10 expression following articular fracture, gene expression was quantified in synovial tissue from knee joints of C57BL/6 mice that develop PTA following articular fracture, and MRL/MpJ mice that are protected from PTA. CXCL10 protein expression was assessed in human cartilage in normal, osteoarthritic (OA), and post-traumatic tissue using immunohistochemistry. The effects of exogenous CXCL10, alone and in combination with IL-1, on porcine cartilage explants were assessed by quantifying the release of catabolic mediators. Synovial tissue gene expression of CXCL10 was upregulated by joint trauma, peaking one day in C57BL/6 mice (25-fold) vs. three days post-fracture in MRL/MpJ mice (15-fold). CXCL10 protein in articular cartilage was most highly expressed following trauma compared with normal and OA tissue. In a dose dependent manner, exogenous CXCL10 significantly reduced total matrix metalloproteinase (MMP) and aggrecanase activity of culture media from cartilage explants. CXCL10 also trended toward a reduction in IL-1α-stimulated total MMP activity (p=0.09) and S-GAG (p=0.09), but not NO release. In conclusion, CXCL10 was upregulated in synovium and chondrocytes following trauma. However, exogenous CXCL10 did not induce a catabolic response in cartilage. CXCL10 may play a role in modulating the chondrocyte response to inflammatory stimuli associated with joint injury and the progression of PTA. This article is protected by copyright. All rights reserved.Item Open Access Immune cell profiles in synovial fluid after anterior cruciate ligament and meniscus injuries.(Arthritis research & therapy, 2021-11) Kim-Wang, Sophia Y; Holt, Abigail G; McGowan, Alyssa M; Danyluk, Stephanie T; Goode, Adam P; Lau, Brian C; Toth, Alison P; Wittstein, Jocelyn R; DeFrate, Louis E; Yi, John S; McNulty, Amy LBackground
Anterior cruciate ligament (ACL) and meniscus tears are common knee injuries. Despite the high rate of post-traumatic osteoarthritis (PTOA) following these injuries, the contributing factors remain unclear. In this study, we characterized the immune cell profiles of normal and injured joints at the time of ACL and meniscal surgeries.Methods
Twenty-nine patients (14 meniscus-injured and 15 ACL-injured) undergoing ACL and/or meniscus surgery but with a normal contralateral knee were recruited. During surgery, synovial fluid was aspirated from both normal and injured knees. Synovial fluid cells were pelleted, washed, and stained with an antibody cocktail consisting of fluorescent antibodies for cell surface proteins. Analysis of immune cells in the synovial fluid was performed by polychromatic flow cytometry. A broad spectrum immune cell panel was used in the first 10 subjects. Based on these results, a T cell-specific panel was used in the subsequent 19 subjects.Results
Using the broad spectrum immune cell panel, we detected significantly more total viable cells and CD3 T cells in the injured compared to the paired normal knees. In addition, there were significantly more injured knees with T cells above a 500-cell threshold. Within the injured knees, CD4 and CD8 T cells were able to be differentiated into subsets. The frequency of total CD4 T cells was significantly different among injury types, but no statistical differences were detected among CD4 and CD8 T cell subsets by injury type.Conclusions
Our findings provide foundational data showing that ACL and meniscus injuries induce an immune cell-rich microenvironment that consists primarily of T cells with multiple T helper phenotypes. Future studies investigating the relationship between immune cells and joint degeneration may provide an enhanced understanding of the pathophysiology of PTOA following joint injury.Item Open Access In vivo cartilage strain increases following medial meniscal tear and correlates with synovial fluid matrix metalloproteinase activity(JOURNAL OF BIOMECHANICS, 2015-06-01) Carter, Teralyn E; Taylor, Kevin A; Spritzer, Charles E; Utturkar, Gangadhar M; Taylor, Dean C; Moorman, Claude T; Garrett, William E; Guilak, Farshid; McNulty, Amy L; DeFrate, Louis EItem Open Access Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis.(Proceedings of the National Academy of Sciences of the United States of America, 2021-03) Lee, Whasil; Nims, Robert J; Savadipour, Alireza; Zhang, Qiaojuan; Leddy, Holly A; Liu, Fang; McNulty, Amy L; Chen, Yong; Guilak, Farshid; Liedtke, Wolfgang BOsteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.Item Open Access Mechanobiology of the meniscus.(J Biomech, 2015-06-01) McNulty, Amy L; Guilak, FarshidThe meniscus plays a critical biomechanical role in the knee, providing load support, joint stability, and congruity. Importantly, growing evidence indicates that the mechanobiologic response of meniscal cells plays a critical role in the physiologic, pathologic, and repair responses of the meniscus. Here we review experimental and theoretical studies that have begun to directly measure the biomechanical effects of joint loading on the meniscus under physiologic and pathologic conditions, showing that the menisci are exposed to high contact stresses, resulting in a complex and nonuniform stress-strain environment within the tissue. By combining microscale measurements of the mechanical properties of meniscal cells and their pericellular and extracellular matrix regions, theoretical and experimental models indicate that the cells in the meniscus are exposed to a complex and inhomogeneous environment of stress, strain, fluid pressure, fluid flow, and a variety of physicochemical factors. Studies across a range of culture systems from isolated cells to tissues have revealed that the biological response of meniscal cells is directly influenced by physical factors, such as tension, compression, and hydrostatic pressure. In addition, these studies have provided new insights into the mechanotransduction mechanisms by which physical signals are converted into metabolic or pro/anti-inflammatory responses. Taken together, these in vivo and in vitro studies show that mechanical factors play an important role in the health, degeneration, and regeneration of the meniscus. A more thorough understanding of the mechanobiologic responses of the meniscus will hopefully lead to therapeutic approaches to prevent degeneration and enhance repair of the meniscus.Item Open Access Meniscus-Derived Matrix Bioscaffolds: Effects of Concentration and Cross-Linking on Meniscus Cellular Responses and Tissue Repair.(International journal of molecular sciences, 2019-12-19) Lyons, Lucas P; Hidalgo Perea, Sofia; Weinberg, J Brice; Wittstein, Jocelyn R; McNulty, Amy LMeniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing.Item Open Access Meniscus-Derived Matrix Scaffolds Promote the Integrative Repair of Meniscal Defects.(Scientific reports, 2019-06-18) Ruprecht, Jacob C; Waanders, Taylor D; Rowland, Christopher R; Nishimuta, James F; Glass, Katherine A; Stencel, Jennifer; DeFrate, Louis E; Guilak, Farshid; Weinberg, J Brice; McNulty, Amy LMeniscal tears have a poor healing capacity, and damage to the meniscus is associated with significant pain, disability, and progressive degenerative changes in the knee joint that lead to osteoarthritis. Therefore, strategies to promote meniscus repair and improve meniscus function are needed. The objective of this study was to generate porcine meniscus-derived matrix (MDM) scaffolds and test their effectiveness in promoting meniscus repair via migration of endogenous meniscus cells from the surrounding meniscus or exogenously seeded human bone marrow-derived mesenchymal stem cells (MSCs). Both endogenous meniscal cells and MSCs infiltrated the MDM scaffolds. In the absence of exogenous cells, the 8% MDM scaffolds promoted the integrative repair of an in vitro meniscal defect. Dehydrothermal crosslinking and concentration of the MDM influenced the biochemical content and shear strength of repair, demonstrating that the MDM can be tailored to promote tissue repair. These findings indicate that native meniscus cells can enhance meniscus healing if a scaffold is provided that promotes cellular infiltration and tissue growth. The high affinity of cells for the MDM and the ability to remodel the scaffold reveals the potential of MDM to integrate with native meniscal tissue to promote long-term repair without necessarily requiring exogenous cells.Item Open Access Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.(Proc Natl Acad Sci U S A, 2014-11-25) Lee, Whasil; Leddy, Holly A; Chen, Yong; Lee, Suk Hee; Zelenski, Nicole A; McNulty, Amy L; Wu, Jason; Beicker, Kellie N; Coles, Jeffrey; Zauscher, Stefan; Grandl, Jörg; Sachs, Frederick; Guilak, Farshid; Liedtke, Wolfgang BDiarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca(2+) signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca(2+) transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.Item Open Access Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.(Pain, 2013-08) Chen, Yong; Williams, Susan H; McNulty, Amy L; Hong, Ji Hee; Lee, Suk Hee; Rothfusz, Nicole E; Parekh, Puja K; Moore, Carlene; Gereau, Robert W; Taylor, Andrea B; Wang, Fan; Guilak, Farshid; Liedtke, WolfgangTemporomandibular joint disorder (TMJD) is known for its mastication-associated pain. TMJD is medically relevant because of its prevalence, severity, chronicity, the therapy-refractoriness of its pain, and its largely elusive pathogenesis. Against this background, we sought to investigate the pathogenetic contributions of the calcium-permeable TRPV4 ion channel, robustly expressed in the trigeminal ganglion sensory neurons, to TMJ inflammation and pain behavior. We demonstrate here that TRPV4 is critical for TMJ-inflammation-evoked pain behavior in mice and that trigeminal ganglion pronociceptive changes are TRPV4-dependent. As a quantitative metric, bite force was recorded as evidence of masticatory sensitization, in keeping with human translational studies. In Trpv4(-/-) mice with TMJ inflammation, attenuation of bite force was significantly less than in wildtype (WT) mice. Similar effects were seen with systemic application of a specific TRPV4 inhibitor. TMJ inflammation and mandibular bony changes were apparent after injections of complete Freund adjuvant but were remarkably independent of the Trpv4 genotype. It was intriguing that, as a result of TMJ inflammation, WT mice exhibited significant upregulation of TRPV4 and phosphorylated extracellular-signal-regulated kinase (ERK) in TMJ-innervating trigeminal sensory neurons, which were absent in Trpv4(-/-) mice. Mice with genetically-impaired MEK/ERK phosphorylation in neurons showed resistance to reduction of bite force similar to that of Trpv4(-/-) mice. Thus, TRPV4 is necessary for masticatory sensitization in TMJ inflammation and probably functions upstream of MEK/ERK phosphorylation in trigeminal ganglion sensory neurons in vivo. TRPV4 therefore represents a novel pronociceptive target in TMJ inflammation and should be considered a target of interest in human TMJD.Item Open Access The Meniscus Cell Phenotype: Effects of Physical, Mechanical, and Inflammatory Environments(2022) Andress, BenjaminThe meniscus of the knee is a fibrocartilaginous structure essential to the biomechanical integrity and function of the knee joint. Millions of people suffer meniscus injuries each year, and meniscus tears are common at all ages and stages of life. Meniscus injury has long-term consequences: loss of meniscus function has been definitively linked to early-onset osteoarthritis. Treatment options for meniscus injury remain limited; although there has been a recent movement to surgically repair the meniscus whenever possible, partial meniscectomy remains one of the most commonly performed orthopedic surgeries. Due to this urgent need, there is currently great interest in methods to stimulate meniscus healing, augment repair, and improve long-term outcomes following meniscus injury using pharmaceutical, biological, or tissue engineering methods. Research on meniscus regenerative medicine, however, is greatly limited by a lack of understanding of meniscus cellular biology.In this dissertation, this gap in knowledge is addressed with a thorough characterization of the meniscus cell phenotype. We have investigated the phenotypic identity of meniscus cells from inner and outer zones of the meniscus by RNA-sequencing, and provide comparisons to articular cartilage and isolated, monolayer cultured meniscus cells. We found that in situ meniscus cells from both the inner and outer zones are strikingly distinct from either articular chondrocytes or monolayer expanded meniscus cells at the transcriptomic level, and that inner and outer zone meniscus cells may be more similar to each other than to chondrocytes or monolayer cultured cells. Differences were also observed between inner and outer zone cells, and this dataset provides a wealth of novel targets for characterizing inner and outer zone cells to better understand regional cell biology of the meniscus. We investigated the role of the physical microenvironment, including native extracellular matrix, monolayer culture, and biomaterial hydrogels, to modulate the meniscus cell transcriptional phenotype and support meniscus cell culture and expansion in vitro. Our findings provide new details on the meniscus cell dedifferentiation process, and demonstrate the utility of bioengineered hydrogels to reverse meniscus cell dedifferentiation for long-term in vitro culture. We also investigated the effect of an injury-relevant inflammatory stimulus (IL-1), and the potential for dynamic mechanical loading to modulate the inflammatory response of meniscus cells in two models of dynamic physiologic loading, cell stretch of isolated meniscus cells and compression of tissue explants. Results of RNA-sequencing, gene set enrichment analysis, and RT-qPCR from both models showed significant modulation of inflammation-related genes and pathways with mechanical stimulation, supporting the potential of mechanotransduction pathways as novel therapeutic targets to improve outcomes following meniscus injury. Overall, this work provides a wealth of data characterizing the meniscus cell phenotype and lays the groundwork for future studies of meniscus regenerative medicine and tissue engineering. Furthermore, this work entailed considerable development and validation of methods for in vitro studies of meniscus cell biology and mechanotransduction, which will be valuable to the field of meniscus research. The work presented in this dissertation represents an enormous step forward in understanding the effects of physical, mechanical, and inflammatory environments on the meniscus cell phenotype, which is essential to the development of effective novel therapies to stimulate meniscus repair and prevent PTOA.