Scholarly Work - Mechanical & Industrial Engineering
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Item Three Decades of Advancements in Osteoarthritis Research: Insights from Transcriptomic, Proteomic, and Metabolomic Studies(Elsevier BV, 2023-12) Farooq Rai, Muhammad; Collins, Kelsey H.; Lang, Annemarie; Maerz, Tristan; Geurts, Jeroen; Ruiz-Romero, Cristina; June, Ronald K.; Ramos, Yolande; Rice, Sarah J.; Ali, Shabana Amanda; Pastrello, Chiara; Jurisica, Igor; Appleton, C. Thomas; Rockel, Jason S.; Kapoor, MohitObjective. Osteoarthritis (OA) is a complex disease involving contributions from both local joint tissues and systemic sources. Patient characteristics, encompassing sociodemographic and clinical variables, are intricately linked with OA rendering its understanding challenging. Technological advancements have allowed for a comprehensive analysis of transcripts, proteomes and metabolomes in OA tissues/fluids through omic analyses. The objective of this review is to highlight the advancements achieved by omic studies in enhancing our understanding of OA pathogenesis over the last three decades. Design. We conducted an extensive literature search focusing on transcriptomics, proteomics and metabolomics within the context of OA. Specifically, we explore how these technologies have identified individual transcripts, proteins, and metabolites, as well as distinctive endotype signatures from various body tissues or fluids of OA patients, including insights at the single-cell level, to advance our understanding of this highly complex disease. Results. Omic studies reveal the description of numerous individual molecules and molecular patterns within OA-associated tissues and fluids. This includes the identification of specific cell (sub)types and associated pathways that contribute to disease mechanisms. However, there remains a necessity to further advance these technologies to delineate the spatial organization of cellular subtypes and molecular patterns within OA-afflicted tissues. Conclusions. Leveraging a multi-omics approach that integrates datasets from diverse molecular detection technologies, combined with patients’ clinical and sociodemographic features, and molecular and regulatory networks, holds promise for identifying unique patient endophenotypes. This holistic approach can illuminate the heterogeneity among OA patients and, in turn, facilitate the development of tailored therapeutic interventions.Item Germ‐Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance(Wiley, 2023-08) Vahidi, Ghazal; Moody, Maya; Welhave, Hope D.; Davidson, Leah; Rezaee, Taraneh; Behzad, Ramina; Karim, Lamya; Roggenbeck, Barbara A.; Walk, Seth T.; Martin, Stephen A.; June, Ronald K.; Heveran, Chelsea M.The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ-free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20- to 21-week-old) C57BL/6J GF and conventionally raised female and male mice (n = 6–10/group). Trabecular microarchitecture and cortical geometry were measured from micro–CT of the femur distal metaphysis and cortical midshaft. Whole-femur strength and estimated material properties were measured using three-point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back-scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole-bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).Item Metabolomic Profiling to Understand Chondrocyte Metabolism(Springer Nature, 2022-11) Brahmachary, Priyanka; Welhaven, Hope D.; June, Ronald K.Metabolism has long been recognized as a critical physiological process necessary to maintain homeostasis in all types of cells including the chondrocytes of articular cartilage. Alterations in metabolism in disease and metabolic adaptation to physiological stimuli such as mechanical loading are increasingly recognized as important for understanding musculoskeletal systems such as synovial joints. Metabolomics is an emerging technique that allows quantitative measurement of thousands of small molecule metabolites that serve as both products and reactants to myriad reactions of cellular biochemistry. This protocol describes procedures to perform metabolomic profiling on chondrocytes and other tissues and fluids within the synovial joint.Item Correlations between metabolites in the synovial fluid and serum: A mouse injury study(Wiley, 2022-03) Wallace, Cameron W.; Hislop, Brady; Hahn, Alyssa K.; Erdogan, Ayten E.; Brahmachary, Priyanka P.; June, Ronald K.Osteoarthritis occurs frequently after joint injury. Currently, osteoarthritis is diagnosed by radiographic changes that are typically found after the disease has progressed to multiple tissues. The primary objective was to compare potential metabolomic biomarkers of joint injury between synovial fluid and serum in a mouse model of posttraumatic osteoarthritis. The secondary objective was to gain insight into the pathophysiology of osteoarthritis by examining metabolomic profiles after joint injury. Twelve-week-old adult female C57BL/6 mice (n = 12) were randomly assigned to control, Day 1, or Day 8 postinjury groups. Randomly selected stifle joints were subjected to a single rapid compression. At Days 1 and 8 postinjury, serum was extracted before mice were euthanized for synovial fluid collection. Metabolomic profiling detected ~2500 metabolites across serum and synovial fluid. Of these, 179 were positively correlated and 51 were negatively correlated between synovial fluid and serum, indicating the potential for the development of metabolomic biomarkers. Synovial fluid captured injury-induced differences in metabolomic profiles at both Days 1 and 8 after injury whereas serum did not. However, synovial fluid and serum were distinct at both time points after injury. In synovial fluid, pathways of interest mapped to amino acid synthesis and degradation, bupropion degradation, and transfer RNA (tRNA) charging. In serum, pathways were amino acid synthesis and degradation, the phospholipase pathway, and nicotine degradation. These results provide a rich picture of the injury response at early time points after joint injury. Furthermore, the correlations between synovial fluid and serum metabolites suggest the potential to gain insight into intra-articular pathophysiology through analysis of serum metabolites.Item A 3-D constitutive model for finite element analyses of agarose with a range of gel concentrations(Elsevier BV, 2020-11) Wang, Xiaogang; June, Ronald K.; Pierce, David M.Hydrogels have seen widespread application across biomedical sciences and there is considerable interest in using hydrogels, including agarose, for creating in vitro three-dimensional environments to grow cells and study mechanobiology and mechanotransduction. Recent advances in the preparation of agarose gels enable successful encapsulation of viable cells at gel concentrations as high as 5%. Agarose with a range of gel concentrations can thus serve as an experimental model mimicking changes in the 3-D microenvironment of cells during disease progression and can facilitate experiments aimed at probing the corresponding mechanobiology, e.g. the evolving mechanobiology of chondrocytes during the progression of osteoarthritis. Importantly, whether stresses (forces) or strains (displacements) drive mechanobiology and mechanotransduction is currently unknown. We can use experiments to quantify mechanical properties of hydrogels, and imaging to estimate microstructure and even strains; however, only computational models can estimate intra-gel stresses in cell-seeded agarose constructs because the required in vitro experiments are currently impossible. Finite element modeling is well-established for (computational) mechanical analyses, but accurate constitutive models for modeling the 3-D mechanical environments of cells within high-stiffness agarose with varying gel concentrations are currently unavailable. In this study we aimed to establish a 3-D constitutive model of high-stiffness agarose with a range of gel concentrations. We applied a multi-step, physics-based optimization approach to separately fit subsets of model parameters and help achieve robust convergence. Our constitutive model, fitted to experimental data on progressive stress-relaxations, was able to predict reaction forces determined from independent experiments on cyclical loading. Our model has broad applications in finite element modeling aimed at interpreting mechanical experiments on agarose specimens seeded with cells, particularly in predicting distributions of intra-gel stresses. Our model and fitted parameters enable more accurate finite element simulations of high-stiffness agarose constructs, and thus better understanding of experiments aimed at mechanobiology, mechanotransduction, or other applications in tissue engineering.Item Metabolomic Profiling to Understand Chondrocyte Metabolism(2021) Brahmachary, Priyanka; Welhaven, Hope D.; June, Ronald K.Metabolism has long been recognized as a critical physiological process necessary to maintain homeostasis in all types of cells including the chondrocytes of articular cartilage. Alterations in metabolism in disease and metabolic adaptation to physiological stimuli such as mechanical loading are increasingly recognized as important for understanding musculoskeletal systems such as synovial joints. Metabolomics is an emerging technique that allows quantitative measurement of thousands of small molecule metabolites that serve as both products and reactants to myriad reactions of cellular biochemistry. This protocol describes procedures to perform metabolomic profiling on chondrocytes and other tissues and fluids within the synovial joint.Item Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis(2019-08) Carlson, Alyssa K.; Rawle, Rachel A.; Wallace, Cameron W.; Brooks, Ellen G.; Adams, Erik; Greenwood, Mark C.; Olmer, Merissa; Lotz, Martin K.; Bothner, Brian; June, Ronald K."Objective Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.Item Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis(2019-03) Haudenschid, Dominik R.; Carlson, Alyssa K.; Zignego, Donald L.; Yik, Jasper H. N.; Hilmer, Jonathan K.; June, Ronald K.OBJECTIVE Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome. DESIGN To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 h after injury. RESULTS We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites. CONCLUSIONS Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage.Item Emerging role of metabolic signaling in synovial joint remodeling and osteoarthritis(2016-12) June, Ronald K.; Liu-Bryan, Ru; Long, Fanxing; Griffin, Timothy M.Obesity and associated metabolic diseases collectively referred to as the metabolic syndrome increase the risk of skeletal and synovial joint diseases, including osteoarthritis (OA). The relationship between obesity and musculoskeletal diseases is complex, involving biomechanical, dietary, genetic, inflammatory, and metabolic factors. Recent findings illustrate how changes in cellular metabolism and metabolic signaling pathways alter skeletal development, remodeling, and homeostasis, especially in response to biomechanical and inflammatory stressors. Consequently, a better understanding of the energy metabolism of diarthrodial joint cells and tissues, including bone, cartilage, and synovium, may lead to new strategies to treat or prevent synovial joint diseases such as OA. This rationale was the basis of a workshop presented at the 2016 Annual ORS Meeting in Orlando, FL on the emerging role of metabolic signaling in synovial joint remodeling and OA. The topics we covered included (i) the relationship between metabolic syndrome and OA in clinical and pre-clinical studies; (ii) the effect of biomechanical loading on chondrocyte metabolism; (iii) the effect of Wnt signaling on osteoblast carbohydrate and amino acid metabolism with respect to bone anabolism; and (iv) the role of AMP-activated protein kinase in chondrocyte energetic and biomechanical stress responses in the context of cartilage injury, aging, and OA. Although challenges exist for measuring in vivo changes in synovial joint tissue metabolism, the findings presented herein provide multiple lines of evidence to support a central role for disrupted cellular energy metabolism in the pathogenesis of OA.Item Physiological dynamic compression regulates central energy metabolism in primary human chondrocytes(2018-02) Salinas, Daniel; Mumey, Brendan M.; June, Ronald K.Chondrocytes use the pathways of central metabolism to synthesize molecular building blocks and energy for cartilage homeostasis. An interesting feature of the in vivo chondrocyte environment is the cyclical loading generated in various activities (e.g., walking). However, it is unknown whether central metabolism is altered by mechanical loading. We hypothesized that physiological dynamic compression alters central metabolism in chondrocytes to promote production of amino acid precursors for matrix synthesis. We measured the expression of central metabolites (e.g., glucose, its derivatives, and relevant co-factors) for primary human osteoarthritic chondrocytes in response to 0–30 minutes of compression. To analyze the data, we used principal components analysis and ANOVA-simultaneous components analysis, as well as metabolic flux analysis. Compression-induced metabolic responses consistent with our hypothesis. Additionally, these data show that chondrocyte samples from different patient donors exhibit different sensitivity to compression. Most importantly, we find that grade IV osteoarthritic chondrocytes are capable of synthesizing non-essential amino acids and precursors in response to mechanical loading. These results suggest that further advances in metabolic engineering of chondrocyte mechanotransduction may yield novel translational strategies for cartilage repair.