Metabolic Profiles of Encapsulated Chondrocytes Exposed to Short-Term Simulated Microgravity.

The mechanism by which chondrocytes respond to reduced mechanical loading environments and the subsequent risk of developing osteoarthritis remains unclear. This is of particular concern for astronauts. In space the reduced joint loading forces during prolonged microgravity (10 -6 g ) exposure could lead to osteoarthritis (OA), compromising quality of life post-spaceflight. In this study, we encapsulated human chondrocytes in an agarose gel of similar stiffness to the pericellular matrix to mimic the cartilage microenvironment. We then exposed agarose-chondrocyte constructs to simulated microgravity (SM) using a rotating wall vessel (RWV) bioreactor to better assess the cartilage health risks associated with spaceflight. Global metabolomic profiling detected a total of 1205 metabolite features across all samples, with 497 significant metabolite features identified by ANOVA (FDR-corrected p-value < 0.05). Specific metabolic shifts detected in response to SM exposure resulted in clusters of co-regulated metabolites, as well as key metabolites identified by variable importance in projection scores. Microgravity-induced metabolic shifts in gel constructs and media were indicative of protein synthesis, energy metabolism, nucleotide metabolism, and oxidative catabolism. The microgravity associated-metabolic shifts were consistent with early osteoarthritic metabolomic profiles in human synovial fluid, which suggests that even short-term exposure to microgravity (or other reduced mechanical loading environments) may lead to the development of OA.

Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2.

Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.

Aging alters the subchondral bone response 7 days after noninvasive traumatic joint injury in C57BL/6JN mice.

Posttraumatic osteoarthritis (PTOA) commonly develops following anterior cruciate ligament (ACL) injuries, affecting around 50% of individuals within 10-20 years. Recent studies have highlighted early changes in subchondral bone structure after ACL injury in adolescent or young adult mice, which could contribute to the development of PTOA. However, ACL injuries do not only occur early in life. Middle-aged and older patients also experience ACL injuries and PTOA, but whether the aged subchondral bone also responds rapidly to injury is unknown. This study utilized a noninvasive, single overload mouse injury model to assess subchondral bone microarchitecture, turnover, and material properties in both young adults (5 months) and early old age (22 months) female C57BL/6JN mice at 7 days after injury. Mice underwent either joint injury (i.e., produces ACL tears) or sham injury procedures on both the loaded and contralateral limbs, allowing evaluation of the impacts of injury versus loading. The subchondral bone response to ACL injury is distinct for young adult and aged mice. While 5-month mice show subchondral bone loss and increased bone resorption postinjury, 22-month mice did not show loss of bone structure and had lower bone resorption. Subchondral bone plate modulus increased with age, but not with injury. Both ages of mice showed several bone measures were altered in the contralateral limb, demonstrating the systemic skeletal response to joint injury. These data motivate further investigation to discern how osteochondral tissues differently respond to injury in aging, such that diagnostics and treatments can be refined for these demographics.

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

Unraveling sex-specific risks of knee osteoarthritis before menopause: Do sex differences start early in life?

OBJECTIVE: Sufficient evidence within the past two decades have shown that osteoarthritis (OA) has a sex-specific component. However, efforts to reveal the biological causes of this disparity have emerged more gradually. In this narrative review, we discuss anatomical differences within the knee, incidence of injuries in youth sports, and metabolic factors that present early in life (childhood and early adulthood) that can contribute to a higher risk of OA in females. DESIGN: We compiled clinical data from multiple tissues within the knee joint-since OA is a whole joint disorder-aiming to reveal relevant factors behind the sex differences from different perspectives. RESULTS: The data gathered in this review indicate that sex differences in articular cartilage, meniscus, and anterior cruciate ligament are detected as early as childhood and are not only explained by sex hormones. Aiming to unveil the biological causes of the uneven sex-specific risks for knee OA, we review the current knowledge of sex differences mostly in young, but also including old populations, from the perspective of (i) human anatomy in both healthy and pathological conditions, (ii) physical activity and response to injury, and (iii) metabolic signatures. CONCLUSIONS: We propose that to close the gap in health disparities, and specifically regarding OA, we should address sex-specific anatomic, biologic, and metabolic factors at early stages in life, as a way to prevent the higher severity and incidence of OA in women later in life.

Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage.

Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, metabolite extracts from healthy (n = 11) and end-stage osteoarthritic cartilage (n = 35) were analyzed using liquid chromatography-mass spectrometry metabolomic profiling. Specific metabolites and metabolic pathways, including lipid and amino acid pathways, were differentially regulated in osteoarthritis-derived and healthy cartilage. The detected alterations in amino acids and lipids highlighted key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in OA-derived cartilage compared to healthy cartilage. Moreover, the metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes, highlighting the heterogenous nature of OA metabolism and the diverse landscape within the joint in patients. The results of this study demonstrate that human cartilage has distinct metabolomic profiles in healthy and end-stage OA patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.

Osteochondral fluid transport in an ex vivo system.

OBJECTIVE: Alterations to bone-to-cartilage fluid transport may contribute to the development of osteoarthritis (OA). Larger biological molecules in bone may transport from bone-to-cartilage (e.g., insulin, 5 kDa). However, many questions remain about fluid transport between these tissues. The objectives of this study were to (1) test for diffusion of 3 kDa molecular tracers from bone-to-cartilage and (2) assess potential differences in bone-to-cartilage fluid transport between different loading conditions. DESIGN: Osteochondral cores extracted from bovine femurs (N = 10 femurs, 10 cores/femur) were subjected to either no-load (i.e., pure diffusion), pre-load only, or cyclic compression (5 ± 2% or 10 ± 2% strain) in a two-chamber bioreactor. The bone was placed into the bone compartment followed by a 3 kDa dextran tracer, and tracer concentrations in the cartilage compartment were measured every 5 min for 120 min. Tracer concentrations were analyzed for differences in beginning, peak, and equilibrium concentrations, loading effects, and time-to-peak tracer concentration. RESULTS: Peak tracer concentration in the cartilage compartment was significantly higher compared to the beginning and equilibrium tracer concentrations. Cartilage-compartment tracer concentration and maximum fluorescent intensity were influenced by strain magnitude. No time-to-peak relationship was found between strain magnitudes and cartilage-compartment tracer concentration. CONCLUSION: This study shows that bone-to-cartilage fluid transport occurs with 3 kDa dextran molecules. These are larger molecules to move between bone and cartilage than previously reported. Further, these results demonstrate the potential impact of cyclic compression on osteochondral fluid transport. Determining the baseline osteochondral fluid transport in healthy tissues is crucial to elucidating the mechanisms OA pathology.

Evolution and advancements in genomics and epigenomics in OA research: How far we have come.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent musculoskeletal disease affecting articulating joint tissues, resulting in local and systemic changes that contribute to increased pain and reduced function. Diverse technological advancements have culminated in the advent of high throughput "omic" technologies, enabling identification of comprehensive changes in molecular mediators associated with the disease. Amongst these technologies, genomics and epigenomics - including methylomics and miRNomics, have emerged as important tools to aid our biological understanding of disease. DESIGN: In this narrative review, we selected articles discussing advancements and applications of these technologies to OA biology and pathology. We discuss how genomics, deoxyribonucleic acid (DNA) methylomics, and miRNomics have uncovered disease-related molecular markers in the local and systemic tissues or fluids of OA patients. RESULTS: Genomics investigations into the genetic links of OA, including using genome-wide association studies, have evolved to identify 100+ genetic susceptibility markers of OA. Epigenomic investigations of gene methylation status have identified the importance of methylation to OA-related catabolic gene expression. Furthermore, miRNomic studies have identified key microRNA signatures in various tissues and fluids related to OA disease. CONCLUSIONS: Sharing of standardized, well-annotated omic datasets in curated repositories will be key to enhancing statistical power to detect smaller and targetable changes in the biological signatures underlying OA pathogenesis. Additionally, continued technological developments and analysis methods, including using computational molecular and regulatory networks, are likely to facilitate improved detection of disease-relevant targets, in-turn, supporting precision medicine approaches and new treatment strategies for OA.

Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage.

Objective: Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, healthy and end-stage osteoarthritic cartilage were compared metabolically to uncover disease-associated profiles, classify OA-specific metabolic endotypes, and identify targets for intervention for the diverse populations of individuals affected by OA. Design: Femoral head cartilage (n=35) from osteoarthritis patients were collected post-total joint arthroplasty. Healthy cartilage (n=11) was obtained from a tissue bank. Metabolites from all cartilage samples were extracted and analyzed using liquid chromatography-mass spectrometry metabolomic profiling. Additionally, cartilage extracts were pooled and underwent fragmentation analysis for biochemical identification of metabolites. Results: Specific metabolites and metabolic pathways, including lipid- and amino acid pathways, were differentially regulated between osteoarthritis-derived and healthy cartilage. The detected alterations of amino acids and lipids highlight key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in osteoarthritis-derived cartilage compared to healthy. Moreover, metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes highlighting the heterogenous nature of OA metabolism and diverse landscape within the joint between patients. Conclusions: The results of this study demonstrate that human cartilage has distinct metabolomic profiles between healthy and end-stage osteoarthritis patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage, and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.

The metabolome of individuals with knee osteoarthritis is influenced by 18-months of an exercise and weight loss intervention and sex: the IDEA trial.

Objective: The Intensive Diet and Exercise for Arthritis (IDEA) trial was conducted to evaluate the effects of diet and exercise on osteoarthritis (OA), the most prevalent form of arthritis. Various risk factors, such as obesity and sex, contribute to the debilitating nature of OA. While diet and exercise are known to improve OA symptoms, cellular and molecular mechanisms underlying these interventions, as well as effects of participant sex, remain elusive. Methods: Serum was obtained at three timepoints from IDEA participants assigned to groups of diet, exercise, or combined diet and exercise (n=10 per group). All serum metabolites were extracted and analyzed via liquid chromatography-mass spectrometry combined with metabolomic profiling. Extracted serum was pooled and fragmentation patterns were analyzed to identify metabolites that statistically differentially regulated between groups. Results: Changes in metabolism across male and female IDEA participants after 18-months of diet, exercise, and combined diet and excise intervention mapped to lipid, amino acid, carbohydrate, vitamin, and matrix metabolism. The diverse metabolic landscape detected across IDEA participants shows that intervention type impacts the serum metabolome of individuals with OA in distinct patterns. Moreover, differences in the serum metabolome corresponded with participant sex. Conclusions: These findings suggest that intensive weight loss among male and female subjects offers potential metabolic benefits for individuals with knee OA. This provides a deeper understanding of dysregulation occurring during OA development that may pave the way for improved interventions, treatments, and quality of life of those impacted by this disease.

Metabolomic profiles of cartilage and bone reflect tissue type, radiography-confirmed osteoarthritis, and spatial location within the joint.

Osteoarthritis is the most common chronic joint disease, characterized by the abnormal remodeling of joint tissues including articular cartilage and subchondral bone. However, there are currently no therapeutic drug targets to slow the progression of disease because disease pathogenesis is largely unknown. Thus, the goals of this study were to identify metabolic differences between articular cartilage and subchondral bone, compare the metabolic shifts in osteoarthritic grade III and IV tissues, and spatially map metabolic shifts across regions of osteoarthritic hip joints. Articular cartilage and subchondral bone from 9 human femoral heads were obtained after total joint arthroplasty, homogenized and metabolites were extracted for liquid chromatography-mass spectrometry analysis. Metabolomic profiling revealed that distinct metabolic endotypes exist between osteoarthritic tissues, late-stage grades, and regions of the diseased joint. The pathways that contributed the most to these differences between tissues were associated with lipid and amino acid metabolism. Differences between grades were associated with nucleotide, lipid, and sugar metabolism. Specific metabolic pathways such as glycosaminoglycan degradation and amino acid metabolism, were spatially constrained to more superior regions of the femoral head. These results suggest that radiography-confirmed grades III and IV osteoarthritis are associated with distinct global metabolic and that metabolic shifts are not uniform across the joint. The results of this study enhance our understanding of osteoarthritis pathogenesis and may lead to potential drug targets to slow, halt, or reverse tissue damage in late stages of osteoarthritis.

Heat Conduction Simulation of Chondrocyte-Embedded Agarose Gels Suggests Negligible Impact of Viscoelastic Dissipation on Temperature Change.

Agarose is commonly used for 3D cell culture and to mimic the stiffness of the pericellular matrix of articular chondrocytes. Although it is known that both temperature and mechanical stimulation affect the metabolism of chondrocytes, little is known about the thermal properties of agarose hydrogels. Thermal properties of agarose are needed to analyze potential heat production by chondrocytes induced by various experimental stimuli (carbon source, cyclical compression, etc). Utilizing ASTM C177, a custom-built thermal conductivity measuring device was constructed and used to calculate the thermal conductivity of 4.5% low gelling temperature agarose hydrogels. Additionally, Differential Scanning Calorimetry was used to calculate the specific heat capacity of the agarose hydrogels. Testing of chondrocyte-embedded agarose hydrogels commonly occurs in Phosphate-Buffered Saline (PBS), and thermal analysis requires the free convection coefficient of PBS. This was calculated using a 2D heat conduction simulation within MATLAB in tandem with experimental data collected for known boundary and initial conditions. The specific heat capacity and thermal conductivity of 4.5% agarose hydrogels was calculated to be 2.85 J/g°C and 0.121 W/mK, respectively. The free convection coefficient of PBS was calculated to be 1000.1 W/m 2 K. The values of specific heat capacity and thermal conductivity for agarose are similar to the reported values for articular cartilage, which are 3.20 J/g°C and 0.21 W/mK (Moghadam, et al. 2014). This suggests that in addition to 4.5% agarose hydrogels mimicking the physiological stiffness of the cartilage PCM, they can also mimic the thermal properties of articular cartilage for in vitro studies.

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Three decades of advancements in osteoarthritis research: insights from transcriptomic, proteomic, and metabolomic studies.

OBJECTIVE: 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.

Metabolic phenotypes reflect patient sex and injury status: A cross-sectional analysis of human synovial fluid.

OBJECTIVE: Osteoarthritis is a heterogeneous disease. The objective was to compare differences in underlying cellular mechanisms and endogenous repair pathways between synovial fluid (SF) from male and female participants with different injuries to improve the current understanding of the pathophysiology of downstream post-traumatic osteoarthritis (PTOA). DESIGN: SF from n = 33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. SF was extracted and analyzed via liquid chromatography-mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies (ligament, meniscal, and combined ligament and meniscal) and patient sex. Samples were pooled and underwent secondary fragmentation to identify metabolites. RESULTS: Different knee injuries uniquely altered SF metabolites and downstream pathways including amino acid, lipid, and inflammatory-associated metabolic pathways. Notably, sexual dimorphic metabolic phenotypes were examined between males and females and within injury pathology. Cervonyl carnitine and other identified metabolites differed in concentrations between sexes. CONCLUSIONS: These results suggest that different injuries and patient sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries, sex, and PTOA development may yield data regarding how endogenous repair pathways differ between male and female injury types. Ongoing metabolomic analysis of SF in injured male and female patients can be performed to monitor PTOA development and progression.

Germ-Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance.

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).

Metabolomic profiling to identify early urinary biomarkers and metabolic pathway alterations in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of numerous fluid-filled cysts that lead to progressive loss of functional nephrons. Currently, there is an unmet need for diagnostic and prognostic indicators of early stages of the disease. Metabolites were extracted from the urine of patients with early-stage ADPKD (n = 48 study participants) and age- and sex-matched normal controls (n = 47) and analyzed by liquid chromatography-mass spectrometry. Orthogonal partial least squares-discriminant analysis was used to generate a global metabolomic profile of early ADPKD for the identification of metabolic pathway alterations and discriminatory metabolites as candidates of diagnostic and prognostic biomarkers. The global metabolomic profile exhibited alterations in steroid hormone biosynthesis and metabolism, fatty acid metabolism, pyruvate metabolism, amino acid metabolism, and the urea cycle. A panel of 46 metabolite features was identified as candidate diagnostic biomarkers. Notable putative identities of candidate diagnostic biomarkers for early detection include creatinine, cAMP, deoxycytidine monophosphate, various androgens (testosterone; 5-α-androstane-3,17,dione; trans-dehydroandrosterone), betaine aldehyde, phosphoric acid, choline, 18-hydroxycorticosterone, and cortisol. Metabolic pathways associated with variable rates of disease progression included steroid hormone biosynthesis and metabolism, vitamin D3 metabolism, fatty acid metabolism, the pentose phosphate pathway, tricarboxylic acid cycle, amino acid metabolism, sialic acid metabolism, and chondroitin sulfate and heparin sulfate degradation. A panel of 41 metabolite features was identified as candidate prognostic biomarkers. Notable putative identities of candidate prognostic biomarkers include ethanolamine, C20:4 anandamide phosphate, progesterone, various androgens (5-α-dihydrotestosterone, androsterone, etiocholanolone, and epiandrosterone), betaine aldehyde, inflammatory lipids (eicosapentaenoic acid, linoleic acid, and stearolic acid), and choline. Our exploratory data support metabolic reprogramming in early ADPKD and demonstrate the ability of liquid chromatography-mass spectrometry-based global metabolomic profiling to detect metabolic pathway alterations as new therapeutic targets and biomarkers for early diagnosis and tracking disease progression of ADPKD.NEW & NOTEWORTHY To our knowledge, this study is the first to generate urinary global metabolomic profiles from individuals with early-stage ADPKD with preserved renal function for biomarker discovery. The exploratory dataset reveals metabolic pathway alterations that may be responsible for early cystogenesis and rapid disease progression and may be potential therapeutic targets and pathway sources for candidate biomarkers. From these results, we generated a panel of candidate diagnostic and prognostic biomarkers of early-stage ADPKD for future validation.

Metabolomic Phenotypes Reflect Patient Sex and Injury Status: A Cross-Sectional Analysis of Human Synovial Fluid.

Background: Post-traumatic osteoarthritis (PTOA) is caused by knee injuries like anterior cruciate ligament (ACL) injuries. Often, ACL injuries are accompanied by damage to other tissues and structures within the knee including the meniscus. Both are known to cause PTOA but underlying cellular mechanisms driving disease remain unknown. Aside from injury, patient sex is a prevalent risk factor associated with PTOA. Hypothesis: Metabolic phenotypes of synovial fluid that differ by knee injury pathology and participant sex will be distinct from each other. Study Design: A cross-sectional study. Methods: Synovial fluid from n=33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. Synovial fluid was extracted and analyzed via liquid chromatography mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies and participant sex. Additionally, samples were pooled and underwent fragmentation to identify metabolites. Results: Metabolite profiles revealed that injury pathology phenotypes were distinct from each other where differences in endogenous repair pathways that are triggered post-injury were detected. Specifically, acute differences in metabolism mapped to amino acid metabolism, lipid-related oxidative metabolism, and inflammatory-associated pathways. Lastly, sexual dimorphic metabolic phenotypes were examined between male and female participants, and within injury pathology. Specifically, Cervonyl Carnitine and other identified metabolites differed in concentration between sexes. Conclusions: The results of this study suggest that different injuries (e.g., ligament vs. meniscus), as well as sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries and PTOA development may yield data regarding how endogenous repair pathways differ between injury types. Furthermore, ongoing metabolomic analysis of synovial fluid in injured male and female patients can be performed to monitor PTOA development and progression. Clinical Relevance: Extension of this work may potentially lead to the identification of biomarkers as well as drug targets that slow, stop, or reverse PTOA progression based on injury type and patient sex.

Utilizing metabolomics to identify potential biomarkers and perturbed metabolic pathways in osteoarthritis: A systematic review.

PURPOSE: Osteoarthritis (OA) is a joint disease that is clinically diagnosed using components of history, physical exam, and characteristic radiographic findings, such as joint space narrowing. Currently, there are no laboratory findings that are specific to a diagnosis of OA. The purpose of this systematic review is to evaluate the state of current studies of metabolomic biomarkers that can aid in the diagnosis and treatment of OA. METHODS: Articles were gathered from PubMed and Web of Science using the search terms "osteoarthritis" and "biomarkers" and "metabolomics". Last search of databases took place December 3rd, 2022. Duplicates were manually screened, along with any other results that were not original journal articles. Only original reports involving populations with diagnosed primary or secondary OA (human participants) or surgically induced OA (animal participants) and a healthy control group for comparison were considered for inclusion. Metabolites and metabolic pathways reported in included articles were then manually extracted and evaluated for importance based on reported a priori p-values and/or area under the receiver-operator curve (AUC). RESULTS: Of the 161 results that were returned in the database searches, 43 unique articles met the inclusion criteria. Articles were categorized based on body fluid analyzed: 6 studies on urine samples, 13 studies on plasma samples, 11 studies on synovial fluid (SF) samples, 11 studies on serum samples, 1 study on both synovial fluid and serum, and 1 study that involved both plasma and synovial fluid. To synthesize results, individual metabolites, as well as metabolic pathways that involve frequently reported metabolites, are presented for each study. Indications as to whether metabolite levels were increased or decreased are also included if this data was included in the original articles. CONCLUSIONS: These studies clearly show that there are a wide range of metabolic pathways perturbed in OA. For this period, there was no consensus on a single metabolite, or panel of metabolites, that would be clinically useful in early diagnosis of OA or distinguishing OA from a healthy control. However, many common metabolic pathways were identified in the studies, including TCA cycle, fatty acid metabolism, amino acid metabolism (notably BCAA metabolism and tryptophan metabolism via kynurenine pathway), nucleotide metabolism, urea cycle, cartilage matrix components, and phospholipid metabolism. Future research is needed to define effective clinical biomarkers of osteoarthritis from metabolomic and other data.

Sex-specific effects of calving season on joint health and biomarkers in Montana ranchers.

BACKGROUND: Agricultural workers have a higher incidence of osteoarthritis (OA), but the etiology behind this phenomenon is unclear. Calving season, which occurs in mid- to late-winter for ranchers, includes physical conditions that may elevate OA risk. Our primary aim was to determine whether OA biomarkers are elevated at the peak of calving season compared to pre-season, and to compare these data with joint health survey information from the subjects. Our secondary aim was to detect biomarker differences between male and female ranchers. METHODS: During collection periods before and during calving season, male (n = 28) and female (n = 10) ranchers completed joint health surveys and provided samples of blood, urine, and saliva for biomarker analysis. Statistical analyses examined associations between mean biomarker levels and survey predictors. Ensemble cluster analysis identified groups having unique biomarker profiles. RESULTS: The number of calvings performed by each rancher positively correlated with plasma IL-6, serum hyaluronic acid (HA) and urinary CTX-I. Thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, was significantly higher during calving season than pre-season and was also correlated with ranchers having more months per year of joint pain. We found evidence of sexual dimorphism in the biomarkers among the ranchers, with leptin being elevated and matrix metalloproteinase-3 diminished in female ranchers. The opposite was detected in males. WOMAC score was positively associated with multiple biomarkers: IL-6, IL-2, HA, leptin, C2C, asymmetric dimethylarginine, and CTX-I. These biomarkers represent enzymatic degradation, inflammation, products of joint destruction, and OA severity. CONCLUSIONS: The positive association between number of calvings performed by each rancher (workload) and both inflammatory and joint tissue catabolism biomarkers establishes that calving season is a risk factor for OA in Montana ranchers. Consistent with the literature, we found important sex differences in OA biomarkers, with female ranchers showing elevated leptin, whereas males showed elevated MMP-3.