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GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

ABSTRACT: OBJECTIVE:The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. METHOD:Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. RESULTS:55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. CONCLUSION:This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

SUBMITTER: Batushansky A

PROVIDER: S-EPMC6702098 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

Batushansky A A Lopes E B P EBP Zhu S S Humphries K M KM Griffin T M TM

Osteoarthritis and cartilage 20190525 9

<h4>Objective</h4>The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage.<h4>Method</h4>Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old sample ...[more]

PMID: 31136803

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Project description:ObjectiveIschemic osteonecrosis of the femoral head (ONFH) in piglets results in an ischemic injury to the immature articular cartilage. The molecular changes in the articular cartilage in response to ONFH have not been investigated using a transcriptomic approach. The purpose of this study was to perform a genome-wide transcriptomic analysis to identify genes that are upregulated in the immature articular cartilage following ONFH.MethodsONFH was induced in the right femoral head of 6-week old piglets. The unoperated femoral head was used as the normal control. At 24 hours (acute ischemic-hypoxic injury), 2 weeks (avascular necrosis in the femoral head) and 4 weeks (early repair) after surgery (n = 4 piglets/time point), RNA was isolated from the articular cartilage of the femoral head. A microarray analysis was performed using Affymetrix Porcine GeneChip Array. An enrichment analysis and functional clustering of the genes upregulated due to ONFH were performed using DAVID and STRING software, respectively. The increased expression of selected genes was confirmed by a real-time qRTPCR analysis.ResultsInduction of ONFH resulted in the upregulation of 383 genes at 24 hours, 122 genes at 2 weeks and 124 genes at 4 weeks compared to the normal controls. At 24 hours, the genes involved in oxidoreductive, cell-survival, and angiogenic responses were significantly enriched among the upregulated genes. These genes were involved in HIF-1, PI3K-Akt, and MAPK signaling pathways. At 2 weeks, secretory and signaling proteins involved in angiogenic and inflammatory responses, PI3K-Akt and matrix-remodeling pathways were significantly enriched. At 4 weeks, genes that represent inflammatory cytokines and chemokine signaling pathways were significantly enriched. Several index genes (genes that are upregulated at more than one time point following ONFH and are known to be important in various biological processes) including HIF-1A, VEGFA, IL-6, IL6R, IL-8, CCL2, FGF2, TGFB2, MMP1, MMP3, ITGA5, FN and Col6A1 were upregulated in the immature articular cartilage following ONFH. A qRTPCR analysis of selected genes confirmed the upregulated expression observed in the microarray analysis.ConclusionImmature articular cartilage responds to ONFH by the upregulation of genes involved in hypoxic stress response, angiogenesis, matrix remodeling and inflammation. This study provides novel insights into the multi-faceted role of immature articular cartilage, with inflammation as a key component, following ONFH in piglets.

Project description:Tissue material properties are crucial to understanding their mechanical function, both in healthy and diseased states. However, in certain circumstances logistical limitations can prevent testing on fresh samples necessitating one or more freeze-thaw cycles. To date, the nature and extent to which the material properties of articular cartilage are altered by repetitive freezing have not been explored. Therefore, the aim of this study is to quantify how articular cartilage mechanical properties, measured by nanoindentation, are affected by multiple freeze-thaw cycles. Canine cartilage plugs (n = 11) from medial and lateral femoral condyles were submerged in phosphate buffered saline, stored at 3-5°C and tested using nanoindentation within 12h. Samples were then frozen at -20°C and later thawed at 3-5°C for 3h before material properties were re-tested and samples re-frozen under the same conditions. This process was repeated for all 11 samples over three freeze-thaw cycles. Overall mean and standard deviation of shear storage modulus decreased from 1.76 ± 0.78 to 1.21 ± 0.77MPa (p = 0.91), shear loss modulus from 0.42 ± 0.19 to 0.39 ± 0.17MPa (p=0.70) and elastic modulus from 5.13 ± 2.28 to 3.52 ± 2.24MPa (p = 0.20) between fresh and three freeze-thaw cycles respectively. The loss factor increased from 0.31 ± 0.38 to 0.71 ± 1.40 (p = 0.18) between fresh and three freeze-thaw cycles. Inter-sample variability spanned as much as 10.47MPa across freezing cycles and this high-level of biological variability across samples likely explains why overall mean "whole-joint" trends do not reach statistical significance across the storage conditions tested. As a result multiple freeze-thaw cycles cannot be explicitly or statistically linked to mechanical changes within the cartilage. However, the changes in material properties observed herein may be sufficient in magnitude to impact on a variety of clinical and scientific studies of cartilage, and should be considered when planning experimental protocols.

Project description:(1) Background: Since the large-scale poultry industry has been established, femoral head necrosis (FHN) has always been a major leg disease in fast-growing broilers worldwide. Previous research suggested that cartilage homeostasis could be taken into consideration in the cause of FHN, but the evidence is insufficient. (2) Methods: One-day-old broiler chickens were randomly divided into three groups, 16 broilers per group. The birds in group L were injected intramuscularly with methylprednisolone (MP) twice a week for four weeks (12.5 mg·kg-1). The birds in group H were injected intramuscularly with MP (20 mg·kg-1·d-1) for 7 d (impulse treatment). The birds in group C were treated with sterile saline as a control group. Broilers were sacrificed at 42 and 56 d. Blood samples were collected from the jugular vein for ELISA and biochemical analysis. Bone samples, including femur, tibia, and humerus, were collected for histopathological analysis, bone parameters detection, and real-time quantitative PCR detection. (3) Results: The FHN broilers in group L and H both showed lower body weight (BW) and reduced bone parameters. In addition, the MP treatment resulted in reduced extracellular matrix (ECM) anabolism and enhanced ECM catabolism. Meanwhile, the autophagy and apoptosis of chondrocytes were enhanced, which led to the destruction of cartilage homeostasis. Moreover, the impulse MP injection increased the portion of birds with severer FHN, whereas the MP injection over a long period caused a more evident change in serum cytokine concentrations and bone metabolism indicators. (4) Conclusions: The imbalance of cartilage homeostasis may play a critical role in the development of FHN in broilers. FHN broilers induced by MP showed a more pronounced production of catabolic factors and suppressed the anabolic factors, which might activate the genes of the WNT signal pathway and hypoxia-inducible factors (HIFs), and then upregulate the transcription expression of ECM to restore homeostasis.

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GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

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GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

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