An official website of the United States government
Abstract Long bone growth requires the precise control of chondrocyte maturation from proliferation to hypertrophy during endochondral ossification, but the bioenergetic program that ensures normal cartilage development is still largely elusive. We show that chondrocytes have unique glucose metabolism signatures in these stages, and they undergo bioenergetic reprogramming from glycolysis to oxidative phosphorylation during maturation, accompanied by an upregulation of the pentose phosphate pathway. Inhibition of either oxidative phosphorylation or the pentose phosphate pathway in murine chondrocytes and bone organ cultures impaired hypertrophic differentiation, suggesting that the appropriate balance of these pathways is required for cartilage development. Insulin-like growth factor 2 (IGF2) deficiency resulted in a profound increase in oxidative phosphorylation in hypertrophic chondrocytes, suggesting that IGF2 is required to prevent overactive glucose metabolism and maintain a proper balance of metabolic pathways. Our results thus provide critical evidence of preference for a bioenergetic pathway in different stages of chondrocytes and highlight its importance as a fundamental mechanism in skeletal development.
more »
« less
Chondrocytes Embedded in Agarose Generate Distinct Metabolic Heat Profiles Based on Media Carbon Sources
Abstract Human chondrocytes are responsible for cartilage repair and homeostasis through metabolic production of precursors to collagen and other matrix components. This metabolism is sensitive both to the availability of media energy sources as well as the local temperature. Central carbon metabolites such as glucose and glutamine are essential not only for producing energetic compounds such as ATP and NADH, but also for assembling collagen and aggrecan from non-essential amino acid precursors. The rate at which this metabolism takes place directly relates to temperature: a moderate increase in temperature results in faster enzyme kinetics and faster metabolic processes. Furthermore, these biological processes are exothermic and will generate heat as a byproduct, further heating the local environment of the cell. Prior studies suggest that mechanical stimuli affect levels of central metabolites in three-dimensionally cultured articular chondrocytes. But these prior studies have not determined if articular chondrocytes produce measurable heat. Thus,the goal of this studyis to determine if three-dimensionally encapsulated chondrocytes are capable of heat production which will improve our knowledge of chondrocyte central metabolism and further validate in vitro methods. Here we show the results of microcalorimetric measurements of heat generated by chondrocytes suspended in agarose hydrogels over a 2-day period in PBS, glucose, and glutamine media. The results show that a significant amount of heat is generated by cells (Cells Only: 3.033 ± 0.574 µJ/cell, Glucose: 2.791 ± 0.819 µJ/cell, Glutamine: 1.900 ± 0.650 µJ/cell) versus the absence of cells (No Cells: 0.374 ± 0.251 µJ/cell). This suggests that cells which have access to carbon sources in the media or as intracellular reserves will generate a significant amount of heat as they process these metabolites, produce cellular energy, and synthesize collagen precursors. The length of the microcalorimeter experiment (48 h) also suggests that the metabolism of articular chondrocytes is slower than many other cells, such as human melanoma cells, which can produce similar quantities of heat in less than an hour. These data broadly suggest that chondrocyte metabolism is sensitive to the available nutrients and has the potential to alter cartilage temperature through metabolic activity.
more »
« less
- Award ID(s):
- 2125748
- PAR ID:
- 10630301
- Publisher / Repository:
- Springer Nature Link
- Date Published:
- Journal Name:
- Annals of Biomedical Engineering
- ISSN:
- 0090-6964
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.more » « less
-
Disorders of cartilage homeostasis and chondrocyte apoptosis are major events in the pathogenesis of osteoarthritis (OA). Herein, we sought to assess the chondroprotective effect and underlying mechanisms of a novel chemically modified curcumin, CMC2.24, in modulating extracellular matrix (ECM) homeostasis and inhibiting chondrocyte apoptosis. Rats underwent the anterior cruciate ligament transection and medial menisci resection were treated by intra-articular injection with CMC2.24. In vitro study, rat chondrocytes were pretreated with CMC2.24 before stimulation with sodium nitroprusside (SNP). The effects of CMC2.24 on cartilage homeostasis and chondrocyte apoptosis were observed. The results from in vivo studies demonstrated that the intra-articular administration of CMC2.24 delayed cartilage degeneration and suppressed chondrocyte apoptosis. CMC2.24 ameliorated osteoarthritic cartilage destruction by promoting collagen 2a1 production and inhibited cartilage degradation and apoptosis by suppressing hypoxia-inducible factor-2a (Hif-2α), matrix metalloproteinase-3 (MMP-3), runt-related transcription factor 2 (RUNX2), cleaved caspase-3, vascular endothelial growth factor (VEGF), and the phosphorylation of IκBα and NF-κB p65. The in vitro results revealed that CMC2.24 exhibited a strong inhibitory effect on SNP-induced chondrocyte catabolism and apoptosis. The SNP-enhanced expression of Hif-2α, catabolic and apoptotic factor, decreased after CMC2.24 treatment in a dose-dependent manner. CMC2.24 pretreatment effectively inhibited SNP-induced IκBα and NF-κB p65 phosphorylation in rat chondrocytes, whereas the pretreatment with NF-κB antagonist BMS-345541 significantly enhanced the effects of CMC2.24. Taken together, these results demonstrated that CMC2.24 attenuates OA progression by modulating ECM homeostasis and chondrocyte apoptosis via suppression of the NF-κB/Hif-2α axis, thus providing a new perspective for the therapeutic strategy of OA.more » « less
-
Human primary (hpBMEC) and induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (hiBMEC) are interchangeably used in blood-brain barrier models to study neurological diseases and drug delivery. Both hpBMEC and hiBMEC use glutamine as a source of carbon and nitrogen to produce metabolites and build proteins essential to cell function and communication. We used metabolomic, transcriptomic, and computational methods to examine how hpBMEC and hiBMEC metabolize glutamine, which may impact their utility in modeling the blood-brain barrier. We found that glutamine metabolism was systemically different between the two cell types. hpBMEC had a higher metabolic rate and produced more glutamate and GABA, while hiBMEC rerouted glutamine to produce more glutathione, fatty acids, and asparagine. Higher glutathione production in hiBMEC correlated with higher oxidative stress compared to hpBMEC. α-ketoglutarate (α-KG) supplementation increased glutamate secretion from hiBMEC to match that of hpBMEC; however, α-KG also decreased hiBMEC glycolytic rate. These fundamental metabolic differences between BMEC types may impactin vitroblood-brain barrier model function, particularly communication between BMEC and surrounding cells, and emphasize the importance of evaluating the metabolic impacts of iPSC-derived cells in disease models.more » « less
-
Abstract To elucidate the mechanisms of cellular mechanotransduction, it is necessary to employ biomaterials that effectively merge biofunctionality with appropriate mechanical characteristics. Agarose and collagen separately are common biopolymers used in cartilage mechanobiology and mechanotransduction studies but lack features that make them ideal for functional engineered cartilage. In this study, agarose is blended with collagen type I to create hydrogels with final concentrations of 4% w/v or 2% w/v agarose with 2 mg/mL collagen. We hypothesized that the addition of collagen into a high-concentration agarose hydrogel does not diminish mechanical properties. Acellular and cell-laden studies were completed to assess rheologic and compressive properties, contraction, and structural homogeneity in addition to cell proliferation and sulfated glycosaminoglycan production. Over 21 days in culture, cellular 4% agarose–2 mg/mL collagen I hydrogels seeded with primary murine chondrocytes displayed structural and bulk mechanical behaviors that did not significantly alter from 4% agarose-only hydrogels, cell proliferation, and continual glycosaminoglycan production, indicating promise toward the development of an effective hydrogel for chondrocyte mechanotransduction and mechanobiology studies.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1007/s10439-025-03755-6
