Background/Purpose: We have recently found that articular chondrocytes from human adults contain long cytoplasmic arms that cross the extracellular matrix and physically connect two chondrocytes located in distant lacuna. Besides, we have also demonstrated that primary chondrocytes from adults are able to communicate with each other via gap junctions (GJ) as evidenced by the intercellular transfer of tracer dyes.

Methods: For immunohistochemistry (IHC) assays, in situ cartilage was frozen immediately using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. For Scanning Electron Microscope (SEM), samples were embedded in cacodylate buffer before dehydratation. Dual Voltage-clamp methods along with the study of glucose and oligonucleotides transference and in situ electroporation were used for the study of gap junctional communication (InSitu Porator^TM Cell Projects Ltd). Transwell co-culture system and mass spectrometry were used for the identification and quantification of transjuctional amino acids. Primary chondrocytes were previously cultured in SILAC^® Dulbecco’s Modiffied Eagle’s Medium containing L-lisina [¹³C₆] and L-arginina [¹³C₆, ¹⁵N₄]. Prior to LC analysis amino acids were derivatized using the tEZ: faastTM kit. Detection of analytes was performed with a Thermo Scientific LTQ-Orbitrap.

Results: IHC, electrophysiology experiments and “metabolic capture” assays revealed that GJs play a metabolic function by exchanging nutrients including glucose and essential amino acids between cells. Microinjection of the fluorescent glucose derivative 2-NBDG showed a progressive fluorescence intensity increase in the contacted recipient cells during 22 and 35 minutes after dye injection. In situ electroporation of donor cells confirmed that adult chondrocytes efficiently exchange glucose between adjacent cells (18-20 cells deep). When cells were pre-incubated for 1 hour with 250µg/ml of the GJ inhibitor GAP27, the transference of glucose was severely reduced (1-2 cells deep). For Transwell co-culture system, donors previously cultured in “heavy medium” were allowed to settle for 4 hours, time enough to form cell projections on the membrane right side up containing receivers. Mass spectrometry analysis revealed the transference of 3,5 pmol/ml of [¹³C₆]-L-lysina and 3 pmol/ml of [¹³C₆, ¹⁵N₄]-L-arginina. The results were confirmed using in situ electroporation and fluorescent labelled amino acids. IHC experiments in cartilage from patients with osteoarthritis (OA) revealed significantly elevated levels of the GJ protein Cx43 in the superficial zone and down through the next 1000 µm of tissue. Besides OA condrocytes contain longer cytoplasmic projections.

Conclusion: The results here presented demonstrated that chondrocytes from human adult cartilage are physically connected to each other forming a three-dimensional cellular network, and are able to transfer nutrients such as glucose and essential amino acids through GJ channels. The levels of Cx43 protein and the length of cells projections suggest alterations in the normal homeostasis control and cell-cell communication between chondrocytes in cartilage of patients with OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interconnected-cellular-projections-and-gap-junctions-mediate-metabolic-coupling-between-chondrocytes-located-in-different-layers-of-the-tissue-cell-to-cell-communication-in-normal-and-osteoarthritic/