Long-distance physical connections between chondrocytes; cell-to-cell communication within hyaline cartilage

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s / Osteoarthritis and Cartilage 20 (2012) S10–S53 S11 3 LONG-DISTANCE PHYSICAL CONNECTIONS BETWEEN CHONDROCYTES; CELL-TO-CELL COMMUNICATION WITHIN HYALINE CARTILAGE M. Mayan , R. Gago , P. Carpintero , P. Filgueira-Fernandez , N. Goyanes , V. Valiunas , P. Brink , F.J. Blanco . Osteoarticular and Aging Res. Lab. INIBIC-CHUAC, A Coruna, Spain; Dept. of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA Purpose: Even chondrocytes are metabolically active cells, responsible of the maintenance of the hyaline cartilage during the whole adult life, it is highly accepted in the field that chondrocytes in cartilage do not connect each other, as they are isolated inside their lacunae separated from each other by a distance between 5 to 60mm. In the same lacuna can co-exit several chondocytes interacting between them. However, how the chondrocytes from different lacuna interact between each other and timely respond to physical or chemicals stimuli, are largely unknown. Thus far, the unique communication between chondrocytes in the superficial layer with chondrocytes in themiddle and deeper layers it is accepted that only occurs through diffusion. Nevertheless, the intercellular communication confers to a tissue the ability to responduniformly to localized stimuli. The purpose of this study was to investigate how chondrocytes communicate each other within the matrix and find for some alteration that could explain the degeneration of the matrix that occurs in patients with OA. Methods: In situ cartilage from human and Sus scrofa were fixed and frozen immediately using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Samples were fixed with acetone for confocal optical microscopy assays. Samples were embedded in cacodylate buffer before dehydratation for Scanning Electron Microscope (SEM). Gold was use for coating. Frozen samples were also used for immunofluorescence and immunohistochemistry assays. For total RNA isolation, the proteins and DNA were removed from cell extracts using TRIZOL Reagent (Invitrogene) before using the RNeasy Kit (Qiagen) which includes DNase treatment (RNAse-Free DNAse Set). Quantifications were done using SYBr green real-time PCR. Virgin Valiunas performed dual voltageclamp method and whole-cell/perforated patch using chondrocytes from healthy donors and patients with OA. Results: By using confocal optical microscopy and Scanning Electron Microscopy (SEM), we have found that chondrocytes are physical connected between each other across the extracellular matrix through large cytoplasmic projections. The projections are between 5 to 150 mm in length in human healthy cartilage and young cartilage from Sus scrofa. However condrocytes projections found in OA cartilage are between 10 to 200 mm in length. High levels of expression for the gap junction protein connexin 43 have been detected by RT-PCR and immunohistochemical experiments. We have also found expression of Cx32 and Cx45, however electrophysiology experiments demonstrated that healthy and OA chondrocytes show voltage-dependent behaviour for channels mainly formed by Cx43. We have performed patch clamp experiments to study the interchange of Lucifer Yellow and small molecules of RNA. Our results demonstrated that healthy and OA chondrocytes are able to interchange Lucifer Yellow and small molecules of RNA through Cx43 channels. In fact, our preliminary results showed that inhibition of channels affect the synthesis of collagen type II, MMP3 andMMP9 suggesting that the activity of channels are actively involved with the maintenance of the cartilage matrix. Conclusions:Wehavefoundcell/cellphysicalconnectionsthroughall layers of cartilage between chondrocytes located at different distance within cartilage. Intercellular communication occurs through channels voltagedependent formedby Cx43. Cytoplasmic projections fromOAchondrocytes are longer than from healthy chondrocytes, therefore the channel activity can be affected by the electrical depolarization of the membrane in OA chondrocytes. We have found that chondrocytes interchange small RNAs and inhibition of channels affect the expression of matrix-related genes. 4 SYNERGISTIC INDUCTION OF ELF3. IN VITRO EFFECT OF LEPTIN AND IL-1 IN HUMAN CHONDROCYTES J. Conde , M. Scotece , M. Otero , R. Gomez , J.J. Gomez-Reino , M.B. Goldring , O. Gualillo . 1 SERGAS, Santiago Univ. Clinical Hosp. NEIRID LAB, Santiago de Compostela, Spain; Hosp. for Special Surgery,