Research Papers: General

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

[+] Author Affiliations
Maria Kuzma-Kuzniarska

University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom

Clarence Yapp

University of Oxford, Structural Genomics Consortium, Oxford OX3 7DQ, United Kingdom

Thomas W. Pearson-Jones

University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom

Andrew K. Jones

Oxford Brookes University, Faculty of Health and Life Sciences, Department of Biological and Medical Sciences Oxford OX3 0BP, United Kingdom

Philippa A. Hulley

University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom

J. Biomed. Opt. 19(1), 015001 (Jan 03, 2014). doi:10.1117/1.JBO.19.1.015001
History: Received July 31, 2013; Revised November 11, 2013; Accepted December 3, 2013
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Abstract.  Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

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© 2014 Society of Photo-Optical Instrumentation Engineers

Citation

Maria Kuzma-Kuzniarska ; Clarence Yapp ; Thomas W. Pearson-Jones ; Andrew K. Jones and Philippa A. Hulley
"Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching", J. Biomed. Opt. 19(1), 015001 (Jan 03, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.1.015001


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