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Research Papers

Moderate Cyclic Tensile Strain Alters the Assembly of Cartilage Extracellular Matrix Proteins In Vitro

[+] Author and Article Information
Judith Bleuel

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany
e-mail: j.bleuel@dshs-koeln.de

Frank Zaucke

Center for Biochemistry,
Medical Faculty,
University of Köln,
Joseph-Stelzmann-Straße 52,
Köln 50931, Germany;
Cologne Center for Musculoskeletal Biomechanics,
Medical Faculty,
University of Köln,
Joseph-Stelzmann-Straße 9,
Köln 50931, Germany
e-mail: frank.zaucke@uni-koeln.de

Gert-Peter Brüggemann

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany;
Cologne Center for Musculoskeletal Biomechanics,
Medical Faculty,
University of Köln,
Joseph-Stelzmann-Straße 9,
Köln 50931, Germany
e-mail: brueggemann@dshs-koeln.de

Juliane Heilig

Center for Biochemistry,
Medical Faculty,
University of Köln,
Joseph-Stelzmann-Straße 52,
Köln 50931, Germany
e-mail: juliane.heilig@uni-koeln.de

Marie-Louise Wolter

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany
e-mail: Mary_LouW@web.de

Nina Hamann

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany
e-mail: hamann@dshs-koeln.de

Sara Firner

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany
e-mail: s.firner@dshs-koeln.de

Anja Niehoff

Institute of Biomechanics and Orthopaedics,
German Sport University Köln,
Am Sportpark Müngersdorf 6,
Köln 50933, Germany;
Cologne Center for Musculoskeletal Biomechanics,
Medical Faculty,
University of Köln,
Joseph-Stelzmann-Straße 9,
Köln 50931, Germany
e-mail: niehoff@dshs-koeln.de

Manuscript received October 6, 2014; final manuscript received March 9, 2015; published online April 14, 2015. Assoc. Editor: Carlijn V. C Bouten.

J Biomech Eng 137(6), 061009 (Jun 01, 2015) (9 pages) Paper No: BIO-14-1497; doi: 10.1115/1.4030053 History: Received October 06, 2014; Revised March 09, 2015; Online April 14, 2015

Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

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Figures

Grahic Jump Location
Fig. 1

Representative Western blots and densitometric data of (a) COMP, (b) collagen IX, (c) matrilin-3, and (d) collagen II in loaded (+) and unloaded (−) chondrocytes. Proteins were detected in the supernatant, the lysate, and the matrix. (d) Collagen II was only detected after digestion with pepsin. Data are presented as mean ± standard deviation of three independent experiments. Unloaded samples of each fraction were set 1. *Significantly (p < 0.05) different between loaded and unloaded cells.

Grahic Jump Location
Fig. 2

Semiquantitative RT-PCR of (a) COMP, (b) collagen IX, (c) matrilin-3, and (d) collagen II in loaded (gray) and unloaded (black) primary mouse chondrocytes at different time-points after loading. Signal intensity was normalized to GAPDH. Data represent means ± standard deviation of three independent experiments. Time point 24 h: n = 2. Unloaded samples of each time point were set 1.

Grahic Jump Location
Fig. 3

Immunofluorescence staining of (a) COMP, (b) collagen IX, and (c) matrilin-3 each costained with collagen II in loaded and unloaded primary mouse chondrocytes. Nuclei were stained with DAPI. Arrows indicate thick collagen II containing fibrils (a, b, c) that were strongly costained for COMP (a) or collagen IX (b) in loaded chondrocytes. White arrows indicate very intense intracellular staining (a, b) in unloaded chondrocytes. Intracellular staining intensity was evaluated quantitatively, whereas higher score values indicate stronger intracellular staining intensity. Experiments were conducted three times. Pictures and data from one representative experiment. *significantly (p < 0.05) different between loaded and unloaded cells.

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