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Mechanical stimulation and mechanobiological characterization of cartilage micropellets with a single and new custom-made device

Abstract : Background. Articular cartilage is a tissue with poor self-repair capacity, which is prone to progressive destruction after injuries. In cartilage, the chondrocytes surrounded by their extracellular matrix are organized in a complex structure that plays an essential weight-bearing role in the joint and can both sense and respond to various mechanical stimuli. Cartilage micropellet is a relevant and widely used in vitro model to study cartilage growth but poorly investigated in terms of mechanical characterization because of its small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. Methods. Alginate beads were made with a solution of 3 % sodium alginate extruded into 0,1 M CaCl2. Collagen microspheres were prepared with type I collagen in hydrofluoroether. Human bone marrow-derived MSCs were differentiated into chondrocytes by culture in micropellets with 10 ng/mL TGFβ3- containing inductive medium for 21 days. The fluidic-based device was designed for the concomitant culture of six micropellets placed into the conical wells of a chamber where they were stimulated by a positive pressure (sinusoidal, square or constant). The sinking of each micropellet into the cone and its deformation were recorded by a camera. Expression of the chondrocyte markers SOX9, AGG and COL2B were quantified 24 hours after stimulation by RT-qPCR. The Young’s modulus was determined using a finite element model employing a neo-Hookean hyperelastic law. Results. Alginate- and collagen-based microspheres were first used to validate the reliability of the device. Repeatability and reproducibility of pressure signals used for mechanical stimulation were demonstrated. The mechanical properties of the microspheres were equivalent to those determined by a conventional compression test and shown to be reproducible. MSC-derived cartilage micropellets were stimulated with sinusoidal, square or constant pressure. Different parameters (amplitude, frequency, duration) of the square pressure signal were tested on the expression of chondrocyte markers. A stimulation of 1 Hz at 7 kPa for 30 min induced a significant increase of chondrocyte markers. The mechanical properties of the micropellets were measured and a Young’s modulus of 47.6 ± 26.2 kPa was determined. Conclusion. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with radius in range of 600 to 1300 μm. Of importance in case of MSC-based cartilage micropellets, mechanical stimulation can be performed in parallel on six microspheres allowing the molecular and mechanical characterization on the same group of samples. In the future, the device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli in a longitudinal study.
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https://hal.archives-ouvertes.fr/hal-03440712
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Submitted on : Monday, November 22, 2021 - 3:12:41 PM
Last modification on : Tuesday, January 4, 2022 - 5:57:01 AM

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  • HAL Id : hal-03440712, version 1

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Noémie Petitjean, Marie Maumus, Gilles Dusfour, Jade Berthelot, Emmanuel Belamie, et al.. Mechanical stimulation and mechanobiological characterization of cartilage micropellets with a single and new custom-made device. 1er congrès français de médecine régénératrice et biothérapies, Oct 2020, Montpellier, France. ⟨hal-03440712⟩

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