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Biomechanical analysis In order to distinguish the mechanical strength of alginate itself, cell containing constructs were prepared and directly taken for mechanical testing as described previously. [40] In short, for mechanical characterization of engineered cartilage constructs after in vivo cell culture, constructs 2.5 mm thick and 5 mm in diameter were used. The samples were placed in close- fitting Ø 5 mm stainless steel cylindrical wells. Mechanical testing was performed with a materials testing machine (Zwick Z005, Ulm, Germany) equipped with a 10 N load cell, a built- in displacement control, and a cylindrical, plane ended, stainless steel indenter (Ø 1.2 mm). During mechanical testing the samples were immersed in PBS. Stress-strain testing was performed: the samples were compressed to a final height of 0.5 mm at a loading rate of 5 mm per minute. An in-house Matlab® script was used to locate the sample surface and measure the sample thickness. Force-displacement curves were then converted to stress- strain curves. Measurements of compressive modulus at 40% strain, E40%, were determined for every sample. Gene-expression analyses For total RNA isolation, alginate was dissolved in ice-cold 55 mM sodium citrate and 20 mM Ethylene Diamintetraacetate (EDTA) in 150 mM NaCl and centrifuged. Each cell-pellet was subsequently suspended in 1 mL RNA-Bee TM (TEL-TEST, USA). For total RNA isolation from pellets, pellets were manually homogenized and suspended in 300 μL/pellet RNA-Bee TM . RNA was extracted with chloroform and purified from the supernatant using the RNAeasy Micro Kit (Qiagen, Germany) according to the manufacturer’s guidelines by on-column DNA- digestion. Extracted total RNA was quantified using NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at 260/280 nm. Total RNA of each sample was reverse transcribed into cDNA using RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, Germany). For quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis, forward and reverse primers were designed using PrimerExpress 2.0 software (Applied Biosystems, USA) to meet TaqMan or SYBR Green requirements. Gene specificity of all primers was guaranteed by Basic Local Alignment Search Tool (BLASTN). Analysed genes are listed in table 2. qRT-PCR was performed using qPCR Mastermix Plus for SYBR Green (Eurogentec, the Netherlands) according to the manufacturers’ guidelines and using ABIPRISM® 7000 with SDS software version 1.7 (Applied Biosystems, The Netherlands). Relative gene expressions were calculated by means of the 2 -ΔCT formula. 92 CHAPTER 5