15502-m-pleumeekers

Biomechanical analysis For mechanical characterization, constructs of 2.0 mm thick and 5 mm in diameter were created. 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, 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). 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. Gene-expression analyses To further evaluate the contribution of each individual cell type (i.e. h BMSCs, b ECs or b NCs) to cartilage matrix formation, species-specific gene-expression analyses was achieved. For total RNA isolation, alginate was dissolved in ice-cold 55 mM sodium citrate and 20 mM EDTA in 150 mM NaCl and centrifuged. Each cell-pellet was subsequently suspended in 1 mL RNA- BeeTM (TEL-TEST, USA). 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. 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. Analysed genes are listed in table 2. RT-PCR was performed using TaqMan® Universal PCR Mastermix (Applied Biosystems) or 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 - ΔΔCT formula. Statistical analysis All data were analyzed with PSAW statistics 20.0 (SPSS Inc., USA). The mean and standard deviation were presented. In-vitro data represents at least three independent donors per condition performed in triplicate. For statistical evaluation of these experiments, a mixed linear model was used followed by Fisher's least significant post-hoc comparisons tests. ‘Condition’ and ‘time point’ were defined as fixed factors in the model. ‘Donor’ and ‘sample number’ were treated as random factors. For the in-vivo experiments six constructs per condition were used, with duplicate samples for three independent donors. For statistical evaluation of these experiments, one-way ANOVA was used followed by Fisher's least significant difference post-hoc comparisons tests. For all tests, values of p <0.05 were considered statistically significant. 113 CO-CULTURE: A PROMISING CELL-BASED THERAPY FOR FACIAL CARTILAGES 6