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previously described. [210] In short, alginate suspensions containing 4x10 6 cells/ml were injected into a custom-designed slab mold consisting of two calcium-permeable membranes (Durapore® 5.0 μm membrane filters, Millipore) rigidly supported by stainless-steel meshes and separated by a stainless-steel casting frame. Part of these constructs were harvested after five weeks of cell-culture for analyses and a part was implanted subcutaneously on the dorsal side of athymic mice. For the in-vivo experiments a total of six constructs per cell source were used, with duplicate samples for three different donors. Subcutaneous implantation in vivo In total, seventeen nine-week old, female NMRI nu/nu mice (Charles River Laboratories, the Netherlands) were used to evaluate the performance of constructs cultured with or without TGFβ1. Mice were placed under general anesthesia using 2.5% isofluorane. Two separate subcutaneous incisions of approximately 1.0 cm were made along the central line of the spine (one at the shoulders and one at the hips), after which four separate subcutaneous pockets were prepared by blunt dissection of the subcutaneous tissue. For implantation the alginate constructs were randomly assigned to these four pockets. Eight weeks after subcutaneous implantation, animals were sacrificed and samples were explanted for histological, biomechanical and biochemical analyses. Animal experiments were carried out with approval of the local Animal Experiments Committee of the Erasmus MC and were approved as outlined in the national Animals Act (EMC 2429). Gene expression analyses For total RNA isolation, alginate was dissolved in ice-cold 55 mM sodium citrate (150 μL/bead) and 20 mM Ethylene Diamintetraacetate (EDTA) in 150 mM NaCl and centrifuged at 2.5 G for 8 minutes. Each pellet was subsequently suspended in 1 mL RNA-Bee TM (TEL-TEST, Fiendswood, USA). RNA was extracted with chloroform and purified from the supernatant using the RNAeasy Micro Kit (Qiagen, Hilden, 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, USA) at 260/280 nm. Total RNA of each sample was reverse transcribed into cDNA using RevertAid TM 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, Foster City, CA) to meet TaqMan or SYBR Green requirements. They were designed to bind separate exons to avoid co-amplification of genomic DNA. Gene specificity of all primers was guaranteed by Basic Local Alignment Search Tool (BLASTN), as listed in table 1. The following genes were analyzed: Aggrecan ( ACAN ), Collagen type IIA1 ( COL2A1 ), Collagen type X ( COL10 ), Alkaline Phosphatase ( ALP ), and Matrix MetalloProteinase-13 ( MMP13 ). GlycerAldehyde 3- Phosphate DeHydrogenase ( GAPDH ), and Hypoxanthine PhosphoRibosylTransferase 1 ( HPRT1 ) were used as housekeeping genes. The expression of GAPDH and HPRT1 did not differ between cell sources and both were used to calculate the best housekeeper index. [211] Using repeated pair-wise correlation analysis, data were normalized by calculating the ‘best housekeeper index’. (Data not shown) Polymerase Chain Reactions were performed using 64 CHAPTER 4