15502-m-pleumeekers

LSAB+System-HRP kit (Dako), which is based on the labeled streptavidin biotin method, was used according to the manufacturer’s protocol. Sections were counterstained with hematoxylin. Gene expression analysis Samples were harvested after 2, 4 and 6 weeks of in vitro culture, snap-frozen and stored at - 80°C until analyzed. For total RNA isolation, frozen constructs were placed in 2 ml microcentrifuge tubes in quadruples and 100 µl of lysis buffer (10 µl β-Mercaptoethanol per 1 ml Buffer RLT; Qiagen, Germany) was added to each tube. The samples were disrupted and homogenized for 2 minutes using a TissueLyser LT (Qiagen). Subsequently, 500 µl of lysis buffer was added to each tube and the cell lysate was used for total RNA isolation using RNeasy Mini Kit (Qiagen), according to manufacturer´s protocol. Total RNA was quantified using a multimode microplate reader (Infinite M200 Pro, Tecan AG) at 260/280 nm. cDNA was synthesized from the extracted RNA using QuantiTect Reverse Transcription Kit (Qiagen), according to manufacturer´s protocol, in a PeqSTAR thermocycler (96 Universal Gradient, PeqLab, Germany). For real-time two-step RT-PCR analysis, the sense and antisense primers used are listed in Table 1. The following genes were analyzed: aggrecan ( ACAN ), collagen type IIA1 ( COL2A1 ), versican ( VCAN ) and collagen type IA1 ( COL1A1 ). Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as housekeeping gene. Real-time two-step RT-PCR was performed using the Real Time ready RNA Virus Master assay and LightCycler ® 2.0 instrument (Roche, Germany). Relative gene expression levels were calculated by means of the 2 -ΔCT formula. UPL probe# Sense primer Antisense primer Target genes ACAN 79 5ʹ-TGCAGCTGTCACTGTAGAAACTT-3ʹ 5ʹ-ATAGCAGGGGATGGTGAGG-3’ COL1A1 15 5ʹ-ATGTTCAGCTTTGTGGACCTC-3ʹ 5ʹ-CTGTACGCAGGTGATTGGTG-3ʹ COL2A1 19 5ʹ-CCCTGGTCTTGGTGGAAAC-3ʹ 5ʹ- TCCTTGCATTACTCCCAACTG-3ʹ VCAN 54 5ʹ-GCACCTGTGTGCCAGGATA-3ʹ 5ʹ-CAGGGATTAGAGTGACATTCATCA-3ʹ Housekeeping gene GAPDH 60 5ʹ-GCTCTCTGCTCCTCCTGTTC-3ʹ 5ʹ-ACGACCAAATCCGTTGACTC-3ʹ Table 1. Sequences of target genes and reference gene for real-time two-step PCR. Cell study II: performance of bilayer BNC scaffolds in vivo Rapid isolation of human nasoseptal chondrocytes and bone marrow mononuclear cells Nasoseptal cartilage was obtained from male and female patients ( n=47 ; mean age 31 years; age range 18-69 years) undergoing routine reconstructive septorhinoplasty at the Department of Otorhinolaryngology of Ulm University Medical Center (Ulm, Germany) as waste material 155 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8