15502-m-pleumeekers

To examine proteoglycans present in the newly synthesized ECM, deparaffinized sections were stained with Safranin O and fast green. To allow the use of the monoclonal mouse antibody collagen type II (II-II6B3, 1:100; DSHB) on constructs which had been implanted in nude mice, we coupled the first and second antibody before applying them on the sections to prevent unwanted binding of the anti-mouse antibodies to mouse immunoglobulins, as described previously [214]. In short, the primary antibody was pre-coupled overnight with goat anti-mouse biotin at 4°C (1:500; Jackson Laboratories, ME, USA), followed by a 2 hour incubation in 0.1% normal mouse serum (CLB, the Netherlands) in order to capture the unbound second antibody. Antigen retrieval was performed through incubation with 0.1% pronase in PBS for 30 minutes at 37°C, followed by a 30 minute incubation with 1% hyaluronidase in PBS at 37°C. Non-specific binding sites were blocked with 10% goat serum in PBS and sections were stained with the pre-treated antibodies for 60 minutes. Sections were then incubated with enzyme-streptavidin conjugate (1:100; Biogenex, California, USA) in PBS/1% BSA, followed by incubation with Neu Fuchsin substrate (Chroma, Germany). Positive staining for type II collagen was confirmed with the use of native ear cartilage. A monoclonal mouse IgG1 antibody (X0931; Dako) was used as a negative control. Biochemical analysis Sulfated glycosaminoglycans (sGAG) were quantified using the 1,9-Dimethylmethylene blue (DMMB) dye-binding assay. First, alginate was dissolved in 55 mM sodium citrate and digested overnight at 56°C in papain (250 µg/ml in 0.2 M NaH2PO4, 0.01 M EDTA, containing 5 mM L- cysteine; pH 6.0). To be suitable for cell cultures containing alginate, the DMMB-pH-level was decreased to pH 1.75, as described previously [353]. The metachromatic reaction of DMMB was monitored using a spectrophotometer. Absorption ratios of 540 and 595 nm were used to determine the sGAG content with chondroitin sulfate C derived from shark as a standard. The amount of sGAG was expressed per tissue wet weight ( n=4 ). Biomechanical analysis Mechanical properties of the retrieved constructs ( n=8 , MNC/NC-seeded and cell-free scaffolds) and non-implanted bilayer BNC scaffolds containing cell-free alginate solution ( n=5, non-implanted group) were assessed with uniaxial materials testing machine (Z005, Zwick GmbH, Germany) equipped with a 10 N load cell, a cylindrical plane-ended stainless steel indenter (Ø 0.35 mm) and a built-in displacement control. Bilayer BNC scaffolds were placed in close-fitting stainless steel cylindrical wells containing PBS supplemented with 1% antibiotic/antimycotic solution. Stress relaxation testing was performed as described previously [354]. Briefly, a preload of 3 mN was first applied on the sample to locate the sample surface and measure sample thickness, and held for 5 minutes. Five successive strain steps were then applied in 5% increments of the original sample thickness, and specimens were left to relax for 20 minutes at each step. The hold time was defined as the time necessary to reach equilibrium. Two locations were tested on each sample (center of the sample, and 1.2 mm off-center). Measurements of maximum stress (σ max ), instantaneous modulus (Ein) and equilibrium modulus (Eeq) were computed from the stress-strain curves, which are normalized for sample thickness. Additionally, a relaxation half-life time (t 1/2 ), defined as the 157 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8