15502-m-pleumeekers

Flat constructs (8 mm diameter; 2 mm height) were processed as previously described. [40] Constructs were either cultured in vitro or directly implanted subcutaneously in mice. In-vitro culture was performed for either three or five weeks in growth-factor-free medium consisting of DMEM supplemented with sodium pyruvate (Gibco), L-proline, supplemented Insulin Transferrine Selenium (B&D Bioscience, USA), dexamethasone, and L-ascorbic acid 2- phosphate. Medium was changed twice a week. After three and five weeks, constructs were processed for biochemical and gene-expression analysis. For in-vivo studies, a total number of sixteen nine-week-old female NMRI nu/nu mice (Charles River Laboratories, the Netherlands) were used. Two separate incisions were made along the central line of the spine, after which four separate subcutaneous dorsal pockets were prepared by blunt dissection. After eight weeks, animals were terminated and samples were explanted for histological, biomechanical and biochemical analyses. Animal experiments were carried out with approval of the Animal Ethical Committee (EMC 2429). Biochemical evaluation of the extracellular matrix Alginate constructs were digested overnight at 56 o C in papain (250 μg/mL in 0.2 M NaH2PO4, 0.01 M EDTA, containing 5 mM L-cystein; pH 6.0). After digestion, samples were subjected to biochemical analyses to determine the DNA, glycosaminoglycan (GAG), and hydroxyproline contents, as described previously. [40] In short, the amount of DNA was determined by Ethidium bromide (GibcoBR1), using calf thymus DNA as a standard. Sulfated GAGs (sGAGs) were quantified by the 1,9-Dimethylmethylene blue (DMMB) dye-binding assay (pH 1.75), using shark chondroitin sulphate C as a standard. For the hydroxyproline content, digests were hydrolysed, dried and redissolved in 150 µL water. Hydroxyproline contents were measured using chloramine-T and dimethylaminobenzaldehyde as reagents and hydroxyproline (Merck, Germany) as a standard. Histological evaluation of the extracellular matrix After eight weeks of subcutaneous implantation, constructs were harvested, set in 2% agarose, fixed in 4% formalin in PBS and embedded in paraffin. Paraffin-embedded sections (6 μm) were deparaffinised and rehydrated. To allow the use of monoclonal mouse antibody collagen type II (II-II6B3 1:100; Developmental Studies Hybridoma Bank, USA) on constructs which had been implanted in mice, the primary antibody was pre-coupled overnight with goat anti-mouse biotin at 4 o C (1:500; Jackson Laboratories, USA), followed by a two-hour incubation in 0.1% normal mouse serum (CLB, the Netherlands), to prevent unwanted binding of the anti-mouse antibodies to mouse immunoglobulins. [214] Antigen retrieval was performed through incubation with 0.1% pronase for 30 minutes at 37 o C, continued with a 30 minutes incubation with 1% hyaluronidase at 37 o C. Non-specific binding sites were blocked with 10% goat serum and sections were stained with the pre- treated antibodies for 60 minutes. Sections were than incubated with enzyme-streptavidin conjugate (Label, 1:100, Biogenex, HK-321-UK, USA) in PBS/1% BSA, followed by incubation with Neu Fuchsin substrate (Chroma, Germany). 112 CHAPTER 6