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spectrofluorometer (Wallac 1420 Victor 2; Perkin-Elmer, Wellesley, USA), using an extinction filter (340 nm) and an emission filter (590 nm). Glycosaminoglycan content Sulfated glycosaminoglycans (sGAGs) were quantified using the 1,9-Dimethylmethylene blue (DMMB) dye-binding assay. To be suitable for cell cultures containing alginate, the DMMB- pH-level was decreased to pH 1.75, as described previously. [212] 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 sulphate C (shark) as a standard. For each sample, the amount of sGAG was corrected for the amount of DNA. Hydroxyproline content The hydroxyproline content was quantified using a method described previously. [213] Briefly, the papain digests were hydrolyzed with equal volumes of 12 M HCL at 108 o C for 18–20 hours. Samples were then dried and redissolved in 150 µL water. Hydroxyproline contents were measured using a colorimetric method (extinction, 570 nm), with chloramine-T and dimethylaminobenzaldehyde as reagents and hydroxyproline (Merck, Damstadt, Germany) as a standard. Histological evaluation of the extracellular matrix After eight weeks of subcutaneous implantation, alginate discs were harvested, set in 2% agarose, fixed in 4% formalin in PBS and embedded in paraffin. Paraffin-embedded sections (6 μm) were deparaffinized and rehydrated. Immunohistochemistry for collagen type II, elastin and human vimentin To allow the use of monoclonal mouse antibodies on constructs which have been implanted in athymic mice, we used a method to couple the first and second antibody before applying them on the sections to prevent unwanted binding of the anti-mouse antibodies to mouse- immunoglobulins. [214] In short, primary antibodies were pre-coupled overnight with goat anti-mouse biotin at 4 o C (Jackson Laboratories, Bar Harbor, USA), followed by a two-hour incubation in 0.1% normal mouse serum (CLB, Amsterdam, the Netherlands) in order to capture the unbound second antibody. Antigen retrieval for the collagen type II (Developmental Studies Hybridoma Bank, Iowa, USA) antibody was performed through incubation with 0.1% pronase in PBS for 30 minutes at 37 o C, continued with a 30 minutes incubation with 1% hyaluronidase in PBS for at 37 o C. Antigen retrieval for elastin (BA4) required incubation with 0.25% trypsin in PBS for 20 minutes at 37 o C. Non-specific binding sites were blocked with 10% goat serum in PBS and sections were stained with the pre-treated primary antibodies against collagen type II (1:100) or elastin (1:1000) for 60 minutes. Sections were than incubated with enzyme-streptavidin conjugate (Label, 1:100, Biogenex, HK-321-UK, California, USA) in PBS/1% BSA, followed by incubation with Neu Fuchsin substrate (Chroma, Köngen, Germany). Positive staining for collagen II and elastin was confirmed with the use of native ear cartilage. A mouse monoclonal negative control antibody (mIgG1: X0931,Dako) was used as an isotype control. 66 CHAPTER 4