15502-m-pleumeekers

to alginate cultures of the other cell types. Again, a 80:20 ratio between h MSCs and b ACs was maintained. (Figure 1C) 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) ; pellets were digested overnight at 56 o C in proteinase K (1 mg/mL in Tris/EDTA buffer containing 185 µg/mL iodoacetamide and 1 µg/mL pepstatin A ; pH 7.6). After digestion, samples were subjected to biochemical analyses to determine DNA, sulfated-glycosaminoglycan (sGAG), 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. sGAGs were quantified by the 1,9- Dimethylmethylene blue (DMMB) dye-binding assay, using shark chondroitin sulphate C as a standard. To be suitable for cell cultures containing alginate, the DMMB-pH-level was adjusted to pH 1.75, as described previously. [212] 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. Collagen content was subsequently estimated from the hydroxyproline content, assuming that one collagen triple helix molecule contains 300 hydroxyproline residues. Histological evaluation After 8 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 evaluate tissue calcification, Von Kossa staining was performed. Slides were immersed in 5% silver nitrate solution for 10 minutes, rinsed in MilliQ and exposed to light for another 10 minutes. Excess silver nitrate was removed with 5% sodium-thiosulphate and slides were rinsed in distilled water afterwards. Sections were counterstained with Nuclear fast red (Merck). 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 2-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). 91 AMSCs OR BMSCs FULFILL A TROPHIC ROLE IN CO-CULTURE 5