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after surgery, with approval of the local medical ethics committee (no. 152/08). The collected nasoseptal cartilage was washed with PBS containing penicillin-streptomycin and stored in standard culture medium at 37°C, 5% CO 2 , until further use. The 47 nasoseptal cartilage biopsies were divided in three pools for the chondrocyte isolations. Bone marrow aspirate was collected from three donors (mean age 70 years, 2 males, 1 female) during total hip replacement surgery, after acquiring written patient consent. The isolations of NCs from the cartilage and MNCs from the bone marrow were performed by CellCoTec (Bilthoven, the Netherlands). Patented clinically applied protocols were used to isolate the cells within the hour [347]. In brief, cartilage pieces were digested enzymatically under mechanical stimulation. Upon rapid digestion, any remaining debris was filtered out with a 100-µm cell strainer. For the collection of MNCs, the bone marrow aspirate was relieved of its erythrocyte content using lysis buffer. Standard cell buffer was used for washing steps. Cell numbers and viability were measured using the Bürker-Türk method with trypan-blue exclusion. Seeding of bilayer BNC scaffolds with MNCs and NCs First, bilayer BNC scaffolds were freeze-dried in order to improve cell uptake during the cell seeding. To further improve the retention of cells in the scaffolds, cells were seeded in 1.1 % w/w alginate solution (CellMed AG). Cells encapsulated in alginate were then seeded in bilayer BNC scaffolds as a combination of 80% freshly isolated human MNCs and 20% freshly isolated human NCs at a total cell concentration of 20 × 10 6 cells/ml alginate (MNC/NC, n=4 ). 200 µl of the cell-alginate suspension was seeded into the porous layer of each scaffold. A cell-free alginate solution acted as a negative control (Cell-free, n=4 ). Subsequently, the alginate was instantaneously crosslinked with sterile 100 mM CaCl 2 for 10 minutes and washed with 0.9% NaCl, followed by high glucose DMEM (Dulbecco's Modified Eagle's Medium). Subcutaneous implantation of constructs in mice To evaluate the stability of the bilayer BNC scaffolds and neocartilage formation in vivo , MNC/NC-seeded and cell-free bilayer BNC scaffolds were implanted subcutaneously on the dorsal side of 9-week-old nude female mice ( n=2 ; NMRI nu/nu, Charles River Laboratories, the Netherlands). Mice were placed under general anesthesia using 2.5% isoflurane. Two separate subcutaneous incisions of approximately 1 cm were made along the central line of the spine (1 at the shoulders and 1 at the hips), after which 4 separate subcutaneous pockets were prepared by blunt dissection of the subcutaneous tissue. The overall behavior and wound healing at the implant sites were assessed macroscopically over the implantation period. Eight weeks after subcutaneous implantation, animals were terminated and samples were explanted. Each sample was cut in half and one part was used for histology and the other part for biomechanical and biochemical analyses. Animal experiments were carried out with approval of the local Animal Experiments Committee of the Erasmus MC, Rotterdam, the Netherlands (EMC 2429). Histological and immunohistochemical analyses After 8 weeks of subcutaneous implantation, constructs were harvested, set in 2% agarose, fixed in 10% neutral buffered formalin solution, embedded in paraffin and sectioned (6 μm). 156 CHAPTER 8