15502-m-pleumeekers

Table 1. Construct conditions. Cell density is displayed as the number of cells (nc) in 1 milliliter of alginate. h AMSC = human Adipose-tissue- derived Mesenchymal Stem Cell ; h BMSC = human Bone-marrow-derived Mesenchymal Stem Cell ; b AC = bovine Articular Chondrocyte. Flat constructs (8 mm diameter ; 2 mm height) were processed as previously described. [40] In short, alginate suspensions were injected into a custom designed slab mold consisting of 2 calcium-permeable membranes (Durapore® 5.0 μm membrane filters, Millipore) rigidly supported by stainless-steel meshes and separated by a stainless-steel casting frame. Alginate was instantaneously gelated for 30 minutes in 102 mM CaCl 2 and thereafter washed with 0.9% NaCl and HG-DMEM. Sterile biopsy punches (Spengler, Asnières sur Seine, France) were used to create alginate constructs suitable for mechanical testing. Constructs were either cultured in vitro or directly implanted subcutaneously in mice. (Figure 1A) In vitro , constructs were cultured in ‘basic medium’ containing serum-free HG-DMEM supplemented with 50 µg/mL gentamycin; 0.5 µg/mL Fungizone; 1 mM sodium pyruvate (Gibco); 40 μg/mL L-proline; supplemented Insulin Transferrine Selenium (ITS+ ; B&D Bioscience, Bedford, MA, USA); 10 -7 M dexamethason; and 25 μg/mL L-ascorbic acid 2- phosphate without the addition of growth factors. For each condition referred to in table 1, 3 independent donors were used in triplicate (total n=54 ). After 3 and 5 weeks, constructs were processed for biochemical and gene-expression analysis. In-vivo studies were completed after 8 weeks of subcutaneous implantation. In total, 10 9-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 (1 at the shoulders and 1 at the hips), after which 4 separate subcutaneous dorsal pockets were prepared by blunt dissection. For each condition referred to in table 1, 3 independent donors were used in duplicate (total n=36 ). Moreover, cell-free constructs were used as controls ( n=4 ). For implantation, alginate constructs were randomly assigned to these 4 pockets. After 8 weeks, animals were sacrificed and samples were explanted for histological, biomechanical and biochemical analyses. Animal experiments were carried out to the guidelines prescribed by the Dutch National Institutes of Health, and were approved by the Dutch equivalent of the Institutional Animal Care and Use Committee, the Erasmus MC Dier Ethische Commissie (protocol # EMC 2429). Human stem cells Bovine chondrocytes Source Cell density (x10 6 ) Source Cell density (x10 6 ) h AMSC h AMSCs 4 nc/mL x x h BMSC h BMSCs 4 nc/mL x x b AC x x b ACs 4 nc/mL h AMSC/ b AC h AMSCs 3.2 nc/mL b ACs 0.8 nc/mL h BMSC/ b AC h BMSCs 3.2 nc/mL b ACs 0.8 nc/mL Control b AC x x b ACs 0.8 nc/mL 88 CHAPTER 5