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Cell study I: performance of bilayer BNC scaffolds in vitro Isolation and expansion of human nasoseptal chondrocytes Nasoseptal cartilage was obtained from 1 female patient (19 years) undergoing routine reconstructive septorhinoplasty at the Department of Otorhinolaryngology of Ulm University Medical Center (Ulm, Germany), as waste material after surgery, with approval of the local medical ethics committee (no. 152/08). The isolation of nasoseptal chondrocytes (NC) from the cartilage was done by enzymatic digestion of the tissue with 0.3% type II collagenase (Worthington Biochemical, NJ, USA) in growth medium (DMEM/Ham’s F-12 supplemented with 10% FBS and 0.5% gentamycin; Biochrom) for 16 hours at 37°C under agitation. Cells were separated by filtration through a 100-µm cell strainer and resuspended in growth medium. Subsequently, cell viability was determined using trypan blue staining and NCs were seeded in culture flasks at a density of 5,000 cells/cm 2 for expansion in monolayer culture. Once a cell confluence of about 85% was reached, the cells were trypsinized and cryopreserved. Cell culture of human chondrocytes in bilayer BNC scaffolds NCs were thawed and expanded one time in growth medium as described above. Once sub- confluent, cells were detached and resuspended in differentiation medium (NH ChondroDiff Medium; Miltenyi Biotec, Germany) supplemented with 0.5% gentamycin. Prior to cell seeding, bilayer BNC scaffolds ( n=30 ) were incubated in differentiation medium for 24 hours. The medium was discarded and 50 µl of cell suspension containing 1.0×10 6 cells was seeded into the porous scaffold layer (10,000 cells/mm 3 ). Cells were allowed to attach to the scaffolds for 4 hours in standard culture conditions (37°C, 5% CO 2 and 95% relative humidity), before transferring the seeded scaffolds to differentiation medium. Cell-seeded bilayer BNC scaffolds were cultured for up to 6 weeks and the medium was changed twice a week. Histological and immunohistochemical analyses During the in-vitro culture, constructs were harvested weekly for qualitative evaluation of neocartilage synthesized by the chondrocytes. The constructs were fixed in 10% neutral buffered formalin solution supplemented with 20 mM CaCl 2 at room temperature overnight, embedded in paraffin and sectioned (5 μm). For assessment of sulfated glycosaminoglycans (sGAG) and cell distribution within the bilayer BNC scaffolds, longitudinal sections were stained with Alcian blue and counterstained with Mayer’s hematoxylin. Furthermore, seeded scaffolds were processed for immunohistochemical staining to detect cartilage specific proteins such as aggrecan (AB1031; Millipore, MA, USA), type II collagen (II-II6B3; DSHB, IA, USA) and the dedifferentiation marker type I collagen (ab34710; Abcam, UK). An enzymatic antigen retrieval step was performed before incubation with primary antibodies. For aggrecan staining, slides were incubated in 0.5 U/ml chondroitinase ABC in PBS for 20 min at 32°C, followed by incubation with primary antibody for 1 hour at a 1/100 dilution. For type II collagen staining, slides were incubated in 1% hyaluronidase in PBS and 0.2% pronase (Calbiochem, Germany) in PBS, each for 15 min at 37°C, followed by incubation with primary antibody for 1 hour at a 1/4000 dilution. For type I collagen staining, slides were incubated in proteinase K (Dako, Germany) for 5 minutes at room temperature, followed by incubation with primary antibody for 1 hour at a 1/400 dilution. For visualization of these markers, the 154 CHAPTER 8