15502-m-pleumeekers

To study whether cells in the alginate constructs harvested after in-vivo implantation were originated from human origin, a monoclonal mouse anti-human vimentin antibody was used (AMF-17b, Developmental Studies Hybridoma Bank, Iowa, USA), as described previously. [56] In short, slides were incubated in 3% aqueous hydrogen peroxidase solution, in order to inhibit endogenous peroxidase and allow for peroxidase-antiperoxidase staining. Antigen retrieval required incubation in Rodent Decloaker® for 60 minutes at 95 o C. Non-specific binding sites were blocked with Rodent Block M® followed by a 30 minute staining with vimentin (1:40, V6630). Thereafter, the MM-polymer-HRP® secondary antibody was used, succeeded by incubation with 3’diaminobenzidine chromogen solution. Tissue specificity was confirmed by the absence of staining on sections of mouse liver tissue. A mouse monoclonal negative control antibody was used as an isotype control. Von Kossa/Thionin/Resorcin-Fuchsin staining To evaluate tissue calcification, a Von Kossa staining was performed. Slides were immersed in 5% silver nitrate solution for 10 minutes, rinsed in MiliQ and exposed to light for another 10 minutes. Sections were counterstained with Nuclear fast red (Merck). sGAGs were visualized using 0.4% Thionin in 0.01 M aqueous sodium acetate (pH 4.5) for 5 minutes at room temperature. To check whether we stained sGAGs rather than the remaining alginate, sections were pre-treated with 20 mM EDTA. As EDTA treatment did not change the intensity and/or localization of Thionin on our slides, we confirmed that alginate did not interfere with our sGAG-staining protocol. The presence, as well as the arrangement of the elastic fibers, were visualized using Weigert’s Resorcin-Fuchsin staining (Klinipath, Duiven, the Netherlands). We used a semi-quantitative scoring system - The Bern Score [215] - to evaluate the chondrogenic capacity of alginate-encapsulated cells after subcutaneous implantation. (Table 2) In short, the scoring system evaluates cartilage formation based on three elements: (1) the uniformity and/or intensity of the Thionin and collagen type II staining; (2) the distance between cells and the extent of matrix produced; and (3) the cellular morphology. Each category has scores ranging from 0 to 3, resulting in a possible minimum collective score of 0 and a maximum of 9. Samples that were either not visible anymore after eight weeks of subcutaneous implantation or were dissolved during formalin fixation were scored 0. 67 CARTILAGE-FORMING CAPACITY OF SEVERAL CELL SOURCES 4