Mixed cells revealed a weaker collagen III signal compared with monocultures PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 (Figure 4C). Deposition of collagen X was detected in BMSC monocultures. In all other culture regimens, there was only poor collagen X staining (Figure 4D). Overall, immunofluorescence staining of ECM deposited collagens I, II, III, and X supported data of collagen quantification with ELISA and dot blot.Biomechanical propertiesYoung's modulus under 40 to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 50 strain tended to be higher in 3 out of 4 samples (Figure 5B). Aggregate modulus at equilibrium was lowered in almost any cases for all co- and tri-cultured samples (Figure 5C). Hydraulic permeability of cartilage co- and tri-cultures was increased in comparison with monocultures in 3 out of 4 samples (Figure 5D). Overall, biomechanical tests showed that ECM in monocultured BMSC and mixed fibrin gel constructs seemed to be stiffer and less porous and thus in general of higher integrity and stability--with respect to Young's modulus, aggregate modulus, and hydraulic permeability--than newly formed matrix in co- and tri-cultures. Mechanical parameters in chondrocyte cocultures were partly superior to monocultures.Quantification of cytokines and glycosaminoglycan in culture supernatantsWe determined mechanical properties and load capacity of newly generated ECM. Unconfined mechanical testing indicated that cocultured BMSCs, chondrocytes, or a mixture of both cell types exhibited a decrease in Young's modulus (0 to 10 strain) in 3 out of 4 samples of each cell type (Figure 5A) and a decrease in 4 out of 4 samples (BMSCs) or 3 out of 4 samples (mixed cultures) at 40 to 50 strain (Figure 5B). In cocultured chondrocytes,To quantify representative pro-inflammatory cytokines released into culture supernatants, we bmjopen-2016-011952 performed ELISAsLeyh et al. Stem Cell Research Therapy 2014, 5:77 http://stemcellres.com/content/5/3/Page 9 ofFigure 4 Immunofluorescence staining of collagens. Representative immunofluorescence staining images of collagens (A) I, (B) II, (C) III, and (D) X detected on cryosections after 28 days of culture. Bone marrow-derived stem cells (BMSCs), mixed cultures (BMSCs and chondrocytes in a ratio of 1:1), or chondrocytes were monocultured (F) or co-/tri-cultured with osteoarthritis cartilage (FC). Nuclei were stained with 4,6-diamidino2-phenylindole (DAPI) (blue color). Scale bar is 100 m. N = 5.for IL-1, IL-6, and IL-8. Results revealed that BMSCs, mixed cultures, and chondrocytes cocultured with cartilage explants secrete significant higher IL-1 levels at day 28 than 2-Chloro-3-methoxyaniline monocultures. At day 7, only mixed tri-cultures secreted significant higher IL-1 levels (Figure 6A). Furthermore, cocultured BMSCs, mixed cultures, and chondrocytes had significantly elevated IL-6 and IL-8 levels at day 7 compared with monocultures. Over the culture time, IL-6 and IL-8 levels decreased significantly from day 7 to 28 in supernatants of cocultures (Figure 6B and C). In addition, we tested supernatants of explants cultured without cells, where we detected low levels of N-BOC-3-Fluoro-D-phenylalanine IL-1 for OA cartilage (1.1 ?0.7 pg/mL at day 7 and 3.0 ?0.7 pg at day 28) and for healthy cartilage (1.6 ?0.6 pg/mL at day 7 and 2.6 ?1.4 pg at day 28). We did not detect IL-6 at days 7 and 28 in cell-free OA cartilage explant supernatants and at day 28 in cell-free healthy cartilage explant supernatant, whereas at day 7 cell-free healthy cartilage supernatant contained some IL-6 (103 ?292 pg). IL-8 in supernatant of healthy cellfree cartilage explants was 4,.