15502-m-pleumeekers

RESULTS Cartilage formation in vitro After three weeks, DNA content significantly decreased over time in constructs containing h BMSCs ( p =0.019), b ECs ( p =0.010) or h BMSC/ b ECs ( p <0.001), though remained stable in b NCs or h BMSC/ b NCs. (Figure 1) Similar results were obtained after 5 weeks. (Supplementary figure 1) Figure 1. Cell content of constructs containing h BMSCs and/or chondrocytes, 3 weeks after in-vitro culture. DNA content was determined at baseline before culture (dotted line), being on average 4.29 ± 0.96 μg DNA per construct, and after 3 weeks of culture. * indicates a p -value smaller than 0.05 compared to baseline. Data are shown as mean ± SD. For statistical evaluation, a mixed model was used followed by a Fisher’s least significant post-hoc comparisons test . h BMSC = human Bone-marrow-derived Mesenchymal Stem Cell ( n=3 experiments with 3 different donors) ; b EC = bovine Ear Chondrocyte ( n=3 experiments with 3 pools of donors) ; b NC = bovine Nasal Chondrocyte ( n=3 experiments with 3 pools of donors). Per experiment, 3 samples were used for analyses. Since constructs were cultured in the absence of chondrogenic factors, constructs containing solely h BMSCs produced very little sGAG (Figure 2A) and collagen (Figure 2B). To demonstrate the additional effect of h BMSCs in mixed-cell-type populations, a control condition - containing similar numbers of chondrocytes without the supplementation of h BMSCs - was evaluated (Figure 2 white lines). The additional effect of h BMSCs in mixed-cell-type populations was dependent on the chondrocyte-source used: the addition of h BMSCs to b NCs demonstrated a significant increase in the production of sGAG ( p =0.012) and collagen ( p =0.007) compared to their controls ; no additional effects were observed in h BMSCs/ b ECs. h BMSC/ b NCs contained significantly more sGAG ( p =0.026) and collagen ( p =0.040) compared to h BMSC/ b ECs. Normalization of the data to their initial number of seeded primary chondrocytes (PCs) revealed more distinct differences between mixed-cell-type and single- cell-type populations: h BMSCs/ b ECs and h BMSC/ b NCs produced more cartilage matrix per initial seeded chondrocyte than chondrocytes only. (Figure 2, right ) Similar results were 115 CO-CULTURE: A PROMISING CELL-BASED THERAPY FOR FACIAL CARTILAGES 6