15502-m-pleumeekers

Cell behavior in co-cultures Using a xenogeneic culture system enabled us to determine the contribution of each individual cell type (i.e. h BMSCs, b ECs or b NCs) to cartilage matrix production using species-specific gene-expression analyses. First, GAPDH- gene expression was analysed after 5 weeks of in- vitro culture. h BMSC/ b ECs and h BMSC/ b NCs contained cells from both bovine (EC or NC) and human (BMSC) origin. (Table 3) Then, chondrogenic gene expression was analysed by the ACAN -gene. In a growth-factor-free environment, h BMSCs hardly expressed hsACAN . Besides, hsACAN was hardly expressed in h BMSC/ b ECs or h BMSC/ b NCs either. Conversely, h BMSC/ b ECs or h BMSC/ b NCs - containing solely 20% bovine chondrocytes - expressed as much or even higher levels of bsACAN compared to their 100% controls: h BMSC/ b ECs vs b ECs 3.96 ± 6.13 fold change; h BMSC/ b NCs vs b NCs 4.56 ± 6.18 fold change. These data indicate that the formed cartilage matrix was from chondrocyte origin, which suggests a more trophic role for h BMSCs herein. hsGAPDH bsGAPDH h BMSC 24.1 ± 2.78 nd b EC nd 25.0 ± 2.4 b NC nd 23.4 ± 1.7 h BMSC/ b EC 24.0 ± 1.7 26.0 ± 2.7 h BMSC/ b NC 24.8 ± 1.7 25.1 ± 2.4 Table 3. Gene-expression analyses, 5 weeks after in vitro culture. Data are shown as mean CT-values ± SD of housekeeping genes. nd = not detected (ct-value > 35.00) ; hsGAPDH = human-specific GAPDH ; bsGAPDH = bovine-specifiec GAPDH ; 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. Cartilage formation in vivo After 8 weeks of implantation all constructs were identified and harvested. Constructs containing b NCs or h BMSC/ b NCs resembled cartilage tissue in both color and texture, while constructs containing h BMSCs, b ECs or h BMSC/ b ECs were still fragile and did not express a cartilaginous appearance. (Figure 3) After subcutaneous implantation, none of the constructs had mineralized or ossified. Collagen type II was abundantly present in constructs containing b EC or b NCs (both single-cell-type and mixed-cell-type populations), but was not visible in constructs containing h BMSCs only. (Figure 3). In vivo , h BMSC/ b ECs and h BMSC/ b NCs contained similar quantities of cartilage matrix as constructs containing chondrocytes only. Moreover h BMSC/ b NCs produced significantly more sGAG ( p =0.004) compared to h BMSC/ b ECs. (Figure 4A) Collagen production demonstrated a similar trend, albeit without statistical significant differences. (Figure 4B) Normalization of the data to their initial number of seeded PCs revealed more distinct 117 CO-CULTURE: A PROMISING CELL-BASED THERAPY FOR FACIAL CARTILAGES 6