title: Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering.
abstract: OBJECTIVE
  To investigate the interaction between mesenchymal stem cells (MSCs) and bone grafts using two different cultivation methods: static and dynamic.
  
  
  METHODS
  MSCs were isolated from rat bone marrow. MSC culture was analyzed according to the morphology, cell differentiation potential, and surface molecular markers. Before cell culture, freeze-dried bone (FDB) was maintained in culture for 3 d in order to verify culture medium pH. MSCs were co-cultured with FDB using two different cultivation methods: static co-culture (two-dimensional) and dynamic co-culture (three-dimensional). After 24 h of cultivation by dynamic or static methods, histological analysis of Cell adhesion on FDB was performed. Cell viability was assessed by the Trypan Blue exclusion method on days 0, 3 and 6 after dynamic or static culture. Adherent cells were detached from FDB surface, stained with Trypan Blue, and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture. Statistical analyses were performed with SPSS and a P < 0.05 was considered significant.
  
  
  RESULTS
  The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures. Rat MSCs were positive for CD44, CD90 and CD29 and negative for CD34, CD45 and CD11bc. FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH (P > 0.05). In histological analysis, there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods (P < 0.05). The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method. On day 0, the cell viability in the dynamic system was significantly higher than in the static system (P < 0.05). There was a statistical difference in cell viability between days 0, 3 and 6 after dynamic culture (P < 0.05). In static culture, cell viability on day 6 was significantly lower than on day 3 and 0 (P < 0.05).
  
  
  CONCLUSIONS
  An alternative cultivation method was developed to improve the MSCs adhesion on FDB, demonstrating that dynamic co-culture provides a superior environment over static conditions.
authors.affiliation: Fabiany da Costa Gonçalves, Ana Helena da Rosa Paz, Priscila Schmidt Lora, Eduardo Pandolfi Passos, Elizabeth Obino Cirne-Lima, Embryology and Cell Differentiation Laboratory, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos 2350, Porto Alegre, RS, 90035-903, Brazil.
authors.forename: Fabiany da Costa
authors.initials: Fda C
authors.lastname: Gonçalves
authors.forename: Ana Helena da Rosa
authors.initials: AH
authors.lastname: Paz
authors.forename: Priscila Schmidt
authors.initials: PS
authors.lastname: Lora
authors.forename: Eduardo Pandolfi
authors.initials: EP
authors.lastname: Passos
authors.forename: Elizabeth Obino
authors.initials: EO
authors.lastname: Cirne-Lima