Original Article
Copyright ©2012 Baishideng. All rights reserved.
World J Stem Cells. Feb 26, 2012; 4(2): 9-16
Published online Feb 26, 2012. doi: 10.4252/wjsc.v4.i2.9
Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering
Fabiany da Costa Gonçalves, Ana Helena da Rosa Paz, Priscila Schmidt Lora, Eduardo Pandolfi Passos, Elizabeth Obino Cirne-Lima
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
Author contributions: Gonçalves FC, Paz AHR and Cirne-Lima EO designed the study; Gonçalves FC performed the research; Gonçalves FC, Paz AHR and Lora PS participated in the interpretation of the data; Gonçalves FC and Paz AHR wrote the paper; Passos EP and Cirne-Lima EO supervised the study design, execution, analysis, and approved the final version.
Supported by Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Correspondence to: Ana Helena da Rosa Paz, PhD, Embryology and Cell Differentiation Laboratory, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos 2350, Porto Alegre, RS, 90035-903, Brazil. anahpaz@gmail.com
Telephone: +55-51-33598989 Fax: +55-51-33598761
Received: September 3, 2011
Revised: December 21, 2011
Accepted: December 28, 2011
Published online: February 26, 2012
Abstract

AIM: 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).

CONCLUSION: 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.

Keywords: Mesenchymal stem cell; Cell culture; Cell adhesion; Bone matrix; Freeze drying