Development of Methods for Studying the Differentiation of Human Mesenchymal Stem Cells Under Cyclic Compressive Strain

• Published in: Tissue engineering. Part C, Methods Methods, 2012-04, Vol.18 (4), p.252-262
• Main Authors: Michalopoulos, Efstathios, Knight, Richard L., Korossis, Sotirios, Kearney, John N., Fisher, John, Ingham, Eileen
• Format: Article
• Language: English
• Subjects: Aged; aggrecan; Aggrecans - genetics; Aggrecans - metabolism; Alginates - pharmacology; Animals; Antibodies; ATP; Biomechanical Phenomena - drug effects; Biomedical research; Cbfa-1 protein; Cell Culture Techniques - methods; Cell Differentiation - drug effects; Cell lineage; Cell number; Cell Proliferation - drug effects; Collagen; Collagen - pharmacology; Differentiation; Embryo cells; Female; Flow Cytometry; Gene expression; Gene Expression Regulation - drug effects; Glucuronic Acid - pharmacology; Hexuronic Acids - pharmacology; Humans; Mesenchymal Stromal Cells - cytology; Mesenchymal Stromal Cells - drug effects; Mesenchymal Stromal Cells - metabolism; Mesenchyme; Middle Aged; Phenotype; Polymerase chain reaction; PPAR gamma - genetics; PPAR gamma - metabolism; Rats; Real-Time Polymerase Chain Reaction; scaffolds; Scientific method; Sox9 protein; SOX9 Transcription Factor - genetics; SOX9 Transcription Factor - metabolism; Stem cells; Stress, Mechanical; Tissue engineering; Tissue Scaffolds - chemistry; Transcription factors; viscoelasticity
• ISSN: 1937-3384; 1937-3392
• DOI: 10.1089/ten.tec.2011.0347

Human mesenchymal stem cells (hMSC) have numerous potential advantages over terminally differentiated cells and embryonic stem cells for use in tissue engineering applications. The aims of this study were to develop methods to test the hypothesis that hMSC could be differentiated using cyclic compressive strain alone. hMSC were successfully isolated, purified using D7-FIB antibody, cloned, and characterized. The cells were subsequently analyzed using fluorescence-activated cell sorting using a panel of antibodies and differentiation into multiple cell lineages. D7FIB-positive cells were then seeded into collagen–alginate scaffolds and subjected to 10% or 15% cyclic compressive strain for 4 out of 24 hours for up to 21 days in a bespoke servo-assisted displacement-controlled device. Cells were analyzed using adenosine triphosphate assay to determine cell number, live–dead cell assay, and quantitative real-time polymerase chain reaction at 7 and 21 days. Cloned D7-FIB-positive hMSCs showed evidence of differentiation to an osteogenic lineage under 10% cyclic compressive strain alone (core binding factor alpha 1 (CBFA-1) was significantly upregulated at 7 and 21 days by a factor of 18.3 and 32.2, respectively) and to an osteo-chondrogenic lineage under 15% cyclic compressive strain alone (increased expression of CBFA-1, Sox9, and aggrecan). A combination of a composite viscoelastic scaffold and controlled cyclic compressive strain may be useful for study of the differentiation of MSC.