A comparative study of seeding techniques and three-dimensional matrices for mesenchymal cell attachment.

Mesenchymal stem cells (MSCs) offer significant potential as a cell source in tissue-engineering applications because of their multipotent ability. The objective of this study was to evaluate the behaviour of MSCs during the seeding phase, using four different seeding techniques (spinner flask, custom vacuum system combined with a perfused bioreactor or with an orbital shaker, and orbital shaker) with four different scaffold materials [polyglycolic acid, poly(lactic acid), calcium phosphate and chitosan-hyaluronic acid]. Scaffolds were selected for their structural and/or chemical similarity with bone or cartilage, and characterized via scanning electron microscopy (SEM) and measurement of fluid retention. Cell attachment was compared between seeding techniques and scaffolds via cell-binding kinetics, cell viability and DNA quantification. SEM was used to evaluate cell distribution throughout the constructs. We discovered from cell suspension kinetics and DNA data that the type of loading (i.e. direct or indirect) mainly influences the delivery of cells to their respective scaffolds, and that dynamic seeding in a spinner flask tended to improve the cellularity of polymer constructs, especially mesh. Regardless of the seeding method, bone marrow-derived MSCs displayed a superior affinity for calcium phosphate scaffolds, which may be related to their hydrophobicity. MSCs tended to aggregate into flat sheets, occluding the external pores of matrices and affecting cell distribution, regardless of seeding technique or scaffold. Taken together, these results provide insight into the design of future experiments using MSCs to engineer functional tissue.