Mesenchymal stem cell behavior in biomaterials assays

Introduction: in the orthopedic perspective, tissue engineering is focused on the development of innovative materials, whose action consists in recruiting bone progenitor cells and in stimulating their proliferation. In this study, we investigated the interaction between human mesenchymal stem cells from adults and two types of biomaterials, which differ for chemical composition and format. To evaluate the effects of porosity on cell adhesion and proliferation comparative analyses were carried out on different forms of two bioceramic materials. Materials and Methods: Bone marrow aspirates from iliac crests were obtained from orthopedic patients who underwent bone marrow harvesting under general anesthesia. Mesenchymal stem cells (hMSCs) were isolated by Ficoll-mediated discontinuous density gradient centrifugation and polystyrene adherence capacity. Cell cultures at the second passage were characterized, by the flow cytometric analysis (FCA) of specific surface antigens. A surface marker-expression pattern, positive (Stro-1, CD29, CD44, CD71, CD73, CD90, CD105) and negative (CD45, CD34, CD235), was employed to characterize the cells obtained. hMSCs were expanded and duplication time evaluated. The AlamarBlue assay was used to evaluate the number of cells attached to the biomaterials and to the control in adhesion and proliferation assay. The quantification of the relative amount of Focal Adhesion Kinase was employed to evaluate the strength of the cell adhesion to the biomaterials after 36 hours from seeding. In order to verify the morphology of the hMSCs attached to bioceramics, stem cells grown on the biomaterials were analyzed by SEM. Moreover, to verify whether the cytoskeletal organization of hMSCs cultured on the analyzed biomaterials was altered, actin fibers were investigated using TRITC conjugated-Phalloidin. Results: the biomaterials assayed in our study provided a favorable environment for hMSCs adhesion and growth, without any evidence of cytotoxic effects. Indeed, hMSCs adhesion and growth assays indicate that shape and porosity influence the cytocompatibility of bioceramics. We may infer that intra- and iter- porosity of biomaterials investigated herein, are determinant factors influencing hMSCs behavior. Besides, the correlation between vitality and a greater amount of Focal Adhesion Kinase at molecular level show how cellular processes may be modulated by structured surface and three dimensional architecture of biomaterials. hMSC SEM analysis showed an elongated spindle-like morphology. The cells adhered to biomaterials with many dendritic projections with some differences in the morphology due to scaffolds structure. Moreover, hMSCs cytoskeletal architecture on biomaterials was indistinguishable from that of hMSCs grown on plastic petri dishes, used as a control. Conclusion: These assays confirm the feasibility to evaluate biomaterials biological features in pre-clinical environment with patients mesenchymal stem cells in order to obtain information about subject specific response. Moreover, the development of new matrices have to consider the possibility to employ bioactive surfaces able to modulate cell adhesion and differentiation processes.