Cytocompatibility study of a natural biomaterial crosslinker--Genipin with therapeutic model cells.

Genipin has been widely used as a natural crosslinker to substitute chemical crosslinkers such as glutaraldehyde to crosslink various biomaterials like gelatin, collagen, and chitosan. However, there are contradicting views on the cytotoxicity and safety of genipin in tissue engineering. Therefore in this study, we aimed to evaluate the toxicity of genipin on skeletal tissues cells-osteoblasts and chondrocytes as they are also representatives of typical anchorage-dependent cells (ADCs) and nontypical ADCs. Results suggest that genipin toxicity is dose dependent and acute but not time dependent on both osteoblasts and chondrocytes. In particular, chondrocytes exhibit substantial alterations in the gene expression when exposed to Maximum nontoxic concentration (MaxNC) of genipin but there were no significant changes in the genes tested in osteoblasts. Since osteoblasts are typical ADCs, cellular focal adhesion assessment was carried out with F-actin being more contracted and unorganized when exposed to minimum toxic concentration (MinTC) of genipin. The mechanisms involved in cell deaths in both cell types are believed to be similar and hence using osteoblast as the model, cells were stained positive for Annexin-V and Reactive oxygen species (ROS) level were elevated at MinTC of genipin. Collectively, genipin induced cell apoptosis via ROS production, and apparently, gene expressions could also be altered at MaxNC. For this reason, we recommend the dose of genipin to be controlled within 0.5 mM.

Cell surface engineering of mesenchymal stem cells.

By leveraging the capacity to promote regeneration, stem cell therapies offer enormous hope for solving some of the most tragic illnesses, diseases, and tissue defects world-wide. However, a significant barrier to the effective implementation of cell therapies is the inability to target a large quantity of viable cells with high efficiency to tissues of interest. Systemic infusion is desired as it minimizes the invasiveness of cell therapy, and maximizes practical aspects of repeated doses. However, cell types such as mesenchymal stem cells exhibit a poor homing capability or lose their capacity to home following culture expansion (i.e. FASEB J 21:3197-3207, 2007; Circulation 108:863-868, 2003; Stroke: A Journal of Cerebral Circulation 32:1005-1011; Blood 104:3581-3587, 2004). To address this challenge, we have developed a simple platform technology to chemically attach cell adhesion molecules to the cell surface to improve the homing efficiency to specific tissues. This chemical approach involves a stepwise process including (1) treatment of cells with sulfonated biotinyl-N-hydroxy-succinimide to introduce biotin groups on the cell surface, (2) addition of streptavidin that binds to the biotin on the cell surface and presents unoccupied binding sites, and (3) attachment of biotinylated targeting ligands that promote adhesive interactions with vascular endothelium. Specifically, in our model system, a biotinylated cell rolling ligand, sialyl Lewisx (SLeX), found on the surface of leukocytes (i.e., the active site of the P-selectin glycoprotein ligand (PSGL-1)), is conjugated on MSC surface. The SLeX engineered MSCs exhibit a rolling response on a P-selectin coated substrate under shear stress conditions. This indicates that this approach can be used to potentially target P-selectin expressing endothelium in the more marrow or at sites of inflammation. Importantly, the surface modification has no adverse impact on MSCs' native phenotype including their multilineage differentiation capacity, viability, proliferation, and adhesion kinetics. We anticipate that the present approach to covalently modify the cell surface and immobilize required ligands is not limited to MSCs or the SLeX ligand. Therefore, this technology should have broad implications on cell therapies that utilize systemic administration and require targeting of cells to specific tissues. The approach may also be useful to promote specific cell-cell interactions. In this protocol, we describe the conjugation of SLeX on MSC surface and methods to study cell rolling behaviors of SLeX-modified MSCs on a P-selectin coated substrate using an in vitro flow chamber assay. We also provide a brief description of cell characterization assays that can be used to examine the impact of the chemical modification regimen.