Effects Of Cell Shape And Surface Chirality On Adhesion And Differentiation Of Stem Cells Revealed Via Material Techniques Of Surface Patterning

Design of new-generaton biomaterials is, in a large extent, dependent upon the comprehensive understanding of the cell-material interactions. Cells live in a material world, if their extracellular matrix (ECM) and even neighboring cells are regarded as "materials". Cells in an organism incessantly sense and interact with those "materials", and cell functioning relies also on their microenvironment. Hence, one of the core missions in biomaterial research is to mimic the cell microenvironment (structure and function of ECM) through material design and processing, which makes tissue repairing rapidly and effectively. To study and reveal the cues related to the cell-material interactions is thus extremely important in the fields of Biomaterial, Cell Biology, Tissue Engineering, and Regenerative Medicine.In the traditional cell culture system, various cues (cell cues, ECM cues and soluble factors) to influence cell-material interactions are usually coupled with each other. With the development of advanced materials, surface patterning technique has afforded a powerful tool to decouple these cues, and is thus much helpful for revealing cell-material interactions.Bone marrow mesenchymal stem cells (MSCs) are an ideal type of seeding cells for Regenerative Medicine and Tissue Engineering due to their multi-lineage potential, self-renewal ability, and also free of ethical problems. To reveal the cues to affect cell differentiation has been one of key issues in both fields of stem cells and biomaterials.In this thesis, we employed MSCs as a model cell type and examined the effects of cell shape, surface molecular chirality and geometric chirality on adhesion and differentiation of the stem cells on micropatterned surfaces. Our micropatterns of arginine-glycine-aspartate (RGD) peptides on poly(ethylene glycol) (PEG) hydrogels were fabricated based upon a developed transfer technique of micropatterns and molecular self-assembly on surfaces.The main innovative achievements and scientific discoveries are summarized as follows:(1) We fabricated the RGD micropatterns on non-fouling PEG hydrogels with cell-adhesion contrast, controlled cell shape on adhesive microislands persistently, and revealed that cell shape is an inherent cue to regulate cell differentiation. A transfer photolithography strategy was developed, by which we successfully fabricated cell-adhesive RGD microislands with different aspect ratios (ARs) on the surface of persistent non-fouling PEG hydrogels. Such a surface patterning on polymers affords a powerful material technique to shape individual cells under the cell culture media for a long time. Based on this unique surface patterning technique, we achieved maintainence of cell shape with pre-designed ARs as long as 19 days, and found that cell shape has significant effects on stem cell differentiation both in the induction medium and growth medium. While the circular shape with AR 1 favored adipogenesis, osteogenesis exhibited a non-monotonic change with AR, and the optimal aspect ratio was about 2. We further discussed the mechanism underlying the shape effect. This fundmental research illustrates unambiguously that the shape effect is an inherent cue to regulate cell differentiation, and proclaims that the material effect can regulate cell differentiation via controlling cell shape even in the absence of external induction factors.(2) Based on preparation of micropatterns of chiral molecules on PEG hydrogels, we studied the effects of surface molecular chirality on stem cells behaviors, and illustrated that stem cell differentiation is adjusted by surface molecular chirality. We self-assembled L-cysteine and D-cysteine on gold surface, and also developed a transfer lithography technique to fabricate surfaces of the chiral molecules on pre-designed microislands. The stem cell culture and induction indicated that the surface molecular chirality has significant effects on stem cell adhesion and differentiation. Besides, this research has proclaimed that it is the cell spreading area (relative to cell tension) influenced by molecular chirality that changed the forthcoming differentiation extent.(3) We designed and fabricated geometrically chiral microislands, and examined the effects of the geometric chirality of cell-adhesivemicroenvironment on stem cells behaviors; we discovered that geometric chirality is one of regulators of cell polarity and differentiation. This research designed and successfully fabricated geometrically chiral microislands on the non-fouling PEG hydrogels using the transfer photolithography technique. Both clockwise (CW) and counter-clockwise (CCW) spiral microislands of RGD peptides on the PEG hydrogels were obtained. Based on the statistical results of cell adhesion and osteogenic/adipogenic differentiation, the geometric chirality of cell-adhesive microenvironment was, for the first time, found to influence stem cell adhesion and differentiation. The follow-up studies revealed that the regulation of stem cell differentiation by the geometric chirality probably owes to the cell tension difference generated by the mutual effect between the induced cell polarity and possible inherent left-right asymmetry of cell cytoskeleton. The stem cells on CW microislands have larger cell tension, which is beneficial for osteogenesis; cells on CCW microislands have lower cell tension, which relatively favors adipogenesis.These fundmental studies have expanded our knowleage of ECM cue effects on cell behaviors, and paved ways to further studies of cell-material interactions. The new insight is also helpful for guiding the development of novel biomaterials.