Study On Micro/Nano-patterning Techniques And Associated Cell Behaviors Such As Critical Areas Of Cell Adhesion

Cell-material interactions have been paid m(?)re attention with the development of biomaterials from the bioinert generation to the (?)oactive generation. The patterned surfaces have been applied to control and study c(?)llular responses to biomaterials and could draw deterministic conclusions for c mplex biological problems. Appropriate patterns are capable to mimic the extracellular matrix in vitro on cellular and molecular levels thanks for the progress of microscale and nanoscale patterning techniques. Micropatterns provide a unique tool for a precise control of the localization, spreading size and shape of cells, and nanopatterns allow a relatively fine control of adhesion complex that mediates the cell-biomaterial interactions. Micro/nano-composite patterns, which combine the features of micro-and nano-patterns, can achieve better control of cells, and yet the patterning technique needs to be developed.Besides exploring some basic cell behaviors such as adhesion areas on micropatterned surfaces, this Ph. D thesis is aimed to improve the techniques of micro/nano composite patterns and extend the space to use patterned surfaces to study cell behaviors including differentiation of stem cells.The main creative achievements in this thesis are as follows:(1) We first introduced the concept of a few critical areas of cell adhesion on micropatterned surfaces and put forward the approaches to determine these critical values experimentally.We defined seven characteristic areas of cell adhesion on micropatterned surfaces (A*, also named Ac1is the critical area from apoptosis to survival; Ac2, the critical area from single-to multi-cell adhesion; A△, the area for one more cell to adhere;Apeak(1), the area with respect to the maximum population among all microislands occupied exactly by a single cell;Apeak(2), the area with respect to the maximum population among all microislands occupied exactly by two cells; AN(1), the area of the microisland occupied by one cell on average; AN(2), the area of the microisland occupied by two cells on average), and justified their existence via cell culture on a micropatterned surface. On the surface, cell-adhesive RGD microislands were covalently linked to the non-fouling PEG hydrogels. We described semi-quantitatively the basic size-dependent behaviors of cell adhesion, and put forward the practical approaches of their determination. The concepts were justified for all of three cell lines we examined. The general relationships between these critical values were also revealed. This work provides a model surface with a preferred given number of cells localized on adhesive microislands, and seems also stimulating for design of cell chips with appropriate dimensions.(2) We extended the techniques of fabricating patterned surfaces appropriate for studies on cells. Not only the nano-then-micro and micro-then-nano strategies were summarized with inputs of some new approaches, but also a nano1-then-micro-then-nano2strategy was suggested to prepare microscale-nested dual nanopatterns.We improved the fabrication technique of micro-on-nano-pattern, a kind of micro/nano composite pattern, and meanwhile put forward a new route named as "micro-then-nano" method. We also applied the micro-on-nano-patterns for resolving the conflict between the controlled localization and free spreading of cells on micropatterned surface, and achieved the "spreading-localization" of single cells. This work extended the application of micro/nano composite patterns in the investigation on cell-biomaterial interactions.We suggested a nano1-then-micro-then-nano2strategy and prepared a new type of micro/nano composite patterns named as "microscale-nested dual-spacing nanopatterns". The dotsize, spacing and regularity of the two nanoarrays could be independently tuned and conveniently reversed. We designed a fabrication route named as "double self-assembly", and successfully obtained such reversible dual-spacing nanopattern. What’s more, we also introduced thiol-reagent-assisted sonication method for fabricating another nested dual nanopattern with uniform lattice but two kinks of sizes of nanodots, denoted as "dual-dotsize nanopattern", and developed a new route via a selective diminishing method, and also successfully fabricated reversible dual-dotsize nanopatterns.(3) We first carried out the investigation on co-differentiation of mesenchymal stem cells on nanopatterned and micro/nano composite patterns.We first co-induced mesenchymal stem cells (MSCs) towards osteogenesis and adipogenesis on nanopatterned surfaces. The effect of nanospacing on the partition ratio between two directions was found.We also examined co-induction of MSCs on micro-on-nano-patterned surfaces. We proposed an assumption to modulate the spatial distribution of osteogenesis and adipogenesis which can be driven by micro/nano features of underlying material surface, and then verified its feasibility via co-induction of MSCs, which also revealed the importance of nano-spacing. This work further broadens the application of micro/nano composite pattern, suggests a new tool to regulate the co-differentiation behaviors of MSCs, and may provide cues for some diseases related to the balance and spatial distribution between different lineage commitments.In summary, this Ph. D thesis has improved the fabrication technique of micro/nano composite patterns, affords new tools for modulating cell behaviors on biomaterials, shed new insights upon cell-biomaterial interactions and basic cell behaviors such as cell adhesion and differentiation on micropatterns, nanopatterns, and micro-nano composite patterns. The fundamental study might be meaningful for design of biomaterials of the new generation.