Effects Of Cell Shape, Spreading Size And Cell Density On Differentiation Of Stem Cells On Micropatterned Surfaces Of A Polymeric Hydrogel

Cell-material interactions are fundamental topics in the fields of biomaterials, cell biology, and regenerative medicine. The corresponding research is important for design of new biomaterials. Cell is the element of life. While single cells afford a good model of cell studies, the efficiency needs to be enhanced. With the help of appropriate micropatterning techniques, one can localize plenty of single cells on a substrate. And single cells experience the same culture condition and microenvironments, which makes the further statistics more reliable.On the other hand, the interaction between cells is also an important topic. The most common parameter is cell density. Again micropatterning techniques can be applied to control cell-cell contact and the aggregation extent of cells on the level of oligocells.Bone marrow stem cells are regarded as an ideal type of seeding cells for tissue engineering and regenerative medicine due to their multipotential differentiation and self-renewal ability. The stem cell research is a frontier field in natural science.In this thesis we employed bone marrow stem cells (MSCs) as model and tried to examine the physical cues in stem cell differentiation on micropatterned surfaces. Our patterning is based on our unique polymer technique. Effects of cell shape and spreading size effects have been revealed on the level of single cells, and cell density effects have been explored on the level of oligocells. The main innovative achievements are listed as follows:1. We prepared a polymeric hydrogel with strong and persistent cell-adhesion contrast, put forward the criteria of appropriate areas of adhesive microislands for cell shape control, set up a semi-quantitative approach for statistics of differentiation of individual stem cells on micropattemed surfaces. RGD microislands on a PEG hydrogel were generated, and cells were demonstrated to be localized well on the microislands. A semi-quantitative method for statistics of osteogenic and adipogenic differentiations of mesenchymal stem cells on micropattemed surfaces was established based upon the micrographs of cells. The two criteria to judge appropriate sizes of adhesive microislands size for single cell adhesion in revealing the cell shape effects was put forward. So, the methodology bases to examine semi-quantitatively differentiation of single stem cells towards various lineage commitments have been set up.2. Based on the unique micropatterning technique, we revealed the effects of cell shapes on cell differentiation and found optimal aspect ratios for differentiation of MSCs. With the help of the micropatternning technique on the surface of PEG hydrogels developed by our group, we achieved the well localized MSCs for a long time and with the designed shape of circle, square, triangle and star, and also rectangle shape with a series of aspect ratios. We found that the circle cells were inclined to differentiate to adipocytes, while the star cells were inclined to differentiate to osteoblasts. While the adipogenesis decreased monotonically with aspect ratio, the osteogenesis exhibited a peak around2:1for the long axis over short axis of MSCs.3. We systematically investigated the effects of cell size, cell density, and cell-cell contact on stem cell differentiation, and revealed the underlying physical cues of cell density on cell differentiation via our micropatternning technique. We achieved different local cell densities on microislands under the same cell seeding density and cell culture conditions, which enables the simultaneous yet decoupling studies of the effects of cell size, cell-cell contact and cell density. Both cell size and cell-cell contact were found to influence the MSC osteogenic and adipogenic differentiation, and the effect of cell density reflects the interplay between cell size effect and cell-cell contact effect. With the increase of cell density, the adipogenesis showed the increased trend with the cooperation of cell size effect and cell-cell contact effect; however, the osteogenesis showed a non-monotonic trend, which was interpreted by the competition of cell size effect and cell-cell contact effect. The cell density was then suggested to be divided into two regions by the critical contact density.