Rong Peng

Cell–Material Interactions Revealed Via Material Techniques of Surface Patterning

Cell-material interactions constitute a key fundamental topic in biomaterials study. Various cell cues and matrix cues as well as soluble factors regulate cell behaviors on materials. These factors are coupled with each other as usual, and thus it is very difficult to unambiguously elucidate the role of each regulator. The recently developed material techniques of surface patterning afford unique ways to reveal the underlying science. This paper reviews the pertinent material techniques to fabricate patterns of microscale and nanoscale resolutions, and corresponding cell studies. Some issues are emphasized, such as cell localization on patterned surfaces of chemical contrast, and effects of cell shape, cell size, cell-cell contact, and seeding density on differentiation of stem cells. Material cues to regulate cell adhesion, cell differentiation and other cell events are further summed up. Effects of some physical properties, such as surface topography and matrix stiffness, on cell behaviors are also discussed; nanoscaled features of substrate surfaces to regulate cell fate are summarized as well. The pertinent work sheds new insight into the cell-material interactions, and is stimulating for biomaterial design in regenerative medicine, tissue engineering, and high-throughput detection, diagnosis, and drug screening.

The regulation of stem cell differentiation by cell-cell contact on micropatterned material surfaces

Using the material technique recently developed by us, we prepared a micropattern on poly(ethylene glycol) (PEG) hydrogel to keep background resistant to cell adhesion for a long time, which made examination of differentiation of localized stem cells available. Our micropattern designed in this paper prevented or ensured contact between cells adhering in arginine-glycine-aspartic acid (RGD) microdomains, and thus afforded a unique way to study the effects of cell-cell contact on the lineage differentiation of stem cells while ruling out the interference of soluble factors or cell seeding concentration etc. As demonstration, mesenchymal stem cells derived from rats were examined in this study, and both osteogenic and adipogenic differentiations were found to be regulated by cell-cell contact. Isolated cells exhibited less significant differentiation than paired or aggregated cells. For those stem cells in contact, the extent of differentiation was fairly linearly related to the extent of contact characterized by coordination number. Additionally, we revealed the existence of some unknown cues besides gap junction responsible for such effects of cell-cell contact.

Effect of cell anisotropy on differentiation of stem cells on micropatterned surfaces through the controlled single cell adhesion

Micropatterns of arginine-glycine-aspartic acid (RGD) on poly(ethylene glycol) (PEG) hydrogels were fabricated. Under an appropriate size of microislands on this strong and persistent non-fouling background, single mesenchymal stem cells (MSCs) from rats were well localized, keeping the same adhesive area but different shapes. The cell shapes influenced the differentiation of MSCs, and the osteogenic and adipogenic differentiations exhibited different trends. According to comparison between square and rectangular cells, optimal adipogenic differentiation occurred at aspect ratio (AR) 1, but the optimal osteogenic differentiation was found when AR was about 2. We further interpreted the optimal ratios as reflecting the inherent global anisotropy of free adipoblasts and osteoblasts on unpatterned culture plates. According to comparison between globally isotropic circular, square, triangular, and star cells, the optimal adipogenic and osteogenic differentiations happened in circular and star cells, respectively. In this case we found that extents of both adipogenic and osteogenic differentiations were linearly related to cell perimeter, which reflects the non-roundness or local anisotropy of cells. Hence, the present study makes semi-quantitative investigations of the effects of cell shape on differentiation of stem cells based on a material technique, and reveals that the shape anisotropy is very important in directing the lineage commitments of stem cells.

The effect of culture conditions on the adipogenic and osteogenic inductions of mesenchymal stem cells on micropatterned surfaces

Herein we examined an adipogenic or osteogenic induction of rat bone marrow mesenchymal stem cells (MSCs) in the corresponding media and a co-induction in a 1:1 mixed medium. The cell size effect and cell-cell contact effect were employed as two demonstrations to check the similarity or difference of the effects under these two induction ways. We seeded cells on a micropatterned surface with cell-adhesive microislands on poly(ethylene glycol) hydrogels. MSCs were well localized on the microislands separated by the strong and persistent non-fouling background, which enabled the observations of individual cells of varied sizes and numbers. We made statistics of adipogenic and osteogenic differentiations of single MSCs of different sizes (170-5600 μm(2)) and also of cell clusters of different aggregation numbers (1, 2, 4 and 8 etc.) with small, medium or large cell sizes. Both sole induction and co-induction led to monotonic cell size effects: small cells favored the adipogenic differentiation, and large cells preferred to the osteogenic differentiation. The effects of cell-cell contact were, however, rather complicated: the aggregation among cells was beneficial for both adipogenic and osteogenic differentiations, as revealed from the sole inductions; but under the co-induction culture in the mixed medium, the inherent enhancement of differentiation by the cell-cell contact encountered competition between the adipogenic and osteogenic commitments. We also examined the effects of cell density, which involved both size and contact effects, and thus exhibited different behaviors under sole induction and co-induction as well. We revealed that the density effect reflected the cooperation (for adipogenic differentiation) or competition (for osteogenic differentiation) between cell size and cell-cell contact effects.

Control of cell nucleus shapes via micropillar patterns

We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion.

Effects of aspect ratios of stem cells on lineage commitments with and without induction media

The present study is aimed to examine the shape effect on lineage commitment of stem cells in growth medium free of external chemical induction factors. Aspect ratios (ARs) of cells were controlled by micropatterns with cell-adhesive microislands of AR 1, 2 and 8 on the potent nonfouling background of poly(ethylene glycol) hydrogels, and the single stem cells were well shaped for 19 days. Mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured in osteogenic medium, adipogenic medium, mixed coinduction medium, and also growth medium; alkaline phosphatase (ALP) and oil droplets were employed as indicators of osteoblasts and adipocytes, respectively. Those indicators were well observed in all of three induction media as early as day 7, and also in growth medium at a longer culture time till day 13. While a significant monotonic decrease of adipogenesis was observed with the increase of AR, a non-monotonic change of osteogenesis was found with optimal AR about 2. The relative gene expressions further verified the above findings. As a result, cell shape itself is an inherent cue to regulate stem cell differentiation, let alone with or without external chemical induction factors. Such a shape effect disappeared upon addition of a microfilament inhibitor cytochalasin D or a Rho-associated protein kinase (ROCK) inhibitor Y-27632. So, formation of cytoskeleton is necessary for the shape effect, and the ROCK-pathway-related cell tension is responsible for the shape effect on the lineage commitment of stem cells even in growth medium.

Effects of surface molecular chirality on adhesion and differentiation of stem cells

Chirality is one of the most fascinating and ubiquitous cues in nature, especially in life. The effects of chiral surfaces on stem cells have, however, not yet been revealed. Herein we examined the molecular chirality effect on stem cell behaviors. Self assembly monolayers of L- or D-cysteine (Cys) were formed on a glass surface coated with gold. Mesenchymal stem cells (MSCs) derived from bone marrow of rats exhibited more adhering preference and thus less cell spreading on the L surface than on the d one at the confluent condition. More protein adsorption was observed on the L surface after immersed in cell culture medium with fetal bovine serum. After osteogenic and adipogenic co-induction at the confluent condition, a larger proportion of cells became osteoblasts on the d surface, while the adipogenic fraction on the L surface was found to be higher than on the D surface. In order to interpret how this chirality effect worked, we fabricated Cys microislands of two sizes on the non-fouling poly(ethylene glycol) hydrogel to pre-define the spreading areas of single cells. Then the differentiation extents did not exhibit a significant difference between L and D surfaces under a given area of microislands, yet very significant differences of osteogenesis and adipogenesis were found between different areas. So, the molecular chirality influenced stem cells, probably via favored adsorption of natural proteins on the L surface, which led to more cell adhesion; and the larger cell spreading area with higher cell tension in turn favored osteogenesis rather than adipogenesis. As a result, this study reveals the molecular chirality on material surfaces as an indirect regulator of stem cells.

Effects of spreading areas and aspect ratios of single cells on dedifferentiation of chondrocytes.

Dedifferentiation of chondrocytes is common in culturing, and seriously affects the restorative efficacy of cartilage repair. The present study examines the effect of initial cell shapes on dedifferentiation of chondrocytes in vitro. The cell shape was controlled with a unique material micropatterning technique. With this technique, a series of microarrays of cell-adhesive peptide arginine-glycine-aspartate (RGD) were generated on a persistent non-fouling poly(ethylene glycol) (PEG) hydrogel. After culturing chondrocytes derived from rats on the micropatterned surfaces, the cell shapes were adapted by the geometries of adhesive microislands with pre-defined diameters (10, 15, 20 and 30 μm) for round ones and aspect ratios (1, 1.2, 1.5, 2, 4 and 6) for elliptical ones. After 10 days, collagen II staining was demonstrated to identify normal chondrocytes and dedifferentiated cells for those single cells on microislands. Furthermore, the gene expression of collagen II, collagen I, aggrecan and SOX9 were detected by qRT-PCR. The statistical results illustrated that dedifferentiation of chondrocytes happened more probably in the cases of larger sizes and higher aspect ratios. The conclusions stand under circumstances of both normoxia (21% O2) and hypoxia (5% O2) atmospheres.

Effects of cell-cell contact and oxygen tension on chondrogenic differentiation of stem cells.

While cell condensation has been thought to enhance chondrogenesis, no direct evidence so far confirms that cell-cell contact itself increases chondrogenic differentiation of stem cells, since the change of cell-cell contact is usually coupled with those of other cell geometry cues and soluble factors in cell culture. The present study semi-quantitatively examined the effect of cell-cell contact in a decoupled way. We fabricated two-dimensional micropatterns with cell-adhesive peptide arginine-glycine-aspartate (RGD) microdomains on a nonfouling poly(ethylene glycol) (PEG) hydrogel. Mesenchymal stem cells (MSCs) were well localized on the microdomains for a long time. Based on our micropattern design, single MSCs or cell clusters with given cell numbers (1, 2, 3, 6 and 15) and a similar spreading area per cell were achieved on the same substrate, thus the interference of soluble factor difference from cell autocrine and that of cell spreading area were ruled out. After 9-day chondrogenic induction, collagen II was stained to characterize the chondrogenic induction results; the mRNA expression levels of SOX9, collagen II, aggrecan, HIF-1α and collagen I were also detected. The statistics confirmed unambiguously that the extent of the chondrogenic differentiation increased with cell-cell contact, and even a linear relation between differentiation extent and contact extent was established within the examined range. The cell-cell contact effect worked under both hypoxia (5% O2) and normoxia (21% O2) conditions, and the hypoxia condition promoted the chondrogenic induction of MSCs on adhesive microdomains more efficiently than the normoxia condition under the same cell-cell contact extents.

Particle-collision and porogen-leaching technique to fabricate polymeric porous scaffolds with microscale roughness of interior surfaces

A facile technique is herein reported to fabricate three-dimensional (3D) polymeric porous scaffolds with interior surfaces of a topographic microstructure favorable for cell adhesion. As demonstration, a well-known biodegradable polymer poly(lactide-co-glycolide) (PLGA) was employed as matrix. Under the porogen-leaching strategy, the large and soft porogens of paraffin were modified by colliding with small and hard salt particles, which generated micropits on the surfaces of paraffin spheres. The eventual PLGA scaffolds after leaching the modified porogens had thus interior surfaces of microscale roughness imprinted by those micropits. The microrough scaffolds were confirmed to benefit adhesion of bone marrow stromal cells (BMSCs) of rats and meanwhile not to hamper the proliferation and osteogenic differentiation of the cells. The insight and technique might be helpful for biomaterial designing in tissue engineering and regenerative medicine.