Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.

Poly(N-isopropylacrylamide) (PNIPAAm)-grafted poly(dimethylsiloxane) (PDMS) offers an inexpensive, biocompatible, oxygen permeable, and easily microtextured thermo-responsive substrate for producing cell sheets. This study introduces a method of grafting PNIPAAm onto microtextured PDMS that is suitable for generating aligned vascular smooth muscle cell (VSMC) sheets. We examined a wide range of processing parameters in order to identify the conditions that led to acceptable sheet growth and detachment behavior. Substrates grafted under these conditions produced confluent cell sheets that fully detached in less than 10 min after lowering the culture temperature from 37 °C to 20 °C. The grafted layer thickness was determined to be 496±8 nm by atomic force microscopy. Surface characterization by Fourier transform infrared spectroscopy showed a relative grafting yield of 0.488±0.10, defined as the ratio of the PNIPAAm 1647 cm(-1) to the PDMS 2962 cm(-1) absorbance peaks. The water contact angle of the substrates was shown to change from 89.6° to 101.0° at 20 °C and 37 °C, respectively. We also found that cell behavior on PNIPAAm-grafted PDMS was not directly related to surface wettability or relative grafting densities.

gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis.

Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. The combined role of TLRs and integrin on hematopoiesis remains unclear. gp96 (grp94, HSP90b1) is an endoplasmic reticulum master chaperone for multiple TLRs. We report herein that gp96 is also essential for expression of 14 hematopoietic system-specific integrins. Genetic deletion of gp96 thus enables us to determine the collective roles of gp96, integrins, and TLRs in hematopoiesis. We found that gp96-null hematopoietic stem cells could support long-term myelopoiesis. B- and T-cell development, however, was severely compromised with transitional block from pro-B to pre-B cells and the inability of thymocytes to develop beyond the CD4(-)CD8(-) stage. These defects were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development.