Photo-induced <i>in situ</i> crosslinking of polymer brushes with dimethyl maleimide moieties for dynamically stimulating stem cell differentiation

<p>We designed photo-crosslinkable polymer brushes with dimethylmaleimide moieties, in order to demonstrate dynamic stimulation of cell differentiation in mesenchymal stem cells (MSCs). The polymer brushes were synthesized by surface-initiated reversible addition fragmentation chain transfer polymerization using dimethylmaleimide ethyl methacrylate and methyl methacrylate on a chain transfer agent-immobilized glass surface. The polymer brushes were crosslinked by photodimerization of the dimethylmaleimide moieties within polymer chains with stem cells present on the surface. In order to evaluate the effects of <i>in situ</i> photo-induced crosslinking of the polymer brushes on gene expression of stem cells, human bone marrow MSCs were cultured under static and dynamic culture conditions for 7 days. Expression of the osteocalcin (<i>Ocn</i>) gene in MSCs was used as an indicator of osteoblast differentiation under dynamic culture conditions. Structural conversion from non-crosslinked polymer brushes to crosslinked polymer brushes increased the expression of <i>Ocn</i> by 1.4-fold in the presence of adhered cells, compared with non-crosslinked polymer brushes under static culture conditions. These results suggest that MSCs recognized surface conversion from non-crosslinked to crosslinked structures, which resulted in altered differentiation lineages. Therefore, photo-crosslinkable surfaces with dimethyl maleimide moieties are potential novel materials for dynamically stimulating MSC differentiation.</p

Hydrophobized Thermoresponsive Copolymer Brushes for Cell Separation by Multistep Temperature Change

For preparing a thermally modulated biointerface that separates cells without the modification of cell surfaces for regenerative medicine and tissue engineering, poly­(<i>N</i>-isopropylacrylamide-<i>co</i>-butyl methacrylate) (P­(IPAAm-<i>co</i>-BMA), thermo-responsive hydrophobic copolymer brushes with various BMA composition were formed on glass substrate through a surface-initiated atom transfer radical polymerization (ATRP). Characterization of the prepared surface was performed by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR), and gel-permeation chromatography (GPC) measurement. Prepared copolymer brush surfaces were characterized by observing the adhesion (37 °C) and detachment (20 or 10 °C) of four types of human cells: human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), human aortic smooth muscle cells (SMCs), and human skeletal muscle myoblast cells (HSMMs). HUVECs and NHDFs exhibited their effective detachment temperature at 20 and 10 °C, respectively. Using cells’ intrinsic temperature sensitivity for detachment from the copolymer brush, a mixture of green fluorescent protein (GFP)-expressing HUVECs (GFP-HUVECs) and NHDFs was separated

Thermoresponsive Cationic Copolymer Brushes for Mesenchymal Stem Cell Separation

Thermoresponsive, cationic, copolymer brushes poly­(<i>N</i>-isopropylacrylamide­(IPAAm)-<i>co</i>-<i>N</i>,<i>N</i>-dimethylaminopropylacrylamide-<i>co</i>-<i>N</i>-<i>tert</i>-butylacrylamide­(tBAAm)) and poly­(IPAAm-<i>co</i>-3-acrylamidopropyl trimethylammonium chloride-<i>co</i>-tBAAm) were prepared on glass substrates through surface-initiated atom transfer radical polymerization. Prepared copolymer brushes were investigated as thermally modulated cell separation materials. Densely packed cationic copolymer brushes were formed on the glass substrates, and the positive charge density was modulated by controlling the composition of cationic moieties and species. During observation of cell adhesion and detachment properties on copolymer brushes, human bone marrow mesenchymal stem cells (hbmMSC) exhibited thermally modulated cell adhesion and detachment, while other bone-marrow-derived cells did not adhere. Using these properties, hbmMSC could be purified from mixtures of human bone-marrow-derived cells simply by changing the external temperature. Therefore, the prepared cationic copolymer brush is useful for separation of hbmMSC

Structure of Gas Phase Monohydrated Nicotine: Implications for Nicotine’s Native Structure in the Acetylcholine Binding Protein

We report a joint experimental–theoretical study of the never reported before structure and infrared spectra of gas phase monohydrated nicotine (NIC) and nornicotine (NOR) and use them to assign their protonation sites. NIC’s biological activity is strongly affected by its protonation site, namely, the pyrrolidine (Pyrro-NICH+, anticipated active form) and pyridine (Pyri-NICH+) forms; however, these have yet to be directly experimentally determined in either the nicotinic acetylcholine receptor (nAChR, no water present) or the acetylcholine-binding protein (AChBP, a single water molecule is present) but can only be inferred to be Pyrro-NICH+ from the intermolecular distance to the neighboring residues (i.e., tryptophan). Our temperature-controlled double ion trap infrared spectroscopic experiments assisted by the collisional stripping method and high-level theoretical calculations yield the protonation ratio of Pyri:Pyrro = 8:2 at 240 K for the gas phase NICH+···(H2O) complex, which resembles the molecular cluster present in the AChBP. Therefore, a single water molecule in the gas phase enhances this ratio in NICH+ relative to the 3:2 for the nonhydrated gas phase NICH+ in a trend that contrasts with the almost exclusive presence of Pyrro-NICH+ in aqueous solution. In contrast, the Pyri-NORH+ protomer is exclusively observed, a fact that may correlate with its weaker biological activity