Supplementary Materialsmaterials-10-00964-s001. a higher concentration of CNTs. The results suggest that the self-rolling, conductive CNT-dopamine-PEG hydrogel could have multiple potentials, including biomedical usage and as a conductive biosensor. = 700), F-127, dopamine, and NN-methylene-bis-acrylamide (MBA) were all ordered from Sigma-Aldrich (Oakville, ON, Canada). Igacure@2959 was ordered from BASF Canada (Mississauga, ON, Canada). All chemicals were used without further purification. Phosphate buffered saline (PBS) 1 powder, Hoechst 33342, BODIPY FL phallacidin, To-Pro-3 iodide, and a Live-Cell staining kit were all purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay kits were from Biotium Inc. (Hayward, CA, USA). Optimal cutting temperature (OCT) compounds were purchased from VWR Canada (Mississauga, ON, Canada). Pristine multi-walled carbon nanotubes (MWCNTs, 95% purity) were purchased from Timesnano (Chengdu, China) and used without further purification. The morphology of the MWCNT was examined under a FEI Talos F200X Transmission electron microscopy (TEM, Hillsboro, OR, USA). 2.2. Synthesis of Dopamine-MBA Crosslinker Deionized water/ethanol (= 4:3) solution was adjusted to pH 6 by using 0.5 M hydrochloric acid in order to protect dopamine SGI-1776 tyrosianse inhibitor from oxidization. 500 mg of MBA (3 mmol) was then dissolved into the above solution to achieve a concentration of 70.1 mg/mL. After that, 475 mg of dopamine (2 mmol) was added to the solution under nitrogen protection to exclude oxygen. The reaction was conducted in darkness in an oil bath at 45 C and under constant stirring for three days. The dopamine-MBA crosslinker was obtained after lyophilization SGI-1776 tyrosianse inhibitor and stored at ?20 C. 2.3. Preparation of MWCNT-Dopamine-PEG Hydrogels 24 mg MWCNTs were dispersed in 10 (= 3) on a SGI-1776 tyrosianse inhibitor microplate reader. Live/Dead staining was used for evaluating the cytotoxicity of the hydrogels. The hydrogels were prepared between two cover slips, according to the method described before. BMSC were cultured on the hydrogels and dyed at certain time intervals. Cells with hydrogels were immersed in PBS mixed with 2 mM calcein AM (acetoxymethyl) and 4 mM ethidium homodimer probes for 20 min. After that, the hydrogels with cells were rinsed with PBS and imaged under a florescence microscope. 2.8. Cell Morphology The morphology of BMSC-laden hydrogels was observed under a florescence microscope. The Rabbit Polyclonal to ANXA2 (phospho-Ser26) cells on hydrogels were immersed in 4% paraformaldehyde solution at room temperature for 30 min. After being washed with PBS, the samples were permeabilized using 0.5% Triton X-100 in PBS solution at room temperature for 5 min. They were then blocked in 1% bovine serum albumin PBS solution at room temperature for 10 min. The samples were incubated in FITC (Fluorescein isothiocyanate) solution for 20 min at room temperature and stained with TO-PRO3 (a nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially) before being examined under a confocal Laser scanning microscopy (CLSM). 2.9. Electrical Conductivity Test All the electrical conductivity tests were conducted on a Multifunction Digital Four-probe Tester (Suzhou Jingge Electronic Co. Ltd, Suzhou, China). 2.10. Quantitative Real-Time PCR Analysis 100 mL BMSCs solution with a concentration of 2 106 cell/mL was seeded into the hydrogel. After two days incubation, the samples were moved into DMEM mixed with 50 mg/mL L-ascorbic acid 1-phosphate, 10 mM b-glycerophosphate, and 100 nM dexamethasone. The medium was refreshed every two days. At a certain time point, the medium was carefully moved out without damaging cells and was rinsed with DPBS for three times. Then the samples with cells were frozen by liquid nitrogen and grounded into small pieces. The total RNA isolation and cDNA sythesis were performed to analyze the osteogenic differentiation based on standard procedures. SYBER Green assays were used to conduct quantitative real-time PCR (quantitative polymerase chain reaction). In this study, we used the control group SGI-1776 tyrosianse inhibitor as a standard sample. For example, the formula of ALP (alkaline phosphatase) gene expression of 0.7 mg/mL WCNT-dopamine-PEG is as follows: ALP?gene?expression =?2?[(Ctarget?CALP)?(Ctarget?0?CALP?0)] in which Ctarget? is the GAPDH gene expression of 0.5 wcnt-gel, CALP. is the ALP SGI-1776 tyrosianse inhibitor gene expression of 0.7 mg/mL WCNT-dopamine-PEG, Ctarget?0. is the GAPDH gene expression of the control group, and CALP?0 is the ALP gene expression of the control group. 3. Result and Discussion 3.1. Characterization of Self-Folding Film TEM images of MWCNTs were shown in Figure 1C. The MWCNTs have an average diameter of 35 nm and an average length of 400 nm..