Access to the full text of the published version may require a subscription. Rights
Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.
! Scheme S3. Synthesis of P-B-CN (4). GENERAL METHODS 1 Materials. Anhydrous DMF, n BuLi (1.6 M in hexanes), and DIBAL-H (1.0 M in hexanes) were purchased from Aldrich and dispensed using air-sensitive techniques. Benzoyl peroxide and NBS were stored at-20 o C. KO t Bu was stored in a desiccator over anhydrous CaSO 4. LiCl was dried at 120 o C for at least 24 h. Anhydrous diethyl ether for lithiation reactions was opened immediately prior to use. Reagent grade THF was used for most reactions; notably the HWE reactions used reagent grade THF. DCM for reactions was dried by refluxing with CaH 2. 1,6-1 The general methods section is also found in the paper and is reproduced here for ease of interpreting the experimentals that follow. Supporting information 4 Bis(hexyloxy)benzene (5) was prepared as previously reported. 1 All other reagents (including compounds 7 and 8) and solvents were used as received. Column chromatography was carried out on standard grade silica gel (60 Å pore size, 40-63 µm particle size), which was purchased and used as received. Hexanes, dichloromethane, and ethyl acetate used for column chromatography were purchased and used as received. Melting points for all compounds were determined by DSC and are found in the main text in Table 2, listed as T iso. NMR Spectroscopy. 1 H (300 and 400 MHz) and 13 C (75, 100 and 150 MHz) NMR spectra were recorded on Bruker spectrometers. Chemical shifts were referenced to residual 1 H or 13 C signals in deuterated solvents (7.27 and 77.0 ppm, respectively, for CHCl 3 and 5.32 and 54.0 ppm, respectively, for CH 2 Cl 2). Mass Spectrometry. HRMS were recorded on EI-quadrupole or ESI-TOF instruments in the Mass Spectrometry Facility of the University of Pittsburgh. Optical Spectroscopy. UV/VIS absorption spectra were recorded in CHCl 3 on a Perkin Elmer Lambda 9 UV/VIS/NIR spectrometer. Solution (CHCl 3) and film emission spectra were recorded on a Varian Cary Eclipse fluorimeter. Films were drop cast on quartz slides from CHCl 3. Thermal Analysis. DSC was performed on a Perkin Elmer Pyris 6 with a heating and cooling rate of 10 o C/min. Electrochemistry. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed on a CHI Electrochemical Workstation Model 430a (Austin, TX) collected using a three electrode system consisting of a glassy carbon disk (3 mm dia.
The reaction between a bulky N-heterocylic carbene (NHC) and C(60) leads to the formation of a thermally stable zwitterionic Lewis acid-base adduct that is connected via a C-C single bond. Low-energy absorption bands with weak oscillator strengths similar to those of n-doped fullerenes were observed for the product, consistent with a net transfer of electron density to the C(60) core. Corroborating information was obtained using UV photoelectron spectroscopy, which revealed that the adduct has an ionization potential ∼1.5 eV lower than that of C(60). Density functional theory calculations showed that the C-C bond is polarized, with a total charge of +0.84e located on the NHC framework and -0.84e delocalized on the C(60) cage. The combination of reactivity, characterization, and theoretical studies demonstrates that fullerenes can behave as Lewis acids that react with C-based Lewis bases and that the overall process describes n-doping via C-C bond formation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.