Characterization of ultrahigh molecular weight poly(ethylene oxide) by size-exclusion chromatography with multiangle light scattering detection.

This study investigated the experimental conditions needed to obtain molecular weight distribution (MWD) of ultrahigh MW poly(ethylene oxide) (PEO) using size exclusion chromatography (SEC) hyphenated with a multiangle light scattering photometer (MALS) and a differential refractive index detector (RI). Ultrahigh MW components yielded non-linear angular dependency in scattered light intensities. The first-order linear fitting using Zimm formalism resulted in significant differences in MW depending on if the signals from detector 4 to 16 were used in the fitting or only five low-angles from 4 to 8 were used. In contrast, no significant differences in MW were obtained for lower MW PEO samples (equal or less than 1000 KDa) between the two fitting approaches. It was thus proposed to use only the five low-angles to derive MW for a sample with broad polydispersity including both ultrahigh and low MW components. The SEC separation was done using one column designed for ultrahigh MW polymer separation connected with another mixed-bed column. The ultrahigh MW column allowed separation and characterization of polymeric components in the MW range between 10 and 50 million Dalton (MDa) and the size range between 300 and 600 nm in radius of gyration (Rg). Online calibration curves were obtained from the linear fittings of MW as a function of elution volume. MW polydispersity was derived from the online calibration curve showing that the ultrahigh MW PEO had higher polydispersity than the lower MW samples. The double logarithmic plot of radius of gyration versus MW indicated that both ultrahigh MW and low MW PEO would adopt expanded coil conformations in the aqueous solution.

Nuclear and cellular alignment of primary corneal epithelial cells on topography.

The basement membrane of the corneal epithelium presents biophysical cues in the form of topography and compliance that can modulate cytoskeletal dynamics, which, in turn, can result in altering cellular and nuclear morphology and alignment. In this study, the effect of topographic patterns of alternating ridges and grooves on nuclear and cellular shape and alignment was determined. Primary corneal epithelial cells were cultured on either planar or topographically patterned (400-4000 nm pitch) substrates. Alignment of individual cell body was correlated with respective nucleus for the analysis of orientation and elongation. A biphasic response in alignment was observed. Cell bodies preferentially aligned perpendicular to the 800 nm pitch; and with increasing pitch, cells increasingly aligned parallel to the substratum. Nuclear orientation largely followed this trend with the exception of those on 400 nm. On this biomimetic size scale, some nuclei oriented perpendicular to the topography while their cytoskeleton elements aligned parallel. Both nuclei and cell bodies were elongated on topography compared to those on flat surfaces. Our data demonstrate that nuclear orientation and shape are differentially altered by topographic features that are not mandated by alignment of the cell body. This novel finding suggests that nuanced differences in alignment of the nucleus versus the cell body exist and that these differences could have consequences on gene and protein regulation that ultimately regulate cell behaviors. A full understanding of these mechanisms could disclose novel pathways that would better inform evolving strategies in cell, stem cell, and tissue engineering as well as the design and fabrication of improved prosthetic devices.

Functionalization of reactive polymer multilayers with RGD and an antifouling motif: RGD density provides control over human corneal epithelial cell-substrate interactions.

Our study demonstrates that substrates fabricated using a "reactive" layer-by-layer approach promote well-defined cell-substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating "reactive" deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell-substrate interactions enables the use of these films in a wide range of biological applications.

The ability of corneal epithelial cells to recognize high aspect ratio nanostructures.

The basement membrane of the human corneal epithelium comprises topographic features including fibers, pores, and elevations with feature dimensions on the order of 20-400 nm. Understanding the impact of sub-micron and nanotopography on corneal cell behavior will contribute to our understanding of biomechanical cues and will assist in the design of improved synthetic corneal implants. We utilized well defined ridge and groove wave-like nanostructures (wave ordered structures, WOS) of 60-140 pitches (30-70 nm ridge widths) and 200 nm depths to assess human corneal epithelial cell (HCEC) contact guidance and to establish HCEC contact acuity defined as the lower limit in feature dimensions at which cells respond to biomimetic topographic cues. Results using the WOS substrates demonstrate that HCEC contact acuity is in the range of 60 nm pitch for cells in a serum-free basal medium (EpiLife) and in the range of 90 nm pitch for cells in epithelial medium. To further investigate the influence of HCEC contact acuity in the presence of larger topographic cues, we fabricated 70 nm pitch WOS-overlaid parallel to the top of the ridges of 800-4000 nm pitch. HCEC cultured in epithelial medium demonstrate a significant increase in the percent of cells aligning to 4000 nm pitch topography with WOS-overlay compared to controls (both flat and 70 nm WOS alone) and 4000 nm pitch topography alone. These results highlight the significance of the lower range of basement membrane scale topographic cues on cell response and allow for improved prosthetic design.