Publications (13)

  • [Show abstract] [Hide abstract] ABSTRACT: In this study, we used well-defined, homogeneous, gradient and patterned substrates to explore the effects of surface chemistry on the supramolecular structure of adsorbed type I collagen. Type I collagen (320microg/mL) was allowed to adsorb onto self-assembled CH(3)-, COOH-, NH(2)- and OH-terminated alkylthiolate monolayers at 37 degrees C. Atomic force microscopy, ellipsometry and phase microscopy indicated that large supramolecular collagen fibril structures (approximately 200nm in diameter, several microns long) assembled only at the hydrophobic CH(3)-terminated surfaces. By varying the surface energy using a mixture of OH- and CH(3)-terminated thiols during monolayer formation, we found that large fibril assembly occurred at surfaces with a water contact angle above 83 degrees , but not on surfaces with a water contact angle below 63 degrees . Examining a surface with a linear hydrophobicity gradient revealed that the assembly of large collagen fibrils requires a hydrophobic surface with a water contact angle of at least 78 degrees . Collagen fibril density increased over a narrow range of surface energy and reached near-maximum density on surfaces with a water contact angle of 87 degrees . These studies confirm that the supramolecular structure of adsorbed collagen is highly dependent on the underlying substrate surface chemistry. We can take advantage of this dependency to pattern areas of fibrillar and non-fibrillar collagen on a single surface. Morphology studies with vascular smooth muscle cells indicated that only collagen films formed on hydrophobic substrates mimicked the biological properties of fibrillar collagen gels.
    Article · Mar 2007 · Biomaterials
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    Ying Mei · John T Elliott · Jack R Smith · [...] · Lori Henderson
    [Show abstract] [Hide abstract] ABSTRACT: Using quantitative fluorescence microscopy in conjunction with a method of gradient substrate assembly established in their group, the authors were able to introduce and measure reproducible changes in cellular morphology and cell density by manipulating polymer grafting density. The mechanism behind this change in cellular behavior was explained by a semiempirical, geometric model that describes the effect of the spatial distribution of the polymer on protein attachment. A 10-fold increase in graft density of poly(2-hydroxyethyl methacrylate) [PHEMA] along the surface of a gradient sample, preexposed to bovine fibronectin, caused a change in the size of fibroblasts on the surface (i.e., cell spreading) from (1238 +/- 704) to (377 +/- 216) microm(2). The results were in quantitative agreement with those obtained on three separate gradient samples. Both cellular response and fibronectin adsorption (as measured via ellipsometry) were found to vary sigmoidally with graft density of PHEMA, demonstrating the high degree of correlation between the two phenomena. A simple, rigid-disk model accounting for the surface coverage of PHEMA was able to predict the amount of adsorbed fibronectin with a correlation coefficient of 0.97. Maximal cell adhesion and cell spreading were found to occur at fibronectin surface densities of 50 and 100 ng/cm(2), respectively. The results demonstrate the role of gradient substrate assembly as a method for quantifying the relationship between protein and cellular response to technologically relevant polymeric materials.
    Full-text available · Article · Dec 2006 · Journal of Biomedical Materials Research Part A
  • Ying Mei · Tao Wu · Chang Xu · [...] · Newell R Washburn
    [Show abstract] [Hide abstract] ABSTRACT: A simple yet versatile method was developed to prepare a low-density polymerization initiator gradient, which was combined with surface-initiated atom transfer radical polymerization (ATRP) to produce a well-defined poly(2-hydroxyethyl methacrylate) (HEMA) gradient substrate. A smooth variation in film thickness was measured across the gradient, ranging from 20 A to over 80 A, but we observed a nonmonotonic variation in water contact angle. Fits of X-ray reflectivity profiles suggested that at the low graft density end, the polymer chain structure was in a "mushroom" regime, while the polymer chains at high graft density were in a "brush" regime. It was found that the "mushroom" region of the gradient could be made adhesive to cells by adsorbing adhesion proteins, and cell adhesion could be tuned by controlling the density of the polymer grafts. Fibroblasts were seeded on gradients precoated with fibronectin to test cellular responses to this novel substrate, but it was found that cell adhesion did not follow the expected trend; instead, saturated cell adhesion and spreading was found at the low grafting density region.
    Article · Jan 2006 · Langmuir
  • Chang Xu · Tao Wu · Ying Mei · [...] · Kathryn L Beers
    [Show abstract] [Hide abstract] ABSTRACT: Tapered copolymer brushes of methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA) were synthesized via surface-initiated atom transfer radical polymerization (ATRP) by gradual addition of HEMA to a reaction mixture that originally only had MMA as monomer. The copolymer brush grew linearly with polymerization time. The tapered copolymer brushes responded to selective solvent treatments. For the same tapered copolymer brush, pretreating the surface with methylene chloride made the surface more hydrophobic; pretreating the surface with methanol increased the surface hydrophilicity. This change in surface properties was reversible and considered to be caused by the solvent induced rearrangement of the polymer brushes, which is supported by atomic force microscopy images of the surface. Our work demonstrates that the properties of the tapered copolymer brush could be finely tuned by careful control of the composition profile.
    Article · Dec 2005 · Langmuir
  • Tao Wu · Ying Mei · Chang Xu · [...] · Kathryn L. Beers
    [Show abstract] [Hide abstract] ABSTRACT: Block copolymers of poly(ethylene oxide-block-2-hydroxypropyl methacrylate) (PEO-b-PHPMA) with a range of molecular masses of the PHPMA block were obtained by controlled radical polymerization on a chip (CRP chip) using a PEO macroinitiator. A series of well-controlled polymerizations were carried out at different pumping rates or reaction times with a constant ratio of monomer to initiator. The stoichiometry of the reactants was also adjusted by varying relative flow rates to change the reactant concentrations.
    Article · Jul 2005 · Macromolecular Rapid Communications
  • Takahiko Nakaoki · Ying Mei · Lisa M. Miller · [...] · Richard A. Gross
    Article · Jun 2005 · Industrial Biotechnology
  • Tao Wu · Ying Mei · João T Cabral · [...] · Kathryn L Beers
    [Show abstract] [Hide abstract] ABSTRACT: While many parallel synthesis methods developed by the pharmaceutical and life science communities are being applied to polymer synthesis, there remains a need to construct "libraries" of polymeric materials that explore a wider range of polymer structures with accuracy, flexibility, and rapid, often small, changes. We report the use of microfluidics to create an environment for continuous controlled radical polymerization. Varying either the flow rate or the relative concentrations of reactants (i.e., stoichiometry) controls the molecular properties of the products. Molecular variables, here molecular weight, can then be varied continuously. Well-defined materials with narrow molecular weight distributions are produced inside the microfluidic reactor and are available for processing, such as further mixing, deposition, or coating on surfaces. Preliminary kinetic data appear to agree well with literature values reported for larger-scale reactions.
    Article · Sep 2004 · Journal of the American Chemical Society
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    Ying Mei · Ajay Kumar · Wei Gao · [...] · John T Elliott
    [Show abstract] [Hide abstract] ABSTRACT: A series of sorbitol-containing polyesters were synthesized via a one-pot lipase-catalyzed condensation polymerization. Thin films were prepared by spin coating on silicon wafers and surfaces were analyzed by tapping mode atomic force microscopy and contact angle measurements. Surface morphologies and surface energies across the series of polyester films, including a poly(epsilon-caprolactone) (PCL) control were nearly indistinguishable. Biocompatibility of the sorbitol-containing polyester series was evaluated against a PCL control by measuring cell spreading and proliferation of a mouse fibroblast 3T3 cell line in vitro. Results confirmed that the sorbitol-containing polyester surfaces elicited cell behavior similar to the PCL control. These results establish the sorbitol-containing polyester series as a promising material for tissue engineering research and development.
    Full-text available · Article · Sep 2004 · Biomaterials
  • [Show abstract] [Hide abstract] ABSTRACT: A versatile methodology to prepare hybrid biomaterials by atom transfer radical polymerization from resin-supported peptides has been established. As an example, we have synthesized a GRGDS-functionalized poly(2-hydroxyethyl methacrylate). The peptide-polymer was characterized by solid-state (13)C NMR and GPC and found to have a number average molecular weight of 4420 and a polydispersity of 1.47. These values are comparable to those obtained from solution-phase syntheses, suggesting the ATRP reaction is successful from a peptide-conjugated solid support. Solid-state (13)C NMR was used to characterize multiple steps in the reaction, and the synthesis was found to be near quantitative. We have performed cell adhesion experiments and observed the GRGDS sequence-promoted cell adhesion, whereas unfunctionalized poly(2-hydroxyethyl methacrylate) did not. By incorporating cell-signaling moieties in materials with defined molecular architecture, it will be possible to control the interactions between polymeric materials and biological systems.
    Article · Apr 2004 · Journal of the American Chemical Society
  • Ying Mei · Ajay Kumar · Richard Gross
    [Show abstract] [Hide abstract] ABSTRACT: Studies of the kinetics and mechanism of Candida antarctica Lipase B (CALB) catalyzed ε-caprolactone (ε-CL) polymerizations in toluene were performed. The kinetic plot of ln ([M]0/[M]t) vs time was carried out to 96% ε-CL conversion and Mn 11 970. The plot is linear (r2 = 0.998), indicating that termination did not occur and the propagation rate is first order with respect to monomer concentration. Changes in the water (e.g., initiator) concentration did not change the polymerization rate but did change the number of chains [R−OH]. Thus, the polymerization is zero order with respect to [R−OH] and initiator concentration. A plot of ln kapp vs ln [enzyme] gave 0.7 as the reaction order of the enzyme concentration. The apparent activation energy for Novozyme-435 catalyzed ε-CL polymerization in toluene is 2.88 kcal mol-1. This is well below 10.3 kcal mol-1, the activation energy for aluminum alkoxide catalyzed ε-CL polymerization in toluene. Upward deviation from linearity for Mn vs fractional ε-CL conversion and decreases in the number of chains was accentuated by low enzyme water contents and high monomer conversion. These results are consistent with a competition between ring-opening chain-end propagation and chain growth by steplike polycondensations. CALB was irreversibly inhibited by modification with paraoxon at the lipase active site (Ser105). The modified enzyme was no longer active for the polymerization. This supports that the polymerizations studied herein occurred by catalysis at the active serine residue (Ser105) and not by other chemical or nonspecific protein-mediated processes.
    Article · Jul 2003 · Macromolecules
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    Ying Mei · Lisa Miller · Wei Gao · Richard A Gross
    [Show abstract] [Hide abstract] ABSTRACT: Synchrotron infrared microspectroscopy (SIRMS) was used for the first time to image the distribution and secondary structure of an enzyme (lipase B from Candida antarctica, CALB) immobilized within a macroporous polymer matrix (poly(methyl methacrylate)) at 10 microm resolution. The beads of this catalyst (Novozyme435) were cut into thin sections (12 microm). SIRMS imaging of these thin sections revealed that the enzyme is localized in an external shell of the bead with a thickness of 80-100 microm. Also, the enzyme was unevenly distributed throughout this shell. Furthermore, by SIRMS-generated spectra, it was found that CALB secondary structure was not altered by immobilization. Unlike CALB, polystyrene molecules of similar molecular weight diffuse easily throughout Novozyme435 beads. Scanning electron micrograph (SEM) images of the Novozyme435 beads showed that the average pore size is 10 times larger than CALB or polystyrene molecules, implying that there is no physical barrier to enzyme or substrate diffusion throughout the bead. Thus, the difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin.
    Full-text available · Article · Jan 2003 · Biomacromolecules
  • [Show abstract] [Hide abstract] ABSTRACT: Polymerizations of ε-CL catalyzed by Novozyme-435 (immobilized Lipase B from Candida antarctica) were studied at temperatures between 20 and 108 °C. The monomer conversion to polymer was remarkably rapid at ambient temperature. At 20 °C by 7 h, ε-CL conversion and product Mn were >97% and 17 800, respectively. Contrary to previous reports, the number of chains formed, as well as the product molecular weight, was almost identical for polymerizations at constant enzyme water content between 60 and 108 °C. Thus, differences in reaction temperature over a 48 °C range did not “free” water from “bound” states so that it could function for chain initiation. At 60 °C, variation in the enzyme water content from 0.6 to 1.9% increased the number of chains formed but did not change the polymerization propagation kinetics. Therefore, the enzyme water content and not the reaction temperature regulated the product molecular weight. In contrast, at 108 °C, an increase in the reaction water content from 0.6 to 1.8% increased both the number of chains and the polymerization propagation kinetics. Explanations for these differences in behavior as a function of temperature and water contents are discussed.
    Article · Jun 2002 · Macromolecules
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    Ying Mei · Tao Wu · Chang Xu · [...] · Lori Henderson
    [Show abstract] [Hide abstract] ABSTRACT: Introduction The control of protein absorption and cell adhesion presents a challenge for various biomedical and biotechnological applications. 1 Surfaces modified with non-fouling polymers such as poly(ethylene glycol) (PEG) are well-known to resist protein absorption and interfere with cell adhesion. Although PEG grafting density is believed to be an important factor to resist protein absorption, the experimental results are not always unambiguous. This is partially due to the fact that grafted PEG layers in many cases are not well characterized and result in poorly controlled grafting density. 1 Our group and others have demonstrated that gradient techniques provide a fast and convenient tool for high throughput screening of polymeric surfaces over a spectrum of material parameters. 2 Similar to PEG, poly(2-hydroxylethyl methacrylate) (HEMA) is a well-established biomaterial, which can resist non-specific protein absorption and cell adhesion. 3 Although the effect of grafting density on protein absorption has been extensively studied, little has been done to further our knowledge on its effects on cellular response, which is critical to many cell-based biomedical and biotechnological applications such as tissue engineering. The goal of this research is to design combinatorial gradient libraries that elicit specific cellular responses as rapid screening tools. Here, we demonstrated the utility of a combinatorial method in preparation of a novel, cell adhesive protein coated poly(HEMA) gradient with variable grafting densities for the investigation of its effect on cell adhesion. Experimental * The details of the preparation and the characterization of poly(HEMA) gradients was available in reference 4. In brief, a self-assembled monolayer of octyltrichlorosilane (OTS) was established on a silica substrate by vapor evaporation. The polymerization initiator solution was slowly pumped into a test tube, which contained the OTS self-assembled monolayer (SAM) covered silicon wafer, to backfill the defects inside the OTS SAM and generate an initiator density gradient. 4 Surface-initiated atom transfer radical polymerization (ATRP) was employed to prepare poly(2-hydroxylethyl methacrylate) (HEMA) grafting density gradient surfaces. 5 The thickness of poly(HEMA) film and protein layer were measured by a variable angle spectroscopic ellipsometer (J. A. Woollam, Inc.). Cell Culture and Cell Imagine Analysis: Substrates were incubated in fibronectin (FN) (25 mg/mL) for at least 5 h at 4 ºC and rinsed with Dulbecco's Phosphate Buffered Saline (DPBS) before use. NIH-3T3 fibroblasts were maintained in Dulbecco's Minimum Essential Media (DMEM) supplemented with nonessential amino acids, glutamine, penicillin, streptomycin and fetal bovine serum, and maintained in a humidified 5 % CO 2 balanced-air atmosphere at 37 °C. Substrates were placed in four-well tissue culture polystyrene plates and NIH-3T3 cells were seeded on the substrates at (2000 cells/cm 2). Substrates were removed from the incubator after 8 h, and fixed for 24 h at room temperature. Cells were stained by Texas Red-C2-Maleimide and 4',6-diamidino-2-phenylindole (DAPI). Images were collected with 1 mm step sizes over the entire area of the gradient samples, and individual cell morphology and cell density were determined with image analysis software. Results and Discussion Synthesis and Characterization of Poly(HEMA) Grafting Density Gradients. In this investigation, we were able to quantify specific biological responses of NIH-3T3 cells on tailored polymeric surfaces. A combinatoral method was employed to make a FN coated poly(HEMA) surface with variable amounts of FN. This was achieved by grafting poly(HEMA) onto silicon wafer, and essentially producing a conformational gradient with both a mushroom and brush regime (see Figure 1) 6 .
    Full-text available · Article ·