Faculty Bibliography

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.

A new technique has been developed that combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. This new technique, termed "electrochemical optical waveguide lightmode spectroscopy" (EC-OWLS), proved efficient in monitoring molecular surface adsorption and layer thickness changes of an adsorbed polymer layer examined in situ as a function of potential applied to a waveguide in a pilot study. For optical sensing, a layer of indium tin oxide (ITO) served as both a high-refractive-index waveguide and a conductive electrode. In addition, an electrochemical flow-through fluid cell was provided, which incorporated working, reference, and counter electrodes, and was compatible with the constraints of optical sensing. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) served as a model, polycation adsorbate. Adsorption of PLL-g-PEG from aqueous buffer solution increased from 125 to 475 ng/cm(2 )along a sigmoidal path as a function of increasing potential between 0 and 1.5 V versus the Ag reference electrode. Upon buffer rinse, adsorption was partially reversible when a potential of >/=0.93 V was maintained on the ITO waveguide. However, reducing the applied potential back to 0 V before rinsing resulted in irreversible polymer adsorption. PLL-g-PEG modified with biotin demonstrated similar adsorption characteristics, but subsequent streptavidin binding was independent of biotin concentration. Applying positive potentials resulted in increased adsorbed mass, presumably due to polymer chain extension and reorganization in the molecular adlayer.

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 microg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.

Alkanethiolates have been widely used as chemisorbates to modify gold surfaces, in spite of their relatively poor oxidative stability. We introduce gold-chemisorbing block copolymers bearing an anchoring block of poly(propylene sulphide) (PPS), selected in the expectation of greater stability. These materials offer a more robust approach to surface modification of gold. As an example, a triblock copolymer with poly(ethylene glycol) (PEG) was selected, with the goal of minimizing biological adsorption and adhesion. The copolymer PEG17-bl-PPS25-bl-PEG9 chemisorbed to form a dense monolayer of 226 +/- 26 ng cm(-2), approximately 2.2 nm thick. The copolymeric adlayer was much more stable to oxidation than commonly used alkanethiolates. Its presence greatly reduced protein adsorption (>95%), even after exposure to whole blood serum (>55 mg x ml(-1)), as well as cell adhesion over long culture durations (>97%). PPS-containing copolymers are an attractive alternative to alkanethiolates, and PEG-bl-PPS-bl-PEG presents a powerful example for use in biodiagnostic and bioanalytical devices.

A novel process for the preparation of water-borne biomaterials for hard tissue repair from injectable precursors is described, where the precursors form crosslinked materials in situ under physiological conditions. The precursors react by means of a Michael-type addition reaction that makes use of addition donors such as pentaerythritol tetrakis 3'-mercaptopropionate (QT) and addition acceptors such as poly(ethylene glycol) diacrylate 570 MW (PEGDA), pentaerythritol triacrylate (TA), and poly(propylene glycol) diacrylate 900 MW (PPODA). These crosslinked materials (at 75 wt% solid), prepared from water dispersions or reverse emulsions, showed ultimate strengths in compression of 1.8 +/- 0.2 and 6.7 +/- 0.5 MPa and ultimate deformations of 35 +/- 2+/- and 37 +/- 2%, respectively. Scanning electron microscopy (SEM) shows that the morphology of the precursors templated the morphology of the final materials. The current study indicates that it is possible to obtain injectable high-modulus materials that have appropriate mechanical properties and gelation kinetics for tissue augmentation and stabilization applications such as mechanical stabilization of the intervertebral disc annulus.

Hyaluronic acid (HA) was derivatized with methacrylic esters used for the preparation of hydrogels via photopolymerization. Poly(ethylene glycol) diacrylate (PEG-DA) with a molecular weight of 570 was also used as a comacromonomer to improve elastic modulus and swelling behavior. The hydrogels were readily degraded by hyaluronidase and their mechanical properties could be modulated by HA molecular weight and concentration of PEG-DA. The incorporation of RGD peptides allowed modulation of the HA properties from cell non-adhesive to adhesive. Human dermal fibroblasts were cultured on the RGD, RDG, and non-functionalized HA hydrogels for up to 7d, showing adhesion and proliferation only with incorporated RGD.