Biomedical subjects

Kristyn S Masters

Publications and source records attributed to Kristyn S Masters.

7 recordsLinked to original sources

PubMed2006

Detection of antigens in biologically complex fluids with photografted whole antibodies.

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

Antibodies↗

PubMed2005

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Acrylates↗

PubMed2005

Serum deprivation improves seeding and repopulation of acellular matrices with valvular interstitial cells.

Cell-extracted valvular tissues (acellular scaffolds, or aScaffolds) offer unique advantages over synthetic polymers for cardiac valve engineering applications in that they retain extracellular matrix molecules to support cellular ingrowth. The extracellular matrix is important in directing many cellular pathways, such as adhesion, proliferation, migration, differentiation, and survival. However, repopulating this type of scaffold often requires high seeding densities or recurrent cell delivery. The optimization of valvular interstitial cell (VIC) seeding onto aScaffolds is reported herein. VICs (the most prevalent cell type in valve leaflets) have maximal growth in 15-20% serum concentrations on tissue-culture polystyrene. Interestingly, after VIC seeding onto aScaffolds, a reduction of serum content, from 15% serum to 5% or less, was found to increase significantly the number of adherent cells, as well as induce transfer of VICs from a tissue-culture polystyrene surface to the aScaffold. aScaffolds seeded and cultured with periods of reduced serum levels were shown to support and enhance VIC viability and attachment, as well as accelerate VIC migration into the aScaffold, leading to a uniformly repopulated valve leaflet construct after 4 weeks of static culture.

Animals↗

PubMed2005

Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells.

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.

Animals↗

PubMed2004

Designing scaffolds for valvular interstitial cells: cell adhesion and function on naturally derived materials.

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.

Animals↗

PubMed2004

Valvular myofibroblast activation by transforming growth factor-beta: implications for pathological extracellular matrix remodeling in heart valve disease.

The pathogenesis of cardiac valve disease correlates with the emergence of muscle-like fibroblasts (myofibroblasts). These cells display prominent stress fibers containing alpha-smooth muscle actin (alpha-SMA) and are believed to differentiate from valvular interstitial cells (VICs). However, the biological factors that initiate myofibroblast differentiation and activation in valves remain unidentified. We show that transforming growth factor-beta1 (TGF-beta1) mediates differentiation of VICs into active myofibroblasts in vitro in a dose-dependent manner, as determined by a significant increase in alpha-SMA and the dramatic augmentation of stress fiber formation and alignment. Additionally, TGF-beta1 and increased mechanical stress function synergistically to enhance contractility. In turn, contractile valve myofibroblasts exert tension on the extracellular matrix, resulting in a dramatic realignment of extracellular fibronectin fibrils. TGF-beta1 also inhibits valve myofibroblast proliferation without enhancing apoptosis. Our results are consistent with activation of a highly contractile myofibroblast phenotype by TGF-beta1 and are the first to connect valve myofibroblast contractility with pathological valve matrix remodeling. We suggest that the activation of contractile myofibroblasts by TGF-beta1 may be a significant first step in promoting alterations to the valve matrix architecture that are evident in valvular heart disease.

Actins↗

PubMed2002

Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering.

Photoactive polyvinyl alcohol hydrogels (PVA) have been investigated for use as tissue engineering scaffolds. These materials allow in situ polymerization for minimally invasive implantation methods. The mechanical properties of these materials can be tailored for a variety of soft tissue applications. The Young's modulus and ultimate tensile strength of PVA hydrogels are increased with increasing polymer concentration, and highly elastic hydrogels can be formed by altering the number of crosslinkable groups per chain. Fibroblasts homogeneously seeded within 3 mm thick PVA hydrogels remained viable throughout 2 weeks in culture, with no differences in viability across the thickness of the hydrogel. Cells seeded within the PVA hydrogels also produce extracellular matrix proteins, as indicated by the production of hydroxyproline during culture. Intrinsically cell non-adhesive, these PVA hydrogels were functionalized with the cell-adhesive peptide RGDS and found to support the attachment and spreading of fibroblasts in a dose-dependent manner. These results suggest that photopolymerizable PVA hydrogels are promising for tissue engineering applications.

Biocompatible Materials↗