Biomedical subjects

Joyce Y Wong

Publications and source records attributed to Joyce Y Wong.

10 recordsLinked to original sources

PubMed2007

Visualization of flow-aligned type I collagen self-assembly in tunable pH gradients.

Collagen is a major component of the extracellular matrix that exhibits unique hierarchical organization at multiple length scales ranging from nano to macroscale. Despite numerous methods to create collagen-based biomaterials, the self-assembly process of collagen ex vivo is poorly understood. Here, we describe a system that uses a microfluidic method to investigate the dynamics of collagen self-assembly. A main inlet stream of semidilute soluble collagen-I is hydrodynamically focused by two side inlet streams, which gradually increases the pH in the main stream. This enables dynamic nonequilibrium investigation of the self-assembly process simultaneously at different positions and therefore different stages in the assembly process within the same system. The device is designed for in situ monitoring and characterization of collagen assembly using polarization microscopy and X-ray diffraction: the continuous extensional flow provides highly ordered phases of the macromolecules over a large distance in the outlet microchannel and allows for data collection without material damage. We further demonstrate that finite element method simulations provide a good description of our experimental results regarding the diffusive phenomena, flow profile, and pH distribution. Our approach has broad impact, since it provides a powerful means of controlling and investigating the dynamic self-assembly process of biomacromolecules.

Animals↗

PubMed2006

A simple microindentation technique for mapping the microscale compliance of soft hydrated materials and tissues.

Several recent studies have shown that cells respond to the elastic modulus and elasticity gradients on soft substrates. However, traditional macroscale methods for measuring elastic modulus cannot resolve elastic gradients or differences between the macroscale and microscale elastic modulus of layered tissues. Here, we present a technique for measurement of the microscale elastic modulus of soft, hydrated gels and tissues. This technique requires less equipment than equivalent atomic force microscopy (AFM) and can easily measure larger samples with high adhesiveness. We validate this technique by measuring the microscale modulus of a hydrogel with elasticity that does not depend on measurement scale. We show that the elastic modulus measured using microindentation correlates with measurements using AFM and the macroscale tensile modulus. We verified the ability of this technique to characterize a hydrogel with an elastic gradient of 2.2 kPa/mm across 19 mm and to measure the microscale elastic modulus of the endothelial side of human greater saphenous vein, which is an order of magnitude less than the whole vein macroscale modulus. This simple, inexpensive system allows the measurement of the spatial organization of microscale elastic properties of fully hydrated, soft gels and tissues as a routine laboratory technique.

Acrylic Resins↗

PubMed2006

Development and characterization of a porous micro-patterned scaffold for vascular tissue engineering applications.

The fabrication of functional small diameter blood vessel analogs has implications in vascular disease treatment. Current 3D models of the medial vessel layer lack micron-scale topographical cues that have shown promise in vitro by recapitulating native vascular smooth muscle cell (VSMC) behavior. A major obstacle to fabricating 3D scaffolds is maintaining adequate nutrient diffusion to cells. We have developed and characterized porous micro-patterned poly-caprolactone (PCL) scaffolds using a novel technique that integrates soft lithography, melt molding and particulate leaching of polylactic-co-glycolic acid (PLGA) micro/nanoparticles. Scanning electron microscopy showed that PLGA-leached scaffolds have circular pores significantly smaller than the size scale of the grooved surface pattern (48 microm grooves; 5 microm deep; 12 microm spacing). Diffusion of media through PLGA-leached scaffolds was six-fold greater than through non-porous scaffolds, indicating successful introduction of through-pores into PCL by the PLGA leaching technique. VSMC alignment on micro-patterned PLGA-leached scaffolds was similar to that on micro-patterned non-porous scaffolds, indicating no loss in cellular organization on PLGA-leached scaffolds. In contrast, cells seeded on micro-patterned sodium bicarbonate-leached scaffolds remained un-aligned. The ability to micro-pattern cells on porous scaffolds may facilitate the transfer of micro-technology from simple 2D substrates to complex 3D architectures, allowing for tight control over cellular organization in fabricated tissues.

Animals↗

PubMed2005

Crosslinked alpha-elastin biomaterials: towards a processable elastin mimetic scaffold.

Elastin is a critical biochemical and biomechanical component of vascular tissue. However, elastin is also highly insoluble and therefore difficult to process into new biomaterials. We present a simple approach for synthesizing elastin-based materials from two commercially available and water-soluble components: alpha-elastin and a diepoxy crosslinker. Reaction pH was shown to modulate the degree of crosslinking, as demonstrated by materials characterized with a range of swelling ratios (approximately 10-25), enzymatic degradation rates (approximately 8-50% per h in 0.1 u/ml elastase), and elastic moduli (approximately 4-120 kPa). Crosslinking with a combination alkaline and neutral pH process results in materials with the highest degree of crosslinks, as indicated by a swelling ratio of 10, slow degradation rate, and high elastic moduli (approximately 120 kPa). Furthermore, the crosslinked alpha-elastin materials support vascular smooth muscle cell (VSMC) adhesion and a decreased proliferation rate compared to polystyrene controls. The functional outcomes of the crosslinking reaction, including the dependence of structure-function properties on reaction pH, are discussed. Our approach towards 'processable' elastin-based materials is versatile and could be integrated into existing tissue engineering methodologies to enhance biomaterial performance by providing a natural elastomeric and biofunctional component.

Animals↗

PubMed2005

Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix.

The in vitro construction of tissue-engineered small diameter (<6mm) blood vessels with sufficient strength and mechanical compliance has evaded researchers. We hypothesize that the high spatial organization of the medial layer of vascular smooth muscle cells (VSMCs) and their surrounding matrix provides high burst strength, compliance, and stability. We investigated the effect of microfabricated polydimethylsiloxane (PDMS) scaffolds with various groove widths on VSMC organization. We found that the presence of these grooved topographical cues significantly enhanced VSMC aspect ratio, alignment, and oriented remodeling of the underlying extracellular matrix. This study suggests that topographical patterning of tissue scaffolds can influence cellular and matrix spatial organization and could provide a framework for achieving the required organization and physical properties for blood vessels.

Actins↗

PubMed2005

Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response.

Polydimethylsiloxane (PDMS) is used extensively to study cell-substrate interactions because its mechanical properties are easily tuned in physiologically relevant ranges. However, changes in mechanical properties also modulate surface chemistry and cell response. Here, we correlate the mechanical and surface properties of PDMS to vascular smooth muscle cell (VSMC) behavior. We find that a 5-fold increase in base:crosslinker ratio leads to approximately 40-fold decrease in elastic modulus but no significant differences in surface wettability. However, when polyelectrolyte multilayers are adsorbed to promote cell adhesion, wettability varies inversely with substrate stiffness. Despite these differences in hydrophobicity, the amount of adsorbed protein remains the same. In the absence of serum, there is a 39% decrease in cell attachment and a 42% decrease in spreading as the elastic modulus decreases from 1.79 to 0.05 MPa. In the presence of serum or adsorbed fibronectin, the differences in attachment and spreading are diminished. This is not the case for the rate of serum-stimulated cell proliferation, which remains inversely dependent on crosslinker concentration. We conclude that for the range of crosslinker concentrations investigated, the surface properties dominate the initial cell attachment and spreading, whereas the mechanical properties influence the long-term cell growth.

Adsorption↗

PubMed2004

Patterning adjacent supported lipid bilayers of desired composition to investigate receptor-ligand binding under shear flow.

To achieve efficient targeting, carriers containing either drugs or imaging agents must have surface properties that promote binding to targets yet at the same time block rapid immune system clearance. Here we describe a versatile technique that allows simultaneous comparison of the effects of carrier surface composition on binding properties under identical flow conditions. Parallel lanes of supported lipid bilayers that mimic the surface of liposomal delivery vehicles are formed using the vesicle fusion method in microfluidic channels created via standard soft lithography techniques. Vesicle stock solutions are premixed and injected into lanes formed by a poly(dimethylsiloxane) (PDMS) stamp reversibly sealed to a glass slide to create adjacent lanes of distinct composition. After removing the stamp, an adsorbed layer of bovine serum albumin (BSA) is used to prevent bilayer spreading before assembling the patterned substrate into a flow chamber for binding studies. Advantages of this method include easy and rapid preparation of bilayers with desired compositions from an unlimited number of lipid types, choice of feature size, time-stable features, and low nonspecific binding. Feature sizes on the order of tens of microns allow multiple compositions to be analyzed in one field of view, thereby reducing the number of experiments, ensuring identical flow conditions, and enabling simultaneous incorporation of controls. We show that the presence of a long poly(ethylene glycol) (PEG) tether (MW 2000) between the lipid and ligand results in higher detachment resistances as compared to a short six-carbon spacer.

Animals↗

PubMed2004

Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata.

Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (sigma) with a maximum at sigma approximately 200 molecules/microm2, whereas the total traction force increases and reaches a plateau as a function of sigma. In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by approximately 200 microm2 over a approximately 40-fold change in sigma. In addition, fibroblasts on FN exert approximately 3-5 times more total force, which reaches a maximum at a value of sigma approximately 5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN (sigma approximately 2000 microm(-2)) and RGD (sigma approximately 200 microm(-2)) have both similar spread area (approximately 600 microm2) and migration speed (approximately 11 microm/h), yet the total force production is five times higher on FN than RGD (approximately 0.05 dyn compared to approximately 0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD.

Adsorption↗

PubMed2003

Influence of type I collagen surface density on fibroblast spreading, motility, and contractility.

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.

Animals↗

PubMed2002

Identification and validation of a novel cell-recognition site (KNEED) on the 8th type III domain of fibronectin.

Interactions between cell-surface integrins and extracellular matrix proteins underlie a versatile recognition system providing cells with anchorage, traction for migration or matrix remodeling, as well as signals for polarity, differentiation and growth. Short peptide sequences of fibronectin (FN), most notably RGD found on a loop in the 10th type III domain, are effective in promoting cell adhesion when immobilized to a biomaterial scaffold. Additional sequences (e.g. PHSRN) have been shown to act synergistically to enhance cell adhesion and other cellular processes. Using bioinformatics, we identified a candidate cell-binding peptide sequence, KNEED, located on the loop region of the 8th domain of FN that from in vitro studies appears to participate in cell attachment and spreading. Computational analysis revealed that KNEED exhibits both high solvent accessibility and sequence conservation values across FN sequences from seven species. We demonstrate the importance of the KNEED sequence using a solution-phase competitive inhibition assay utilizing soluble peptides. Results indicate that the presence of soluble KNEED peptides inhibits the attachment and spreading of 3T3 balb/c fibroblasts on FN-coated surfaces in a concentration-dependent manner. As more sequence and crystallographic data become available, computational approaches may aid in the identification of new targets for applications where biorecognition plays a key role.

3T3 Cells↗