Faculty Bibliography

We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices (E=322+/-64.2 Pa, stoichiometric ratio: 0.8), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices (E=4,036+/-419.6 Pa, stoichiometric ratio: 1.2) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (alpha(5)beta(1,) alpha(v)beta(3)) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications.

A growing body of evidence suggests that the sensory information from the cytoskeleton and integrins may be responsible for guiding migration during mechano- and haptotaxis. However, the dual function of these subcellular structures as mechano-sensors and -actuators is only partially understood. Using a new cell chamber described in the preceding companion paper (Ref to part I, Raeber et al. 2007a) we investigated the migration response of adhesion-dependent fibroblasts embedded 3-dimensionally within synthetic protease-sensitive poly(ethylene glycol) hydrogels to stepwise and cyclic mechanical loads. To that end, we developed a spatially and temporally resolved migration analysis technique capable of providing estimates of statistical cell migration parameters along and perpendicular to the main strain direction. Fibroblasts reoriented themselves in the direction of principal strain, increased their proteolytic migration activity and moved preferentially parallel to the principal strain axis. These results point to a possible correlation between planes of iso-strain and migration direction.

Photocatalytic lithography couples light with photoreactive coated mask materials to pattern surface chemistry. We excite porphyrins to create radical species that photocatalytically oxidize, and thereby pattern, chemistries in the local vicinity. The technique advantageously is suited for use with a wide variety of substrates. It is fast and robust, and the wavelength of light does not limit the resolution of patterned features. We have patterned proteins and cells to demonstrate the utility of photocatalytic lithography in life science applications.

The slow interstitial flow that drains fluid from the blood capillaries into the lymphatic capillaries provides transport of macromolecular nutrients to cells in the interstitium. We discuss herein how this flow also provides continuous access to immune cells residing in the lymph nodes of antigens from self or from pathogens residing in the interstitium. We also address mechanisms by which dendritic cells in the periphery sense interstitial flow to home efficiently into the lymphatics after activation, and how lymphatic endothelium can be activated by this flow, including how it can act as a lymphatic morphoregulator. Further, we present concepts on how interstitial flow can be exploited with biomaterial systems to deliver antigen and adjuvant molecules directly into the lymphatics, to target dendritic cells residing in the lymph nodes rather than in the peripheral tissues, using particles that are small enough to be carried along by flow through the network structure of the interstitium. Finally, we present recent work on lymphatic and lymphoid tissue engineering, including how interstitial flow can be used as a design principle. Thus, an understanding of the physiological processes that govern transport in the interstitium guides new understanding of both immune cell interactions with the lymphatics as well as therapeutic interventions exploiting the lymphatics as a target.

We here present an evaluation of the carrier performance of nanoparticles that are biofunctional, i.e. derivatized to provide a controlled biological activity, and environmentally responsive, since they respond to the presence of oxidants. In particular, we focus on the possibilities (a) to make the nanoparticles detectable and (b) to control their uptake in phagocytic cells, which determines their lifetime in vivo. We first describe techniques for labeling selectively the nanoparticle surface or bulk with imaging moieties (fluorophores or gold). We then show how surface composition and size, which are both controlled through the use of PEG derivatives, influence uptake by macrophages in vitro and blood circulation in vivo: for example, in vitro uptake is negligible for small (40 nm) particles but not for larger (100 nm) ones and, correspondingly, in vivo blood circulation half-life time decreases from 6.0 to 2.9 h. However, upon decoration with RGD peptides also the small particles can be significantly internalized.

A major challenge for therapeutic delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF(121), TG-VEGF(121) that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare endothelial activation and long-term neovascularization by actively released TG-VEGF(121)versus passively released, diffusible wild-type VEGF(121) in subcutaneous fibrin implants. Monitoring in real-time over 3 weeks of luciferase signal driven by the VEGFR2 promoter revealed endothelial activation in skin exposed to wild-type VEGF(121), but no detectable elevation over fibrin alone by TG-VEGF(121). Histology at 3 weeks, however, demonstrated that TG-VEGF(121) promoted vessel growth significantly more effectively and reliably than wild-type VEGF(121). The majority of vessels surviving to 3 weeks contained stabilizing smooth muscle cells. Yet, by 6 weeks, no extra vessels induced by exogenous VEGF were left. In conclusion, release of fibrin-conjugated variant TG-VEGF(121) elicited lower VEGFR2-luc activation than wild-type VEGF(121) yet significantly more vascularization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, sufficiently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II alpha1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse.

We present a microfluidic device where micro- and nanoparticles can be continuously functionalized in flow. This device relies on an element called "particle exchanger", which allows for particles to be taken from one medium and exposed to some reagent while minimizing mixing of the two liquids. In the exchanger, two liquids are brought in contact and particles are pushed from one to the other by the application of a dielectrophoretic force. We determined the maximum flow velocity at which all the particles are exchanged for a range of particle sizes. We also present a simple theory that accounts for the behaviour of the device when the particle size is scaled. Diffusion mixing in the exchanger is also evaluated. Finally, we demonstrate particle functionalization within the microfluidic device by coupling a fluorescent tag to avidin-modified 880 nm particles. The concept presented in this paper has been developed for synthesis of modified particles but is also applicable to on-chip bead-based chemistry or cellular biology.