Spatially localizing and temporally controlling growth factor presentation for angiogenesis can create spatially organized tissues.

Growth factors have been widely used in strategies to regenerate and repair diseased tissues, but current therapies that go directly from bench to bedside have had limited clinical success. We hypothesize that engineering successful therapies with recombinant proteins will often require specific quantitative information of the spatiotemporal role of the factors and the development of sophisticated delivery approaches that provide appropriate tissue exposures. This hypothesis was tested in the context of therapeutic angiogenesis. An in vitro model of angiogenesis was adapted to quantify the role of the concentration/gradient of vascular endothelial growth factor [VEGF(165)] on microvascular endothelial cells, and a delivery system was then designed, based on a mathematical model, to provide the desired profile in ischemic mice hindlimbs. This system significantly enhanced blood vessel formation, and perfusion and recovery from severe ischemia. This general approach may be broadly applicable to growth factor therapies.

Nature frequently utilizes opposing factors to create a stable activator gradient to robustly control pattern formation. This study employs a biomimicry approach, by delivery of both angiogenic and antiangiogenic factors from spatially restricted zones of a synthetic polymer to achieve temporally stable and spatially restricted angiogenic zones in vivo. The simultaneous release of the two spatially separated agents leads to a spatially sharp angiogenic region that is sustained over 3 wk. Further, the contradictory action of the two agents leads to a stable level of proangiogenic stimulation in this region, in spite of significant variations in the individual release rates over time. The resulting spatially restrictive and temporally sustained profiles of active signaling allow the creation of a spatially heterogeneous and functional vasculature.controlled drug delivery | peripheral ischemia | VEGF antibody | diffusion reaction | Turing pattern

Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-β1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-β1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.

Bacterial adhesion, the fi rst step of biofi lm formation, is of fundamental signifi cance for multiple industries (e.g., petroleum recovery, food processing, drinking water, medicine and healthcare, shipping, or pulp and paper production) due to the huge economic costs associated with biofi lm formation. Assays to monitor bacterial adhesion are the key to elucidate mechanisms of colonization and biofi lm formation. However, the existing microscopy tools typically used to monitor bacterial adhesion are based on observations of bacterial colony formation. In particular, the current optical toolboxes qualitatively defi ne cell adhesion as a simply physically stable association. They neither allow for studying the three-dimensional distribution of bacteria associated with a surface nor for molecular-level analysis of the bacterial interactions that mediate close contact. While some effort has been made in recent years to monitor the adhesion of bacteria populations in real-time using surface plasmon resonance, those techniques do not allow for individual cells to be tracked and provide no quantitative information regarding contact distance so far. [ 1 ] Here, we demonstrate the possibility of obtaining precise information on the three-dimensional distribution of bacteria coming into contact with a surface and propose this new optical technique as a method to monitor and probe individual bacterial adhesion.In our experimental approach ( Figure 1 a), a fl at gold surface of 35 nm thickness was used as the basic substrate for cell adhesion and was prepared by a standard electron beam lithographic technique. Previous reports showed that fi eld enhancement is maximal for 30-60-nm-thick gold layers, and the thickness (35 nm) used in our studies was in this range. [ 2 ] The morphologies of the glass substrate and gold surfaces were investigated using atomic force microscopy (AFM), and the root-mean-square (RMS) roughness of the gold-coated surface was similar to the one of the glass substrate ( Figure S1 in the Supporting Information). The membranes of Pseudomonas aeruginosa strains (PA14 wild type, PA14 pilB , or PA14 fl gK ::Tn5Tet) were labeled with FM4-64, a red fl uorescent dye, as it provided the most uniform and even staining of the various dyes we examined (Table S1 in the Supporting Information). [ 3 ] Wildtype PA14 in close contact with a gold substrate showed a strong increase of FM4-64 fl uorescence compared to cells in contact with the adjacent glass substrate (Figure 1 b and demonstration movies in the Supporting Information). The fl uorescence enhancement was hypothesized to mirror the distance between the cell and the gold surface and to provide a tool to quantitatively monitor bacterial proximity to surfaces. The behavior of individual bacteria on gold surfaces was analyzed next. First, the residence time of the bacteria, defi ned by the length of time the bacteria exhibited enhanced fl uorescence, was analyzed, and a strong correlation was noted between the fl uorescence intensity of the wild-type cells a...