Problem#3Mimicking nature by codelivery of stimulant and inhibitor

Perfusion in the normal hind limb of the same animal

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Unformatted text preview: l hind limb of the same animal. Results System Development. The relation between VEGF and anti-VEGF concentrations on angiogenesis was first evaluated using a common in vitro sprouting assay, in order to quantitatively determine the appropriate doses of the two factors for subsequent in vivo studies. VEGF induced angiogenic sprouting, an analog to the initial stage of angiogenesis, whereas anti-VEGF reduced the angiogenic effects of VEGF (Fig. S2), as expected. The dosedependent effects of anti-VEGF at a constant VEGF of 50 ng∕mL were analyzed, and an anti-VEGF concentration 50-fold greater than that of VEGF effectively eliminated the angiogenic effects of VEGF (Fig. S2b). To allow local and sustained delivery of VEGF and anti-VEGF, the proteins were incorporated into poly(lactide-co-glycolide) scaffolds that have been commonly utilized in the past for delivery of single stimulatory factors (18). However, in this situation, three-layer PLG scaffolds were fabricated, and the different proteins were localized into the distinct layers. Protein that was incorporated into each layer of the scaffold remained confined to that layer, as demonstrated previously (20). Radiolabeled tracers were used to model the release of the two proteins from the scaffolds, and there was a sustained release of the proteins over several weeks (Fig. 1). Approximately 60% and 75%, respectively, of VEGF and anti-VEGF were released in the first 3 d. Notice that the initial burst release for anti-VEGF was greater than that of VEGF. Over the next 11 d, the release rates varied between 0.5% to 3% per day, and from day 14 to 31, only 1–2% of the proteins were released. Computational Model of Protein Distribution. In order to design appropriate encapsulated doses of VEGF and anti-VEGF to create spatially defined angiogenic regions, mass transport PDEs of the proteins in the scaffolds and the underlying tissues were simulated. Parameters for the models were obtained from empirical release kinetics and diffusion and degradation coefficients from literature. To validate the model, predictions of the model were compared to experimentally determined VEGF concentration Fig. 1. In vitro cumulative release kinetics of anti-VEGF antibody and VEGF from scaffolds. Initial mass of proteins incorporated were 4 μg of VEGF and 20 μg of anti-VEGF. Values represent mean, and error bars represent standard deviations (n ¼ 5). Yuen et al. Sðx;y;zÞ ¼  1 for ½VEGFf Š > 5 ng∕mL : 0 for ½VEGFf Š ≤ 5 ng∕mL Sðx;y;zÞ ¼ 1 indicated that angiogenesis was promoted at said coordinate and local S ¼ 0 indicated that angiogenesis was inhibited. Plots of S vs. x (Fig. 2H) demonstrated that angiogenic promotion was restricted with this system in the approximate 1-mm central region, which was defined as the angiogenicpromoting region (APR). This spatial restriction was maintained for 3 wk, demonstrating a highly stable environment, although the APR first broadened then contracted slowly. In...
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This document was uploaded on 09/21/2013.

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