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Unformatted text preview: elopmental biology.
This study was based on the hypothesis that clear demarcation
of stimulatory zones for regeneration can be achieved via appropriate codelivery of stimulatory and inhibitory factors. This
hypothesis was examined in the context of VEGF-driven angiogenesis, using delivery of both recombinant human VEGF and an
angiogenic inhibitor, anti-VEGF antibody (anti-VEGF) (17), and
utilizing a biodegradable polymer scaffold system to allow local
and sustained release of the two factors. The ability of this
approach to spatially regulate angiogenesis was examined in a
model of hind-limb ischemia (18), due to its relevance to clinical
situations requiring revascularization interventions.
Materials and Methods
Cell Culture and in Vitro Sprouting Assay. Dermal human vascular endothelial
cells were seeded onto microcarrier beads and subsequently embedded in
fibrin gels. Detailed protocols are included in SI Text. Gels were incubated
at 37 °C for 30 min, and media of experimental conditions were placed on
top of the gel. Experimental media were prepared by adding appropriate
concentrations of VEGF and anti-VEGF to EGM-2MV without the growth
factor supplements, but with the addition of 10 ng∕mL hepatocyte growth
factor for all conditions. After 4 d, the gels were fixed with 4% paraformaldehyde. Subsequent to fixing, samples were stained with DAPI and visualized
at 10× objective magnification with an Olympus IX2 microscope. Sprouts
were identified as continuous multicellular structures extended from the
microcarrier beads with a minimum of two cells in the structure.
Scaffold Fabrication. An 85∶15, 120-kD copolymer of D,L-lactide and glycolide
(PLG) (Alkermes) was used in a gas-foaming process to form macroporous
PLG matrix scaffolds (19). All scaffolds were cylinders 4.2 mm in diameter
and 3 mm in thickness. Detailed protocol for constructing scaffolds is included in SI Text. Four types of scaffolds were fabricated: (i) blank scaffolds
without protein incorporation (B), (ii) scaffolds with 4 μg of VEGF (V),
(iii) 3-layered scaffolds with a 1-mm central layer containing 4 μg of VEGF
and two surrounding 1-mm layers without protein incorporation (BVB for
Blank-VEGF-Blank), and (iv) three-layered scaffolds with a 1-mm central layer
containing 4 μg of VEGF and two surrounding 1-mm layers each incorporating 20 μg of anti-VEGF (R&D Systems AB-293-NA) (AVA for anti-VEGF-VEGFanti-VEGF).
Quantification of Protein Release Kinetics. The release kinetics of anti-VEGF
and VEGF from each layer of the scaffold were determined using 0.11 μCi
125 I-labeled anti-mouse IgG (PerkinElmer) and 0.11 μCi 125 I-labeled human
VEGF (PerkinElmer), respectively, as tracers. The tracers were entrapped
in scaffolds using an identical process with the remaining bulk quantities
consisting of unlabeled anti-VEGF and unlabeled VEGF, respectively. The total Author contributions: W.W.Y. designed research; W.W.Y., N.R.D., C.H.C., and E.A.S.
performed research; W.W.Y. and...
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- Spring '13