EXTRACELLULAR MATRIX–BASED SCAFFOLDS FROM SCRATCH - Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine - page 393

transforming growth factor-beta (TGF- β ), and platelet-derived
growth factor (PDGF). 37 In vivo , growth factors are stored in the
ECM. One of the functions of glycosaminoglycans is to stabilize
growth factors and to protect them from proteolytic degradation
by their interactions, thereby providing a depot of growth factors. 38
Whengrowthfactorsareboundtoscaffoldsviaglycosaminoglycans,
a sustained release isfound. 35
To bind growth factors to the scaffold, a scaffold containing gly-
cosaminoglycans is incubated in a growth factor solution in phos-
phate buffered saline (PBS) at ambient temperature, followed by
washings with PBS to remove unbound growth factors. The binding
and release characteristics of the growth factors can be determined
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE), Western blotting, or autoradiography in case of the use of
radiolabeled growth factors. The localization can be visualized with
immunostainings. An example of the in vivo effect of growth fac-
torsboundtoacellularcollagen-glycosaminoglycanscaffoldsisgiven
next.
20.4 Acellular Scaffolds for in vivo Tissue Regeneration
Inordertosystematicallystudytheeffectofgrowthfactorsboundto
collagen scaffolds via the glycosaminoglycan heparin, we prepared
five different porous scaffolds composed of 1) cross-linked colla-
gen only (COL), 2) cross-linked collagen with covalently incorpo-
rated heparin (COLH), 3) collagen, heparin, and bound FGF2 (1.6 ±
0.2 μ g/mg scaffold, COLHF), 4) collagen, heparin, and bound VEGF
(1.0 ± 0.3 μ g/mg scaffold, COLHV), and 5) collagen, heparin, bound
FGF2, and VEGF (1.0 ± 0.2 and 0.7 ± 0.3 μ g/mg scaffold, COLHFV).
Fullycharacterizedscaffoldsweresubcutaneouslyimplantedinrats
and analyzedafter 3, 7, and21 days. 39
Angiogenesis and numbers of hypoxic cells were analyzed.
From seven days on, angiogenesis could be observed, both macro-
scopically (Fig. 20.5a) and by type IV collagen immunostaining
(Fig. 20.5b). Most blood vessels were observed in the scaffold with
both growth factors (COLHFV).