Microengineered PEG hydrogels: 3D scaffolds for guided cell growth.

Designing three-dimensional (3D) scaffolds for selective manipulation of cell growth is of high relevance for applications in regenerative medicine. Especially, scaffolds with oriented morphologies bear high potential to guide the restoration of specific tissues. The fabrication of hydrogel scaffolds that support long-term survival, proliferation, and unidirectional growth of embedded cells is presented here. Parallel channel structures are introduced into the bulk hydrogels by uniaxial freezing, providing stable, and uniform porosity suitable for cell invasion (pore diameters of 5-15 µm). In vitro assessment of the scaffolds with murine fibroblasts (NIH L929) shows a remarkable unidirectional movement along the channels, with the cells traveling several millimeters through the hydrogel.

Structural evidence of a fourth Gla residue in fish osteocalcin: biological implications.

Osteocalcin is a small (45 amino acids) secreted protein found to accumulate in bone and dentin of many organisms by interacting with calcium and hydroxyapatite, through the presence of three gamma-carboxylated residues. In this work, we describe the first X-ray crystal structure for a nonmammalian osteocalcin, obtained at 1.4 A resolution, purified from the marine teleost fish Argyrosomus regius. The three-dimensional fit between the A. regius structure and that of the only other known X-ray structure, the porcine osteocalcin, revealed a superposition of the Calpha atoms of their metal chelating residues, Gla and Asp, showing that their spatial distribution is consistent with the interatomic distances of calcium cations in the hydroxyapatite crystals. In both structures, the protein forms a tight globular arrangement of their three alpha-helices while the remaining residues, at N- and C-terminal regions, have essentially no secondary structure characteristics. This study revealed the presence of a fourth gamma-carboxylation at Glu(25), not previously detected in the structure of the porcine osteocalcin or in any other of the sequentially characterized mammalian osteocalcins (human, cow, and rat). A confirmation of the fourth Gla residue in A. regius osteocalcin was achieved via LC-MS analysis. These four doubly charged residues are, together with Asp(24), concentrated in a common surface region located on the same side of the molecule. This further suggests that the known high affinity of osteocalcin for bone mineral may be derived from the clustering of calcium binding sites on this surface of the molecules.