Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

Competition between metal-amido and metal-imido chemistries in the alkaline earth series: an experimental and theoretical study of BaNH.

The pure rotational spectrum of BaNH in its X(1)Sigma(+) ground electronic state has been recorded using millimeter/submillimeter direct absorption methods; data for the deuterium and barium 137 isotopomers have been measured as well. The molecules were produced by the reaction of ammonia or ND(3) and barium vapor in the presence of a dc discharge. Transitions arising from the ground vibrational state and the excited vibrational bending (01(1)0) and heavy atom stretching (100) modes were measured. The rotational spectrum indicates a linear structure, with B(0)(BaNH) = 7984.549 MHz and B(0)(BaND) = 7060.446 MHz. An r(m)((1)) structure has been determined, yielding r(BaN) = 2.077 +/- 0.002 Angstroms and r(NH) = 1.0116 +/- 0.0006 Angstroms. Density functional calculations using an extensive Slater-type basis set with inclusion of scalar relativistic effects gives geometrical parameters and vibrational frequencies for BaNH in excellent agreement with those determined by experiment. The molecular orbital and natural bond order analyses of the BaNH wave function show Ba-N pi bonds formed by electron donation from the formally filled N 2p orbitals of the imido group to the empty Ba 5d orbitals. The multiple bonding between Ba and N stabilizes the linear geometry and, along with the relative ease of oxidation of the Ba atom, favors formation of the metal-imido species over that of the metal-amido species that have been found from similar studies with Mg, Ca, and Sr atoms in this group.