Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND: The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS: Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS: After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS: Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean meal diets.

Two experiments were conducted with pigs 1) to determine the effect of supplemental Zn on growth performance, bone Zn, and plasma Zn in pigs fed Zn-unsupplemented, corn-soybean meal diets and 2) to assess bioavailability of Zn from inorganic and organic Zn sources. In both experiments, weanling pigs were fed a diet with no supplemental Zn for 5 wk to deplete their Zn stores. In Exp. 1, 192 pigs were fed a corn-soybean meal diet (growing diet, 32 mg/kg of Zn; finishing diet, 27 mg/kg of Zn) supplemented with feed-grade ZnSO4.H2O to provide 0, 5, 10, 20, 40, and 80 mg/kg of supplemental Zn. Supplemental Zn did not affect weight gain, feed intake, or gain/feed during either the growing or the finishing period (P > .05). However, bone and plasma Zn concentrations increased linearly (P < .01) in response to supplemental Zn at dietary Zn levels between 27 mg/kg (basal) and 47 mg/kg (breakpoint). In Exp. 2, three levels of supplemental Zn from ZnSO4.H2O (0, 7.5, and 15 mg/kg of supplemental Zn) were used to construct a standard curve (metacarpal, coccygeal vertebrae, and plasma Zn concentrations regressed on supplemental Zn intake; R2 = .93, .89, and .82, respectively). From the standard curve, the bone and plasma Zn concentrations obtained from pigs fed 15 mg/kg of supplemental Zn from ZnO and 7.5 mg/kg of supplemental Zn from Zn-methionine (ZnMET) and Zn-lysine (ZnLYS) were used to calculate bioavailable Zn via multiple linear regression, slope-ratio analysis. The estimates of Zn bioavailability differed depending on which variable was used. Overall trends indicated the following rankings: ZnSO4.H2O > ZnMet > ZnO > ZnLys.

Vanadium metabolism in sheep. II. Effect of dietary vanadium on performance, vanadium excretion and bone deposition in sheep.

Sixteen ram lambs were fed 0, 50 or 200 ppm supplemental vanadium daily as NH4VO3 for 90 d and coccygeal vertebrae were sampled at 0, 15, 30, 60 and 90 d after the imposition of dietary treatments. Vertebral vanadium content was elevated (P less than .05) after exposure to 200 ppm added vanadium in the diet of 15 d, but nonsignificant increases occurred thereafter. Increasing dietary vanadium increased bone ash vanadium concentration from .4 to 1.7 and 3.8 ppm in sheep fed 0, 50 and 200 ppm supplemental vanadium, respectively. In a balance study, urinary concentrations of the element also were related directly to dietary intake. A trend toward elevated urine volume with increased intake of vanadium was also observed.