Background: The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions by forming colloidal protein-mineral complexes called calciprotein particles (CPP). CPP are initially spherical, amorphous and soft, and are referred to as primary CPP. These particles spontaneously convert into secondary CPP, which are larger, oblongate, more crystalline, and less soluble. CPP mediate excess mineral transport and clearance from circulation.Methods: We studied by intravital two-photon microscopy the clearance of primary vs. secondary CPP by injecting i.v. synthetic fluorescent CPP in mice. We analyzed CPP organ distribution and identified CPP endocytosing cells by immunofluorescence. Cellular clearance was studied using bone marrow-derived mouse wildtype and scavenger receptor A (SRA)-deficient macrophages, as well as human umbilical cord endothelial cells (HUVEC), monocyte-derived macrophages (hMDM), and human aortic endothelial cells (haEC). We employed mouse wildtype and mutant immortalized macrophages to analyze CPP-induced inflammasome activation and cytokine secretion.Results: In live mice, only primary CPP were rapidly cleared by liver sinusoidal endothelial cells (LSEC), whereas primary and secondary CPP were cleared by Kupffer cells. Scavenger receptor A (SRA)-deficient bone marrow macrophages endocytosed secondary CPP less well than did wildtype macrophages. In contrast, primary CPP endocytosis did not depend on the presence of SRA, suggesting involvement of an alternative clearance pathway. CPP triggered TLR4 dependent TNFα and IL-1β secretion in cultured macrophages. Calcium content-matched primary CPP caused twice more IL-1β secretion than did secondary CPP, which was associated with increased calcium-dependent inflammasome activation, suggesting that intracellular CPP dissolution and calcium overload may cause this inflammation.Conclusions: Secondary CPP are endocytosed by macrophages in liver and spleen via SRA. In contrast, our results suggest that primary CPP are cleared by LSEC via an alternative pathway. CPP induced TLR4-dependent TNFα and inflammasome-dependent IL-1β secretion in macrophages suggesting that inflammation and calcification may be considered consequences of prolonged CPP presence and clearance.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

In vitro multilayered tissues with mimetic architectures resembling native tissues are valuable tools for application in medical research. In this study, an advanced bioprinting strategy is presented for aligning collagen fibers contained in functional bioinks. Streptavidin-coated iron nanoparticles are embedded in printable bioinks with varying concentrations of low gelling temperature agarose and type I collagen. By applying a straightforward magnetic-based mechanism in hydrogels during bioprinting, it is possible to align collagen fibers in less concentrated hydrogel blends with a maximum agarose concentration of 0.5 w/v%. Conversely, more elevated concentrations of agarose in printable blends show random collagen fiber distribution. Interestingly, hydrogel blends with unidirectionally aligned collagen fibers show significantly higher compression moduli compared to hydrogel blends including random fibers. Considering its application in the field of cartilage tissue engineering, bioprinted constructs with alternating layers of aligned and random fibers are fabricated. After 21 days of culture, cell-loaded constructs with alternating layers of aligned and random fibers express markedly more collagen II in comparison to solely randomly oriented fiber constructs. These encouraging results translate the importance of the structure and architecture of bioinks used in bioprinting in light of their use for tissue engineering and personalized medical applications.