åTitle: Harnessing The Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular matrix (ECM) derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. In this study, a chitosan/alginate (C/A) hydrogel was developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) were cultured with these constructs and proliferation and cytotoxicity assays confirmed that these C/A hydrogels were bioactive. Sequential z-axis fluorescent imaging visualized hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining showed hDPCs cultured with the hydrogels displayed increased calcium ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity was increased, as was expression of transforming growth factor-beta (TGF-ß) as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrated that both dentin and pulp ECM molecules acted as chemoattractants for hDPCs. Data from this study demonstrated that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulated dental tissue repair processes in vitro. This article is protected by copyright. All rights reserved.

Biodegradable methacrylated casein for cardiac tissue engineering applications.

Casein is a naturally derived amino group (-NH2) rich protein, that enables surface functionalization leading to hydrophilicity, which in turn facilitates better cell adhesion. Casein obtained from either commercial ß-casein rich skim milk (A2 milk) or dissolved air flotation (DAF) technology was tested for its potential for tissue engineering applications in a comparative study. A novel biodegradable biomaterial was synthesized from casein by chemically modifying with methacrylic anhydride (MA) and combined with polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blend. The resulting methacrylated casein (CasMA) with the two polymers was processed into porous scaffolds with low and high MA concentrations to demonstrate CasMA's ease of modification and reproducibility. Fourier Transform Infrared Microscopy (FTIR) and Proton Nuclear Magnetic Resonance (1H NMR) revealed the presence of all the components and the successful modification of casein. The rheological and morphological analysis presented viscous behaviour and columnar hollow tube-like microstructures in agreement with the biomaterials' swelling and biodegradation behaviour. The live/dead in vitro assay showed high cell viability that agreed with the cell proliferation (MTT) assay in vitro, which indicated increased proliferation upon casein modification at appropriate biomaterial concentrations and volumes. This study not only showed a possible mechanism of casein methacrylation but also presented the potential use of waste materials like DAF-casein as a value-added product for tissue engineering applications.

Interstitial cells of Cajal are present in human extrahepatic bile ducts.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. METHODS: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post-mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin-embedded sections (3 microm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c-kit antibodies and mast cell tryptase was used to distinguish c-kit-positive cells with typical ICC morphology from c-kit-positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. RESULTS: c-kit-positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. CONCLUSION: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle.

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB1 determines its cellular location.

Critical Role for Cold Shock Protein YB-1 in Cytokinesis.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, ß-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

Development and characterization of a xenograft material from New Zealand sourced bovine cancellous bone.

A xenograft (bovine hydroxyapatite [BHA]) was developed from New Zealand sourced bovine cancellous bone by a successful defatting and deproteinizing procedure. The BHA was chemically, compositionally and structurally characterized. Fourier transform infrared spectroscopy confirmed the removal of organic matter from the bone matrix and the presence of carbonate ( CO32-), hydroxyl (OH- ), and phosphate ( PO43-) functional groups. X-ray diffraction analysis suggested that the processed bone corresponds characteristically to hydroxyapatite (HA). SEM analysis showed that the BHA has an interconnected porous architecture with a pore diameter ranging from 100 to 700 µm while µCT analysis calculated the total porosity as 73.46% ± 1.08. Furthermore, the BHA was stable up to 1000°C and lost only 1.8% of its weight. The Ca/P molar ratio of the BHA was 1.58, which is comparable with commercially available natural HA-Endobon® . After 28 days of incubation in simulated body fluid (SBF), the pH value only fluctuated between 7.1 and 7.5 and the BHA scaffold did not degrade significantly by weight indicating the scaffold had excellent chemical and structural stability. In vitro studies showed the BHA was cytocompatible and supported the proliferative growth of Saos-2 osteoblast cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1054-1062, 2017.