Engineering of an Osteoinductive and Growth Factor-Free Injectable Bone-Like Microgel for Bone Regeneration.

Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein-loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor-free bone-like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro-constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical-sized defects in-vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.

Peptide microarray patterning for controlling and monitoring cell growth.

The fate of cells is influenced by their microenvironment and many cell types undergo differentiation when stimulated by extracellular cues, such as soluble growth factors and the insoluble extracellular matrix (ECM). Stimulating differentiation by insoluble or "immobilized" cues is a particularly attractive method because it allows for the induction of differentiation in a spatially-defined cohort of cells within a larger subpopulation. To improve the design of de novo screening of such insoluble factors, we describe a methodology for producing high-density peptide microarrays suitable for extended cell culture and fluorescence microscopy. As a model, we used a murine mammary gland cell line (NMuMG) that undergoes epithelial to mesenchymal transition (EMT) in response to soluble transforming growth factor beta (TGF-ß) and surface-immobilized peptides that target TGF-ß receptors (TGFßRI/II). We repurposed a well-established DNA microarray printing technique to produce arrays of micropatterned surfaces that displayed TGFßRI/II-binding peptides and integrin binding peptides. Upon long-term culture on these arrays, only NMuMG cells residing on EMT-stimulating areas exhibited growth arrest and decreased E-cadherin expression. We believe that the methodology created in this report will aid the development of peptide-decorated surfaces that can locally stimulate defined cell surface receptors and control EMT and other well-characterized differentiation events. STATEMENT OF SIGNIFICANCE: Scope of work: This manuscript aims to accelerate the development of instructive biomaterials decorated with specific ligands that target cell-surface receptors and induce specific differentiation of cells upon contact. These materials can be used for practical applications, such as fabricating synthetic materials for large scale, stem cell culture, or investigating differentiation and asymmetric division in stem cells. Specifically, in this manuscript, we repurposed a DNA microarray printer to produce microarrays of peptide-terminated self-assembled monolayers (SAMs). To demonstrate the utility of these arrays in phenotypic assays with mammalian cells, we monitored the induction of epithelial to mesenchymal transition (EMT) in murine mammary epithelial cells using specific peptide ligands printed on these arrays. Novelty: We, and others, have published several strategies for producing peptide-based arrays suitable for long-term phenotypic assays. Many reports relied on patterning steps that made adaptation difficult. The use of a DNA microarray printer as the sole production tool simplified the production of peptide microarrays and increased the throughput of this technology. We confirmed that simplification in production did not compromise the performance of the array; it is still possible to study short-term adhesion, long-term growth, and complex phenotypic responses, such as EMT, in the cells. EMT was studied using immunofluorescent staining after four days of culture. IMPACT: This methodology will serve as a foundation for future screening of instructive biomaterials in our research group. As DNA printers are broadly available in academic institutions, we foresee rapid adaptation of this approach by academic researchers.

Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins­ConA, LcH, and PSA­that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Fluorinated pickering emulsions impede interfacial transport and form rigid interface for the growth of anchorage-dependent cells.

This study describes the design and synthesis of amphiphilic silica nanoparticles for the stabilization of aqueous drops in fluorinated oils for applications in droplet microfluidics. The success of droplet microfluidics has thus far relied on one type of surfactant for the stabilization of drops. However, surfactants are known to have two key limitations: (1) interdrop molecular transport leads to cross-contamination of droplet contents, and (2) the incompatibility with the growth of adherent mammalian cells as the liquid-liquid interface is too soft for cell adhesion. The use of nanoparticles as emulsifiers overcomes these two limitations. Particles are effective in mitigating undesirable interdrop molecular transport as they are irreversibly adsorbed to the liquid-liquid interface. They do not form micelles as surfactants do, and thus, a major pathway for interdrop transport is eliminated. In addition, particles at the droplet interface provide a rigid solid-like interface to which cells could adhere and spread, and are thus compatible with the proliferation of adherent mammalian cells such as fibroblasts and breast cancer cells. The particles described in this work can enable new applications for high-fidelity assays and for the culture of anchorage-dependent cells in droplet microfluidics, and they have the potential to become a competitive alternative to current surfactant systems for the stabilization of drops critical for the success of the technology.

Flow-through synthesis on Teflon-patterned paper to produce peptide arrays for cell-based assays.

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60% purity for the majority of the peptides (>95% yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell-based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation.

Reference values for exposure to PAH contaminants: Comparison of fish from Ohio and Mid-Atlantic streams.

Reference values for background exposures of benzo[a]pyrene (BAP)- and naphthalene (NAPH)-type metabolites were calculated for white sucker, northern hog sucker, and rock bass from the mid-Atlantic region based on the 90th percentile of a probability-based sample. Preliminary findings are presented for common carp. Bile was collected from fish and assayed for polycyclic aromatic hydrocarbon metabolites using the fluorescent method of Lin et al. Exposure reference values for white sucker were 0.3 microg/mg protein for BAP-type metabolites and 40 microg/mg protein for NAPH-type metabolites. Values from the Mid-Atlantic region were similar to those previously reported for the Eastern Corn Belt Plains (ECBP) ecoregion. Exposure reference values of BAP-type metabolites for rock bass were 0.3 microg/mg protein and 0.4 microg/mg protein for northern hog sucker. Exposure reference values of NAPH-type metabolites for rock bass were 30 microg/mg protein for rock bass and 50 microg/mg protein for northern hog sucker. Provisional values for common carp based on 39 observations are 0.4 microg/mg protein for BAP-type metabolites and 120 microg/mg protein for NAPH-type metabolites. Small streams exhibited the greatest range of exposures, 0.015-0.689 microg/mg protein for BAP-type metabolites and from 5.7 to 159 microg/mg protein for NAPH-type metabolites. Larger streams had 0.11-0.859 microg/mg protein for BAP-type metabolites and 10.2-286.5 microg/mg protein for NAPH-type metabolites. Exposure reference values for BAP and NAPH-type metabolites could be used as a basis of comparison of exposure to PAH contamination.

Determining probable causes of ecological impairment in the Little Scioto River, Ohio, USA: part 1. Listing candidate causes and analyzing evidence.

The Little Scioto River in north-central Ohio, USA, is considered to be biologically impaired based on the results of fish and invertebrate surveys. The causes for these impairments were evaluated by means of a formal method. Two of the impairments identified on the stream reach were characterized in detail to support the causal assessment. A list of six candidate causes was developed that included habitat alteration, polycyclic aromatic hydrocarbon contamination, metals contamination, low dissolved oxygen, ammonia toxicity, and nutrient enrichment. Evidence for the causal evaluation was developed with data from the site that associated each candidate cause with the biological responses. Evidence was also developed that drew on data from other locations and laboratory studies, including comparisons of site exposures with screening values and criteria. The formal method increased the transparency of the assessment; candidate causes were clearly listed and the pathways by which they may have produced effects were shown. Analysis of the evidence maximized the utility of available data, which were collected as part of monitoring and research programs rather than to specifically support a causal assessment. This case study illustrates how the stressor identification method can be used to draw conclusions from available data about the most likely causes of impairment and to show what additional studies would be useful.