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3D printing of biological tissues has been of increasing interest to the biomaterials community in part because of its potential to produce spatially heterogeneous constructs. Such technology is particularly promising for orthopedic applications, which require the generation of complex geometries to match patient anatomy and complex microstructures to produce spatial heterogeneity and anisotropy. Prior research has demonstrated the capacity to create precisely shaped 3D printed constructs using biocompatible alginate hydrogels. However, alginate is extremely compliant and brittle, and high-density collagen hydrogels could be a preferable option for load-bearing applications. This research focused on developing and evaluating a method of printing soft tissue implants with high-density collagen hydrogels using a commercially available 3D printer, modified for tissue-engineering purposes. The tissue constructs, seeded with primary meniscal fibrochondrocytes, were evaluated using measures of geometric fidelity, cell viability, mechanical properties, and fiber microstructure. The constructs were found to be mechanically stable and were able to support and maintain cell growth. Furthermore, heterogeneous 3D-printed constructs were generated, consisting of discrete domains with distinct mechanical properties.

ABSTRACTAccumulating evidence suggests a connection between asthma development and colonization with nontypeable Haemophilus influenzae(NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associated FOXP3gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

Arterial hemodynamic shear stress and blood vessel stiffening both significantly influence the arterial endothelial cell (EC) phenotype and atherosclerosis progression, and both have been shown to signal through cell-matrix adhesions. However, the cooperative effects of fluid shear stress and matrix stiffness on ECs remain unknown. To investigate these cooperative effects, we cultured bovine aortic ECs on hydrogels matching the elasticity of the intima of compliant, young, or stiff, aging arteries. The cells were then exposed to laminar fluid shear stress of 12 dyn/cm2. Cells grown on more compliant matrices displayed increased elongation and tighter EC-cell junctions. Notably, cells cultured on more compliant substrates also showed decreased RhoA activation under laminar shear stress. Additionally, endothelial nitric oxide synthase and extracellular signal-regulated kinase phosphorylation in response to fluid shear stress occurred more rapidly in ECs cultured on more compliant substrates, and nitric oxide production was enhanced. Together, our results demonstrate that a signaling cross talk between stiffness and fluid shear stress exists within the vascular microenvironment, and, importantly, matrices mimicking young and healthy blood vessels can promote and augment the atheroprotective signals induced by fluid shear stress. These data suggest that targeting intimal stiffening and/or the EC response to intima stiffening clinically may improve vascular health.

In this report we describe a preparation of silver wires (SWs) on gold mirrors and its application to surface enhanced fluorescence (SEF) using a new methodology. Silica protected gold mirrors were drop-coated with a solution of silver triangular nanoprisms. The triangular nanoprisms were slowly air-dried to get silver wires that self-assembled on the gold mirrors. Fluorescence enhancement was studied using methyl azadioxatriangulenium chloride (Me-ADOTA·Cl) dye in PVA spin-coated on a clean glass coverslip. New Plasmonic Platforms (PPs) were assembled by placing a mirror with SWs in contact with a glass coverslip spin-coated with a uniform Me-ADOTA·Cl film. It was shown that surface enhanced fluorescence is a real phenomenon, not just an enhancement of the fluorescence signal due to an accumulation of the fluorophore on rough nanostructure surfaces. The average fluorescence enhancement was found to be about 15-fold. The lifetime of Me-ADOTA·Cl dye was significantly reduced (∼4 times) in the presence of SWs. Moreover, fluorescence enhancement and lifetime did not show any dependence on the excitation light polarization.

Background:We previously showed that intra-amniotic lipopolysaccharide (LPS) amplifies alveolar hypoplasia induced by postnatal hyperoxia. We determined whether the priming effect of intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity (AHR).Methods:LPS or normal saline was injected into the amniotic cavities of pregnant rats at the 20th day of gestation. After birth, rat pups were exposed to 60% O2or air for 14 d. On postnatal day 14, rat pups underwent forced oscillometry, which included a challenge with nebulized methacholine, and the lungs were harvested for morphological studies.Results:Hyperoxia significantly increased airway reactivity and decreased compliance. Intra-amniotic LPS further increased hyperoxia-induced AHR but did not further impair respiratory system compliance. Hyperoxia-induced changes in lung parenchymal and small airway morphology were not further altered by intra-amniotic LPS. However, combined exposure to intra-amniotic LPS and hyperoxia increased the proportion of degranulating mast cells in the hilar airways.Conclusion:Intra-amniotic LPS amplified postnatal hyperoxia-induced AHR. This was associated with increased airway mast cell degranulation, which has previously been linked with hyperoxia-induced AHR. There were no morphologic changes of parenchyma or airways that would account for the LPS augmentation of hyperoxia-induced AHR.

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.

Objectives: To document levels of cardiovascular disease (CVD), diagnosed and undiagnosed risk factors and clinical management of CVD risk in rural Māori.

We assessed the ability of a gamma-secretase inhibitor to promote the in vitrodifferentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.

Premature infants are at increased risk of developing airway hyper-reactivity (AHR) after oxidative stress and inflammation. Mast cells contribute to AHR partly by mediator release, so we sought to determine whether blocking mast cell degranulation or recruitment prevents hyperoxia-induced AHR, mast cell accumulation, and airway smooth muscle (ASM) changes. Rats were exposed at birth to air or 60% O2for 14 d, inducing significantly increased AHR in the latter group, induced by nebulized methacholine challenge and measured by forced oscillometry. Daily treatment (postnatal d 1–14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal d 5–14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell.

Development of an alternative source of functional, transplantable β-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of β-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective β-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating β-cells and facilitate β-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive’ and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive β-cell population from dissociated precursor cells.

Continuing advances in islet cell transplantation have been promising; however, several limitations, including severe shortage of transplantable islets, hinder the widespread use of this therapy. Pancreatic precursor cells are one alternative to cadaveric donor islets. These cells found in the developing pancreatic buds are capable of self-renewal and also have the innate ability to become insulin-producing β-cells. For this work, bioinert polyethylene glycol (PEG) hydrogels were chosen as the supportive three-dimensional matrix for encapsulation of dissociated pancreatic precursor cells obtained from the dorsal pancreatic bud of day-15 rat embryos. This culture system was selected in order to eliminate cell–extracellular matrix and cell–cell signal heterogeneity present when intact pancreatic buds are embedded in protein-based gels, the typical in vitroculture conditions used to study this cell population. In this study it was found that (1) dissociated precursor cells maintain a robust viability for 7 days in PEG hydrogel culture, (2) encapsulated cells selectively differentiate into insulin-expressing β-cells, and (3) differentiated β-cells have releasable insulin stores, but are not achieving a mature, glucose responsive phenotype. These findings suggest that encapsulating dissociated pancreatic precursor cells in an environment designed to minimize the heterogeneous signaling cues present during development or in standard culture conditions generates a population highly enriched in pancreatic β-cells; however, future efforts must focus on achieving glucose responsiveness in this cell population. Further, these results indicate that differentiation down a β-cell lineage may be the default pathway in pancreatic development.

Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther318:657–665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [3H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [3H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.

The electrochemical (platinum and glassy carbon electrodes, cyclic and rotating disk electrode voltammetry, bulk electrolysis, voltammetric simulation) and chemical oxidation (NO+) of the organoamidoplatinum(ii) complex, [Pt{((p-HC₆F₄)NCH₂)₂}(py)₂], has been studied in acetonitrile and acetone. The initial process is a complicated function of concentration, temperature, and method of oxidation. The products formed have been probed by in situ spectroelectrochemical techniques (UV/Visible and EPR spectroscopy), and ex situ by 1H, 19F and 195Pt NMR spectroscopy and electrospray mass spectrometry. Under conditions of cyclic voltammetry, the initial oxidation process (at high concentration) is an overall irreversible one-electron process, complicated by crossover of current on the reverse scan. This and many (but not all) features are simulated by a sequence of electron transfer steps and coupled chemical reactions which requires the formation of two structurally different dinuclear intermediates. Regeneration of [Pt{((p-HC₆F₄)NCH₂)₂}(py)₂] by reduction is possible under short timescale conditions, but not long timescale conditions, implying that the finally observed product from long timescale experiments may be oligomeric. The observation of moderately stable diamagnetic diplatinum(iii) compounds is attributed to the formation of bridged complexes containing Pt–Pt bonds. Oxidation of all platinum(iii) intermediates gives rise to the same or very closely related platinum(iv) complexes. Features of the organoamide ligand that enable the formation of moderately stable platinum(iii) complexes are considered.

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D1receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D1receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D1-EGFP). D1-EGFP was similar to the untagged D1receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D1-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D1receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D1receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

This paper describes the development and application of scaling laws relating bulk-degradation behavior of poly(lactic acid)−poly(ethylene glycol)−poly(lactic acid) (PLA-b-PEG-b-PLA) copolymer hydrogels to the ever-changing transport properties during network degradation. Specifically, the relationships developed were used to extend previous work and to predict the effects of cross-linking density and hydrolytic degradation rate on the controlled release of high molecular weight proteins from these hydrogels. Theoretical drug release profiles, obtained using scaling laws, agree qualitatively with experimental observations and show a decrease in the protein release rate with an increase in protein size, extent of macromer functionalization, and macromer concentration. Quantitative predictions of release behavior were possible under certain experimental conditions.

Background: Ethanol withdrawal seizure‐prone (WSP) and ‐resistant (WSR) mice have been genetically selected for differences in handling‐induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug‐naïve mice from these selected lines also differ in handling‐induced convulsion severity. Different N‐methyl‐D‐aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice.

Abstract:To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Kmof transport from 0.61 µMto 0.34 µMwithout significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.

The clinical application of computerized continuous-infusion pressure-monitored sialography using an aqueous contrast medium during the investigation of 296 salivary glands is described. A comparison between the pressure changes and the sialographic features revealed a significant (P less than 0.001) correlation between raised filling pressure (greater than 60 mmHg) and the presence of an obstructive lesion (stricture, mucous plug, sialolith). Occasionally a raised filling pressure was observed although the associated sialography films were normal. The sensitivity of the technique was 86.4%. It is concluded that the monitoring of pressure changes occurring during sialography can alert the clinician to the presence of high filling pressure and may also provide additional evidence of obstructive changes within salivary glands.

The microbial degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 was studied, and two major products were isolated and identified as 7 alpha, 12 beta-dihydroxyandrosta-1,4-diene-3,17-dione and 7 alpha, 12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid. Four minor products were isolated and evidence is given for the following structures: 7 alpha, 12 alpha-dihydroxyandrosta-1,4-diene-3,17-dione, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 7 alpha, 12 beta, 17 beta-trihydroxyandrosta-1,4-dien-3-one and 7 alpha, 12 alpha-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid. The significance of the production of the steroid products is discussed, along with the possible enzymic mechanisms responsible for their production.

CONSCIOUSNESS is a word with many meanings. Schiller1 remarked that it may connote attending, sensing, being alive, being responsive to stimuli, having insight, being able to communicate, and the psychoanalysts give it another interpretation. It can also be described as a state of passive awareness independent of speech or movement. Such a definition applies to crude as distinct from discriminative consciousness and will be the definition used here.Le Gros Clarke2 has drawn attention to the function of the thalamic and brain-stem reticular system as the possible structural basis of what may be called a general background of consciousness. Interest in the reticular system as the activator of consciousness originates largely from animal experimental work of Magoun3 and others. Clinical evidence in support of this hypothesis is rare, and it is partly for this reason that a case admitted to the Royal Edinburgh Hospital for Mental and

1) An attempt is made to clarify and standardize the technique and modification of electro-convulsive therapy.2) The literature is briefly reviewed.3) A technique suitable for outpatient, hospital or private practice use is suggested and discussed. Kindly and proper handling of the patient, the time element, nursing requirements, and the training of medical staff were among the factors considered in the description of this technique.4) A total of 1863 treatments were given to 246 female patients by the author over an arbitrary period and under exactly similar conditions. The limitation of the trial to females is coincidental.5) For the first time 8 different muscle relaxants are compared and assessed in the same paper. Six of these relaxants were given to 25 of the patients in a partially unsuccessful attempt to give each of these patients a trial with each relaxant on at least one occasion.6) It is concluded that physical disease or age do not contra-indicate the use of E.C.T. provided this is modified.7) It is further concluded that suxethonium bromide is at least as satisfactory, and in most cases more satisfactory, than the other relaxant drugs used for the modification of electro-shock therapy. The drugs were assessed for this purpose in terms of occurrence of fracture, degree of apnoea produced, contra-indications, antidotes, effect upon blood pressure and pulse, production of histamine-like effects, excessive secretions, cyanosis, subjective ill effects, extent of muscle fasciculations, degree of relaxation, modification of the convulsion, time required, and finally—ease of handling.