Phosphomimetics at Ser199/Ser202/Thr205 in Tau Impairs Axonal Transport in Rat Hippocampal Neurons.

Our understanding of the biological functions of the tau protein now includes its role as a scaffolding protein involved in signaling regulation, which also has implications for tau-mediated dysfunction and degeneration in Alzheimer's disease and other tauopathies. Recently, we found that pseudophosphorylation at sites linked to the pathology-associated AT8 phosphoepitope of tau disrupts normal fast axonal transport through a protein phosphatase 1 (PP1)-dependent pathway in squid axoplasm. Activation of the pathway and the resulting transport deficits required tau's N-terminal phosphatase-activating domain (PAD) and PP1 but the connection between tau and PP1 was not well defined. Here, we studied functional interactions between tau and PP1 isoforms and their effects on axonal transport in mammalian neurons. First, we found that wild-type tau interacted with PP1α and PP1γ primarily through its microtubule-binding repeat domain. Pseudophosphorylation of tau at S199/S202/T205 (psTau) increased PAD exposure, enhanced interactions with PP1γ, and increased active PP1γ levels in mammalian cells. Expression of psTau also significantly impaired axonal transport in primary rat hippocampal neurons. Deletion of PAD in psTau significantly reduced the interaction with PP1γ, eliminated increases of active PP1γ levels, and rescued axonal transport impairment in neurons. These data suggest that a functional consequence of phosphorylation within S199-T205 in tau, which occurs in AD and several other tauopathies, may be aberrant interaction with and activation of PP1γ and subsequent axonal transport disruption in a PAD-dependent fashion.

Gene electrotransfer of FGF2 enhances collagen scaffold biocompatibility.

Tendon injuries are a common athletic injury that have been increasing in prevalence. While there are current clinical treatments for tendon injuries, they have relatively long recovery times and often do not restore native function of the tendon. In the current study, gene electrotransfer (GET) parameters for delivery to the skin were optimized with monophasic and biphasic pulses with reporter and effector genes towards optimizing underlying tendon healing. Tissue twitching and damage, as well as gene expression and distribution were evaluated. Bioprinted collagen scaffolds, mimicking healthy tendon structure were then implanted subcutaneously for biocompatibility and angiogenesis analyses when combined with GET to accelerate healing. GET of human fibroblast FGF2 significantly increased angiogenesis and biocompatibility of the bioprinted implants when compared to implant only sites. The combination of bioprinted collagen fibers and angiogenic GET therapy may lead to better graft biocompatibility in tendon repair.

Reference intervals for end-tidal carbon monoxide of preterm neonates.

OBJECTIVES: We constructed reference intervals for end-tidal carbon monoxide (ETCOc) levels of neonates 28 0/7 to 34 6/7 weeks gestation in order to assess hemolytic rate. STUDY DESIGN: This is a prospective four-NICU study in Bangkok, Thailand, and Utah, USA. RESULTS: Of 226 attempted measurements, 92% were successful. Values from day 1 through 28 were charted and upper (>95th percentile) reference interval limits calculated. During the entire 28 days, the ETCOc upper reference intervals from babies in Bangkok were higher than those in Utah (p < 0.01). No differences were found due to sex, or earliest vs. latest gestation at birth (both p > 0.1). Similar to term neonates, preterm neonates in Bangkok and Utah had higher ETCOc values during the first 48 h after birth than thereafter (p < 0.01). CONCLUSIONS: Using this methodology, and the reference interval chart, the hemolytic rate of preterm infants ≥28 weeks can be assessed.

Assembled Cell-Decorated Collagen (AC-DC) Fiber Bioprinted Implants with Musculoskeletal Tissue Properties Promote Functional Recovery in Volumetric Muscle Loss.

Musculoskeletal tissue injuries, including volumetric muscle loss (VML), are commonplace and often lead to permanent disability and deformation. Addressing this healthcare need, an advanced biomanufacturing platform, assembled cell-decorated collagen (AC-DC) bioprinting, is invented to rapidly and reproducibly create living biomaterial implants, using clinically relevant cells and strong, microfluidic wet-extruded collagen microfibers. Quantitative analysis shows that the directionality and distribution of cells throughout AC-DC implants mimic native musculoskeletal tissue. AC-DC bioprinted implants further approximate or exceed the strength and stiffness of human musculoskeletal tissue and exceed collagen hydrogel tensile properties by orders of magnitude. In vivo, AC-DC implants are assessed in a critically sized muscle injury in the hindlimb, with limb torque generation potential measured over 12 weeks. Both acellular and cellular implants promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery. Histological analysis and automated image processing of explanted muscle cross-sections reveal increased total muscle fiber count, median muscle fiber size, and increased cellularization for injuries repaired with cellularized implants. These studies introduce an advanced bioprinting method for generating musculoskeletal tissue analogs with near-native biological and biomechanical properties with the potential to repair myriad challenging musculoskeletal injuries.

Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, ß, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1ß, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

Pathogenic MAST3 Variants in the STK Domain Are Associated with Epilepsy.

OBJECTIVE: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum. METHODS: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells. RESULTS: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally. INTERPRETATION: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284.

cAMP-regulated phosphoproteins DARPP-32, ARPP16/19, and RCS modulate striatal signal transduction through protein kinases and phosphatases.

Decades of research led by Paul Greengard identified protein phosphorylation as a ubiquitous and vital post-translational modification involved in many neuronal signaling pathways. In particular, his discovery that second messenger-regulated protein phosphorylation plays a central role in the propagation and transduction of signals in the nervous system has been essential in understanding the molecular mechanisms of neuronal communication. The establishment of dopamine (DA) as an essential neurotransmitter in the central nervous system, combined with observations that DA activates G-protein-coupled receptors to control the production of cyclic adenosine monophosphate (cAMP) in postsynaptic neurons, has provided fundamental insight into the regulation of neurotransmission. Notably, DA signaling in the striatum is involved in many neurological functions such as control of locomotion, reward, addiction, and learning, among others. This review focuses on the history, characterization, and function of cAMP-mediated regulation of serine/threonine protein phosphatases and their role in DA-mediated signaling in striatal neurons. Several small, heat- and acid-stable proteins, including DARPP-32, RCS, and ARPP-16/19, were discovered by the Greengard laboratory to be regulated by DA- and cAMP signaling, and found to undergo a complex but coordinated sequence of phosphorylation and dephosphorylation events. These studies have contributed significantly to the establishment of protein phosphorylation as a ubiquitous and vital process in signal propagation in neurons, paradigm shifting discoveries at the time. Understanding DA-mediated signaling in the context of signal propagation has led to numerous insights into human conditions and the development of treatments and therapies.

Phosphodiesterase PDE4D Is Decreased in Frontal Cortex of Aged Rats and Positively Correlated With Working Memory Performance and Inversely Correlated With PKA Phosphorylation of Tau.

Age is the largest risk factor for Alzheimer's disease (AD) and contributes to cognitive impairment in otherwise healthy individuals. Thus, it is critical that we better understand the risk aging presents to vulnerable regions of the brain and carefully design therapeutics to address those effects. In this study we examined age-related changes in cAMP-regulatory protein, phosphodiesterase 4D (PDE4D). Inhibition of PDE4D is currently under investigation as a therapeutic target for AD based on memory-enhancing effects in rodent hippocampus. Therefore, it is important to understand the role of PDE4D in brain regions particularly vulnerable to disease such as the frontal association cortex (FC), where cAMP signaling can impair working memory via opening of potassium channels. We found that PDE4D protein level was decreased in the FC of both moderately and extremely aged rats, and that PDE4D level was correlated with performance on a FC-dependent working memory task. In extremely aged rats, PDE4D was also inversely correlated with levels of phosphorylated tau at serine 214 (S214), a site phosphorylated by protein kinase A. In vitro studies of the PDE4D inhibitor, GEBR-7b, further illustrated that inhibition of PDE4D activity enhanced phosphorylation of tau. pS214-tau phosphorylation is associated with early AD tau pathology, promotes tau dissociation from microtubules and primes subsequent tau hyperphosphorylation at other critical AD-related sites. Age-related loss of PDE4D may thus contribute to the specific vulnerability of the FC to degeneration in AD, and play a critical role in normal cAMP regulation, cautioning against the use of pan-PDE4D inhibitors as therapeutics.

Pathogenic tau modifications occur in axons before the somatodendritic compartment in mossy fiber and Schaffer collateral pathways.

The deposition of tau pathology in Alzheimer's disease (AD) may occur first in axons of neurons and then progress back into the cell bodies to form neurofibrillary tangles, however, studies have not directly analyzed this relationship in relatively discrete circuits within the human hippocampus. In the early phases of tau deposition, both AT8 phosphorylation and exposure of the amino terminus of tau occurs in tauopathies, and these modifications are linked to mechanisms of synaptic and axonal dysfunction. Here, we examined the localization of these tau pathologies in well-characterized post-mortem human tissue samples from the hippocampus of 44 cases ranging between non-demented and mild cognitively impaired to capture a time at which intrahippocampal pathways show a range in the extent of tau deposition. The tissue sections were analyzed for AT8 (AT8 antibody), amino terminus exposure (TNT2 antibody), and amyloid-ß (MOAB2 antibody) pathology in hippocampal strata containing the axons and neuronal cell bodies of the CA3-Schaffer collateral and dentate granule-mossy fiber pathways. We show that tau pathology first appears in the axonal compartment of affected neurons in the absence of observable tau pathology in the corresponding cell bodies in several cases. Additionally, deposition of tau in these intrahippocampal pathways was independent of the presence of Aß plaques. We confirmed that the majority of tau pathology positive neuropil threads were axonal in origin and not dendritic using an axonal marker (i.e. SMI312 antibody) and somatodendritic marker (i.e. MAP2 antibody). Taken together, these results support the hypothesis that AT8 phosphorylation and amino terminus exposure are early pathological events and that the deposition of tau pathology, at least in the studied pathways, occurs first in the axonal compartment prior to observable pathology in the somata. These findings highlight the importance on targeting tau deposition, ideally in the initial phases of its deposition in axons.

Impact of moderate prenatal alcohol exposure on histaminergic neurons, histidine decarboxylase levels and histamine H2 receptors in adult rat offspring.

We have reported that moderate prenatal alcohol exposure (PAE) elevates histamine H3 receptor-mediated inhibition of glutamatergic neurotransmission in dentate gyrus (DG), and that the H3 receptor antagonist ABT-239 ameliorates PAE-induced deficits in DG long-term potentiation. Here, we investigated whether PAE alters other markers of histaminergic neurotransmission. Long-Evans rat dams voluntarily consumed either a 0% or a 5% ethanol solution 4 h each day throughout gestation. Young adult female offspring from each prenatal treatment group were used in histidine decarboxylase (HDC) immunohistochemical studies of histamine neuron number in ventral hypothalamus, quantitative Western blotting studies of HDC expression in multiple brain regions, radiohistochemical studies of H2 receptor density in multiple brain regions, and in biochemical studies of H2 receptor-effector coupling in dentate gyrus. Rat dams consumed a mean of 1.90 g of ethanol/kg/day during pregnancy. This level of consumption did not affect maternal weight gain, offspring birth weight, or litter size. PAE did not affect the number of HDC-positive neurons in ventral hypothalamus. However, HDC expression was reduced in frontal cortex, dentate gyrus, and cerebellum of PAE rats compared to controls. Specific [125I]-iodoaminopotentidine binding to H2 receptors was not altered in any of the brain regions measured, nor was basal or H2 receptor agonist-stimulated cAMP accumulation in DG altered in PAE rats compared to controls. These results suggest that not all markers of histaminergic neurotransmission are altered by PAE. However, the observation that HDC levels were reduced in the same brain regions where elevated H3 receptor-effector coupling was observed previously raises the question of whether a cause-effect relationship exists between HDC expression and H3 receptor function in affected brain regions of PAE rats. This relationship, along with the question of why these effects occur in some, but not all brain regions, requires more-detailed investigation.

Effects of printing-induced interfaces on localized strain within 3D printed hydrogel structures.

Additive manufacturing, or 3D printing, is a promising approach for the fabrication of biological structures for regenerative medicine applications using tissue-like materials such as hydrogels. Herein, inkjet printing is implemented as a model droplet-based 3D printing technology for which interfaces have been shown to form between printed lines within printed layers of hydrogel structures. Experimental samples with interfaces in two orientations are fabricated by inkjet printing and control samples with and without interfaces are fabricated by extrusion printing and casting, respectively. The formation of partial and full interfaces is modeled in terms of printing conditions and gelation parameters, and an approach to predicting the ratio of interfacial area to the total contact area between two adjacent lines is presented. Digital image correlation is used to determine strain distributions and identify regions of increased localized deformation for samples under uniaxial tension. Despite the presence of interfaces in inkjet-printed samples, strain distributions are found to be homogeneous regardless of interface orientation, which may be attributed to the multi-layer nature of samples. Conversely, single-layer extrusion-printed samples exhibit localized regions of increased deformation between printed lines, indicating delamination along interfaces. The effective stiffness, failure strength, and failure strain of inkjet-printed samples are found to be dependent on the orientation of interfaces within layers. Specifically, inkjet-printed samples in which tensile forces pull apart interfaces exhibit significantly decreased mechanical properties compared to cast samples.

Axonal Degeneration in Tauopathies: Disease Relevance and Underlying Mechanisms.

Tauopathies are a diverse group of diseases featuring progressive dying-back neurodegeneration of specific neuronal populations in association with accumulation of abnormal forms of the microtubule-associated protein tau. It is well-established that the clinical symptoms characteristic of tauopathies correlate with deficits in synaptic function and neuritic connectivity early in the course of disease, but mechanisms underlying these critical pathogenic events are not fully understood. Biochemical in vitro evidence fueled the widespread notion that microtubule stabilization represents tau's primary biological role and that the marked atrophy of neurites observed in tauopathies results from loss of microtubule stability. However, this notion contrasts with the mild phenotype associated with tau deletion. Instead, an analysis of cellular hallmarks common to different tauopathies, including aberrant patterns of protein phosphorylation and early degeneration of axons, suggests that alterations in kinase-based signaling pathways and deficits in axonal transport (AT) associated with such alterations contribute to the loss of neuronal connectivity triggered by pathogenic forms of tau. Here, we review a body of literature providing evidence that axonal pathology represents an early and common pathogenic event among human tauopathies. Observations of axonal degeneration in animal models of specific tauopathies are discussed and similarities to human disease highlighted. Finally, we discuss potential mechanistic pathways other than microtubule destabilization by which disease-related forms of tau may promote axonopathy.

Promotion of Civic Engagement with the Family Leadership Training Institute.

In this efficacy study, both quantitative and qualitative data were used to gauge the effects of the Family Leadership Training Institute (FLTI) on civic knowledge and empowerment, civic engagement, and community health. The sample of 847 FLTI participants and 166 comparison adults completed pretest and posttest surveys. Medium to very large short-term effects were observed in civic literacy, empowerment, and engagement. Results mapping interviews were conducted with a stratified random sample of FLTI graduates (n = 52) to assess long-term (M = 2.73 years) program impact. Most FLTI graduates (86%) sustained meaningful, sometimes transformative, levels of civic engagement after program completion. This engagement involved multiple forms of leadership, most often advocacy, program implementation, and media campaigns; 63% of graduates directed at least some of their activities to marginalized populations. Content analyses of graduates' civic (capstone) projects and results mapping story maps indicated that 81-90% of community activities aligned with public health priorities. Thus, one promising means to promote community health is to empower families to develop leadership skills, become engaged in civic life, and forge connections with diverse constituents.

Inkjet Bioprinting of 3D Silk Fibroin Cellular Constructs Using Sacrificial Alginate.

Silk fibroin is a natural protein which has shown great promise for tissue engineering but is not printable due to slow gelation or harsh gelation conditions which are not cell-friendly. In this study, a two-step gelation process is proposed for the printing of silk fibroin, which utilizes alginate as a sacrificial hydrogel during an inkjetting-based process. A cell-laden blend of alginate with silk fibroin is utilized to achieve rapid gelation by calcium alginate formation during printing; it is followed by horseradish peroxidase (HRP) catalyzed covalent cross-linking of the fibroin protein at tyrosine residues after printing. This two-step gelation process successfully enables 3D bioprinting of well-defined cell-laden silk fibroin constructs suitable for long-term culture. The constructs remain intact after calcium chelation to liquefy the alginate component, demonstrating the formation of silk fibroin hydrogel. NIH 3T3 fibroblasts proliferate and spread through the hydrogel after printing. Increasing metabolic activity is observed for 5 weeks after printing, and histology shows dense cell populations in cultured constructs. The proposed two-step gelation technique is expected to enable 3D silk fibroin printing for various applications.

In Situ Printing-then-Mixing for Biological Structure Fabrication Using Intersecting Jets.

Although traditional three-dimensional bioprinting technology is suitable for many tissue engineering applications, various biomaterials and constructs call for bioprinting innovations. There is a need for the fabrication of complex structures from reactive biomaterials as well as heterogeneous structures with controlled material compositions. In particular, during reactive material printing, reactive solutions/suspensions that undergo changes in rheological properties or cytocompatibility are not printable using traditional bioprinting approaches that require all components of bioinks to be mixed before deposition. The objective of this study is to develop and implement an intersecting jets-based inkjet bioprinting approach, which enables voxel-resolution printing-then-mixing for the fabrication of biological structures using reactive materials as well as structures having a compositional gradient. Inkjetting is implemented herein as a versatile technique to simultaneously deposit droplets of disparate materials at controlled locations where active collision, mixing, and coalescence occur. For reactive material printing, neural stem cell (NSC) spheres are fabricated from reactive PuraMatrix hydrogel solution and physiological cell suspension, and cell-laden alginate structures are also printed in air directly from reactive sodium alginate and calcium chloride solutions. For heterogeneous structure printing, collagen sheets with a hydroxyapatite (HAP) content gradient are fabricated to demonstrate the unique online control of material composition throughout a structure. It is demonstrated that the proposed bioprinting approach is feasible for applications that utilize reactive materials or require heterogeneous compositions.

Tryptophan, kynurenine, and kynurenine metabolites: Relationship to lifetime aggression and inflammatory markers in human subjects.

Inflammatory proteins are thought to be causally involved in the generation of aggression, possibly due to direct effects of cytokines in the central nervous system and/or by generation of inflammatory metabolites along the tryptophan-kynurenine (TRP/KYN) pathway, including KYN and its active metabolites kynurenic acid (KA), quinolinic acid (QA), and picolinic acid (PA). We examined plasma levels of TRP, KYN, KA, QA, and PA in 172 medication-free, medically healthy, human subjects to determine if plasma levels of these substances are altered as a function of trait aggression, and if they correlate with current plasma levels of inflammatory markers. Plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6), and soluble interleukin-1 receptor-II (sIL-1RII) protein were also available in these subjects. We found normal levels of TRP but reduced plasma levels of KYN (by 48%), QA (by 6%), and a QA/KA (by 5%) ratio in subjects with Intermittent Explosive Disorder (IED) compared to healthy controls and psychiatric controls. Moreover, the metabolites were not associated with any of the inflammatory markers studied. These data do not support the hypothesis that elevated levels of KYN metabolites would be present in plasma of subjects with IED, and associated with plasma inflammation. However, our data do point to a dysregulation of the KYN pathway metabolites in these subjects. Further work will be necessary to replicate these findings and to understand their role in inflammation and aggression in these subjects.

Intensive OutPatient Therapy for Clergy Burnout: How Much Difference Can a Week Make?

A pre-test and post-test quasi-experimental matched pairs design was used to assess the effectiveness of a week-long multi-therapist intensive outpatient intervention process with clergy suffering from depression and burnout. Participants (n = 23) in the "Clergy in Kairos" program of the Pastoral Institute (Muse in J Pastor Care Couns 61(3):183-195, 2007) constituted the experimental variable. Clergy surveyed from United Methodist and Presbyterian denominations (n = 121) provided a control group from which 23 respondents were selected whose pre-test scores in depression and burnout were statistically equivalent to those in the experimental group. The treatment group consisted of clergy from three denominations who self-selected (or in some cases were referred by denominational officials) into the program. At the outset, clergy in both groups reported equivalent levels of conflict, emotional exhaustion, depersonalization, and depression. At the 6-months follow-up, clergy in the experimental group showed significant improvement of depression, emotional exhaustion, and depersonalization scores. By contrast, there was no change in the burnout and depression scores in the control group at 6-months post-test. Findings suggest the usefulness of a week-long multi-therapist intensive outpatient intervention in reducing burnout and depression.

Freeform inkjet printing of cellular structures with bifurcations.

Organ printing offers a great potential for the freeform layer-by-layer fabrication of three-dimensional (3D) living organs using cellular spheroids or bioinks as building blocks. Vascularization is often identified as a main technological barrier for building 3D organs. As such, the fabrication of 3D biological vascular trees is of great importance for the overall feasibility of the envisioned organ printing approach. In this study, vascular-like cellular structures are fabricated using a liquid support-based inkjet printing approach, which utilizes a calcium chloride solution as both a cross-linking agent and support material. This solution enables the freeform printing of spanning and overhang features by providing a buoyant force. A heuristic approach is implemented to compensate for the axially-varying deformation of horizontal tubular structures to achieve a uniform diameter along their axial directions. Vascular-like structures with both horizontal and vertical bifurcations have been successfully printed from sodium alginate only as well as mouse fibroblast-based alginate bioinks. The post-printing fibroblast cell viability of printed cellular tubes was found to be above 90% even after a 24 h incubation, considering the control effect.

A double-blind placebo-controlled comparison of a novel formulation of intravenous diclofenac and ketorolac for postoperative third molar extraction pain.

Dyloject is a novel formulation of diclofenac intended for intravenous (IV) administration. This formulation employs the solubilizing agent hydroxypropyl-ß-cyclodextrin to permit bolus IV administration. The efficacy and safety of 5 dose levels of IV diclofenac were compared with IV ketorolac and placebo following third molar extraction. This was a single-dose, randomized, double-blind, placebo- and comparator-controlled, parallel-group study. A total of 353 subjects with moderate to severe pain received placebo; ketorolac 30 mg; or IV diclofenac 3.75, 9.4, 18.75, 37.5, or 75 mg (N  =  51 for all groups, except N  =  47 for ketorolac). The primary endpoint was total pain relief over 6 hours (TOTPAR6) as measured by the visual analog scale (VAS). Secondary endpoints included multiple measures of pain intensity and relief; patient global evaluation; and times to pain relief and rescue medication. Dropouts and adverse effects (AEs) were also monitored. IV diclofenac was superior to placebo as measured by TOTPAR6 (P < .0001 for all doses except 3.75 mg, for which P  =  .0341). IV diclofenac 3.75 mg was statistically superior to placebo for TOTPAR2 and TOTPAR4. IV diclofenac at both 37.5 and 75 mg was superior to placebo (P < .05) at the earliest (5 minute) assessments of pain intensity and pain relief, but ketorolac was not. The proportion of patients reporting 30% or greater pain relief at 5 minutes was significantly greater after IV diclofenac 37.5 and 75 mg than after ketorolac 30 mg or placebo. Secondary endpoints confirmed the primary findings. Treatment-related AEs were generally mild to moderate and were typical for nonsteroidal anti-inflammatory drugs (NSAIDs). The more rapid onset of action of IV diclofenac compared with the reference injectable NSAID ketorolac suggests additional clinical benefit. If confirmed in larger series, these findings may improve the safety and efficacy of postoperative NSAID analgesia.

A single-tablet fixed-dose combination of racemic ibuprofen/paracetamol in the management of moderate to severe postoperative dental pain in adult and adolescent patients: a multicenter, two-stage, randomized, double-blind, parallel-group, placebo-controlled, factorial study.

BACKGROUND: The combination of ibuprofen and paracetamol may confer analgesic benefits over monotherapy with either agent. In a previous study, an ibuprofen/paracetamol combination provided significantly better analgesic efficacy than comparable doses of ibuprofen or paracetamol alone in patients experiencing moderate to severe acute postoperative pain after extraction of impacted third molars. OBJECTIVE: This study compared the efficacy and tolerability of 3 doses of a single-tablet fixed-dose combination (FDC) of ibuprofen and paracetamol (ibuprofen/paracetamol doses of 100 mg/250 mg, 200 mg/500 mg, and 400 mg/1000 mg) with comparable doses of ibuprofen (200 or 400 mg) monotherapy, paracetamol (500 or 1000 mg) monotherapy, and placebo in the 8 hours after surgical removal of 3 to 4 impacted third molars (stage 1) and with placebo over the next 72 hours (stage 2). METHODS: This was a multicenter, 2-stage, randomized, double-blind, parallel-group, placebo-controlled, factorial study. Male or female outpatients were eligible for the study if they were aged >or=16 years, were referred for surgical removal of at least 3 impacted third molars (2 of which had to be mandibular impacted molars), and gave written informed consent. In stage 1, patients were randomly assigned to ibuprofen 200 mg, ibuprofen 400 mg, paracetamol 500 mg, paracetamol 1000 mg, FDC ibuprofen 100 mg/paracetamol 250 mg, FDC ibuprofen 200 mg/paracetamol 500 mg, FDC ibuprofen 400 mg/paracetamol 1000 mg, or placebo. In stage 2, patients who had been taking FDC therapy or placebo continued the same treatment, whereas those taking monotherapy received FDC therapy, incorporating the same dose of active monotherapy from stage 1. First-line rescue medication (hydrocodone 7.5 mg and paracetamol 500 mg) was available at any time after dosing; however, in stage 1, any patient who required rescue medication within 60 minutes of receipt of study medication was considered a "dropout" from therapy, and, in stage 2, patients who required >2 doses of first-line rescue medication in a 24-hour period were considered treatment failures. In stage 1, the primary efficacy end points were the sum of pain relief and pain intensity differences over an 8-hour follow-up period (SPRID8) and the pain relief and pain intensity difference scores at each time point (15, 30, 45, 60, 90, 120, 180, 240, 300, 360, 420, and 480 minutes after dosing). Several secondary variables were also measured, including the patient's global assessment of the study medication. The 2-stopwatch method was used for measures of perceptible and meaningful pain relief. In stage 2, the primary efficacy end point was the number of completed 24-hour periods (as 0, 1, 2, or 3) in which the patient required no more than one dose of rescue medication and rated the treatment as "good," "very good," or "excellent." The tolerability of study medications was assessed in relation to the frequency, nature, and severity of adverse events (AEs), as well as their relationship to study medication; vital signs were also measured. AEs were assessed at 8 hours after dosing in stage 1, at 72 hours after dosing in stage 2, and at the follow-up visit (7-10 days after surgery); in addition, patients were instructed to report any AE that occurred between scheduled assessments. RESULTS: Of 735 patients randomly assigned in stage 1, a total of 715 (97.3%) entered and 678 (92.2%) completed stage 2. Most patients were female (62.6% [460/735]) and white (91.3% [671/735]); the mean (SD) age was 20.3 (3.5) years and the mean weight was 69.7 (15.7) kg. The mean total impaction score was 11.1 (1.4), and the mean baseline pain score on the visual analog scale was 76.9 (12.0). Overall, 422 of 735 patients (57.4%) reported severe pain at baseline and 313 of 735 (42.6%) reported moderate pain. For the primary efficacy end point (SPRID8), FDC ibuprofen 400 mg/paracetamol 1000 mg was significantly more effective than ibuprofen 400 mg (P = 0.02) and paracetamol 1000 mg (P < 0.001) in the immediate postoperative period (stage 1), and FDC ibuprofen 200 mg/paracetamol 500 mg was significantly more effective than ibuprofen 200 mg (P < 0.001) and paracetamol 500 mg (P < 0.001). The FDC ibuprofen 200 mg/paracetamol 500 mg was also significantly more effective than paracetamol 1000 mg (P < 0.001), but failed to reach statistical significance in comparison with ibuprofen 400 mg. Continued postoperative therapy with all 3 FDCs on an as-needed basis was significantly more effective than placebo during the subsequent 72 hours (all, P < 0.001). During stage 1, rescue medication was required by 53 of 73 (72.6%), 29 of 75 (38.7%), 21 of 74 (28.4%), 56 of 76 (73.7%), and 51 of 74 (68.9%) patients in the placebo group and the ibuprofen 200-mg, ibuprofen 400-mg, paracetamol 500-mg, and paracetamol 1000-mg treatment groups, respectively. In the FDC treatment groups, the need for rescue medication included 27 of 71 (38.0%), 40 of 143 (28.0%), and 32 of 149 (21.5%) patients in the ibuprofen 100-mg/paracetamol 250-mg, ibuprofen 200-mg/paracetamol 500-mg, and ibuprofen 400-mg/paracetamol 1000-mg groups, respectively. The rates of treatment-related AEs between the first and second doses of study medication were significantly lower for FDC ibuprofen 400 mg/paracetamol 1000 mg (8/149 patients [5.4%]) than for placebo (14/73 [19.2%]; P < 0.01) and paracetamol 1000 mg (10/74 [13.5%]; P < 0.05); and also lower for FDC ibuprofen 200 mg/paracetamol 500 mg (9/143 [6.3%]) compared with ibuprofen 200 mg (12/75 [16.0%]; P < 0.05) and paracetamol 500 mg (17/76 [22.4%]; P < 0.001). CONCLUSION: FDC ibuprofen 200 mg/paracetamol 500 mg and ibuprofen 400 mg/paracetamol 1000 mg were significantly more effective in this population than were comparable doses of ibuprofen or paracetamol alone in moderate to severe acute dental pain and were significantly more effective than placebo in providing sustained pain relief.