3D Bioprinting of Collagen-based Microfluidics for Engineering Fully-biologic Tissue Systems.

Microfluidic and organ-on-a-chip devices have improved the physiologic and translational relevance of in vitro systems in applications ranging from disease modeling to drug discovery and pharmacology. However, current manufacturing approaches have limitations in terms of materials used, non-native mechanical properties, patterning of extracellular matrix (ECM) and cells in 3D, and remodeling by cells into more complex tissues. We present a method to 3D bioprint ECM and cells into microfluidic collagen-based high-resolution internally perfusable scaffolds (CHIPS) that address these limitations, expand design complexity, and simplify fabrication. Additionally, CHIPS enable size-dependent diffusion of molecules out of perfusable channels into the surrounding device to support cell migration and remodeling, formation of capillary-like networks, and integration of secretory cell types to form a glucose-responsive, insulin-secreting pancreatic-like microphysiological system.

Rapid model-guided design of organ-scale synthetic vasculature for biomanufacturing.

Our ability to produce human-scale bio-manufactured organs is critically limited by the need for vascularization and perfusion. For tissues of variable size and shape, including arbitrarily complex geometries, designing and printing vasculature capable of adequate perfusion has posed a major hurdle. Here, we introduce a model-driven design pipeline combining accelerated optimization methods for fast synthetic vascular tree generation and computational hemodynamics models. We demonstrate rapid generation, simulation, and 3D printing of synthetic vasculature in complex geometries, from small tissue constructs to organ scale networks. We introduce key algorithmic advances that all together accelerate synthetic vascular generation by more than 230 -fold compared to standard methods and enable their use in arbitrarily complex shapes through localized implicit functions. Furthermore, we provide techniques for joining vascular trees into watertight networks suitable for hemodynamic CFD and 3D fabrication. We demonstrate that organ-scale vascular network models can be generated in silico within minutes and can be used to perfuse engineered and anatomic models including a bioreactor, annulus, bi-ventricular heart, and gyrus. We further show that this flexible pipeline can be applied to two common modes of bioprinting with free-form reversible embedding of suspended hydrogels and writing into soft matter. Our synthetic vascular tree generation pipeline enables rapid, scalable vascular model generation and fluid analysis for bio-manufactured tissues necessary for future scale up and production.

FRESH 3D Bioprinting a Ventricle-like Cardiac Construct Using Human Stem Cell-Derived Cardiomyocytes.

Here we describe a method to engineer a contractile ventricle-like chamber composed of human stem cell-derived cardiomyocytes using freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. To do this, we print a support structure using a collagen type I ink and a cellular component using a high-density cell ink supplemented with fibrinogen. The gelation of the collagen and the fibrinogen into fibrin is initiated by pH change and enzymatic crosslinking, respectively. Fabrication of the ventricle-like chamber is completed in three distinct phases: (i) materials preparation, (ii) bioprinting, and (iii) tissue maturation. In this protocol, we describe the method to print the construct from a high-density cell ink composed of human stem cell-derived cardiomyocytes and primary fibroblasts (~300 × 106 cells/mL) using our open-source dual-extruder bioprinter. Additional details are provided on FRESH support preparation, bioink preparation, dual-extruder needle alignment, print parameter selection, and post-processing. This protocol can also be adapted by altering the 3D model design, cell concentration, or cell type to FRESH 3D bioprint other cardiac tissue constructs.

Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication.

In tissue engineering, an unresolved challenge is how to build complex 3D scaffolds in order to recreate the structure and function of human tissues and organs. Additive manufacturing techniques, such as 3D bioprinting, have the potential to build biological material with unprecedented spatial control; however, printing soft biological materials in air often results in poor fidelity. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) is an embedded printing approach that solves this problem by extruding bioinks within a yield-stress support bath that holds the bioinks in place until cured. In this Perspective, we discuss the challenges of 3D printing soft and liquid-like bioinks and the emergence for FRESH and related embedded printing techniques as a solution. This includes the development of FRESH and embedded 3D printing within the bioprinting field and the rapid growth in adoption, as well as the advantages of FRESH printing for biofabrication and the new research results this has enabled. Specific focus is on the customizability of the FRESH printing technique where the chemical composition of the yield-stress support bath and aqueous phase crosslinker can all be tailored for printing a wide range of bioinks in complex 3D structures. Finally, we look ahead at the future of FRESH printing, discussing both the challenges and the opportunities that we see as the biofabrication field develops.

Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties.

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 µm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.

Pediatrician attitudes concerning school-located vaccination clinics for seasonal influenza.

OBJECTIVE: Vaccinating all children aged 6 months to 18 years every year has potentially large ramifications for office-based primary care pediatricians. We determined the degree to which pediatricians support routine annual influenza vaccination outside the medical home, especially in school-located mass influenza vaccination clinics. METHODS: Internet-based survey sent in May and June 2009 to all 623 currently practicing primary care general pediatricians who were members of the Maryland Chapter of the American Academy of Pediatrics. RESULTS: Of those surveyed, 193 (31%) responded. Approximately 67% reported they vaccinated more than half the children in their practice with at least one dose in the 2008-2009 influenza season, and about half anticipated that, in their office, they would not attain ≥75% coverage of all patients older than 5 months of age. Approximately 27% of respondents predicted they would likely have difficulty obtaining sufficient vaccine to cover commercially insured patients, and 32% were likely to have difficulty getting sufficient vaccine to cover Medicaid, underinsured, and uninsured patients because of ordering or distribution problems. Approximately 78% of respondents cited borderline or poor reimbursement for influenza vaccinations, and 53% had unused vaccine at the end of the 2008-2009 influenza season. Ninety-six percent of respondents supported school-located influenza vaccination programs in their community for their patients. CONCLUSIONS: These results indicate awareness by primary care pediatricians in Maryland of the potential difficulties involved in implementing universal influenza vaccinations in their practice and their support of school-located vaccination programs managed by the local health department in their community.

Nonsteroidal 2,3-dihydroquinoline glucocorticoid receptor agonists with reduced PEPCK activation.

Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.

Tetrahydroquinolin-3-yl carbamate glucocorticoid receptor agonists with reduced PEPCK activation.

Continuing studies on tetrahydroquinoline glucocorticoid receptor anti-inflammatory agents lead to the identification of several tetrahydroquinolin-3-yl carbamates that exhibited steroid-like activity in in vitro transrepression assays with reduced transactivation of phosphoenol pyruvate carboxykinase (PEPCK), a key enzyme in the gluconeogenesis pathway.

Discovery of orally available tetrahydroquinoline-based glucocorticoid receptor agonists.

A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity.

Tetrahydroquinoline glucocorticoid receptor agonists: discovery of a 3-hydroxyl for improving receptor selectivity.

We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure-activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists.

Recent developments in the discovery of selective glucocorticoid receptor modulators (SGRMs).

Steroidal glucocorticoids are widely prescribed for the treatment of a variety of inflammatory and autoimmune diseases. Although they are effective, the side-effects associated with chronic glucocorticoid treatment, such as osteoporosis and hyperglycemia, can severely limit their long-term use. Hence, there is a need to develop new effective anti-inflammatory agents for systemic use which are dissociated from their unwanted side effects.

Synthesis and characterization of nonsteroidal glucocorticoid receptor modulators for multiple myeloma.

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

5(Z)-benzylidene-1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes as non-steroidal glucocorticoid receptor modulators.

A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure-activity relationships for this series of compounds are presented.

Chromium(0)-promoted multicomponent cycloaddition of tethered diynes with cyclic trienes: application to the total synthesis of 9-epi-pentalenic acid.

An efficient protecting group controlled regioselective chromium(0)-mediated three-component higher order cycloaddition of tethered diynes with cyclic trienes that generates five rings and six stereogenic centers in one step is described. Following a sequence of reactions featuring a chemoselective Baeyer-Villiger rearrangement and a regioselective cyclopropane hydrogenolysis, the total synthesis of 9-epi-pentalenic acid was achieved.