Arti Ahluwalia

Microsyringe-Based Deposition of Two-Dimensional and Three-Dimensional Polymer Scaffolds with a Well-Defined Geometry for Application to Tissue Engineering

A technique for controlled deposition of biomaterials and cells in specific and complex architectures is described. It employs a highly accurate three-dimensional micropositioning system with a pressure-controlled syringe to deposit biopolymer structures with a lateral resolution of 5 microm. The pressure-activated microsyringe is equipped with a fine-bore exit needle and a wide variety of two- and three-dimensional patterns on which cells to be deposited can adhere. The system has been characterized in terms of deposition parameters such as applied pressure, motor speed, line width and height, and polymer viscosity, and a fluid dynamic model simulating the deposition process has been developed, allowing an accurate prediction of the topological characteristics of the polymer structures.

Criticality of the Biological and Physical Stimuli Array Inducing Resident Cardiac Stem Cell Determination

The replacement of injured cardiac contractile cells with stem cell‐derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine‐tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues.

Acute retinal ganglion cell injury caused by intraocular pressure spikes is mediated by endogenous extracellular ATP

Elevated intraocular pressure may lead to retinal ganglion cell injury and consequent visual deficits. Chronic intraocular pressure increase is a major risk factor for glaucoma, a leading blinding disease, and permanent visual deficits can also occur following acute pressure increments due to trauma, acute glaucoma or refractive surgery. How pressure affects retinal neurons is not firmly established. Mechanical damage at the optic nerve head, reduced blood supply, inflammation and cytotoxic factors have all been called into play. Reasoning that the analysis of retinal neurons soon after pressure elevation would provide useful cues, we imaged individual ganglion cells in isolated rat retinas before and after short hydrostatic pressure increments. We found that slowly rising pressure to peaks observed in trauma, acute glaucoma or refractive surgery (50–90 mmHg) did not damage ganglion cells, whereas a rapid 1 min pulse to 50 mmHg injured 30% of these cells within 1 h. The severity of damage and the number of affected cells increased with stronger or repeated insults. Degrading extracellular ATP or blocking the P2X receptors for ATP prevented acute pressure‐induced damage in ganglion cells. Similar effects were observed in vivo. A short intraocular pressure transient increased extracellular ATP levels in the eye fluids and damaged ganglion cells within 1 h. Reducing extracellular ATP in the eye prevented damage to ganglion cells and accelerated recovery of their response to light. These data show that rapid pressure transients induce acute ganglion cell injury and unveil the causal role of extracellular ATP elevation in such injury.

Microfabricated PLGA scaffolds: a comparative study for application to tissue engineering

A variety of techniques for the manufacture of biodegradable, three-dimensional scaffolds for tissue engineering have been developed in recent years. In this study, we report and compare two simple methods for fabricating poly(DL-lactide-co-glycolide) (PLGA) scaffolds with feature sizes of 10–200 Am, which have been developed in our laboratories. The first technique is based on the use of a microsyringe that makes use of a computer-controlled, three-axis micropositioner, which allows the control of motor speeds and position. A PLGA solution is drawn from the needle of the syringe by the application of a constant pressure of 10–300 mm Hg resulting in controlled polymer deposition of 10–600 Am in diameter. The second technique is based on ‘‘soft lithographic’’ approaches that utilizes a Poly(dimethylsiloxane) (PDMS) mold. The polymer solution is cast on the mold under vacuum. Polymer concentration, solvent composition, and casting conditions influence the integrity and the lateral resolution of the resulting scaffold. Both techniques allow the possibility of constructing three-dimensional architectures that permit the study of cell behaviour in an environment similar to that in vivo, and may provide tools for the construction of engineered tissue. D 2002 Elsevier Science B.V. All rights reserved.

An android for enhancing social skills and emotion recognition in people with autism

It is well documented that the processing of social and emotional information is impaired in people with autism. Recent studies have shown that individuals, particularly those with high functioning autism, can learn to cope with common social situations if they are made to enact possible scenarios they may encounter in real life during therapy. The main aim of this work is to describe an interactive life-like facial display (FACE) and a supporting therapeutic protocol that will enable us to verify if the system can help children with autism to learn, identify, interpret, and use emotional information and extend these skills in a socially appropriate, flexible, and adaptive context. The therapeutic setup consists of a specially equipped room in which the subject, under the supervision of a therapist, can interact with FACE. The android display and associated control system has automatic facial tracking, expression recognition, and eye tracking. The treatment scheme is based on a series of therapist-guided sessions in which a patient communicates with FACE through an interactive console. Preliminary data regarding the exposure to FACE of two children are reported.

Modular bioreactor for primary human hepatocyte culture: Medium flow stimulates expression and activity of detoxification genes

Down‐regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long‐standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250–500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7–21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP‐glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S‐transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up‐regulated by medium flow as compared with static controls in all cultures tested. In 2‐week‐old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up‐regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down‐regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.

A Low Shear Stress Modular Bioreactor for Connected Cell Culture Under High Flow Rates

A generic “system on a plate” modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using “milli‐molding,” a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 µL/min, corresponding to wall shear stresses of 10−5 Pa or less at the cell culture surface. Biotechnol. Bioeng. 2010; 106: 127–137.

A phase diagram for microfabrication of geometrically controlled hydrogel scaffolds

Hydrogels are considered as excellent candidates for tissue substitutes by virtue of their high water content and biphasic nature. However, the fact that they are soft, wet and floppy renders them difficult to process and use as custom-designed scaffolds. To address this problem alginate hydrogels were modeled and characterized by measuring stress-strain and creep behavior as well as viscosity as a function of sodium alginate concentration, cross-linking time and calcium ion concentration. The gels were then microfabricated into scaffolds using the pressure-assisted microsyringe. The mechanical and viscous characteristics were used to generate a processing window in the form of a phase diagram which describes the fidelity of the scaffolds as a function of the material and machine parameters. The approach can be applied to a variety of microfabrication methods and biomaterials in order to design well-controlled custom scaffolds.

Connected Culture of Murine Hepatocytes and Human Umbilical Vein Endothelial Cells in a Multicompartmental Bioreactor

A multicompartmental bioreactor was conceived and designed to mimic cross talk between cells in different culture chambers connected only by flow, such that cell-cell interaction is mediated by soluble ligands as occurs in the body. The system was tested with a connected culture of murine hepatocytes and human umbilical vein endothelial cells. Metabolites such as albumin, urea, lactate and viability were monitored during the course of the experiments and compared with monoculture conditions in the bioreactor. When the two cell types are placed in connected culture, there is an increase in endothelial cell viability and hepatic glucose synthesis as well as albumin and urea production, while overall lactate production in the system is downregulated. The results show that the multicompartmental bioreactor enhances cell function, effectively combining both heterotypic interactions with increased nutrient availability.

Cooperation of Biological and Mechanical Signals in Cardiac Progenitor Cell Differentiation

Dr. S. Pagliari , Dr. G. Forte , Dr. F. Pagliari , Dr. M. Minieri , Prof. P. Di Nardo Laboratory of Molecular and Cellular Cardiology Department of Internal Medicine University of Rome “Tor Vergata”Rome 00133, Italy E-mail: dinardo@uniroma2.it Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoJapanese-Italian Tissue Engineering Laboratory (JITEL) Tokyo Women’s Medical University-Waseda University Joint Institution for Advanced Biomedical Sciences (TWIns) Tokyo, Japan Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoItalian Institute for Cardiovascular Research (INRC) 40126 Bologna, Italy Prof. A. C. Vilela-Silva Instituto de Ciencias Biomedicas and Laboratorio de Tecido Conjuntivo Hospital Universitario Clementino Fraga Filho Rio de Janeiro, Brazil Dr. C. Mandoli , Prof. E. Traversa International Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS) 1–1 Namiki, Tsukuba, Ibaraki 305–0044, Japan E-mail: TRAVERSA.Enrico@nims.go.jp Dr. S. Pietronave , Prof. M. Prat Department of Medical Sciences University “A. Avogadro” of Piemonte Orientale 28100 Novara, Italy Dr. G. Vozzi , Prof. A. Ahluwalia Interdepartmental Research Center “E. Piaggio” University of Pisa56126 Pisa, Italy Prof. S. Licoccia , Prof. E. Traversa NAST Centre & Department of Chemical Science and Technology University of Rome “Tor Vergata” Roma 00133, Italy [†] S.P. and A.C.V.S. contributed equally to this work.