Design and Validation of an Open-Hardware Print-Head for Bioprinting Application

In the last decades drop-on-demand inkjet technology played an increasing role in industrial and medical applications. This is due to the ability to deposit a small amount of material in precisely defined position. In the field of Biofabrication, inkjet printers are used to build 2D and 3D scaffolds and gels with biological molecules, including living cells. Several works, including seminal papers on inkjet bioprinting, were carried out with modified office printers. These printers have fixed structural characteristics and operating size, especially on the print-head, limiting the range of materials that can be dispensed. The aim of the present work is the design and fabrication of an open-source piezoelectric inkjet print-head, optimized for the bioprinting field. This low-cost, reproducible, reliable, versatile and biocompatible device will enable various research laboratories to work with a shared device; the open source allowing for parts to be modified to suit specific needs. The design was carried out by Finite Element (FE) modelling of the piezoelectric, mechanical, fluid dynamics and their coupling. The design was optimized for shear rate, which we minimized in order to be able to print cells. The mechanical frame of the printer was designed and built using a low-cost 3D printer. The nozzle plate was fabricated from a polycarbonate disc coated with biocompatible silicone, to increase the hydrophobicity of the outer surface of the disc, preventing ink adhesion on the edge of the nozzle; the refilling system, and the electronic control were also part of the project and will be freely available to download. The FE models were validated with ad-hoc experiments, printing water, gelatin solution, and cell culture media, by modulating the wave power in amplitude, frequency and duty cycle. The tests showed a large working window both respect to viscosity and to surface tension. Finally Human Skin Fibroblasts (ATCC-CRL- 2522, Teddington UK), suspended in culture media, were printed. Cell viability, assessed by CellTiter-Blue and LIVE / DEAD tests, resulted comparable with the control, demonstrating the validity of the first open source piezoelectric inkjet print-head for biofabrication

Preliminary experimental analysis of Reservoir Computing approach for balance assessment

Evaluation of balance stability in elderly people is of prominent relevance in the field of health monitoring. Recently, the use of Wii Balance Board has been proposed as valid alternative to clinical balance tests, such as the widely used Berg Balance Scale (BBS) test, allowing to measure and analyze static features such as the duration or the speed of assessment of patients' center of pressure. In an innovative way, in this paper we propose to take into consideration the whole temporal information generated by the balance board, analyzing it by means of dynamical neural networks. In particular, using Recurrent Neural Networks implemented according to the Reservoir Computing paradigm, we propose to estimate the BBS score from the temporal data generated by the execution of one simple exercise on the balance board. Preliminary experimental assessments of the proposed approach on a real-world dataset show promising results

Integration of Biomechanical and Biological Characterization in the Development of Porous Poly(Caprolactone)-Based Membranes for Abdominal Wall Hernia Treatment.

AIMS: Synthetic meshes are the long-standing choice for the clinical treatment of abdominal wall hernias: the associated long-term complications have stimulated the development of a new-generation of bio-resorbable prostheses. In this work, polycaprolactone (PCL) porous membranes prepared by solvent casting/porogen leaching of PCL/poly(ethylene glycol) (PEG) blends with different compositions (different PCL/PEG weight ratio and PEG molecular weight) were investigated to be applied in the field. An optimal porous membrane structure was selected based on the evaluation of physicochemical, biomechanical and in-vitro biological properties, compared to a reference commercially available hernia mesh (CMC). FINDINGS: Selected PCL7-2i membranes (derived from PCL/PEG 70/30, PCL: Mw 70,000-90,000 Da; PEG: 35,000 Da) showed suitable pore size for the application, intermediate surface hydrophilicity and biomimetic mechanical properties. In-vitro cell tests performed on PCL7-2i membranes showed their cytocompatibility, high cell growth during 21 days, a reduced production of pro-inflammatory IL-6 respect to CMC and a significant secretion of Collagen Type I. CONCLUSIONS: PCL7-2i membranes showed biomimetic biomechanical properties and in-vitro biological properties similar to or even better than - in the case of anti-inflammatory behavior and collagen production - CMC, a commercially available product, suggesting potentially improved integration in the host tissue

Biological Effects of Transforming Growth Factor Beta in Human Cholangiocytes

: TGF-β is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-β in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-β was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-β stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-β treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-β treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins (CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin) involved in Ca2+ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-β. These results suggested that the effects of TGF-β in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-β signaling

Smart environments and context-awareness for lifestyle management in a healthy active ageing framework

Health trends of elderly in Europe motivate the need for technological solutions aimed at preventing the main causes of morbidity and premature mortality. In this framework, the DOREMI project addresses three important causes of morbidity and mortality in the elderly by devising an ICT-based home care services for aging people to contrast cognitive decline, sedentariness and unhealthy dietary habits. In this paper, we present the general architecture of DOREMI, focusing on its aspects of human activity recognition and reasoning

Three-dimensional reconstruction of coronary arteries and plaque morphology using CT angiography - comparison and registration with IVUS

Background: The aim of this study is to present a new methodology for three-dimensional (3D) reconstruction of coronary arteries and plaque morphology using Computed Tomography Angiography (CTA). Methods: The methodology is summarized in six stages: 1) pre-processing of the initial raw images, 2) rough estimation of the lumen and outer vessel wall borders and approximation of the vessel's centerline, 3) manual adaptation of plaque parameters, 4) accurate extraction of the luminal centerline, 5) detection of the lumen - outer vessel wall borders and calcium plaque region, and 6) finally 3D surface construction. Results: The methodology was compared to the estimations of a recently presented Intravascular Ultrasound (IVUS) plaque characterization method. The correlation coefficients for calcium volume, surface area, length and angle vessel were 0.79, 0.86, 0.95 and 0.88, respectively. Additionally, when comparing the inner and outer vessel wall volumes of the reconstructed arteries produced by IVUS and CTA the observed correlation was 0.87 and 0.83, respectively. Conclusions: The results indicated that the proposed methodology is fast and accurate and thus it is likely in the future to have applications in research and clinical arena

Non-enhanced micro-CT of paraffin embedded coronary vessels: a tool for experimental atherosclerosis

Purpose. To assess and quantify morphometric parameters of full-length coronary arteries in three dimensions at high resolution by micro-CT (?CT), and to evaluate the possible integration with in vivo information by invasive coronary angiography (ICA) and and intravascular ultrasound (IVUS). Methods and Materials. Excised left coronary artery (LMCA and LAD) and underlying myocardium from pigs on a high-cholesterol diet were fixed in formalin, dehydrated and embedded in paraffin as a whole sample inside a Falcon-type test tube. In vivo information obtained by ICA and IVUS were also available for the same animals. After the ?CT scan, the samples were further processed in order to perform standard histological stains. Results. For each sample, we have obtained a reconstructed volume of 512x512x1400 isotropic voxels, with a voxel size of 57.4^3 ?m^3. The ?CT images provided good discrimination between fat, muscle and calcium, allowing a reliable segmentation of walls and lesions. The profiles of total area, wall area and calcium along the entire coronary artery allowed a comprehensive evaluation of the progress of atherosclerosis in terms of intimal thickening, plaque formation and calcification. Microcalcifications down to 0.2 nl (nanoliters) were localized and displayed in 3D. The integration with IVUS allowed also to recover the lumen area under in vivo physiological state. Conclusion. Micro-CT allows a comprehensive evaluation in 3D and at high resolution of plaque evolution in an experimental model of coronary artery disease in pigs. The integration with ICA and IVUS can lead to the full characterization of disease progression

Three-dimensional reconstruction of coronary arteries and plaque morphology using CT angiography – comparison and registration with IVUS

BACKGROUND: The aim of this study is to present a new methodology for three-dimensional (3D) reconstruction of coronary arteries and plaque morphology using Computed Tomography Angiography (CTA). METHODS: The methodology is summarized in six stages: 1) pre-processing of the initial raw images, 2) rough estimation of the lumen and outer vessel wall borders and approximation of the vessel’s centerline, 3) manual adaptation of plaque parameters, 4) accurate extraction of the luminal centerline, 5) detection of the lumen - outer vessel wall borders and calcium plaque region, and 6) finally 3D surface construction. RESULTS: The methodology was compared to the estimations of a recently presented Intravascular Ultrasound (IVUS) plaque characterization method. The correlation coefficients for calcium volume, surface area, length and angle vessel were 0.79, 0.86, 0.95 and 0.88, respectively. Additionally, when comparing the inner and outer vessel wall volumes of the reconstructed arteries produced by IVUS and CTA the observed correlation was 0.87 and 0.83, respectively. CONCLUSIONS: The results indicated that the proposed methodology is fast and accurate and thus it is likely in the future to have applications in research and clinical arena

New bioreactors systems for pharmacological screening

Il lavoro di dottorato si è basato sullo sviluppo di nuovi sistemi di coltura cellulare, definiti bioreattori, capaci di ricreare condizioni di vita cellulare maggiormente fisiologiche. Lo scopo è quello di poter analizzare i processi cellulari (crescita, differenziazione, metabolismo) in presenza di stimoli fisici e chimici tali da ricreare un ambiente di vita simil-corporeo. I sistemi sviluppati sono stati 3: il Laminar flow bioreactor, sviluppato per ricreare condizioni di shear stress presenti nel sistema vascolare ed analizzarne gli effetti su colture di cellule endoteliali; l’Array Bioreactor, sistema abile a riprodurre i gradienti di concentrazione di sostanze bioattive per valutarne il loro effetto su cellule di varia tipologia; il Multi-Compartimental Bioreactor, una sistema per lo studio del cross-talk intercellulare progettato tramite l’utilizzo di leggi allometriche e realizzato tramite l’utilizzo di di 4 diversi tipi cellulari connessi tra loro tramite flusso di terreno di coltura

New bioreactors systems for pharmacological screening

Bioreactors, biotechnological devices for in vitro cell cultures with dynamic conditions, have the potential to provide information on local cell behavior and function. The development of bioreactors could lead to a multitude of applications from drug testing and development, tissue engineering and basic research to the identification of new and alternative therapies for many disorders. High quality, reliable, in vitro data also provide a shift in focus from large scale animal testing to the analysis of specific and local cell responses in controlled environments. In order to reduce, in the future, use of animals in drug testing for different reasons (economic, ethical and scientific), aim of this work is the development of these new biotechnological tools, the bioreactors. These technogical supports for research, designed at the Interdepartmental Research Centre "E. Piaggio" (University of Pisa) and tested at CNR Institute of Clinical Physiology (Pisa), are composed of: a main cell culture chamber, where cells, directly or by use of a support, are seeded; a mixing chamber for gas exchange and for pH and temperature measurement; an electronic and software control system for control and setting of experimental conditions. In three years of work, our attention was focused on optimization of cell life conditions and recreation of a physiological cell environment. In accordance with these guidelines, we have designed and realized different types of bioreactors to approach and simulate different biological conditions and, also, to elucidate, in a manner much more consistent, the basic working mechanisms of cells. We have customized our bioreactors in order to reproduce physical stimuli (Laminar Flow Bioreactor), chemical stimuli (Array Bioreactor) and metabolic conditions (Multi-Compartmental Bioreactor). For each bioreactor, we have observed their good functionality and the possibility to recreate the physical, chemical and biological environment for which they are designed