Carmelo De Maria

Substrate stiffness influences high resolution printing of living cells with an ink-jet system.

The adaptation of inkjet printing technology for the realisation of controlled micro- and nano-scaled biological structures is of great potential in tissue and biomaterial engineering. In this paper we present the Olivetti BioJet system and its applications in tissue engineering and cell printing. BioJet, which employs a thermal inkjet cartridge, was used to print biomolecules and living cells. It is well known that high stresses and forces are developed during the inkjet printing process. When printing living particles (i.e., cell suspensions) the mechanical loading profile can dramatically damage the processed cells. Therefore computational models were developed to predict the velocity profile and the mechanical load acting on a droplet during the printing process. The model was used to investigate the role of the stiffness of the deposition substrate during droplet impact and compared with experimental investigations on cell viability after printing on different materials. The computational model and the experimental results confirm that impact forces are highly dependent on the deposition substrate and that soft and viscous surfaces can reduce the forces acting on the droplet, preventing cell damage. These results have high relevance for cell bioprinting; substrates should be designed to have a good compromise between substrate stiffness to conserve spatial patterning without droplet coalescence but soft enough to absorb the kinetic energy of droplets in order to maintain cell viability.

The PAM2 system: a multilevel approach for fabrication of complex three-dimensional structures

Purpose – The traditional tissue engineering approach employs rapid prototyping systems to realise microstructures (i.e. scaffolds) which recapitulate the function and organization of native tissues. The purpose of this paper is to describe a new rapid prototyping system (PAM‐modular micro‐fabrication system, PAM2) able to fabricate microstructures using materials with different properties in a controlled environment.Design/methodology/approach – Computer‐aided technologies were used to design multi‐scale biological models. Scaffolds with specific features were then designed using custom software and manufactured using suitable modules. In particular, several manufacturing modules were realised to enlarge the PAM2 processing material window, controlling physical parameters such as pressure, force, temperature and light. These modules were integrated in PAM2, allowing a precise control of fabrication parameters through a modular approach and hardware configuration.Findings – Synthetic and natural polymeric...

Biofabrication: A Guide to Technology and Terminology

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient.

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells’ morphogenesis, and they range in a three‐dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffness. Copyright

Combining Inkjet Printing and Sol-Gel Chemistry for Making pH-Sensitive Surfaces

Today biomedical sciences are experiencing the importance of imaging biological parameters with luminescence methods. Studying 2D pH distribution with those methods allows building knowledge about complex cellular processes. Immobilizing pH sensitive nanoparticles inside hydrogel matrixes, in order to guarantee a proper SNR, could easily make stable and biocompatible 2D sensors. Inkjet printing is also well known as tool for printing images onto porous surfaces. Recently it has been used as a free-form fabrication method for building three-dimensional parts, and now is being explored as a way of printing electrical and optical devices. Inkjet printing was used either as a rapid prototyping method for custom biosensors. Sol-gel method is naturally bound with inkjet, because the picoliter-sized ink droplets evaporate quickly, thus allowing quick sol-gel transitions on the printed surface. In this work will be shown how to merge those technologies, in order to make a nanoparticles doped printable hydrogel, which could be used for making 2D/3D smart scaffolds able to monitor cell activities. An automated image analysis system was developed in order to quickly have the pH measurements from pH nanosensors fluorescence images.

A new path to platelet production through matrix sensing

Megakaryocytes (MK) in the bone marrow (BM) are immersed in a network of extracellular matrix components that regulates platelet release into the circulation. Combining biological and bioengineering approaches, we found that the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4), a mechano-sensitive ion channel, is induced upon MK adhesion on softer matrices. This response promoted platelet production by triggering a cascade of events that lead to calcium influx, β1 integrin activation and internalization, and Akt phosphorylation, responses not found on stiffer matrices. Lysyl oxidase (LOX) is a physiological modulator of BM matrix stiffness via collagen crosslinking. In vivo inhibition of LOX and consequent matrix softening lead to TRPV4 activation cascade and increased platelet levels. At the same time, in vitro proplatelet formation was reduced on a recombinant enzyme-mediated stiffer collagen. These results suggest a novel mechanism by which MKs, through TRPV4, sense extracellular matrix environmental rigidity and release platelets accordingly.

Realisation and characterization of conductive hollow fibers for neuronal tissue engineering

In this article, conductive hollow fibers have been fabricated using melt spinning technique. Multiwalled carbon nanotubes (MWNTs) and poly(3-hexylthiophene-2,5-diyl) (P3HT) have been used to fabricate conductive poly-caprolactone (PCL) composites. The hollow fibers have inner and outer diameter in the range of 300 µm and 500 µm, respectively. Critical parameters to tune the dimension of hollow fibers have been defined following two-dimensions mathematical model. Evaluation of the mechanical properties showed that the incorporation of 1-3 wt % MWNTs and 5-8 wt % P3HT increased Young Modulus of 10% and 20% respectively, compared with pure PCL. The electrical property assessment demonstrated that a minimum incorporation of 3 wt % MWNT and 8 wt % P3HT in PCL matrix transformed composite materials into conductive materials. In addition, SH-SY5Y human neuroblastoma cells were seeded on the fabricated samples an their adhesion, proliferation and neurite length growth were analysed. In particular we observed that these materials promoted cell activities and in particular on MWNT/PCL composites there was a significant increase of neurite growth.

The influence of mesh topology in the abdominal wall repair process

The tissue integration and the formation of adhesions in the repair of abdominal wall defects are principally led to the topology and the mechanical properties of implanted prosthesis. In this study we analyzed the influence of the topology of the meshes for abdominal wall repair, made of polypropylene (PP), evaluating its ability to prevent and to minimize the formation of adhesions, and to promote tissue ingrowth. Two series of in vivo studies were performed. In the first, two types of PP meshes, a lightweight macroporous mesh (LWM) and a heavyweight microporous mesh (HWM) were compared with determine the optimal porosity for tissue integration. In the second, a composite mesh, Clear Mesh Composite (CMC), made of a LWM sewn on a PP planar smooth film, was compared with a PP planar film, to demonstrate how two different topologies of same material are able to induce different tissue integration with the abdominal wall and different adhesion with internal organs. In both studies, the prostheses were implanted in Wistar rats and histological analysis and mechanical characterization of tissue coupled with the implants were performed. LWM showed better host tissue ingrowth in comparison to HWM. CMC prosthesis showed no adhesions to the viscera and no strong foreign body reaction, moreover its elasticity and anisotropy index were more similar to that of natural tissue. These results demonstrated that the surface morphology of PP surgical meshes allowed to modulate their repair ability.

Development of a novel micro-ablation system to realise micrometric and well-defined hydrogel structures for tissue engineering applications

Purpose – This paper aims to develop a novel micro-ablation system to realise micrometric and well-defined hydrogel structures. To engineer a tissue it is necessary to evaluate several aspects, such as cell-cell and cell-substrate interactions, its micro-architecture and mechanical stimuli that act on it. For this reason, it is important to fabricate a substrate which presents a microtopology similar to natural tissue and has chemical and mechanical properties able to promote cell functions. In this paper, well-defined hydrogel structures embedding cells were microfabricated using a purposely developed technique, micro-laser ablation, based on a thulium laser. Its working parameters (laser power emission, stepper motor velocity) were optimised to produce shaded “serpentine” pattern on a hydrogel film. Design/methodology/approach – In this study, initially, swelling/contraction tests on agarose and alginate hydrogel in different solutions of main components of cell culture medium were performed and were co...

ADMET: ADipocyte METabolism mathematical model

White fat cells have an important physiological role in maintaining triglyceride and free fatty acid levels due to their fundamental storage property, as well as determining insulin resistance. ADipocyte METabolism is a mathematical model that mimics the main metabolic pathways of human white fat cell, connecting inputs (composition of culture medium) to outputs (glycerol and free fatty acid release). It is based on a set of nonlinear differential equations, implemented in Simulink® and controlled by cellular energetic state. The validation of this model is based on a comparison between the simulation results and a set of experimental data collected from the literature.