Andrea Barbetta

Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink

A novel bioink and a dispensing technique for 3D tissue-engineering applications are presented. The technique incorporates a coaxial extrusion needle using a low-viscosity cell-laden bioink to produce highly defined 3D biostructures. The extrusion system is then coupled to a microfluidic device to control the bioink arrangement deposition, demonstrating the versatility of the bioprinting technique. This low-viscosity cell-responsive bioink promotes cell migration and alignment within each fiber organizing the encapsulated cells.

Porous alginate hydrogels: synthetic methods for tailoring the porous texture.

Alginate is a versatile, renewable biopolymer that has found numerous applications in diverse areas such as adsorbent materials of water pollutants and scaffolds for tissue engineering. In such kinds of applications the most convenient physical form of alginate-based materials is as porous matrices. The pore scale dimension has to be carefully engineered to meet the requirements posed by the specific application. The aim of this paper is to describe two synthetic methodologies that allow the preparation of alginate porous materials characterized by pores lying in well separated dimension ranges. One process is based on emulsion templating, which consists of dispersing an organic phase into an aqueous solution of alginate in the presence of a suitable emulsion stabilizer and locking in the structure of the continuous phase by chemical cross-linking. This approach required the preliminary degradation of alginate to reduce its molecular weight and, hence, the viscosity of the external phase of the concentrated emulsion. Porous matrices were characterized by pores and interconnects of about 10-20 and 2-5 microm, respectively, and a surface area of 230 m(2)/g. The second process consisted of replacing the organic, internal phase with a gas, namely, CO(2), generated in situ the aqueous solution of alginate. The chemical reaction for CO(2) generation, nature of the surfactant, and cross-linking method were carefully selected to give highly porous, stable matrices with pores and interconnects of the order of 300 and 80 microm, respectively.

Porous gelatin hydrogels by gas-in-liquid foam templating

In the present work, porous gelatin scaffolds were prepared by insufflating an inert gas (argon) inside a concentrated solution of gelatin in the presence of a suitable polymeric surfactant in association with sodium dodecyl sulfate. The implementation of such an approach involved the design and manufacturing of a specially dedicated reactor. Foams were prepared at a temperature of 50 °C and then let gel at 4 °C. After purification, they were auto-cross-linked with EDC and freeze-dried. The scaffolds synthesised with this technique present a morphology characterised by pores of spherical symmetry highly interconnected by a plurality of interconnections and, as a consequence, are particularly suited for cell culturing. The dosage of the volume of the insufflated gas permits to modulate the scaffold pore and interconnect dimensions. In this way matrices characterised by void and interconnect average diameters ranging from 250 to 360 μm and from 80 to 150 μm, respectively, can be successfully obtained.

Emulsion templated scaffolds that include gelatin and glycosaminoglycans.

Gelatin is one of the most commonly used biopolymer for creating cellular scaffolds due to its innocuous nature. To create stable gelatin scaffolds at physiological temperature (37 degrees C), chemical cross-linking is a necessary step. In a previous paper (Biomacromolecules 2006, 7, 3059-3068), cross-linking was carried out by either radical polymerization of the methacrylated derivative of gelatin (GMA) or through the formation of isopeptide bonds catalyzed by transglutaminase. The method of scaffold production was based on emulsion templating in which an organic phase is dispersed in the form of discrete droplets into a continuous aqueous solution of the biopolymer. Both kinds of scaffolds were tested as culture medium for hepatocytes. It turned out that the enzymatic cross-linked scaffold performed superiorily in this respect, even though it was mechanically less stable than the GMA scaffold. In the present paper, in an attempt to improve the biocompatibility of the GMA-based scaffold, biopolymers present in the extracellular matrix (ECM) were included in scaffold formulation, namely, chondroitin sulfate and hyaluronic acid. These biopolymers were derivatized with methacrylic moieties to undergo radical polymerization together with GMA. The morphology of the scaffolds was tuned to some extent by varying the volume fraction of the internal phase and to a larger extent by inducing a controlled destabilization of the precursor emulsion through the use of additives. In this way, scaffolds with 44% of the void volume attributable to voids with a diameter exceeding 60 microm and with 79% of the interconnect area attributable to interconnects with a diameter exceeding 20 microm in diameter could be successfully synthesized. To test whether the inclusion of ECM components into scaffold formulation resolves in an improvement of their biocompatibility with respect to GMA scaffolds, hepatocytes were seeded on both kinds of scaffolds and cell viability and function assays were carried out and compared.

3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage formation.

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>10(7) cells ml(-1)), high cell viability (85 ÷ 90%) and high printing resolution (≈100 μm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Human cardiosphere-seeded gelatin and collagen scaffolds as cardiogenic engineered bioconstructs

Cardiac tissue engineering (CTE) aims at regenerating damaged myocardium by combining cells to a biocompatible and/or bioactive matrix. Collagen and gelatin are among the most suitable materials used today for CTE approaches. In this study we compared the structural and biological features of collagen (C-RGD) or gelatin (G-FOAM)-based bioconstructs, seeded with human adult cardiac progenitor cells in the form of cardiospheres (CSps). The different morphology between C-RGD (fibrous ball-of-thread-like) and G-FOAM (trabecular sponge-like) was evidenced by SEM analysis and X-ray micro-tomography, and was reflected by their different mechanical characteristics. Seeded cells were viable and proliferating after 1 week in culture, and a reduced expression of cell-stress markers versus standard CSp culture was detected by realtime PCR. Cell engraftment inside the scaffolds was assessed by SEM microscopy and histology, evidencing more relevant cell migration and production of extracellular matrix in C-RGD versus G-FOAM. Immunofluorescence and realtime PCR analysis showed down-regulation of vascular and stemness markers, while early-to-late cardiac markers were consistently and significantly upregulated in G-FOAM and C-RGD compared to standard CSps culture, suggesting selective commitment towards cardiomyocytes. Overall our results suggest that CSp-bioconstructs have suitable mechanical properties and improved survival and cardiogenic properties, representing promising tools for CTE.

Gas-in-Liquid Foam Templating as a Method for the Production of Highly Porous Scaffolds

In the present work, a novel synthetic methodology for the preparation of scaffold of biopolymeric nature is described. In particular, a porous gelatin scaffold was prepared by foam templating. The gas phase, nitrogen, was generated by means of the reaction between sulfamic acid and sodium nitrite in situ a concentrated solution of gelatin and in the presence of a suitable polymeric surfactant in association with sodium dodecyl sulfate. The foam was prepared at a temperature of 45 degrees C and then let gel at 5 degrees C. After purification, the physical gel was auto-cross-linked with EDC and freeze-dried. The scaffold synthesized with this technique presents a morphology characterized by voids of spherical symmetry highly interconnected by a plurality of interconnects, and, as a consequence, is particularly suited for cell culturing. In more quantitative terms, voids and interconnects are characterized by an average diameter of 230 and 90 microm, respectively. Preliminary tests of cell culturing demonstrated the suitability of such a scaffold for tissue engineering applications.

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application

•  Introduction •  Lessons from cell therapy •  Cardiac tissue engineering ‐  In vivo CTE applications ‐  In vitro CTE applications •  Conclusions

Morphological comparison of PVA scaffolds obtained by gas foaming and microfluidic foaming techniques.

In this article, we have exploited a microfluidic foaming technique for the generation of highly monodisperse gas-in-liquid bubbles as a templating system for scaffolds characterized by an ordered and homogeneous porous texture. An aqueous poly(vinyl alcohol) (PVA) solution (containing a surfactant) and a gas (argon) are injected simultaneously at constant flow rates in a flow-focusing device (FFD), in which the gas thread breaks up to form monodisperse bubbles. Immediately after its formation, the foam is collected and frozen in liquid nitrogen, freeze-dried, and cross-linked with glutaraldehyde. In order to highlight the superior morphological quality of the obtained porous material, a comparison between this scaffold and another one, also constituted of PVA but obtained with a traditional gas foaming technique, was carried out. Such a comparison has been conducted by analyzing electron microscopy and X-ray microtomographic images of the two samples. It turned out that the microfluidic produced scaffold was characterized by much more uniform porous texture than the gas-foaming one as witnessed by narrower pore size, interconnection, and wall thickness distributions. On the other side, scarce pore interconnectivity, relatively low pore volume, and limited production rate represent, by now, the principal disadvantages of microfluidic foaming as scaffold fabrication method, emphasizing the kind of improvement that this technique needs to undergo.

Rapid prototyping of chitosan-coated alginate scaffolds through the use of a 3D fiber deposition technique†

Three dimensional, periodic scaffolds of chitosan-coated alginate are fabricated in a layer-by-layer fashion by rapid prototyping. A novel dispensing system based on two coaxial needles delivers simultaneously alginate and calcium chloride solutions permitting the direct deposition of alginate fibers according to any designed pattern. Coating of the alginate fiber with chitosan and subsequent cross-linking with EDC and genipin assured the endurance of the scaffold in the culture environment for a prolonged period of time. The cross-linking protocol adopted imparted to the scaffold a hierarchical chemical structure as evidenced by Confocal Laser Microscopy and FTIR spectroscopy. The core of the fibers making up the scaffold is represented by alginate chains cross-linked by ester bonds only, the periphery of the fiber is constituted by an inter-polyelectrolyte complex of alginate and chitosan cross-linked in all pair combinations. Fibers belonging to adjacent layers are glued together by the chitosan coating. Mechanical behavior of the scaffolds characterized by different layouts of deposition was determined revealing anisotropic properties. The biocompatibility and capability of the scaffolds to sustain hepatocyte (HepaRG) cultures were demonstrated. Typical hepatic functions such as albumin and urea secretion and induction of CYP3A4 enzyme activity following drug administration were excellent, thus proving the potential of these constructs in monitoring the liver specific function.