Mariella Dentini

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.

The influence of side-chains on the dilute-solution properties of three structurally related, bacterial anionic polysaccharides

Abstract Viscometric, calorimetric, and chiroptical data collected by using dilute aqueous solutions of the extracellular polysaccharides [Me4N+ salt form] from Pseudomonas elodea (S-60), Alcaligenes (ATCC 31555) (S-130), and Alcaligenes (ATCC 31961) (S-194) over a range of ionic strength (Me4NCl concentrations) and temperature are reported. The data demonstrate that increasing the ionic strength of dilute solutions of the nonbranched S-60 at 25° promotes a distinctly co-operative, conformational transition over a narrow range of added salt concentration. Moreover, over the range of ionic strength examined, S-60 also undergoes a thermally reversible, conformational transition which exhibits no hysteresis: the temperatures at the midpoint of the sigmoidal transitions (TM) increase moderately with increasing ionic strength. This conformational ordering has been monitored under conditions where S-60 does not gel. However, because both higher concentrations of S-60 at the same ionic strength, and higher ionic strengths at the same S-60 concentration, lead to gel formation, it is likely that the conformational ordering we have observed is the one involved in the gelation of S-60. The data also indicate that the two different modes of branching along the S-60 backbone present in S-130 (α- l -rhamnose and α- l -mannose linked to O-3 of the d -glucose after the d -glucuronic acid in the tetrasaccharide repeating-unit) and S-194 [β- d -glucopyranosyl-(1 → 6)-α- d -glucopyranosyl units linked to O-6 of the d -glucosyl residue before the d -glucosyluronic acid] mediate against conformational ordering. This is consistent with the fact that S-60 forms firm aqueous gels, whereas S-130 and S-194 are non-gel-forming, but give stable, highly viscous solutions. In addition, the measurements indicate that the carboxyl group in S-30 is in a screened, molecular environment. It is suggested that this screening could arise from hydrogen bonding involving the l -rhamnose- l -mannose side-chains.

Comparative analysis of the behavior of gellan gum (S-60) and welan gum (S-130) in dilute aqueous solution☆

Abstract Optical rotation, circular dichroism, and microcalorimetric data clearly and consistently show that gellan gum, S-60 (Me 4 N + form), undergoes in water at 25° a rather sharp conformational transition upon increasing the concentration of added Me 4 NCl. Similar data show that S-60 behaves anomalously upon addition of Ca 2+ ions with, eventually, formation of aggregates and/or gels. The Me 4 NCl-induced conformational change of S-60 is thermally reversible with no hysteresis. In contrast, with welan gum, S-130 (Me 4 N + form), no evidence could be found for a dependence of chain conformation of the main external variables considered. Comparison of the circular-dichroism spectra of the two polysaccharides suggests that S-130 in water might be present in a stiff conformation similar to that assumed by S-60 in aqueous Me 4 NCl.

Chitosan-coated PLGA nanoparticles: a sustained drug release strategy for cell cultures.

A recently patented one-step methodology was used for the formulation of chitosan (CS) coated polylactic-co-glycolic acid (PLGA) nanoparticles containing dexamethasone (DXM) as a model drug. SEM investigations showed that nanoparticles (NPs) were spherical in shape with smooth surface. CS coating switched NPs ζ-potential from negative to positive, without modifying particle size distribution. Moreover, CS coating allowed a significant modulation of in vitro drug release, providing a sustained drug delivery in cultured cells. The uptake of fluorescent CS-coated PLGA NPs by hepatocytes (C3A) and fibroblasts (3T6) as well as the fate of internalized NPs were investigated by confocal microscopy. 3T6 and C3A cells were treated with DXM-loaded NPs and experiments were addressed to analyze the specific cell response to DXM, in order to evaluate its functional efficiency in comparison with conventional addition to culture medium. CS-coating of DXM loaded PLGA NPs allowed their uptake by cultured cells without inducing cytotoxicity.

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.

Synthesis and preliminary characterisation of charged derivatives and hydrogels from scleroglucan

The synthesis of negatively and positively charged polyelectrolytes from scleroglucan is described. Polycarboxylates were synthesised through nucleophilic substitution with chloroacetic acid or through a selective 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO)-mediated oxidation of the primary alcohol groups. Amine groups were introduced through nucleophilic substitution with 2-chloroethylamine or 3-chloropropylamine. Reaction conditions were varied to obtain insight into the influence of variables on the degree of substitution. The conformational behaviour of the obtained polyelectrolytes was studied as a function of pH, temperature and solvent. For the products with a low degree of modification, evidence of an ordered conformation was found, whereas the polymers with a higher degree of modification behaved as random coils in solution. The negatively charged polymers were reticulated using the Ugi four-component condensation, obtaining negatively charged hydrogels. The positively charged polymers were reticulated using diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dion, DES) to obtain positively charged hydrogels.

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.