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Tissue engineering represents a possible approach to replace the lost or defective muscle. The purpose of this study was to assess a new scaffold design for muscle tissue engineering, by comparing the growth of C2C12 mouse cells on different polymers. Polymeric substrata were prepared with a soft lithography technique (replica molding) from three different masters (a milled aluminium plate, a laser-processed alumina sheet, and a microgrooved modeling paste mould obtained from a home-made fibers array). Each master imprinted with an elastomeric material (Elastosilw RT601 Wacker), was in turn used as a mould to prepare the microgrooved polymeric substrata by solvent-casting. Polymer selected was a medical grade biodegradable poly(L-lactic acid) /trimethylencarbonate (PLLA/TMC 68:32, Boerhinger Ingelheim). Four different groups based on fiber spacing (30–35, 50–55, 70–75, and 90–95 mm) were evaluated. We compared 3-week growth of C2C12 cells cultured on scaffolds alone, or supplementing the scaffold with two key cytokines involved in muscle regeneration, bFGF(30G120 pM) and HGF(70G280 pM). Both methods facilitated cell attachment, growth, and viability. The cells lined the inner and outer surfaces of the scaffold, filling the pores, as demonstrated by scanning electron microscopy and histology. BrdUrd incorporation confirmed high levels of cellular proliferation. Yet, the combination of both cytokines showed a strong synergistic stimulation of C2C12 myoblast proliferation and chemotactic activity. After 21 days in the HGF-treated samples large areas of well-formed and aligned myotube fascicles were present which appeared to be more frequent than in the scaffold-alone samples and to show higher expression of differentiation-associated proteins. Our results show that the combination of PLLA/TMC polymer as a vehicle for myoblasts and exogenous HGF as a proliferative stimulator is a good candidate for the generation of skeletal muscle in vitro.

The possibility of refining the grain structure of commercial Al-Mg-Si alloys was evaluated using the asymmetric rolling technique in the severe plastic deformation (SPD) regime. Bars of annealed alloy having a thickness of 10 mm were rolled at room temperature down to a thickness of 0,2 mm with a laboratory rolling mill featuring the possibility of independently modifying the rotational speed of its two rolls. Trials were performed with speed ratio ranging from 1 (conventional rolling) to 2 (the upper roll rotates at a speed that is twice as that of the lower roll). Analyses on the severely deformed samples were mainly focused on the distribution of hardness and the evolution of microstructure along their thickness as a function of rolling conditions. Experimental tests were carried out by optical and transmission electron microscopy (TEM) and hardness microindentations. The investigation demonstrated that asymmetric rolling can readily promote the achievement Of an ultrafine grained structure (UFG) in the Al-Mg-Si alloy with an improved homogeneity of hardness and microstructure along thickness. This rolling technique therefore represents a promising route for the production of UFG long products such as strips, plates and bars in continuous industrial processing plants.

Silk fibroin (SF)-based or -coated biomaterials are likely to be endowed with structural and surface properties that render them particularly apt for biomedical applications. In this work we investigated the behavior of four different strains of normal human adult fibroblasts that had been seeded onto membranes made up of poly(carbonate) urethane (PCU), the surfaces of which had or had not been homogeneously coated with SF. Cell adhesion within 3h to the SF-coated PCU films was 2.2-fold that to their uncoated homologues. After 30 days of incubation in vitro, 2.5-fold more cells had grown on the SF-coated specimens than on the uncoated ones. This enhanced cell adherence and hence growth on the SF-coated surfaces was coupled with higher cumulative rates of D-glucose (but not L-glutamine) uptake and of both lactate and interleukin-6 (IL-6) cumulative secretion. Conversely, human fibroblasts cultured on either type of PCU scaffolds never secreted any ELISA-assayable amount of three main proinflammatory cytokines, namely interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1). Finally, when the metabolic activities were compared on a per 10(5) cells basis, it became clear that the adhesion to SF favored an initially higher consumption of D-glucose, a late higher release of IL-6, and an at-first more intense, but declining, extracellular assembly of type I collagen fibers. Overall, these results show that SF-coated PCU membranes represent a novel type of biomaterial that favors the adhesion, the growth and performance of specific metabolic tasks by normal human adult fibroblasts without eliciting any concurrent secretion of some of the chief proinflammatory cytokines.

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

Chitosan is a natural polymer widely investigated and used due to its antibacterial activity, mucoadhesive, analgesic, and hemostatic properties. Its biocompatibility makes chitosan a favorable candidate for different applications in tissue engineering (TE), such as skin, bone, and cartilage tissue regeneration. Despite promising results obtained with chitosan 3D scaffolds, significant challenges persist in fabricating hydrogel structures with ordered architectures and biological properties to mimic native tissues. In this work, chitosan has been investigated aiming at designing and fabricating uniaxial scaffolds which can be proposed for the regeneration of anisotropic tissues (i.e., skin, skeletal muscle, myocardium) by 3D printing technology. Chitosan was blended with gelatin to form a polyelectrolyte complex in two different ratios, to improve printability and shape retention. After the optimization of the printing process parameters, different crosslinking conditions were investigated, and the 3D printed samples were characterized. Tripolyphosphate (TPP) was used as crosslinker for chitosan-based scaffolds. For the optimization of the printing temperature, the sol-gel temperature of the chitosan-gelatin blend was determined by rheological measurements and extrusion temperature was set to 20°C (i.e., below sol-gel temperature). The shape fidelity and surface morphology of the 3D printed scaffolds after crosslinking was dependent on crosslinking conditions. Interestingly, mechanical properties of the scaffolds were also significantly affected by the crosslinking conditions, nonetheless the stability of the scaffolds was strongly determined by the content of gelatin in the blend. Lastly, in vitro cytocompatibility test was performed to evaluate the interactions between L929 cells and the 3D printed samples. 2% w/v chitosan and 4% w/v gelatin hydrogel scaffolds crosslinked with 10% TPP, 30 min at 4°C following 30 min at 37°C have shown cytocompatible and stable characteristics, compared to all other tested conditions, showing suitable properties for the regeneration of anisotropic tissues.

New porous scaffolds, with a suitable hydrolytic and enzymatic degradation, useful for tissue engineering applications have been obtained by a carbodiimide mediated reaction between hyaluronan (HA) and a synthetic polymer with a polyaminoacid structure such as α,β-polyaspartylhydrazide (PAHy). Scaffolds with a different molar ratio between PAHy repeating units and HA repeating units have been prepared and characterized from a chemical and physicochemical point of view. Tests of indirect and direct cytotoxicity, cell adhesion, and spreading on these biomaterials have been performed by using murine L929 fibroblasts. The new biomaterials showed a good cell compatibility and ability to allow cell migration into the scaffolds as well as spreading on their surface. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008