New Bioengineering Breakthroughs and Enabling Tools in Regenerative Medicine.

PURPOSE OF REVIEW: In this review, we provide a general overview of recent bioengineering breakthroughs and enabling tools that are transforming the field of regenerative medicine (RM). We focus on five key areas that are evolving and increasingly interacting including mechanobiology, biomaterials and scaffolds, intracellular delivery strategies, imaging techniques, and computational and mathematical modeling. RECENT FINDINGS: Mechanobiology plays an increasingly important role in tissue regeneration and design of therapies. This knowledge is aiding the design of more precise and effective biomaterials and scaffolds. Likewise, this enhanced precision is enabling ways to communicate with and stimulate cells down to their genome. Novel imaging technologies are permitting visualization and monitoring of all these events with increasing resolution from the research stages up to the clinic. Finally, algorithmic mining of data and soft matter physics and engineering are creating growing opportunities to predict biological scenarios, device performance, and therapeutic outcomes. SUMMARY: We have found that the development of these areas is not only leading to revolutionary technological advances but also enabling a conceptual leap focused on targeting regenerative strategies in a holistic manner. This approach is bringing us ever more closer to the reality of personalized and precise RM

Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour

Live-imaging techniques are at the forefront of biology research to explore behaviour and function from sub-cellular to whole organism scales. These methods rely on intracellular fluorescent probes to label specific proteins, which are commonly assumed to only introduce artefacts at concentrations far-exceeding routine use. Lifeact, a small peptide with affinity for actin microfilaments has become a gold standard in live cell imaging of the cytoskeleton. Nevertheless, recent reports have raised concerns on Lifeact-associated artefacts at the molecular and whole organism level. We show here that Lifeact induces dose-response artefacts at the cellular level, impacting stress fibre dynamics and actin cytoskeleton architecture. These effects extend to the microtubule and intermediate filament networks as well as the nucleus, and ultimately lead to altered subcellular localization of YAP, reduced cell migration and abnormal mechanical properties. Our results suggest that reduced binding of cofilin to actin filaments may be the underlying cause of the observed Lifeact-induced cellular artefacts

Methodology of the Virtual Reconstruction of Arquitectonic Heritage: Ambassador Vich's Palace in Valencia

The 19th century was disastrous as far as the conservation of architectonic heritage is concerned. The awareness of the importance of preserving monuments that has prevailed since the end of the last century was dazzlingly absent in the previous, leading both to the disappearance of representative heritage works and the plundering of many others. The present study establishes the methodological basis to proceed with the virtual reconstruction of many disappeared architectures, representative of emblematic architectonic typologies. A method based on the combination of deduction and induction allows benchmarks to be created that signify a starting point to which the key and specific elements of each building are later incorporated, from the data extracted from the conserved parts and the graphic, literary and archive documents. The result is the virtual recovery of the general outlines of the architecture: morphology of the plot, volumetry, exterior and interior facades, and the functional layout. The good results obtained in the study of the disappeared Ambassador Vich's Palace, allow the methodology to be extended to the analysis of other similar examples, serving investigators as a tool to carry out an arduous task of deciphering a trail that is increasingly fading with the passing of time.Galiana Agullo, M.; Mas Tomas, MDLA.; Lerma Elvira, C.; Peñalver Martínez, MJ.; Conesa Tejada, S. (2014). Methodology of the Virtual Reconstruction of Arquitectonic Heritage: Ambassador Vich's Palace in Valencia. International Journal of Architectural Heritage. 8(1):94-123. doi:10.1080/15583058.2012.672623S9412381Boix, V. 1979.Historical and topographic Valencia[in Spanish]. Vol. I261 S. A. Printing J. Rius.Estaban Chapapría, J. (2001). Impostación del patio del Embajador Vich en el ex-convento del Carmen (Valencia). Loggia, Arquitectura & Restauración, (12), 26. doi:10.4995/loggia.2001.3605Morrish, S. W., & Laefer, D. F. (2010). Web-Enabling of Architectural Heritage Inventories. International Journal of Architectural Heritage, 4(1), 16-37. doi:10.1080/15583050902731056Lotz, W. 1995.Architecture in Italy 1500–1600 [in Italian]35–37. ed. RizzoliYale University Press.Lourenço, P. B., Peña, F., & Amado, M. (2010). A Document Management System for the Conservation of Cultural Heritage Buildings. International Journal of Architectural Heritage, 5(1), 101-121. doi:10.1080/15583050903318382Vila Ferrer, S. (2001). La recuperación del patio del palacio del Embajador Vich (Valencia). Loggia, Arquitectura & Restauración, (12), 44. doi:10.4995/loggia.2001.3606Zonta, D., Pozzi, M., & Zanon, P. (2008). Managing the Historical Heritage Using Distributed Technologies. International Journal of Architectural Heritage, 2(3), 200-225. doi:10.1080/1558305080206369

From Cheese Whey Permeate To An Anti-Listeria Food Packaging Device: Bacterial Cellulose Nanocrystals/Sakacin-A Conjugates (Nanosak)

In the present project cheese whey permeate (CWP), the residual by-product obtained by extraction of whey proteins from cheese whey, was used as substrate for the growth of bacterial species that produce two appealing molecules: the anti-listerial bacteriocin sakacin-A and bacterial cellulose (BC). BC is then turned into nanocrystals (BCNCs) that are finally conjugated with sakacin-A to obtain an innovative antimicrobial device for food which could support Listeria monocytogenes growth. Sakacin-A was produced by Lactobacillus sakei DSMZ 6333 in liquid cultures. The highest bacteriocin production (around 300 AU/mL) was achieved after 9 h at 26\ub0C; a food-grade, salt-free enriched sakacin-A extract was obtained by using a gravity reverse phase chromatography. BC was produced by Komagataeibacter xylinus DSMZ 2325 by static fermentation of CWP in presence of 0.5 U/mL of \u3b2-galactosidase at 30\ub0C; after 7 days, BC yield was around 7 g/L. BCNCs were then obtained by acid hydrolysis mediated by sulfuric acid, with the goal of removing the amorphous regions of BC and introduce a net negative charge by esterification on the hydroxyl group on C6. BCNCs/sakacin-A conjugates were prepared by exploiting their opposite charge: enriched sakacin-A extract was mixed with BCNCs and, after incubation, conjugates collected by centrifugation have a specific activity of 100 AU/mg BCNCs. Among all peptides present in the enriched sample, sakacin-A appears to preferentially absorb onto BCNCs, thus allowing its further purification. Sakacin-A as well its BCNCs conjugates were then included in a hydroxypropil-cellulose coating spread onto paper sheets at a concentration of 5 and 25 AU/cm2. The addition of the coating did not bring any significant change in the oxygen barrier properties of the cellulosic substrate. In a similar way, the static contact angle of both uncoated and coated substrate was of approximately 130\ub0. However, the presence of BCNCs seemed to increase the swelling phenomenon of the coating. Sakacin A was also included in whey, caseine and cellulose derived matrices to prepare films and coatings with diverse results. The kinetics of Sakacin-A released from active films to aqueous food was analyzed by immersion of samples in water (as simulant) and measuring the anti-Listeria activity of the simulant after increasing times of exposure. In vitro and in vivo antimicrobial trials were carried out on real food products demonstrated their anti-listerial effectiveness, proving that the developed devices can contribute to increase shelf life, quality and safety of perishable foods

Portal Vein Embolization is Associated with Reduced Liver Failure and Mortality in High-Risk Resections for Perihilar Cholangiocarcinoma

Background Preoperative portal vein embolization (PVE) is frequently used to improve future liver remnant volume (FLRV) and to reduce the risk of liver failure after major liver resection. Objective This paper aimed to assess postoperative outcomes after PVE and resection for suspected perihilar cholangiocarcinoma (PHC) in an international, multicentric cohort. Methods Patients undergoing resection for suspected PHC across 20 centers worldwide, from the year 2000, were included. Liver failure, biliary leakage, and hemorrhage were classified according to the respective International Study Group of Liver Surgery criteria. Using propensity scoring, two equal cohorts were generated using matching parameters, i.e. age, sex, American Society of Anesthesiologists classification, jaundice, type of biliary drainage, baseline FLRV, resection type, and portal vein resection. Results A total of 1667 patients were treated for suspected PHC during the study period. In 298 patients who underwent preoperative PVE, the overall incidence of liver failure and 90-day mortality was 27% and 18%, respectively, as opposed to 14% and 12%, respectively, in patients without PVE (p < 0.001 and p = 0.005). After propensity score matching, 98 patients were enrolled in each cohort, resulting in similar baseline and operative characteristics. Liver failure was lower in the PVE group (8% vs. 36%, p < 0.001), as was biliary leakage (10% vs. 35%, p < 0.01), intra-abdominal abscesses (19% vs. 34%, p = 0.01), and 90-day mortality (7% vs. 18%, p = 0.03). Conclusion PVE before major liver resection for PHC is associated with a lower incidence of liver failure, biliary leakage, abscess formation, and mortality. These results demonstrate the importance of PVE as an integral component in the surgical treatment of PHC

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate, but Not FAK

BACKGROUND: Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. PRINCIPAL FINDINGS: Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. CONCLUSIONS: These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background: Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood. Principal Findings: In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize. Conclusions: Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin

Portal Vein Embolization is Associated with Reduced Liver Failure and Mortality in High-Risk Resections for Perihilar Cholangiocarcinoma

Background: Preoperative portal vein embolization (PVE) is frequently used to improve future liver remnant volume (FLRV) and to reduce the risk of liver failure after major liver resection. Objective: This paper aimed to assess postoperative outcomes after PVE and resection for suspected perihilar cholangiocarcinoma (PHC) in an international, multicentric cohort. Methods: Patients undergoing resection for suspected PHC across 20 centers worldwide, from the year 2000, were included. Liver failure, biliary leakage, and hemorrhage were classified according to the respective International Study Group of Liver Surgery criteria. Using propensity scoring, two equal cohorts were generated using matching parameters, i.e. age, sex, American Society of Anesthesiologists classification, jaundice, type of biliary drainage, baseline FLRV, resection type, and portal vein resection. Results: A total of 1667 patients were treated for suspected PHC during the study period. In 298 patients who underwent preoperative PVE, the overall incidence of liver failure and 90-day mortality was 27% and 18%, respectively, as opposed to 14% and 12%, respectively, in patients without PVE (p < 0.001 and p = 0.005). After propensity score matching, 98 patients were enrolled in each cohort, resulting in similar baseline and operative characteristics. Liver failure was lower in the PVE group (8% vs. 36%, p < 0.001), as was biliary leakage (10% vs. 35%, p < 0.01), intra-abdominal abscesses (19% vs. 34%, p = 0.01), and 90-day mortality (7% vs. 18%, p = 0.03). Conclusion: PVE before major liver resection for PHC is associated with a lower incidence of liver failure, biliary leakage, abscess formation, and mortality. These results demonstrate the importance of PVE as an integral component in the surgical treatment of PHC