Influence of Initial Surface Roughness on LIPSS Formation and Its Consecutive Impact on Cell/Bacteria Attachment for TiAl6V4 Surfaces

The influence of the initial surface roughness of TiAl6V4 samples on the orientation and periodicity of the resulting laser-induced periodic surface structures (LIPSS), as well as the surface wettability and chemistry is reported here. Before LIPSS fabrication, initial sample surface roughness is adjusted by variations of finial polishing steps with polishing grain sizes of 18.3, 8.4, 5, and 0.5 µm. A 3 × 3 irradiation matrix was defined and lasered on all samples by changing the laser power and distance between consecutive laser scans. The resulting structures were characterized by scanning electron microscopy (SEM), atomic force microscopy, Raman spectroscopy, and contact angle measurements. As a further step, three representative generated structures were chosen to explore their bone implant viability by resazurin assays, alkaline phosphatase activity, and direct SEM imaging of the induced cells (MG63) and bacteria (Escherichia coli and Staphylococcus aureus). Results show that initial surface roughness has big influence on the wettability of the resulting surface, as well as inducing small variations on the orientation of the generated LIPSS. Structures generated with a higher integrated fluence have also shown to enhance cell differentiation while reducing bacterial activity, making them a great candidate for improved bone implant compatibility and durability

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of β-catenin.

Cardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.CDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.Results revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.Combined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders

Investigating Potential Effects of Ultra-Short Laser-Textured Porous Poly-ε-Caprolactone Scaffolds on Bacterial Adhesion and Bone Cell Metabolism

Developing antimicrobial surfaces that combat implant-associated infections while promoting host cell response is a key strategy for improving current therapies for orthopaedic injuries. In this paper, we present the application of ultra-short laser irradiation for patterning the surface of a 3D biodegradable synthetic polymer in order to affect the adhesion and proliferation of bone cells and reject bacterial cells. The surfaces of 3D-printed polycaprolactone (PCL) scaffolds were processed with a femtosecond laser (λ = 800 nm; τ = 130 fs) for the production of patterns resembling microchannels or microprotrusions. MG63 osteoblastic cells, as well as S. aureus and E. coli, were cultured on fs-laser-treated samples. Their attachment, proliferation, and metabolic activity were monitored via colorimetric assays and scanning electron microscopy. The microchannels improved the wettability, stimulating the attachment, spreading, and proliferation of osteoblastic cells. The same topography induced cell-pattern orientation and promoted the expression of alkaline phosphatase in cells growing in an osteogenic medium. The microchannels exerted an inhibitory effect on S. aureus as after 48 h cells appeared shrunk and disrupted. In comparison, E. coli formed an abundant biofilm over both the laser-treated and control samples; however, the film was dense and adhesive on the control PCL but unattached over the microchannels

Preoperative risk factors for massive transfusion, prolonged ventilation requirements, and mortality in patients undergoing liver transplantation

BackgroundDespite improvements in techniques and management of liver transplant patients, numerous perioperative complications that contribute to perioperative mortality remain. Models to predict intraoperative massive blood transfusion, prolonged mechanical ventilation, or in-hospital mortality in liver transplant recipients have not been identified. In this study we aim to identify preoperative factors associated with the above mentioned complications.MethodsA retrospective observational analysis was conducted on data collected from 124 orthotopic liver transplants performed at a single institution between 2014 and 2017. A multivariable logistic regression using backwards elimination was performed for three defined outcomes (massive transfusion ≥ 10 units packed red blood cells (PRBC), prolonged mechanical ventilation > 24 h, and in-hospital mortality) to identify associations with preoperative characteristics.ResultsStatistically significant (P < 0.05) associations with massive transfusion ≥ 10 units PRBC were hepatocellular carcinoma and preoperative transfusion of PRBC. Significant associations with prolonged mechanical ventilation > 24 h were hepatitis C, alcoholic hepatitis, elevated preoperative ALT, and hepatorenal syndrome. Male gender was protective for requiring prolonged mechanical ventilation. End-stage renal disease and hepatitis B were significantly associated with increased in-hospital mortality.ConclusionsThis study identified risk factors associated with common perioperative complications of liver transplantation. These factors may assist practitioners in risk stratification and may form the basis for further investigations of potential interventions to mitigate these risks

Toward Smart Biomimetic Apatite-Based Bone Scaffolds with Spatially Controlled Ion Substitutions

Biomimetic apatites exhibit a high reactivity allowing ion substitutions to modulate their in vivo response. We developed a novel approach combining several bioactive ions in a spatially controlled way in view of subsequent releases to address the sequence of events occurring after implantation, including potential microorganisms’ colonization. Innovative micron-sized core-shell particles were designed with an external shell enriched with an antibacterial ion and an internal core substituted with a pro-angiogenic or osteogenic ion. After developing the proof of concept, two ions were particularly considered, Ag+ in the outer shell and Cu2+ in the inner core. In vitro evaluations confirmed the cytocompatibility through Ag-/Cu-substituting and the antibacterial properties provided by Ag+. Then, these multifunctional “smart” particles were embedded in a polymeric matrix by freeze-casting to prepare 3D porous scaffolds for bone engineering. This approach envisions the development of a new generation of scaffolds with tailored sequential properties for optimal bone regeneration

Aza and AA concentration determination.

<p><b>(A)</b> Aza concentration determination by LDH cytotoxicity assay. CDCs were treated at five concentrations, (1 μM, 5 μM, 10 μM, 20 μM and 50 μM) of Aza. Significant toxicity of Aza was observed at 20 μM and 50 μM on both day 1 and day 2. Data is presented as Mean ± SEM and the statistical significance (<i>p</i> ≤ 0.001) is calculated using Two-way Anova. <b>(B)</b> Aza effect on cell proliferation of CDCs determined by Alamar Blue assay. Aza at the concentrations of 1, 5 and 10 μM concentrations did not show distinct difference in proliferation (<i>p</i> = ns). Data is presented as Mean ± SEM using Two-way Anova. <b>(C)</b> AA concentration determination by proliferation assay. CDCs were treated with 10, 50, 100, 250 and 500 μM concentrations of AA and proliferation rate were assessed using hemocytometer. CDCs displayed increased trend in proliferation from 3 to 7 days at the concentrations, 10, 50 and 100 μM respectively. Data is presented as Mean ± SEM and the statistical significance is calculated using Two-way Anova.</p

Culture and characterization of CEOCs and CDCs.

<p><b>(A)</b> Phase contrast microscopic images representing culture and derivation of cardiosphere derived cells. <b>a</b> On day 2 of culture, fibroblast like cells started shedding from the edge of cardiac explants. <b>b</b> On day 7 of culture, phase bright round cells were found on top of fibroblast like monolayer around the explants. <b>c</b> Robust production of round cells from the explants on day 10 of the culture. <b>d</b> Round cells found to be confluent around the explant on 14 days post culture. <b>e</b> Formation of Cardiospheres derived from CEOCs. <b>f</b> Monolayer culture of CDCs. Arrows indicate the mentioned cells in respective panels. <b>(B)</b> Represents immunofluorescence analysis of CEOCs surrounding the explant. Z stacks were acquired for 8 images at 76 μm optical section thickness. CPCs were present in the outermost layer of the explant and were stained positively for c-kit marker (red). While the other cells in the explant were stained with DAPI alone. <b>(C)</b> Representative images of CEOCs for c-kit marker and CDCs for c-kit and CD 105 markers. CEOCs showed prominent expression of c-kit whereas CDCs for CD 105 marker. Scale bar = 20μm. Arrows indicate cells positive for the respective marker.</p

Protein expression in control, Aza and Aza+AA treated CDCs.

<p>(<b>A)</b> Representation of protein expression by Western blot analysis - α-Sarcomeric actinin, GATA 4, non-phospho β-catenin, phospho β-catenin and Cyclin D1. Lane 1 = control, Lane 2 = Aza 7 days, Lane 3 = Aza 14 days, Lane 4 = Aza+AA 7 days, Lane 5 = Aza+AA 14 days. (<b>B-E)</b> Graphical representation of the respective proteins expression in comparison to control. α-Sarcomeric actinin showed significant upregulation in Aza+AA group (<i>p</i> ≤ 0.001). GATA 4 displayed marked upregulation in expression on 14^th day of Aza+AA group (<i>p</i> = 0.0242). Wnt proteins, non-phospho β-catenin and Cyclin D1 showed significant down regulation by 14 days in both the treatment groups, especially in Aza+AA group (<i>p</i> ≤ 0.05) while phospho β-catenin showed significant up regulation on 14^th day in Aza+AA treatment group (<i>p</i> = 0.0001) when compared to Aza. # represents overall significance with respect to control. * represents significance of student’s t-test analysis. #<i>p</i> < 0.05 Vs control, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.001, ***<i>p</i> ≤ 0.0001.</p

Real time PCR analysis of control, Aza and Aza+AA treated CDCs.

<p>(<b>A)</b> CD 105 showed distinct down regulation in Aza+AA treated CDCs when compared to control on 14^th day (#<i>p</i> < 0.0241). (<b>B</b>-<b>C)</b> Nkx 2.5 and GATA 4 expression were relatively upregulated in Aza+AA group when compared to control on 14^th day (#<i>p</i> < 0.0156 and <i>p</i> < 0.0087 respectively). (<b>D-E)</b> β-catenin and Cyclin D1expression were distinctly down regulated in Aza+AA group when compared to control with <i>p</i> values, 0.0107 and 0.0116 respectively. # represents overall significance with respect to control as analyzed by One way Anova with Kruskal Wallis test. * represents significant values from student’s <i>t</i>-test analysis. #, <i>p</i> < 0.05 Vs control, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Schematic representation of CDCs differentiation towards cardiomyogenic lineage.

<p>Aza+AA efficiently differentiated CDCs to cardiomyocyte like cells with distinct upregulation of cardiac specific genes. Wnt pathway was negatively regulated in this process via phosphorylation of β-catenin.</p