Magnetically-actuated microcages for cells entrapment, fabricated by laser direct writing via two photon polymerization.

The manipulation of biological materials at cellular level constitutes a sine qua non and provocative research area regarding the development of micro/nano-medicine. In this study, we report on 3D superparamagnetic microcage-like structures that, in conjunction with an externally applied static magnetic field, were highly efficient in entrapping cells. The microcage-like structures were fabricated using Laser Direct Writing via Two-Photon Polymerization (LDW via TPP) of IP-L780 biocompatible photopolymer/iron oxide superparamagnetic nanoparticles (MNPs) composite. The unique properties of LDW via TPP technique enabled the reproduction of the complex architecture of the 3D structures, with a very high accuracy i.e., about 90 nm lateral resolution. 3D hyperspectral microscopy was employed to investigate the structural and compositional characteristics of the microcage-like structures. Scanning Electron Microscopy coupled with Energy Dispersive X-Ray Spectroscopy was used to prove the unique features regarding the morphology and the functionality of the 3D structures seeded with MG-63 osteoblast-like cells. Comparative studies were made on microcage-like structures made of IP-L780 photopolymer alone (i.e., without superparamagnetic properties). We found that the cell-seeded structures made by IP-L780/MNPs composite actuated by static magnetic fields of 1.3 T were 13.66 ± 5.11 folds (p < 0.01) more efficient in terms of cells entrapment than the structures made by IP-L780 photopolymer alone (i.e., that could not be actuated magnetically). The unique 3D architecture of the microcage-like superparamagnetic structures and their actuation by external static magnetic fields acted in synergy for entrapping osteoblast-like cells, showing a significant potential for bone tissue engineering applications.

A Review on Stimuli-Actuated 3D Micro/Nanostructures for Tissue Engineering and the Potential of Laser-Direct Writing via Two-Photon Polymerization for Structure Fabrication.

In this review, we present the most recent and relevant research that has been done regarding the fabrication of 3D micro/nanostructures for tissue engineering applications. First, we make an overview of 3D micro/nanostructures that act as backbone constructs where the seeded cells can attach, proliferate and differentiate towards the formation of new tissue. Then, we describe the fabrication of 3D micro/nanostructures that are able to control the cellular processes leading to faster tissue regeneration, by actuation using topographical, mechanical, chemical, electric or magnetic stimuli. An in-depth analysis of the actuation of the 3D micro/nanostructures using each of the above-mentioned stimuli for controlling the behavior of the seeded cells is provided. For each type of stimulus, a particular recent application is presented and discussed, such as controlling the cell proliferation and avoiding the formation of a necrotic core (topographic stimulation), controlling the cell adhesion (nanostructuring), supporting the cell differentiation via nuclei deformation (mechanical stimulation), improving the osteogenesis (chemical and magnetic stimulation), controlled drug-delivery systems (electric stimulation) and fastening tissue formation (magnetic stimulation). The existing techniques used for the fabrication of such stimuli-actuated 3D micro/nanostructures, are briefly summarized. Special attention is dedicated to structures' fabrication using laser-assisted technologies. The performances of stimuli-actuated 3D micro/nanostructures fabricated by laser-direct writing via two-photon polymerization are particularly emphasized.

Innovative Technical Solution Using the Renal Artery Stump after Nephrectomy as an Inflow Artery for Lower Limb Revascularization-A Case Report.

We present the case of a 56-year-old patient admitted to the vascular unit of the Targu Mures County Emergency Clinical Hospital after a computed tomography angiography performed for critical limb ischemia showed a tumor of the right kidney of 11.3/12/11 cm anteroposterior/later-lateral/craniocaudal, accompanied by an abdominal aortic aneurysm (AAA) (3 cm diameter) and right iliac artery occlusion. An interdisciplinary team formed of urological and vascular surgeons decided and performed a one-step operation. The right kidney was removed, and the limb revascularization was achieved by performing a bypass that used the right renal arterial stump as an inflow artery, thus called a reno-femoral bypass. The AAA had no indication for reconstruction. The final pathology interpretation of the kidney tumor revealed a clear cell renal cell carcinoma, excised with oncological safety margins. A short-term follow-up found the patient without ischemic symptomatology and a fully functional graft.

Laser Direct Writing of Dual-Scale 3D Structures for Cell Repelling at High Cellular Density.

The fabrication of complex, reproducible, and accurate micro-and nanostructured interfaces that impede the interaction between material's surface and different cell types represents an important objective in the development of medical devices. This can be achieved by topographical means such as dual-scale structures, mainly represented by microstructures with surface nanopatterning. Fabrication via laser irradiation of materials seems promising. However, laser-assisted fabrication of dual-scale structures, i.e., ripples relies on stochastic processes deriving from laser-matter interaction, limiting the control over the structures' topography. In this paper, we report on laser fabrication of cell-repellent dual-scale 3D structures with fully reproducible and high spatial accuracy topographies. Structures were designed as micrometric "mushrooms" decorated with fingerprint-like nanometric features with heights and periodicities close to those of the calamistrum, i.e., 200-300 nm. They were fabricated by Laser Direct Writing via Two-Photon Polymerization of IP-Dip photoresist. Design and laser writing parameters were optimized for conferring cell-repellent properties to the structures, even for high cellular densities in the culture medium. The structures were most efficient in repelling the cells when the fingerprint-like features had periodicities and heights of ≅200 nm, fairly close to the repellent surfaces of the calamistrum. Laser power was the most important parameter for the optimization protocol.

Fascin-1 and its role as a serological marker in prostate cancer: a prospective case-control study.

AIM: This study aims to investigate any modification of serological FSCN1 in prostate cancer patients compared with patients without neoplasia. MATERIAL & METHODS: Clinical data and blood specimens from patients with and without prostate cancer were obtained. A quantitative sandwich ELISA method was used to determine serological values of FSCN1. RESULTS: Although serum values of FSCN1 were dissimilar in the two cohorts of patients (6.90 vs 7.33 ng/ml), the difference was not statistically significant (p = 0.20). Serum values of FSCN1 stratified for Gleason score groups were not significantly distinguishable (p = 0.65). A negative correlation (rho = -0.331; p = 0.009) was reported between FSCN1 and age. CONCLUSION: Further studies are required to evaluate a possible diagnostic role of FSCN1 in prostate cancer.

Laser Direct Writing via Two-Photon Polymerization of 3D Hierarchical Structures with Cells-Antiadhesive Properties.

We report the design and fabrication by laser direct writing via two photons polymerization of innovative hierarchical structures with cell-repellency capability. The structures were designed in the shape of "mushrooms", consisting of an underside (mushroom's leg) acting as a support structure and a top side (mushroom's hat) decorated with micro- and nanostructures. A ripple-like pattern was created on top of the mushrooms, over length scales ranging from several µm (microstructured mushroom-like pillars, MMP) to tens of nm (nanostructured mushroom-like pillars, NMP). The MMP and NMP structures were hydrophobic, with contact angles of (127 ± 2)° and (128 ± 4)°, respectively, whereas flat polymer surfaces were hydrophilic, with a contact angle of (43 ± 1)°. The cell attachment on NMP structures was reduced by 55% as compared to the controls, whereas for the MMP, a reduction of only 21% was observed. Moreover, the MMP structures preserved the native spindle-like with phyllopodia cellular shape, whereas the cells from NMP structures showed a round shape and absence of phyllopodia. Overall, the NMP structures were more effective in impeding the cellular attachment and affected the cell shape to a greater extent than the MMP structures. The influence of the wettability on cell adhesion and shape was less important, the cellular behavior being mainly governed by structures' topography.

Heterogeneity of Antibiotics Multidrug-Resistance Profile of Uropathogens in Romanian Population.

Urinary tract infections (UTIs) are a leading cause of morbidity for both males and females. The overconsumption of antibiotics in general medicine, veterinary, or agriculture has led to a spike in drug-resistant microorganisms; obtaining standardized results is imposed by standard definitions for various categories of drug-resistant bacteria-such as multiple-drug resistant (MDR), extensive drug-resistant (XDR), and pan drug-resistant (PDR). This retrospective study conducted in three university teaching hospitals in Romania has analyzed urine probes from 15,231 patients, of which 698 (4.58%) presented multidrug-resistant strains. Escherichia coli was the leading uropathogen 283 (40.54%), presenting the highest resistance to quinolones (R = 72.08%) and penicillin (R = 66.78%) with the most important patterns of resistance for penicillin, sulfonamides, and quinolones (12.01%) and aminoglycosides, aztreonam, cephalosporins, and quinolones (9.89%). Klebsiella spp. followed-260 (37.24%) with the highest resistance to amoxicillin-clavulanate (R = 94.61%) and cephalosporins (R = 94.23%); the leading patterns were observed for aminoglycosides, aminopenicillins + ß-lactams inhibitor, sulfonamides, and cephalosporins (12.69%) and aminoglycosides, aztreonam, cephalosporins, quinolones (9.23%). The insufficient research of MDR strains on the Romanian population is promoting these findings as an important tool for any clinician treating MDR-UTIs.

Chemical hemorrhagic cystitis: Diagnostic and therapeutic pitfalls (Review).

Chemical cystitis (CC) is an inflammation of the bladder caused by various chemical agents ingested intentionally or accidentally. It is linked to chemotherapeutic agents such as cyclophosphamide, therapeutic agents for diverse diseases, and anesthetic agents consumed abusively for recreational effects such as ketamine, or can be linked to environmental and surrounding factors such as soaps, gels, spermicides, and dyes. CC is a pathology with an increasing incidence that is inadequately treated due to its infectious cystitis-like symptoms. The hemorrhagic form can have a rampant evolution. Treatment options of CC and its complications are under continuous research with no accepted standardized sequence. In many situations, the treatments are difficult to obtain, administer, and follow-up. In addition, the lack of experience of the physician may pose other obstacles in delivering treatment to the patient. In conclusion, CC is a disease with an increasing incidence, challenging to diagnose, which is frequently mistreated, and has multiple treatment modalities that still require standardization in administration and sequencing.

Collagen/Chitosan Functionalization of Complex 3D Structures Fabricated by Laser Direct Writing via Two-Photon Polymerization for Enhanced Osteogenesis.

The fabrication of 3D microstructures is under continuous development for engineering bone substitutes. Collagen/chitosan (Col/CT) blends emerge as biomaterials that meet the mechanical and biological requirements associated with bone tissue. In this work, we optimize the osteogenic effect of 3D microstructures by their functionalization with Col/CT blends with different blending ratios. The structures were fabricated by laser direct writing via two-photons polymerization of IP-L780 photopolymer. They comprised of hexagonal and ellipsoidal units 80 µm in length, 40 µm in width and 14 µm height, separated by 20 µm pillars. Structures' functionalization was achieved via dip coating in Col/CT blends with specific blending ratios. The osteogenic role of Col/CT functionalization of the 3D structures was confirmed by biological assays concerning the expression of alkaline phosphatase (ALP) and osteocalcin secretion as osteogenic markers and Alizarin Red (AR) as dye for mineral deposits in osteoblast-like cells seeded on the structures. The structures having ellipsoidal units showed the best results, but the trends were similar for both ellipsoidal and hexagonal units. The strongest osteogenic effect was obtained for Col/CT blending ratio of 20/80, as demonstrated by the highest ALP activity, osteocalcin secretion and AR staining intensity in the seeded cells compared to all the other samples.

Spectrum and Antibiotic Resistance of Uropathogens in Romanian Females.

Urinary tract infections (UTIs) in women represent a common bacteriological finding, with negligible recent and consistent research on antimicrobial resistance (AMR) in the female population. We designed a retrospective study to observe the incidence of frequent uropathogens and their resistance rates to common antibiotics. We elaborated multicenter research in three different teaching hospitals in Romania, analyzing 13,081 urine samples, of which 1588 met the criteria of inclusion. Escherichia coli (58.37%) was the most frequent Gram-negative uropathogen, presenting high resistance rates to levofloxacin (R = 29.66%), amoxicillin-clavulanic ac. (R = 14.13%), and ceftazidime (R = 6.68%). We found good sensitivity to imipenem and meropenem (both 98.16%), amikacin (S = 96.0%), and fosfomycin (S = 90.39%). The second most prevalent uropathogen was Klebsiella (16.93%), with the highest resistance quota to amoxicillin-clavulanic ac. (R = 28.62%), levofloxacin and nitrofurantoin (both R = 15.61%), and ceftazidime (R = 15.24%), and good sensitivity to imipenem (S = 93.93%), meropenem (S = 91.91%), and amikacin (S = 88.47%). Enterococcus (13.35%) was the most encountered Gram-positive pathogen. It proved the highest resistance to levofloxacin (R = 32.07%), penicillin (R = 32.07%), and ampicillin (R = 14.62%) and good sensitivity to vancomycin (S = 91.98%), fosfomycin (S = 94.4%), and nitrofurantoin (S = 89.15%). Considering the lack of recent and consistent data on this topic, we find our survey a valuable starting research study in this area with high significance for an accurate clinical approach.

A Clinical Perspective on the Antimicrobial Resistance Spectrum of Uropathogens in a Romanian Male Population.

Considering urinary tract infections (UTIs), a significant public health problem with negligible recent research, especially on the male eastern European population, we aimed to determine the antimicrobial resistance rates of uropathogens for the most commonly used antibiotics in urological practice in our country. We conducted a multicenter retrospective study in two different teaching hospitals in Romania, analyzing urine samples from 7719 patients to determine the frequency of incriminating pathogens and their resistance to different antibiotics, in a comparative approach. We determined Escherichia coli (35.98%) to be the most common pathogen with the highest sensitivity to amikacin (S = 91.72%), meropenem (S = 97.17%) and fosfomycin (S = 86.60%) and important resistance to amoxicillin-clavulanic ac. (R = 28.03%) and levofloxacin (R = 37.69%), followed by Klebsiella spp. (22.98%) with the highest sensitivity to amikacin (S = 78.04%) and meropenem (S = 81.35%) and important resistance to amoxicillin-clavulanic ac. (R = 65.58%) and levofloxacin (R = 45.36%); the most frequent Gram-positive pathogen was Enterococcus spp. (19.73%) with the highest sensitivity for vancomycin (S = 93.75%) and fosfomycin (S = 87.5%) and considerable resistance to penicillin (R = 33.52%) and levofloxacin (R = 42.04%). The findings are an important tool in managing UTIs and should be acknowledged as reference research not only for clinicians from Romania but for all physicians treating male UTIs.

State of the Art in Fertility Preservation for Female Patients Prior to Oncologic Therapies.

Quality of life improvement stands as one of the main goals of the medical sciences. Increasing cancer survival rates associated with better early detection and extended therapeutic options led to the specific modeling of patients' choices, comprising aspects of reproductive life that correlated with the evolution of modern society, and requires better assessment. Of these, fertility preservation and ovarian function conservation for pre-menopause female oncologic patients pose a contemporary challenge due to procreation age advance in evolved societies and to the growing expectations regarding cancer treatment. Progress made in cell and tissue-freezing technologies brought hope and shed new light on the onco-fertility field. Additionally, crossing roads with general fertility and senescence studies proved highly beneficial due to the enlarged scope and better synergies and funding. We here strive to bring attention to this domain of care and to sensitize all medical specialties towards a more cohesive approach and to better communication among caregivers and patients.

3D Superparamagnetic Scaffolds for Bone Mineralization under Static Magnetic Field Stimulation.

We reported on three-dimensional (3D) superparamagnetic scaffolds that enhanced the mineralization of magnetic nanoparticle-free osteoblast cells. The scaffolds were fabricated with submicronic resolution by laser direct writing via two photons polymerization of Ormocore/magnetic nanoparticles (MNPs) composites and possessed complex and reproducible architectures. MNPs with a diameter of 4.9 ± 1.5 nm and saturation magnetization of 30 emu/g were added to Ormocore, in concentrations of 0, 2 and 4 mg/mL. The homogenous distribution and the concentration of the MNPs from the unpolymerized Ormocore/MNPs composite were preserved after the photopolymerization process. The MNPs in the scaffolds retained their superparamagnetic behavior. The specific magnetizations of the scaffolds with 2 and 4 mg/mL MNPs concentrations were of 14 emu/g and 17 emu/g, respectively. The MNPs reduced the shrinkage of the structures from 80.2 ± 5.3% for scaffolds without MNPs to 20.7 ± 4.7% for scaffolds with 4 mg/mL MNPs. Osteoblast cells seeded on scaffolds exposed to static magnetic field of 1.3 T deformed the regular architecture of the scaffolds and evoked faster mineralization in comparison to unstimulated samples. Scaffolds deformation and extracellular matrix mineralization under static magnetic field (SMF) exposure increased with increasing MNPs concentration. The results are discussed in the frame of gradient magnetic fields of ~3 × 10-4 T/m generated by MNPs over the cells bodies.

High Repetition Rate UV versus VIS Picosecond Laser Fabrication of 3D Microfluidic Channels Embedded in Photosensitive Glass.

Glass is an alternative solution to polymer for the fabrication of three-dimensional (3D) microfluidic biochips. Femtosecond (fs) lasers are nowadays the most promising tools for transparent glass processing. Specifically, the multiphoton process induced by fs pulses enables fabrication of embedded 3D channels with high precision. The subtractive fabrication process creating 3D hollow structures in glass, known as fs laser-assisted etching (FLAE), is based on selective removal of the laser-modified regions by successive chemical etching in diluted hydrofluoric acid solutions. In this work we demonstrate the possibility to generate embedded hollow channels in photosensitive Foturan glass volume by high repetition rate picosecond (ps) laser-assisted etching (PLAE). In particular, the influence of the critical irradiation doses and etching rates are discussed in comparison of two different wavelengths of ultraviolet (355 nm) and visible (532 nm) ranges. Fast and controlled fabrication of a basic structure composed of an embedded micro-channel connected with two open reservoirs, commonly used in the biochip design, are achieved inside glass. Distinct advantages such as good aspect-ratio, reduced processing time for large areas, and lower fabrication cost are evidenced.

Comparison of 10-year overall survival between patients with G1 and G2 grade Ta bladder tumors.

To compare long-term overall survival (OS) in patients with G1 and G2 grade Ta bladder cancer after transurethral resection of bladder tumors (TURBTs). Secondary aim was to investigate clinical and pathologic prognostic factors for OS of Ta patients, except G3/high grade (HG).A total of 243 patients, retrospectively selected, with Ta nonmuscle invasive bladder cancer (NMIBC) underwent TURBT between January 2006 and December 2008 (median follow-up 109 months). Inclusion criteria were: Ta at first manifestation, G1 or G2 grade with no associated carcinoma in situ (CIS). Seventy-nine patients were excluded due to concomitant CIS (1), G3/HG tumors (47), and lost to follow-up (31). Ethical approval was obtained from the Ethical Committee of the Mures County Hospital. Statistical analysis was performed using STATA 11.0.Following inclusion criteria, 164 patients with primary G1 or G2 Ta tumors, were enrolled. Recurrence was observed in 26 (15.8%) and progression in 5 (3%) patients. Ten-year survival in G1 patients was 67.8% (CI 54.3-78.1) and in G2 patients 59% (CI 49-67.3) (P = .31). Univariable and multivariable logistic regression analysis underlined that advanced age at diagnosis (hazard ratio [HR] 1.10) and no Bacillus Calmette-Guerin (BCG) treatment (HR 0.24 and 0.29) were independent predictors for death at 10 years after diagnosis.Long-term analysis confirms that patients with well differentiated (G1) and moderately well differentiated (G2) Ta tumors have similar OS. A longer OS was even reported in those who underwent BCG adjuvant therapy.

Laser fabrication of diffractive optical elements based on detour-phase computer-generated holograms for two-dimensional Airy beams.

We have designed, fabricated, and tested an amplitude diffractive optical element for generation of two-dimensional (2D) Airy beams. The design is based on a detour-phase computer-generated hologram. Using laser ablation of metallic films, we obtained a 2 mm×2 mm diffractive optical element with a pixel of 5 µm×5 µm and demonstrated a fast, cheap, and reliable fabrication process. This device can modulate 2D Airy beams or it can be used as a UV lithography mask to fabricate a series of phase holograms for higher energy efficiency. Tests according to the premise and an analysis of the transverse profile and propagation are presented.

3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis.

We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20-40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

Laser-direct writing by two-photon polymerization of 3D honeycomb-like structures for bone regeneration.

A major limitation of existing 3D implantable structures for bone tissue engineering is that most of the cells rapidly attach on the outer edges of the structure, restricting the cells penetration into the inner parts and causing the formation of a necrotic core. Furthermore, these structures generally possess a random spatial arrangement and do not preserve the isotropy on the whole volume. Here, we report on the fabrication and testing of an innovative 3D hierarchical, honeycomb-like structure (HS), with reproducible and isotropic arhitecture, that allows in 'volume' migration of osteoblasts. In particular, we demonstrate the possibility to control the 3D spatial cells growth inside these complex architectures by adjusting the free spaces inside the structures. The structures were made of vertical microtubes arranged in a mulitlayered configuration, fabricated via laser direct writing by two photons polymerization of the IP-L780 photopolymer. In vitro tests performed in MG-63 osteoblast-like cells demonstrated that the cells migration inside the 3D structures is conducted by the separation space between the microtubes layers. Specifically, for layers separation between 2 and 10 µm, the cells gradually penetrated between the microtubes. Furthermore, these structures induced the strongest cells osteogenic differentiation and mineralization, with ALP activity 1.5 times stronger, amount of calcified minerals 1.3 times higher and osteocalcin secretion increased by 2.3 times compared to the other structures. On the opposite, for layers separation less than 2 µm and above 10 µm, the cells were not able to make interconnections and exhibited poor mineralization ability.

Molecular symbiosis of CHOP and C/EBP beta isoform LIP contributes to endoplasmic reticulum stress-induced apoptosis.

Induction of the transcription factor CHOP (CCAAT-binding homologous protein; GADD 153) is a critical cellular response for the transcriptional control of endoplasmic reticulum (ER) stress-induced apoptosis. Upon nuclear translocation, CHOP upregulates the transcription of proapoptotic factors and downregulates antiapoptotic genes. Transcriptional activation by CHOP involves heterodimerization with other members of the basic leucine zipper transcription factor (bZIP) family. We show that the bZIP protein C/EBP beta isoform LIP is required for nuclear translocation of CHOP during ER stress. In early ER stress, LIP undergoes proteasomal degradation in the cytoplasmic compartment. During later ER stress, LIP binds CHOP in both cytoplasmic and nuclear compartments and contributes to its nuclear import. By using CHOP-deficient cells and transfections of LIP-expressing vectors in C/EBP beta(-/-) mouse embryonic fibroblasts (MEFs), we show that the LIP-CHOP interaction has a stabilizing role for LIP. At the same time, CHOP uses LIP as a vehicle for nuclear import. LIP-expressing C/EBP beta(-/-) MEFs showed enhanced ER stress-induced apoptosis compared to C/EBP beta-null cells, a finding in agreement with the decreased levels of Bcl-2, a known transcriptional control target of CHOP. In view of the positive effect of CHOP-LIP interaction in mediating their proapoptotic functions, we propose this functional cooperativity as molecular symbiosis between proteins.