Polysaccharide-based layer-by-layer nanoarchitectonics with sulfated chitosan for tuning anti-thrombogenic properties.

The development of blood-interacting surfaces is critical to fabricate biomaterials for medical use, such as prostheses, implants, biosensors, and membranes. For instance, thrombosis is one of the leading clinical problems when polymer-based materials interact with blood. To overcome this limitation is necessary to develop strategies that limit platelets adhesion and activation. In this work, hyaluronan (HA)/chitosan (Chi) based-films, recently reported in the literature as platforms for tumor cell capture, were developed and, subsequently, functionalized with sulfated chitosan (ChiS) using a layer-by-layer technique. ChiS, when compared to native Chi, presents the unique abilities to confer anti-thrombogenic properties, to reduce protein adsorption, and also to limit calcification. Film physicochemical characterization was carried out using FTIR and XPS for chemical composition assessment, AFM for the surface morphology, and contact angle for hydrophilicity evaluation. The deposition of ChiS monolayer promoted a decrease in both roughness and hydrophilicity of the HA/Chi films. In addition, the appearance of sulfur in the chemical composition of ChiS-functionalized films confirmed the film modification. Biological assay indicated that the incorporation of sulfated groups limited platelet adhesion, mainly because a significant reduction of platelets adhesion to ChiS-functionalized films was observed compared to HA/Chi films. On balance, this work provides a new insight for the development of novel antithrombogenic biomaterials, opening up new possibilities for devising blood-interaction surfaces.

Controlling antimicrobial activity and drug loading capacity of chitosan-based layer-by-layer films.

We report on layer-by-layer (LbL) films of chitosans (CHI) and hyaluronic acid (HA) whose properties could be controlled by employing chitosans with different degrees of deacetylation (DD¯ ≈ 85%; 65%; 40%) and high average molecular weight (ca. 106 g/mol). In spite of their high molecular weight, these chitosans are soluble within a wide pH range, including physiological pH. HA/CHI LbL films produced from polymer solutions at pH 4.5 were thinner, smoother, more hydrophilic than those prepared at pH 7.2. This is attributed to the more extended conformation adopted by chitosan due to its very high charge density at low pH, favoring a compact chain packing during the film formation and resulting in lower film thickness and roughness. The smoother HA/CHI LbL films obtained at pH 4.5 were effective against Escherichia coli, while the thicker, rougher LbL films fabricated at pH 7.2 could be used in the controlled released of Rose Bengal dye. In summary, the tuning of only two parameters, i.e. solution pH and DD¯ of chitosans, provides access to a library of HA/CHI LbL films for tailored, diversified applications.

Computational Fluid Dynamics Using a Porous Media Setting Predicts Outcome after Flow-Diverter Treatment.

BACKGROUND AND PURPOSE: Knowledge about predictors of the outcome of flow-diverter treatment is limited. The aim of this study was to predict the angiographic occlusion status after flow-diverter treatment with computational fluid dynamics using porous media modeling for decision-making in the treatment of large wide-neck aneurysms. MATERIALS AND METHODS: A total of 27 patients treated with flow-diverter stents were retrospectively analyzed through computational fluid dynamics using pretreatment patient-specific 3D rotational angiography. These patients were classified into no-filling and contrast-filling groups based on the O'Kelly-Marotta scale. The patient characteristics, morphologic variables, and hemodynamic parameters were evaluated for understanding the outcomes of the flow-diverter treatment. RESULTS: The patient characteristics and morphologic variables were similar between the 2 groups. Flow velocity, wall shear stress, shear rate, modified aneurysmal inflow rate coefficient, and residual flow volume were significantly lower in the no-filling group. A novel parameter, called the normalized residual flow volume, was developed and defined as the residual flow volume normalized by the dome volume. The receiver operating characteristic curve analyses demonstrated that the normalized residual flow volume with an average flow velocity of ≥8.0 cm/s in the aneurysmal dome was the most effective in predicting the flow-diverter treatment outcomes. CONCLUSIONS: It was established in this study that the hemodynamic parameters could predict the angiographic occlusion status after flow-diverter treatment.

Engineering the surface of prostate tumor cells and hyaluronan/chitosan multilayer films to modulate cell-substrate adhesion properties.

This paper explores different film assembly conditions of the polyelectrolyte solutions of hyaluronan (HA) and chitosan (CHI), as well as both substrate and cell surface modifications, to investigate PC3 cells adhesion properties. UV-Visible, AFM-IR and Zeta potential techniques indicate that the solution ionic strength is a relevant parameter to modulate the free carboxylic groups of HA on the film surface. In addition, capacitive coupling measurements suggest that assembly conditions that favor surface charge mobility inhibit cell adhesion due to polymer rearrangements that support non-specific electrostatic interactions of positively charged CHI residues and the negatively charged cell moieties, rather than specific CD44-hyaluronan interactions. Moreover, the PC3 cells treatment with hyaluronidase and anti-CD44 antibody also highlighted the importance of CD44 binding site availability on the tumor cell adhesion properties. Finally, the conjugation of wheat germ agglutinin on the film surface proved to be a suitable strategy to boost the PC3 cell adhesion properties. Our results reveal the remarkable capacity of HA/CHI films to modulate cell-substrate properties, which pave the road for the development of surfaces suitable for several applications based on biosensing.

Quantitative Analysis of Conebeam CT for Delineating Stents in Stent-Assisted Coil Embolization.

BACKGROUND AND PURPOSE: Innovative techniques and device-related advances have improved the outcomes of neuroendovascular treatment. 3D imaging has previously used 2 × 2 binning, but 1 × 1 binning has recently been made available. The aim of this study was to evaluate the quantitative ability of conebeam CT for stent delineation and to investigate its effectiveness in the clinical environment. MATERIALS AND METHODS: Four acquisition groups of 3D MIP images acquired using conebeam CT with varying conditions (acquisition time, 10 or 20 seconds and binning, 1 × 1 or 2 × 2) were compared. Two methods of analysis were performed, a phantom study and an analysis of 28 randomly selected patients. The phantom study assessed the contrast-to-noise ratio and full width at half maximum values in conebeam CT images of intracranial stent struts. In the clinical subjects, we assessed contrast-to-noise ratio, full width at half maximum, and dose-area product. RESULTS: In the phantom study, the contrast-to-noise ratio was not considerably different between 10- and 20-second acquisition times at equivalent binning settings. Additionally, the contrast-to-noise ratio at equivalent acquisition times did not differ considerably by binning setting. For the full width at half maximum results, equivalent acquisition times differed significantly by binning setting. In the clinical analyses, the 10-second/1 × 1 group (versus 20 second/2 × 2) showed a higher contrast-to-noise ratio (P < .05) and a dose-area product reduced by approximately 70% (P < .05), but the difference in full width at half maximum was not significant (P = .20). CONCLUSIONS: For stent-assisted coil embolization, quantitative assessment of conebeam CT showed that 10 second/1 × 1 was equivalent to 20 second/2 × 2 for imaging deployed intracranial stents. Furthermore, the 10-second/1 × 1 settings resulted in a much smaller DAP.

Antibacterial and non-cytotoxic ultra-thin polyethylenimine film.

In recent years, a common strategy, to obtain more uniform and controlled synthesis of polyelectrolytes multilayers (PEMs), relies on a previous polyethylenimine (PEI) coating of the substrate surface. PEI is a synthetic cationic polymer which provides a positive charge distribution on the materials to be covered with PEMs. Despite being an important step, this pre-layer deposition is frequently overlooked and no comprehensive characterizations or deep discussions are reported in literature. In that sense, this work reports on the synthesis of a typical PEI film that works as a precursor for PEMs, and its detailed physicochemical characterization. As many PEMs are produced for antibacterial and biomedical applications, the cytotoxicity of the film was also tested using fibroblasts, and its antibacterial activity was studied using Staphylococcus aureus and Pseudomonas aeruginosa. Our results present the formation of an ultra-thin film of PEI with a thickness around 3.5nm, and with a significant percent of NH3+ (35% of the total amount of N) in its chemical structure; NH3+ is a key chemical group because it is considered an important bacterial killer agent. The film was stable and did not present important cytotoxic effect for fibroblasts up to 7days, contrary to other reports. Finally, the PEI film showed high antibacterial activity against the S. aureus strain: reductions in cell density were higher than 95% up to 24h.

How often and when Fisher syndrome is overlapped by Guillain-Barré syndrome or Bickerstaff brainstem encephalitis?

BACKGROUND AND PURPOSE: Fisher syndrome (FS) may overlap with Guillain-Barré syndrome (GBS), in particular the pharyngeal-cervical-brachial variant form (PCB-GBS), or Bickerstaff brainstem encephalitis (BBE). Our aim was to elucidate the frequency of this overlap and the patterns of clinical progression in patients with FS. METHODS: Sixty consecutive patients with FS were studied. FS/PCB-GBS was diagnosed when the patients developed pharyngeal, cervical and/or brachial weakness. Patients with flaccid tetraparesis were diagnosed as having FS/conventional GBS. FS/BBE was defined as the development of consciousness disturbances. RESULTS: All 60 patients initially developed the FS clinical triad alone (pure FS). Of these, 30 (50%) patients had pure FS throughout their course, whereas the remaining 50% of patients showed an overlap: PCB-GBS in 14 (23%) patients, conventional GBS in nine (15%) patients and BBE in seven (12%) patients. The median (range) durations from FS onset to progression to FS/PCB-GBS, FS/GBS or FS/BBE were 5 (1-7), 3 (1-4) and 3 (1-5) days, respectively. Patients with overlap syndromes more frequently received immune-modulating treatment, and the outcomes were generally favourable. The frequencies of positivity for anti-GQ1b, GT1a, GD1a, GD1b, GalNAc-GD1a and GM1 antibodies were not significantly different amongst the four groups. CONCLUSIONS: Of the patients with pure FS, 50% later developed an overlap with PCB-GBS, conventional GBS or BBE. The overlap occurred within 7 days of FS onset; thus, physicians should pay attention to the possible development of this overlap during the first week after FS onset.

Hyaluronan/chitosan nanofilms assembled layer-by-layer and their antibacterial effect: A study using Staphylococcus aureus and Pseudomonas aeruginosa.

In the last few years, chitosan-based coatings have been proposed as antibacterial surfaces for biomedical devices in order to prevent nosocomial infections. In that sense, this work reports the optimized synthesis of hyaluronan/chitosan (HA/CHI) nanofilms assembled layer-by-layer in order to maximize the antibacterial effect for two important human pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa. In this assembly, HA forms a soft, highly hydrated, and nontoxic film, whereas CHI shows the antimicrobial characteristics. Our HA/CHI nanofilm synthesis optimization was based on changing pH values of the biopolymer stem-solutions and the consequent variation of their ionization degree. Furthermore, the surface density of primary amino groups, which are related to the antibacterial effect, was also enhanced by increasing the number of HA/CHI bilayers. The antibacterial effect of HA/CHI nanofilms was evaluated by the spread plate counting method for both bacteria. These results were correlated with the morphology of nanofilms (characterized using SEM and AFM), as well as with their chemical properties studied by UV-vis, Kelvin Probe Force microscopy and XPS spectroscopy.

Vascular endothelial growth factor as a predictive marker for POEMS syndrome treatment response: retrospective cohort study.

OBJECTIVE: POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes) syndrome is a rare multisystem disease characterised by plasma cell dyscrasia and overproduction of vascular endothelial growth factor (VEGF). VEGF is assumed to be useful in monitoring disease activity, because VEGF levels usually decrease after treatment. However, there is no study to investigate whether the extent of decrease in VEGF correlates with clinical outcome. We tested the predictive efficacy of serum VEGF levels in POEMS syndrome. METHOD: This was an institutional review board approved retrospective observational cohort study of 20 patients with POEMS monitored regularly for more than 12 months (median follow-up, 87 months) after treatment onset using our prospectively accumulated database of POEMS from 1999 to 2015. Patients were treated by autologous peripheral blood stem cell transplantation or thalidomide administration. Serum VEGF was measured by ELISA. Outcome measures included clinical and laboratory findings and relapse-free survival. RESULTS: Serum VEGF levels decreased rapidly after treatment, and stabilised by 6 months post treatment. Patients with normalised serum VEGF levels (<1040 pg/mL) at 6 months showed prolonged relapse-free survival (HR=12.81, 95% CI 2.691 to 90.96; p=0.0001) and greater later clinical improvement. The rate of serum VEGF reduction over the first 6 months post treatment correlated with increased grip strength, serum albumin levels, and compound muscle action potential amplitudes at 12 months. CONCLUSIONS: Serum VEGF level at 6 months post treatment is a predicative biomarker for disease activity and prognosis in POEMS syndrome. Serum VEGF could be used as a surrogate endpoint for relapse-free survival or clinical or laboratory improvement of POEMS syndrome for clinical trials.

Ultrasonographic findings in cubital tunnel syndrome caused by a cubitus varus deformity.

We have retrospectively reviewed the clinical, preoperative ultrasonographic, and operative findings of eight patients who had tardy ulnar nerve palsy caused by a cubitus varus deformity. The mean varus angle on the affected side was 23°. With preoperative ultrasonography, the anterior dislocation of the ulnar nerve from the medial epicondyle was detected in dynamic scanning of short-axis images, and long-axis images revealed nerve compression and kinking in the proximal border of the flexor carpi ulnaris. Operative findings revealed compression of the ulnar nerve by a fibrous band, which was released in all cases. The cause of the tardy ulnar nerve palsy in this series of patients was constriction by a fibrous band and kinking in the proximal border of the flexor carpi ulnaris due to ulnar nerve dislocation from compression resulting from the forward movement of the medial head of the triceps brachii muscle.

Acetabular and proximal femoral alignment in patients with osteoarthritis of the dysplastic hip and its influence on the progression of disease.

In order to evaluate the relationship between acetabular and proximal femoral alignment in the initiation and evolution of osteoarthritis of the dysplastic hip, the acetabular and femoral angles were calculated geometrically from radiographs of 62 patients with pre-arthrosis and early osteoarthritis. The sum of the lateral opening angle of the acetabulum and the neck-shaft angle was defined as the lateral instability index (LII), and the sum of the anterior opening angle of the acetabulum and the anteversion angle of the femoral neck as the anterior instability index (AII). These two indices were compared in dysplastic and unaffected hips. A total of 22 unilateral hips with pre-arthrosis were followed for at least 15 years to determine whether the two indices were associated with the progression of osteoarthritis. The LII of the affected hips (197.4 (sd 6.0)) was significantly greater than that of the unaffected hips (1830 (sd 6.9)). A follow-up study of 22 hips with pre-arthrosis showed that only the LII was associated with progression of the disease, and an LII of 196 was the threshold value for this progression.

Combination joint-preserving surgery for forefoot deformity in patients with rheumatoid arthritis.

Proximal osteotomies for forefoot deformity in patients with rheumatoid arthritis have hitherto not been described. We evaluated combination joint-preserving surgery involving three different proximal osteotomies for such deformities. A total of 30 patients (39 feet) with a mean age of 55.6 years (45 to 67) underwent combined first tarsometatarsal fusion and distal realignment, shortening oblique osteotomies of the bases of the second to fourth metatarsals and a fifth-ray osteotomy. The mean follow-up was 36 months (24 to 68). The mean foot function index scores for pain, disability and activity subscales were 18, 23, and 16 respectively. The mean Japanese Society for Surgery of the Foot score improved significantly from 52.2 (41 to 68) to 89.6 (78 to 97). Post-operatively, 14 patients had forefoot stiffness, but had no disability. Most patients reported highly satisfactory walking ability. Residual deformity and callosities were absent. The mean hallux valgus and intermetatarsal angles decreased from 47.0 degrees (20 degrees to 67 degrees) to 9.0 degrees (2 degrees to 23 degrees) and from 14.1 degrees (9 degrees to 20 degrees) to 4.6 degrees (1 degree to 10 degrees), respectively. Four patients had further surgery including removal of hardware in three and a fifth-ray osteotomy in one. With good peri-operative medical management of rheumatoid arthritis, surgical repositioning of the metatarsophalangeal joint by metatarsal shortening and consequent relaxing of surrounding soft tissues can be successful. In early to intermediate stages of the disease, it can be performed in preference to joint-sacrificing procedures.

Gravitational unloading inhibits the regenerative potential of atrophied soleus muscle in mice.

AIM: The present study was performed to investigate the influence of unloading on the regeneration of atrophied and injured skeletal muscle. METHODS: Male mice (C57BL/6J), aged 8 weeks, were used. Cardiotoxin (CTX) was injected into soleus muscles bilaterally. Gravitational unloading on soleus muscle was performed by hind limb suspension for 2 weeks before and additionally 6 weeks after CTX injection in one group. Soleus muscles in the remaining groups were loaded keeping the mice in the cages and were dissected 14, 28 and 42 days after the injection. RESULTS: Recovery of the wet weight and protein content of soleus in the CTX-injected group was inhibited by unloading. Increase in satellite cell number, induced by CTX injection and loading, was also inhibited by unloading. Disappearance of infiltration of mononucleated cells into the necrotic area was also delayed. This phenomenon suggests that regeneration, which is indicated by the appearance of fibres with central nuclei, was inhibited by unloading. CONCLUSION: Results suggested that loading plays an important role in the activation of the regenerating potential of injured skeletal muscle.

Evaluation of batch adsorption of chromium ions on natural and crosslinked chitosan membranes.

Removal of chromium ions from aqueous solutions by using natural and crosslinked chitosan membranes was achieved using batch adsorption experiments. The effect of pH (6.0 and 2.0), concentration of chromium ions and crosslinking agents (glutaraldehyde: GLA and epichlorohydrin: ECH) on the adsorption properties of chitosan membranes was analyzed. The experimental equilibrium data was fitted to Langmuir and Freundlich models. Through the model curves, it was possible to observe that the amount of chromium ions adsorbed was significantly higher for crosslinked membranes compared to non-crosslinked chitosan. The maximum adsorbed amount was about 1400 mg g(-1) for ECH-crosslinked chitosan at pH 6.0. The adsorption rates for crosslinked chitosan membranes with glutaraldehyde and epichlorohydrin were similar for natural chitosan. Desorption study using NaCl (1 mol L(-1)) solution was performed on chitosan membranes, in order to recover chromium ions and to determine the suitable number of cycles for repeated use of these membranes without considerable decrease in their adsorption capacity. The desorption results showed that chromium ions could be more effectively removed at pH 2.0 than pH 6.0, mainly for ECH-crosslinked chitosan.

Positron emission tomography with 18F-FDG in osteoarthritic knee.

OBJECTIVES: To evaluate osteoarthritis (OA) of the knee using positron emission tomography (PET) with 2-(18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) as a tracer. MATERIALS AND METHODS: Fifteen patients with medial-type knee OA and three healthy subjects were enrolled in the study. After clinical examination and conventional radiography, (18)F-FDG PET and magnetic resonance imaging (MRI) were performed. (18)F-FDG uptake was quantified as a standardized uptake value (SUV) and the localization of (18)F-FDG uptake was identified using fusion images created with MRI scans. RESULTS: (18)F-FDG generally accumulated in periarticular lesions and was absent in the articular cartilage. SUVs of the whole knee were higher in OA than in controls, and those in the medial condyle were higher than in the lateral condyle in OA. Prominent (18)F-FDG uptake was found in the intercondylar notch in OA and extended along the posterior cruciate ligament (PCL) in some cases. Periosteophytic accumulation was found in one-half of cases with definite osteophytes. Accumulation was also found in subchondral lesions and bone marrow, which corresponded with bone edema diagnosed by MRI. No significant correlation was found between SUV and clinical manifestations. CONCLUSIONS: (18)F-FDG uptake was upregulated in OA and generally accumulated in periarticular lesions. Increased uptake was found in the intercondylar notch extending along the PCL, periosteophytic lesions, and bone marrow. These results provide in vivo pathognomonic insights into OA.

Change in the cross-sectional area of a patellar tendon graft after anterior cruciate ligament reconstruction.

The purpose of this study was to clarify the change in the cross-sectional area (CSA) of a patellar tendon graft after anterior cruciate ligament (ACL) reconstruction, and its relationship with postoperative knee laxity. Forty patients (25 men and 15 women) were included in this study. Intraoperative CSA measurements were performed with an instrumented areamicrometer, while a magnetic resonance imaging (MRI) evaluation was utilized for the assessment postoperatively. For intraoperative measurement, the average CSA of a 10-mm wide patellar tendon graft was 32.3 +/- 7.0 mm2, while the average CSA measured at follow-up (mean: 14.8 months) was 48.8 mm2, showing a significant mean increase ratio of 49.4%. This value corresponded to 115% of the native ACL. The average CSA measured in 30 patients at 6 months was 49.7 mm2, almost equal to the value at the final follow-up (49.8 mm2) in the same patient group. Among potentially influential factors, postoperative notch width (available space for the ACL graft) had significant correlation with the CSA of the graft at follow-up. Finally, both intra- and postoperative CSA values did not correlate with postoperative knee laxity, indicating that a bigger graft does not guarantee a better laxity.

Tryptase enhances release of vascular endothelial growth factor from human osteoarthritic chondrocytes.

OBJECTIVE: A contribution of mast cells and its mediators in the pathogenesis of arthritis has been postulated. We aimed to clarify the role of mast cell-derived serine protease tryptase and proteinase activated receptor (PAR)-2-mediated signaling in chondrocytes. METHODS: Human articular cartilage specimens were obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA) and with traumatic fracture without arthritis (PT; as controls) who underwent joint surgery. Isolated chondrocytes were cultured in vitro by monolayer, and confluent cells were incubated with recombinant human lung Beta tryptase or with a PAR-2 agonist peptide. The secreted level of vascular endothelial growth factor (VEGF) in culture supernatant was measured using commercially available ELISA kits, and expression of VEGF mRNA was analyzed using real-time PCR. RESULTS: The tryptase-stimulated chondrocytes from OA or RA, but not from PT patients, produced significantly higher amount of VEGF in their supernatants. The response was blocked by a G-protein receptor inhibitor pertussis toxin, however, was not reproduced by incubation of cells with the PAR-2 agonist, suggesting a presence of non-PAR-2 dependent signals for the VEGF induction. In addition, actinomycin D and cycloheximide did not exert significant inhibition, indicating a regulation of VEGF release by tryptase. CONCLUSION: The inflammatory mediator, mast cell-derived protease tryptase may modulate chondrocyte metabolism through induction of VEGF release.

High expression levels of nuclear factor kappaB, IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity.

BACKGROUND: It has been reported that the transcription factor nuclear factor kappaB (NF-kappaB) is involved in the growth, invasion, and antiapoptotic activity of cultured tumor cells. METHODS: The authors used immunohistochemistry to examine the expression of NF-kappaB and the signaling molecules leading to NF-kappaB activation in 36 untreated biopsy specimens from patients with squamous cell carcinoma (SCC) and in 15 specimens from patients with epithelial dysplasia of the oral cavity. RESULTS: Among the molecules examined, the p65 subunit of NF-kappaB (p65) and IkappaB kinase alpha (IKKalpha) were expressed highly in almost all SCC specimens examined, whereas the samples of normal squamous epithelia adjacent to tumors as well as epithelial dysplasia specimens were negative in for immunohistochemical staining. The invasiveness and metastasis of SCC seemed to correlate with the degree of staining degree in the molecules. Moreover, phosphorylated Akt kinase, which may be associated with antiapoptosis signaling of NF-kappaB, was detected in the same areas where IKKalpha existed in large amounts. CONCLUSIONS: The results suggest that high expression levels of p65 and IKKalpha contribute to malignant behavior and antiapoptotic activity in SCC of the oral squamous epithelium.

Determination of urinary pyrraline by solid-phase extraction and high performance liquid chromatography.

Pyrraline is one of the advanced glycation end products formed under non-enzymatic and non-oxidative conditions in vivo. In this study, we developed a novel method for determination of urinary pyrraline using solid-phase extraction as a pretreatment procedure prior to determination by high performance liquid chromatography (HPLC). The Oasis HLB solid-phase extraction cartridge was used for pretreatment of urine samples without hydrolysis. The chromatogram obtained clearly revealed the peak for urinary pyrraline owing to prior removal of interfering substances in urine samples. The recovery rate of pyrraline was 97.2+/-3.3% (n=6). The mean excretion level of urinary pyrraline in healthy control (20-77 years old, n = 30) was 1.42+/-0.65 micromol/mmol creatinine, and the daily variation in the excretion level was considered to be insignificant. We propose the above procedure as a simple, rapid, and accurate method for determination of pyrraline levels in urine.