Effect of peptide secondary structure on adsorption and adsorbed film properties on end-grafted polyethylene oxide layers

Poly-l-lysine (PLL), in α-helix or β-sheet configuration, was used as a model peptide for investigating the effect of secondary structures on adsorption events to poly(ethylene oxide) (PEO) modified surfaces formed using θ solvents. Circular dichroism results showed that the secondary structure of PLL persisted upon adsorption to Au and PEO modified Au surfaces. Quartz crystal microbalance with dissipation (QCM-D) was used to characterize the chemisorbed PEO layer in different solvents (θ and good solvents), as well as the sequential adsorption of PLL in different secondary structures (α-helix or β-sheet). QCM-D results suggest that chemisorption of PEO 750 and 2000 from θ solutions led to brushes 3.8 ± 0.1 and 4.5 ± 0.1 nm thick with layer viscosities of 9.2 ± 0.8 and 4.8 ± 0.5 cP, respectively. The average number of H₂O per ethylene oxides, while in θ solvent, was determined as ∼0.9 and ∼1.2 for the PEO 750 and 2000 layers, respectively. Upon immersion in good solvent (as used for PLL adsorption experiments), the number of H₂O per ethylene oxides increased to ∼1.5 and ∼2.0 for PEO 750 and 2000 films, respectively. PLL adsorbed masses for α-helix and β-sheet on Au sensors was 231 ± 5 and 1087 ± 14 ng cm⁻², with layer viscosities of 2.3 ± 0.1 and 1.2 ± 0.1 cP, respectively; suggesting that the α-helix layer was more rigid, despite a smaller adsorbed mass, than that of β-sheet layers. The PEO 750 layer reduced PLL adsorbed amounts to ∼10 and 12% of that on Au for α-helices and β-sheets respectively. The PLL adsorbed mass to PEO 2000 layers dropped to ∼12% and 4% of that on Au, for α-helix and β-sheet respectively. No significant differences existed for the viscosities of adsorbed α-helix and β-sheet PLL on PEO surfaces. These results provide new insights into the fundamental understanding of the effects of secondary structures of peptides and proteins on their surface adsorption.     

Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.     

Biofilm formation on bone grafts and bone graft substitutes: Comparison of different materials by a standard in vitro test and microcalorimetry

We analyzed the initial adhesion and biofilm formation of Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) on various bone grafts and bone graft substitutes under standardized in vitro conditions. In parallel, microcalorimetry was evaluated as a real-time microbiological assay in the investigation of biofilm formation and material science research. The materials β-tricalcium phosphate (β-TCP), processed human spongiosa (Tutoplast?) and poly(methyl methacrylate) (PMMA) were investigated and compared with polyethylene (PE). Bacterial counts (log₁₀ cfu per sample) were highest on β-TCP (S. aureus 7.67 ± 0.17; S. epidermidis 8.14 ± 0.05) while bacterial density (log₁₀ cfu per surface) was highest on PMMA (S. aureus 6.12 ± 0.2, S. epidermidis 7.65 ± 0.13). Detection time for S. aureus biofilms was shorter for the porous materials (β-TCP and processed human spongiosa, p < 0.001) compared to the smooth materials (PMMA and PE), with no differences between β-TCP and processed human spongiosa (p > 0.05) or PMMA and PE (p > 0.05). In contrast, for S. epidermidis biofilms the detection time was different (p < 0.001) between all materials except between processed human spongiosa and PE (p > 0.05). The quantitative analysis by quantitative culture after washing and sonication of the material demonstrated the importance of monitoring factors like specific surface or porosity of the test materials. Isothermal microcalorimetry proved to be a suitable tool for an accurate, non-invasive and real-time microbiological assay, allowing the detection of bacterial biomass without removing the biofilm from the surface.     

Anatomic proximity of the peroneal nerve to the posterolateral corner of the knee determined by MR imaging

BackgroundThe pie crusting technique has been extensively used to release the lateral soft tissue in total knee arthroplasty. However, it may place the peroneal nerve at direct injury risk when performed in a valgus knee. The aim of this study was to determine the anatomic proximity of the peroneal nerve to the posterolateral corner of the knee.MethodsOne hundred knees were measured on axial MR images for the proximity of peroneal nerve to the closest edge of the inner surface of joint capsule or the posterolateral corner of proximal tibia at the level of the joint line and the level of the tibial cut respectively.ResultsThe distance between the peroneal nerve and the closest edge of the inner surface of joint capsule at the level of the joint line was 15.0 ± 2.6 mm (range, 8.5-22.3 mm), and the distance between the peroneal nerve and the posterolateral corner of proximal tibia was 14.0 ± 2.7 mm (range, 8.0-23.2 mm). These distances were correlated with the anteroposterior diameter of the soft tissue of the knee, but not correlated with the size of the tibia.ConclusionsThese results suggest that it is safe enough providing that the scalpel blade does not pierce more than 8 mm deep. However, patients with smaller legs are at greater risk of direct peroneal nerve injury.     

Serum vitamin D status is associated with the presence but not the severity of primary open angle glaucoma

ObjectivesVitamin D is involved in visual health and function. Our objective was to determine whether age-related vitamin D insufficiency was associated with the presence and the severity of primary open angle glaucoma (POAG) in a case-control study of older adults.Study designCase-control study.Main outcome measures. One hundred fifty cases diagnosed with moderate-to-severe POAG (mean, 75.1 ± 8.5 years; 42.0% female) and 164 healthy controls (mean, 73.0 ± 7.9 years; 59.8% female) were included. POAG diagnosis was based on classical diagnostic criteria of optic nerve cupping and/or RNFL thinning, measured with optical coherence tomography. Severe POAG was defined as Humphrey visual field mean deviation (MD) worse than −12 dB. Vitamin D insufficiency was defined as serum 25OHD ≤ 75 nmol/L. Age, gender, mean arterial pressure, vitamin D supplementation, visual acuity, and intraocular pressure were used as potential confounders.ResultsPOAG cases had lower mean serum 25OHD concentration than controls (42.9 ± 25.7 nmol/L versus 49.4 ± 29.5 nmol/L, P = 0.039) and a greater prevalence of vitamin D insufficiency (90.7% versus 82.3%, P = 0.032). Increased mean serum 25OHD concentrations were associated with lower POAG frequency, even after adjustment for potential confounders (OR = 0.89 per 10 nmol/L of 25OHD, P = 0.045). Similarly, vitamin D insufficiency was associated with POAG (OR = 2.09, P = 0.034). Among POAG cases, no 25OHD difference was observed between moderate and severe POAG cases (respectively, 39.2 ± 23.3 nmol/L versus 45.1 ± 26.7 nmol/L, P = 0.188); and no between-group difference regarding the prevalence of vitamin D insufficiency (88.9% versus 94.0%, P = 0.313).ConclusionsDecreased serum 25OHD concentration was associated with POAG. There was no 25OHD difference between moderate and severe POAG.     

Peripheral tactile sensory perception of older adults improved using subsensory electrical noise stimulation

Loss of tactile sensory function is common with aging and can lead to numbness and difficulty with balance and gait. In previous work we found that subsensory electrical noise stimulation (SENS) applied to the tibial nerve improved tactile perception in the soles of the feet of healthy adults. In this work we aimed to determine if SENS remained effective in an older adult population with significant levels of sensory loss.Older adult subjects (N = 8, female = 4, aged 65–80) had SENS applied via surface electrodes placed proximally to the medial and lateral malleoli. Vibration perception thresholds (VPTs) were assessed in six conditions, two control conditions (no SENS) and four SENS conditions (zero mean ± 15 µA, 30 µA, 45 µA and 60 µA SD). VPT was assessed at three sites on the plantar aspect of the foot.Vibration perception was significantly improved in the presence of ± 30 µA SENS and by 16.2 ± 2.4% (mean ± s.e.m.) when optimised for each subject. The improvement in perception was similar across all VPT test sites.     

Synergistic effects of electrospun PLLA fiber dimension and pattern on neonatal mouse cerebellum C17.2 stem cells

Topographical features, including fiber dimensions and pattern, are important aspects in developing fibrous scaffolds for tissue engineering. In this study aligned poly(l-lactide) (PLLA) fibers with diameters of 307 ± 47, 500 ± 53, 679 ± 72 and 917 ± 84 nm and random fibers with diameters of 327 ± 40, 545 ± 54, 746 ± 82 and 1150 ± 109 nm were obtained by optimizing the electrospinning parameters. We cultured neonatal mouse cerebellum C17.2 cells on the PLLA fibers. These neural stem cells (NSCs) exhibited significantly different growth and differentiation depending upon fiber dimension and pattern. On aligned fibers cell viability and proliferation was best on 500 nm fibers, and reduced on smaller or larger fibers. However, on random fibers cell viability and proliferation was best with the smallest (350 nm) and largest (1150 nm) diameter fibers. Polarized and elongated cells were orientated along the fiber direction on the aligned fibers, with focal contacts bridging the cell body and aligned fibers. Cells of spindle and polygonal morphologies were randomly distributed on the random fibers, with no focal contacts observed. Moreover, longer neurites were obtained on the aligned fibers than random fibers within the same diameter range. Thus, the surface topographic morphologies of fibrous scaffolds, including fiber pattern, dimensions and mesh size, play roles in regulating the viability, proliferation and neurite outgrowth of NSCs. Nevertheless, our results indicated that aligned 500 nm fiber are most promising for fine tuning the design of a nerve scaffold.     

A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts

A major barrier to the development of a clinically useful small diameter tissue engineered vascular graft (TEVG) is the scaffold component. Scaffold requirements include matching the mechanical and structural properties with those of native vessels and optimizing the microenvironment to foster cell integration, adhesion and growth. We have developed a small diameter, bilayered, biodegradable, elastomeric scaffold based on a synthetic, biodegradable elastomer. The scaffold incorporates a highly porous inner layer, allowing cell integration and growth, and an external, fibrous reinforcing layer deposited by electrospinning. Scaffold morphology and mechanical properties were assessed, quantified and compared with those of native vessels. Scaffolds were then seeded with adult stem cells using a rotational vacuum seeding device to obtain a TEVG, cultured under dynamic conditions for 7 days and evaluated for cellularity. The scaffold showed firm integration of the two polymeric layers with no delamination. Mechanical properties were physiologically consistent, showing anisotropy, an elastic modulus (1.4 ± 0.4 MPa) and an ultimate tensile stress (8.3 ± 1.7 MPa) comparable with native vessels. The compliance and suture retention forces were 4.6 ± 0.5 × 10^?4 mmHg^?1 and 3.4 ± 0.3 N, respectively. Seeding resulted in a rapid, uniform, bulk integration of cells, with a seeding efficiency of 92 ± 1%. The scaffolds maintained a high level of cellular density throughout dynamic culture. This approach, combining artery-like mechanical properties and a rapid and efficient cellularization, might contribute to the future clinical translation of TEVGs.     

Engineering surfaces for site-specific vascular differentiation of mouse embryonic stem cells

Differentiation of stem and progenitor cells routinely relies on the application of soluble growth factors, an approach that enables temporal control of cell fate but enables no spatial control of the differentiation process. Angiogenic progenitor cells derived from mouse embryonic stem cells (ESCs) were differentiated here according to the pattern of immobilized vascular endothelial growth factor-A (VEGF). Mouse ESCs engineered to express green fluorescent protein (eGFP) under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ angiogenic progenitors were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF₁₆₅ was covalently immobilized onto collagen IV (ColIV) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF–ColIV was stamped as 100 μm wide lanes on top of the chitosan layer and the Flk1+ angiogenic progenitors were seeded for site-specific differentiation. Lanes stamped with only ColIV served as controls. The results presented here demonstrate that the cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53 ± 13% CD31 positive and 17 ± 2% smooth muscle actin positive), whereas surfaces without VEGF favored vascular smooth muscle-like cell differentiation (26 ± 17% CD31 positive and 38 ± 9% smooth muscle actin positive).     

Phagocytizing activity of PMN from severe trauma patients in different post-traumatic phases during the 10-days post-injury course

ObjectivePhagocytizing leukocytes (granulocytes and monocytes) play a fundamental role in immunological defense against pathogens and clearance of cellular debris after tissue injury due to trauma. According to the “two-hit hypothesis”, phagocytes become primed due to/after trauma. Subsequently, a secondary stimulus may lead to their exaggerated response. This immune dysfunction can result in serious infectious complications, also depending on trauma injury pattern. Here, we investigated the phagocytizing capacity of leukocytes, and its correlation to trauma injury pattern.Material/methodsPeripheral whole blood was taken daily from 29 severely injured trauma patients (TP, Injury Severity Score, ISS ≥ 28) for ten days (1–10) following admission to the emergency department (ED). Sixteen healthy volunteers served as controls (HV). Samples were incubated with opsonized Staphylococcus aureus labelled with pHrodo fluorescent reagent and the percentage of phagocytizing activity was assessed by flow cytometry. Abbreviated Injury Scales (AIS) ≥ 3 of head, chest and extremities were used for injury pattern analysis.ResultsOverall distribution of active phagocytes (out of 100% phagocytizing leukocytes) in TP included granulocytes with 28.6 ± 1.5% and monocytes with 59.3 ± 1.9% at ED, and was comparable to HV (31.5 ± 1.6% granulocytes and 60.1 ± 1.6% monocytes). The percentage of phagocytizing granulocytes increased significantly after D2 (39.1 ± 1.2%), while the percentage of phagocytizing monocytes (52.0 ± 1.2%, p < 0.05) decreased after D2. These changes persisted during the whole time course. Phagocytizing activity of granulocytes (27.9 ± 2.8%) and monocytes (55.2 ± 3.3%) was significantly decreased at ED compared to HV (42.4 ± 4.1% and 78.1 ± 3.1%, respectively). After D2 up to D10, phagocytizing activity was significantly enhanced in granulocytes. Phagocytizing activity of monocytes remained decreased on D1 and has risen continuously during the ten days time course to values comparable to HV. No significant differences in phagocytosis could be associated to certain injury pattern.ConclusionsOur data demonstrate that the increasing percentage of phagocytizing granulocytes may indicate their enhanced mobilization out of bone marrow persisting until post-injury day 10. Furthermore, an initially decreased phagocytizing activity of granulocytes is strongly increased in the 10-days post-injury course. The altered activity of phagocytes due to injury could not be linked to any trauma injury pattern, and emerged rather as a general characteristic of phagocytes after severe trauma.     

In vivo evaluation of femoral and tibial graft tunnel placement following all-inside arthroscopic tibial inlay reconstruction of the posterior cruciate ligament

BackgroundThe arthroscopic all-inside tibial inlay technique represents a novel procedure for posterior cruciate ligament (PCL) reconstruction. However, in vivo investigations that evaluate the accuracy of this technique regarding anatomic graft tunnel placement are few. The objective of this study was to analyse the femoral and tibial tunnel apertures using computed tomography (CT) and compare these findings to recommendations in the literature.MethodsCT scans were obtained in 45 patients following single-bundle PCL reconstruction. The centres of the tibial and femoral tunnel apertures were correlated to measurement grid systems used as a radiographic reference.ResultsThe centre of the femoral tunnel aperture was located at 42.9% ± 9.4% of the total intercondylar depth and at 12.9% ± 7.2% of the total intercondylar height. The angle α for the femoral tunnel position was measured at 64.2° ± 10.0°. The centre of the tibial tunnel aperture was found at 51.8% ± 4.1% of the total mediolateral diameter of the tibial plateau. The superoinferior distance of the tibial tunnel aperture to the joint line was 9.6 mm ± 4.4 mm on frontal and 9.3 mm ± 3.4 mm on sagittal 3D-CT scans. The distance of the tibial tunnel aperture to the former physis line averaged to 0.8 mm ± 3.4 mm. Comparison to the corresponding reference values revealed no statistically significant difference.ConclusionArthroscopic tibial inlay reconstruction is an efficient procedure for precise replication of the anatomical footprint of the PCL.Level of evidenceIV, prospective case series     

Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout

Acute toxicity of the fungicide, captan, to juvenile rainbow trout was evaluated under static-renewal test condition. Actual concentrations of captan ranged from 0.05 to 1.00 mg/L. The concentrations of captan that killed 50% of the rainbow trout (3.11 ± 0.8 g) within 24 (24 h; LC₅₀), 48, 72 and 96 h were 0.57 ± 0.09, 0.49 ± 0.10, 0.44 ± 0.11 and 0.38 ± 0.13 mg/L (95% confidence limits), respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to captan (≥0.65 mg/L). Hypertrophy, separation of epithelium from lamellae, lamellar fusion, and epithelial cell necrosis were observed on captan exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to fungicide had inflammation and necrosis in liver, trunk kidney and spleen. In order, the most affected organs were gill, trunk kidney and liver.     

Preparation and characterization of collagen-based ADSC-carrier sheets for cardiovascular application

The use of scaffolds composed of natural biodegradable matrices represents an attractive strategy to circumvent the lack of cell engraftment, a major limitation of stem cell therapy in cardiovascular diseases. Bovine-derived non-porous collagen scaffolds with different degrees of cross-linking (C0, C2, C5 and C10) were produced and tested for their mechanical behavior, in vitro biocompatibility with adipose-derived stem cells (ADSCs) and tissue adhesion and inflammatory reaction. Uniaxial tensile tests revealed an anisotropic behavior of collagen scaffolds (2 × 0.5 cm) and statistically significant differences in the mechanical behavior between cross-linked and non-cross-linked scaffolds (n = 5). In vitro, ADSCs adhered homogenously and showed a similar degree of proliferation on all four types of scaffolds (cells × 10³ cm^?2 at day 7: C0: 94.7 ± 37.1; C2: 91.7 ± 25.6; C5: 88.2 ± 6.8; C10: 72.8 ± 10.7; P = n.s.; n = 3). In order to test the in vivo biocompatibility, a chronic myocardial infarction model was performed in rats and 1.2 × 1.2 cm size collagen scaffolds implanted onto the heart 1 month post-infarction. Six animals per group were killed 2, 7 and 30 days after transplant. Complete and long-lasting adhesion to the heart was only observed with the non-cross-linked scaffolds with almost total degradation 1 month post-transplantation. After 7 and 30 days post-implantation, the degree of inflammation was significantly lower in the hearts treated with non-cross-linked scaffolds (day 7: C0: 10.2 ± 2.1%; C2: 16.3 ± 2.9%; C5: 15.9 ± 4.8%; C10: 17.4 ± 4.1%; P < 0.05 vs. C0; day 30: C0: 1.3 ± 1.3%; C2: 9.4 ± 3.0%; C5: 7.0 ± 2.1%; C10: 9.8 ± 2.5%; P < 0.01 vs. C0). In view of the results, the non-cross-linked scaffold (C0) was chosen as an ADSC-carrier sheet and tested in vivo. One week post-implantation, 25.3 ± 7.0% of the cells transplanted were detected in those animals receiving the cell-carrier sheet whereas no cells were found in animals receiving cells alone (n = 3 animals/group).We conclude that the biocompatibility and mechanical properties of the non-cross-linked collagen scaffolds make them a useful cell carrier that greatly favors tissue cell engraftment and may be exploited for cell transplantation in models of cardiac disease.     

A computer-aided diagnosis approach for emphysema recognition in chest radiography

The purpose of this work is twofold: (i) to develop a CAD system for the assessment of emphysema by digital chest radiography and (ii) to test it against CT imaging. The system is based on the analysis of the shape of lung silhouette as imaged in standard chest examination. Postero-anterior and lateral views are processed to extract the contours of the lung fields automatically. Subsequently, the shape of lung silhouettes is described by polyline approximation and the computed feature-set processed by a neural network to estimate the probability of emphysema.Images of radiographic studies from 225 patients were collected and properly annotated to build an experimental dataset named EMPH. Each patient had undergone a standard two-views chest radiography and CT for diagnostic purposes. In addition, the images (247) from JSRT dataset were used to evaluate lung segmentation in postero-anterior view.System performances were assessed by: (i) analyzing the quality of the automatic segmentation of the lung silhouette against manual tracing and (ii) measuring the capabilities of emphysema recognition. As to step i, on JSRT dataset, we obtained overlap percentage (Ω) 92.7 ± 3.3%, Dice Similarity Coefficient (DSC) 95.5 ± 3.7% and average contour distance (ACD) 1.73 ± 0.87 mm. On EMPH dataset we had Ω = 93.1 ± 2.9%, DSC = 96.1 ± 3.5% and ACD = 1.62 ± 0.92 mm, for the postero-anterior view, while we had Ω = 94.5 ± 4.6%, DSC = 91.0 ± 6.3% and ACD = 2.22 ± 0.86 mm, for the lateral view. As to step ii, accuracy of emphysema recognition was 95.4%, with sensitivity and specificity 94.5% and 96.1% respectively. According to experimental results our system allows reliable and inexpensive recognition of emphysema on digital chest radiography.     

Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery

We have previously reported the initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3490 ± 892 mmHg, n = 230) were higher than native saphenous vein (SV) (1599 ± 877 mmHg, n = 7), and not significantly different from native internal mammary artery (IMA) (3196 ± 1264 mmHg, n = 16). Suture retention strength for the TEBVs (152 ± 50 gmf) was also not significantly different than IMA (138 ± 50 gmf). Compliance for the TEBVs prior to implantation (3.4 ± 1.6%/100 mmHg) was lower than IMA (11.5 ± 3.9%/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8 ± 4.2%/100 mmHg, n = 5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A–V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels.     

Zinc-containing apatite layers on external fixation rods promoting cell activity

Zinc-containing apatite layers were successfully formed on commercially available anodically oxidized Ti external fixation rods using ZnCl₂-containing supersaturated calcium phosphate solutions. With an increase in concentration of ZnCl₂ in the supersaturated calcium phosphate solutions, the amounts of zinc that precipitated on the Ti external fixation rods increased (from 0 to 0.195 ± 0.020 μg cm^?2); meanwhile, the amounts of calcium and phosphorus that precipitated on the Ti external fixation rods decreased (from 11.2 ± 1.5 and 4.8 ± 0.5 μg cm^?2 to 2.9 ± 1.6 and 1.3 ± 0.9 μg cm^?2, respectively). The zinc-containing apatite layers precipitated on the Ti external fixation rods caused a significant increase in fibroblastic proliferation, osteoblastic proliferation and differentiation in vitro. The Ti external fixation rods coated with zinc-containing apatite layers are expected to be more effective in accelerating the tissue regeneration around the surgical site than those coated with an apatite layer.     

Synaptic degradation of cardiac autonomic nerves in streptozotocin-induced diabetic rats

Background: Cardiac autonomic neuropathy (CAN) is a common complication in type I diabetes mellitus (DM). Nevertheless, the relationship between functional and structural disturbances of cardiac autonomic nerves remains unclear. Methods and results: To clarify this relationship, we studied heart rate variability (HRV) and ultrastructural changes of cardiac autonomic nerves in streptozotocin (STZ)-induced DM in rats. STZ was injected (65 mg/kg intravenous) into the tail vein of male Wistar rats to destroy β cells in the pancreatic islets. After STZ injection, fasting blood sugar (FBS) increased from baseline values of 75 ± 3 mg/dl up to 328 ± 12 mg/dl within 1 week and it reached up to 353 ± 24 mg/dl within 17 weeks. HR in these rats was decreased within 20 days and low HR was maintained for the observation period. TP and HF power started decreasing 20 days after STZ injection, and this decrease progressed throughout the observation period. The L/H power ratio was decreased 80 days after STZ. Electron microscopic findings indicated a depletion of neurotransmitter vesicles and degradation of parasympathetic nerve endings but not of sympathetic ones in the SA node region of the heart in the early stages of DM. In the late stages of DM, the same region showed degradation of both sympathetic and parasympathetic nerve endings. Conclusion: Synaptic degradation in parasympathetic nerves immediately after the onset of DM, and in sympathetic nerves much later in the development of DM is consistent with functional derangements in cardiac autonomic nerve activities assessed by HRV analysis.     

Aortic elastic properties and cognitive function in elderly individuals: The Ikaria Study

BackgroundThe aim of this work was to evaluate the association between aortic elastic properties and cognitive function in elderly individuals, permanent inhabitants of Ikaria Island.MethodsIn 535 individuals (75 ± 6 years, 53% males) aortic distensibility (AoD) was non-invasively calculated from the aortic diameters measured with echocardiography and brachial artery pressure using the formula by Stefanadis et al.; cognitive status was evaluated using the Mini Mental State Examination (MMSE).Results88% of the elders had normal values of MMSE score (i.e., ≥24). Elders who achieved MMSE score ≥24 had higher values of AoD (1.90 ± 2.06 vs. 1.08 ± 1.42, p < 0.001), as well as were more physically active (85% vs. 69%, p = 0.05), had higher educational status (8.5 ± 2.8 years vs. 6 ± 2 years, p = 0.001), higher creatinine clearance levels (70 ± 21 vs. 63 ± 23, p = 0.05) and lower pulse pressure (PP) values (63 ± 16 vs. 68 ± 18, p = 0.06), as compared with those individuals with MMSE < 24. Logistic regression analysis showed that for every unit increase in AoD there was a 25% higher likelihood of having MMSE ≥ 24 (OR per 1000 × mmHg^?1 = 1.25, 95%CI 0.99–1.58), after adjustments for age, gender, current smoking, cardiovascular disease, creatinine clearance, hypertension, diabetes mellitus, obesity, physical activity status and education status. Furthermore having PP levels in the upper tertile (>70 mmHg), increases by 55% the likelihood of having MMSE < 24 (OR for above 70 mmHg = 0.45, 95%CI 0.22, 0.92), after the same adjustments were made.ConclusionArterial aging seems to affect cognitive function; a finding that states a novel research hypothesis about the pathophysiological mechanisms of mental functioning.     

Contribution of nitric oxide synthase (NOS) in blood–brain barrier disruption during acute focal cerebral ischemia in normal rat

Endogenous level of nitric oxide (NO) is increased in the brain following the stroke, and deactivation of NO synthase has been shown to attenuate its destructive actions in animal stroke models using middle cerebral artery occlusion (MCAO) procedures. However, little is known about the effects of NO in cerebral vascular integrity and edema during acute cerebral ischemia. Here we investigated whether NO plays any role in the progression of blood–brain barrier (BBB) disruption and edema formation in ischemia/reperfusion injury. Intraperitoneal administration of NO substrate l-arginine (300 mg/kg), or NOS inhibitor (l-NAME, 1 mg/kg), was done in normal rats at 20 min before a 60-min MCAO. Mean arterial blood pressures (MAP) and regional cerebral blood flow (rCBF) were continuously recorded during experiment. Neurological deficit score (NDS) was evaluated 12 h after termination of MCAO followed with evaluations of cerebral infarction volume (CIV), edema formation and cerebral vascular permeability (CVP), as determined by the Evans blue dye extravasations (EBE) technique. No significant changes were observed in the values of MAP and rCBF with l-arginine or l-NAME during ischemia or reperfusion periods. There was a 75–85% reduction in rCBF in during MCAO which returned back to its pre-occlusion level during reperfusion. Acute cerebral ischemia with or without l-arginine augmented NDS (4.00 ± 0.44 and 3.00 ± 0.30), in conjunction with increased CIV (518 ± 57 mm³ and 461 ± 65 mm³), provoked edema (3.09 ± 0.45% and 3.30 ± 0.49%), and elevated EBE (8.28 ± 2.04 μg/g and 5.09 ± 1.41 μg/g). Inhibition of NO production by l-NAME significantly improved NDS (1.50 ± 0.22), diminished CIV (248 ± 56 mm³), edema (1.18 ± 0.58%) and EBE (1.37 ± 0.12 μg/g). This study reconfirms the cerebroprotective properties of reduced tissue NO during acute ischemic stroke, and it also validates the deleterious actions of increased NOS activity on the disruption of cerebral microvascular integrity and edema formation of ischemia/reperfusion injuries in normal rat, without changing arterial blood pressure or blood flows to ischemic regions.     

Elevated neopterin levels in non-allergic asthma

Neopterin is synthesized by human monocyte-derived macrophages upon stimulation with interferon-γ (IFN-γ). Measurement of neopterin concentration is useful to monitor cell-mediated (Th1-type) immune activation.In this study, we aimed to analyze the behaviour of neopterin in long lasting asthma considering its role as a marker of the Th1 environment and to establish the distinction between patients belonging either to the allergic or the non-allergic population, particularly in the elderly where asthma is often under diagnosed. Therefore we evaluated allergic parameters such as skin prick tests, IgE and hemogram (eosinophils count), and we compared our findings with neopterin values found in an age-matched control population.A group of individuals older than 65 was selected. It included 64 asthmatic patients (mean age 72 ± 5 years) and 41 healthy individuals (mean age 79 ± 7 years). In our study population, 42 patients presented positive skin tests, mainly to house dust mites. All patients were clinically stable and presented an average percentage of predicted forced expiratory volume in the first second (FEV1) of 73.6 ± 25.3 and predicted median expiratory flow percentage (MEF50) of 38.8 ± 26.7.Blood cell counts showed statistically different mean values of eosinophils between allergic and non-allergic controls (5.42 ± 4.7% versus 2.8 ± 2.8%; p < 0.04). IgE values were increased in allergic asthmatic patients when compared with non-allergic asthmatic patients (493.2 ± 549.8 IU/ml versus 85.3 ± 194.4 IU/ml; p = 0.000).Allergic asthmatic patients presented mean neopterin levels similar to those found in the control group (2.4 ± 2.8 ng/ml versus 2.1 ± 1.9 ng/ml). In contrast, in non-allergic asthmatic patients these values were higher when compared with the control group (4.0 ± 4.7 ng/ml versus 2.1 ± 1.9 ng/ml). Neopterin levels were lower in allergic asthmatic patients when compared with non-allergic asthmatic patients (2.4 ± 2.8 ng/ml versus 4.0 ± 4.7 ng/ml).Within asthmatic patients, those with higher neopterin values (>2.1 ng/ml) presented lower mean IgE values (IgE ≤ 336.58 IU) than those with lower neopterin values (≤2.1 ng/ml) who presented mean IgE values of 402.70 IU.Our initial findings may lead to a better understanding of the immunoinflammatory pathways in asthma. Further studies will probably show that serum neopterin could became a useful marker for asthma classification including in elderly patients with long lasting disease.