Solvent system effects on the physical and mechanical properties of electrospun Poly(ε-caprolactone) scaffolds for in vitro lung models.

Mechanical properties are among the key considerations for the design and fabrication of complex tissue models and implants. In addition to the choice of material and the processing technique, the solvent system can significantly influence the mechanical properties of scaffolds. Poly(ε-caprolactone) (PCL) has been abundantly used to develop constructs, fibrous in particular, for pharmaceutical and biomedical research due to the flexibility offered by PCL-based fibrous matrices. The effect of solvent type on the morphological features of electrospun fibres has been extensively studied. Nevertheless, comprehensive studies on the impact of the solvent system on the mechanical properties of electrospun PCL fibres are lacking. This study elucidates the relationship between topographical, physical and mechanical properties of electrospun PCL fibrous meshes upon using various solvent systems. The results of the mechanical investigation highlight the significance of inter-fibre bonds on the mechanical properties of the bulk membranes and that the option of altering the solvent system composition could be considered for tuning the mechanical properties of the PCL scaffolds to serve specific biomedical application requirements. The applicability of the developed membranes as artificial ECM (Extracellular matrix) in the lung will then be investigated and compared to the commercial Polycarbonate (PC) membranes that are often used for in vitro lung models.

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation.

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.

Autologous bone graft harvesting: a review of grafts and surgical techniques.

Spinal fusion with or without instrumentation often requires the use of bone graft. Bone graft may be autogenous or exogenous. There are various forms of bone graft which may be acquired from numerous sites. Knowledge of fusion biology is imperative for understanding the benefits and limitations of these grafts. Equally as important is the knowledge of outcome measures, management of donor-site morbidity, and potential reconstruction. This review details the methods of obtaining bone graft and details the properties of each, as well as discusses observed outcomes, donor-site morbidities, and reconstruction techniques.

Dynamic response signatures of a scaled model platform for floating wind turbines in an ocean wave basin.

Understanding of dynamic behaviour of offshore wind floating substructures is extremely important in relation to design, operation, maintenance and management of floating wind farms. This paper presents assessment of nonlinear signatures of dynamic responses of a scaled tension-leg platform (TLP) in a wave tank exposed to different regular wave conditions and sea states characterized by the Bretschneider, the Pierson-Moskowitz and the JONSWAP spectra. Dynamic responses of the TLP were monitored at different locations using load cells, a camera-based motion recognition system and a laser Doppler vibrometer. The analysis of variability of the TLP responses and statistical quantification of their linearity or nonlinearity, as non-destructive means of structural monitoring from the output-only condition, remains a challenging problem. In this study, the delay vector variance (DVV) method is used to statistically study the degree of nonlinearity of measured response signals from a TLP. DVV is observed to create a marker estimating the degree to which a change in signal nonlinearity reflects real-time behaviour of the structure and also to establish the sensitivity of the instruments employed to these changes. The findings can be helpful in establishing monitoring strategies and control strategies for undesirable levels or types of dynamic response and can help to better estimate changes in system characteristics over the life cycle of the structure.

Polygodial: a contact active antifouling biocide.

Ongoing investigation of the candidate antifouling (AF) biocide polygodial (PG) has revealed that this compound may be contact active, whereby it can confer effect while remaining bound within a stable matrix. To test this hypothesis, the AF activity of PG-laced coatings was compared to that of seawater in which PG-laced coatings had been soaked. Four coating types spanning high to low affinity for PG were examined and AF activity was assessed based on inhibition of settlement and metamorphosis of larvae of three fouling organisms: Ciona savignyi Herdman, Mytilus galloprovincialis Lamarck and Spirobranchus caraniferus Gray. Direct exposure to the coatings had a significantly greater impact on larval metamorphosis than indirect exposure to seawater in which the coatings had been soaked. In particular, metamorphosis was almost completely inhibited by high-affinity coatings containing ≥ 200 ng of PG per replicate, while corresponding soaking waters had no detectable effect. These findings support the assertion that PG is contact active.

Investigation of a small-diameter decellularised artery as a potential scaffold for vascular tissue engineering; biomechanical evaluation and preliminary cell seeding.

The development of a small-diameter tissue engineered blood vessel (TEBV), with equivalent mechanical properties to the vessel being replaced, may provide a potential solution to the limitations associated with natural and synthetic bypass grafts of small-diameter vessels. This study presents the biomechanical properties of small-diameter (<4mm) porcine coronary arteries (PCA) and the corresponding natural matrix scaffold of the artery achieved through short-term decellularisation. Tubular segments, up to 50mm in length, of PCA were perfused with 0.1М sodium hydroxide (NaOH) for 3 to 12h to achieve the natural matrix scaffold. Uniaxial tensile, inflation and permeability tests were performed on non-decellularised and decellularised sections within 24h of slaughter to determine the alteration in mechanical properties as a result of decellularisation. A treatment time of 9h achieved decellularisation as all cell nuclei were appropriately disrupted and there was an absence of smooth muscle in the vascular wall. Uniaxial tensile and inflation tests confirmed the scaffold maintains its non-linear response, however a less stiff, more distensible low-load response and stiffer high-load response was found compared to non-decellularised sections. Vascular smooth muscle cells were successfully seeded to the lumen, abluminal side and lateral edges of decellularised sections and attachment and infiltration of the xenogeneic cells after 15 days confirmed the viability of the PCA scaffold as a suitable environment for cell growth and infiltration. An extended decellularisation treatment time increased the porosity whilst maintaining the mechanical integrity of the scaffold and this may optimise the repopulation of the scaffold. This study provides valuable information for the development of an optimum TEBV, while also establishing the potential of this natural matrix scaffold to be used as a graft or vascular tissue engineering scaffold.

[Sagittal balance and compensatory mechanism after segmental instrumentation for adolescent idiopathic scoliosis]. / Sagittale Balance und Kompensationsmechanismen nach operativer Versorgung bei idiopathischer Skoliose mittels segmentaler Pedikelschraubeninstrumentation.

PURPOSE: Previous studies have demonstrated good ability to improve the sagittal profile with the use of segmental instrumentation for adolescent idiopathic scoliosis (AIS) by analysing the sagittal vertical axis (SVA). However, several authors recommend analysing the spinopelvic relation in addition to the SVA. While compensatory mechanisms may neutralise a positive SVA, these mechanisms are energy consuming and may lead to unphysiological biomechanics, which can negatively influence the quality of life. The purpose of this radiographic analysis was to analyse global sagittal balance and to identify potential compensatory mechanisms after segmental instrumentation for AIS. METHODS: From a prospectively collected multicentre database, patients with segmental instrumentation for AIS and a minimum follow-up of 2 years were identified. An additional inclusion criterion was the ability to measure lumbar lordosis (LL), pelvic tilt (PT), pelvic incidence (PI), and SVA on each X-ray (preoperative, 1st erect, and the 2-year follow-up visits). LL was analysed in relation to PI in order to identify iatrogenic changes. Changes in LL were correlated to changes in PT and SVA. RESULTS: 91 patients were included. On the 1st erect X-ray, LL significantly decreased from pre-op but the SVA remained stable due to a significant increase of PT. At two years follow-up, PT recurred to preoperative values, accompanied by a significant increase of SVA despite a compensatory hyperlordosis below the instrumentation. The temporary increase of PT on the 1st erect was significantly correlated to an iatrogenic decrease of LL. An iatrogenic decrease of LL was significantly correlated to an increase of SVA on the 1st erect and at 2 years follow-up. CONCLUSION: An iatrogenic decrease of LL after segmental instrumentation for AIS is initially compensated for by pelvic retroversion and later by a hyperlordosis below the instrumentation.

[Non-operative treatment for severe forms of infantile idiopathic scoliosis]. / Therapieerfolg der konservativen Therapie bei schweren infantilen idiopathischen Skoliosen.

INTRODUCTION: Infantile idiopathic scoliosis (IIS) is a rare orthopaedic condition. Braces and casts are popular options in the treatment of IIS but there is a paucity of studies commenting on the outcome of non-operative treatment. The purpose of this study was to analyse failure and success after non-operative treatment for severe forms of IIS. METHODS: We retrospectively reviewed the data of all children who had been treated for IIS between 2003 and 2009 at a single institution. After calculating the failure and success rates, we additionally performed a risk factor analysis for patients who failed non-operative treatment. Chi (2) and T tests were used for statistical analysis with significance set at p < 0.05. RESULTS: 25 children with an average age of 11 months and an Cobb angle of 46 degrees at presentation were analysed. Seven (28 %) patients were considered as having failed non-operative treatment after an average follow-up of 28 months. The pretreatment Cobb angle was identified as single significant risk factor for failure (55 versus 42) while neither age, gender, nor RVAD seem to influence the outcome. In children who were considered as successfully treated, the Cobb angle decreased from 42 to 18 degrees. CONCLUSION: Non-operative treatment for IIS is successful in 3 out of 4 patients.

Vertebral body stapling as an alternative in the treatment of idiopathic scoliosis.

OBJECTIVE: Unilateral vertebral body stapling as a fusionless alternative to control curvature progression in patients with idiopathic scoliosis (IS). INDICATIONS: Skeletally immature patients (Risser 0 or 1) with IS measuring 20-45° and correction of the curvature <20° on side-bending X-rays. CONTRAINDICATIONS: Congenital scoliosis, curvature above T4 or below L4, thoracic kyphosis >40°. SURGICAL TECHNIQUE: Unilateral disc-sparing staples are placed at the convex side. A thoracoscopic approach can be used for thoracic curves and a mini-open retroperitoneal approach for lumbar curves. POSTOPERATIVE MANAGEMENT: Early ambulation on postoperative day 1 is encouraged. There are no absolute activity restrictions, and no bracing beyond 4 weeks is required. RESULTS: A total of 28 patients (4 males, 24 females; average age at operation 9.4 years) met all inclusion criteria and had a minimum 2-year follow-up (range 2-5.3 years). The success rate (improvement or stabilization of the curvature) was 86% for thoracic curves <35° and all lumbar curves meeting the indications.

A method to develop mock arteries suitable for cell seeding and in-vitro cell culture experiments.

Sylgard((R)) is a biocompatible elastomer which has been widely used in biomedical applications including in simulations of the mechanical response of soft tissues and mechanotransduction investigations. In this study the effect of fabrication parameters including base to curing agent ratio and curing time on the mechanical response of Sylgard((R)) was investigated and a novel fabrication technique for the production of mock arteries with highly uniform thickness, which is essential for mechanotransduction studies, is described. Finally a method for the surface treatment of Sylgard((R)) using sulphuric acid and fibronectin to enhance smooth muscle cell (SMC) adhesion is proposed and examined in vitro. Sylgard((R)) mock coronary arteries fabricated using the proposed technique exhibited a mechanical response close to arterial tissue with cell adhesion enhanced using the surface treatment techniques described.

Cell encapsulation within PVA-based hydrogels via freeze-thawing: a one-step scaffold formation and cell storage technique.

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA-based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue-engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA-gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze-thaw process can be utilized to prepare and store cell-laden hydrogels with adjustable mechanical properties.

Physically crosslinked composite hydrogels of PVA with natural macromolecules: structure, mechanical properties, and endothelial cell compatibility.

Polyvinyl alcohol (PVA) hydrogels have been considered potentially suitable for applications as engineered blood vessels because of their structure and mechanical properties. However, PVA's hydrophilicity hinders its capacity to act as a substrate for cell attachment. As a remedy, PVA was blended with chitosan, gelatin, or starch, and hydrogels were formed by subjecting the solutions to freeze-thaw cycles followed by coagulation bath immersion. The structure-property relationships for these hydrogels were examined by measurement of their swelling, rehydration, degradation, and mechanical properties. For the case of pure PVA hydrogels, the equilibrium swelling ratio was used to predict the effect of freeze thaw cycles and coagulation bath on average molecular weights between crosslinks and on mesh size. For all hydrogels, trends for the reswelling ratio, which is indicative of the crosslinked polymer fraction, were consistent with relative tensile properties. The coagulation bath treatment increased the degradation resistance of the hydrogels significantly. The suitability of each hydrogel for cell attachment and proliferation was examined by protein adsorption and bovine vascular endothelial cell culture experiments. Protein adsorption and cell proliferation was highest on the PVA/gelatin hydrogels. This study demonstrates that the potential of PVA hydrogels for artificial blood vessel applications can be improved by the addition of natural polymers, and that freeze-thawing and coagulation bath treatment can be utilized for fine adjustment of the physical characteristics.

An analysis of the strain field in biaxial Flexcell membranes for different waveforms and frequencies.

Mechanical stimuli have been shown to affect cell behaviour in terms of proliferation, apoptosis, and protein expression. In terms of cardiovascular diseases, for example, endothelial and smooth muscle cells exposed to an abnormal strain environment have been associated with atherosclerosis and in-stent restenosis. The FX-4000 system (Flexercell Tension Plus System, Flexcell Corporation, McKeesport, Pennsylvania, USA) is an in-vitro system that is widely used to strain cells in order to evaluate their response to strain. The precision, accuracy, and repeatability of the strains controlled by the system are therefore crucial to analyse and interpret the results confidently. The aim of this study was to investigate the mechanical behaviour of the FX-4000 Flexercell six-well-plate silicon membranes for static and dynamic cyclic strains by measuring the maximum peak strain and analysing the change in the membrane deformation after cyclic strain for 0 h, 24 h, and 48 h at different strain amplitudes and frequencies. The results of the tests conducted demonstrate notable differences between the measured strains of the membranes in comparison with both the inputs and the outputs of the Flexcell software. The calibration method used by Flexcell International assumes that the strain values determined for a given vacuum pressure on the silicone membranes are reliable for different waveforms and frequencies. The data reported here clearly indicate that this is not the case. The results indicate that a unique calibration pressure-strain curve must be determined for each test given the viscoelastic nature of the Flexcell system. A new method to calibrate the machine in house was applied using new pressure-strain equations. This new calibration method has been presented and should enable researchers using the Flexcell machine to set up their cell experiments more accurately.

Vascular cell viability on polyvinyl alcohol hydrogels modified with water-soluble and -insoluble chitosan.

Polyvinyl alcohol (PVA) hydrogels blended with chitosan or other biological macromolecules have shown promise for cell culture and tissue engineering. This study investigates the attachment and growth of bovine aortic endothelial (BAEC) and smooth muscle cells (BASMC) on the PVA hydrogels modified with water soluble and water insoluble chitosan. Cell adhesion on the surface of the membranes was examined by phase contrast microscopy while cell morphologies were studied using immunocytochemistry staining with EC and SMC specific biomarkers (F-actin and alpha actin respectively). Cells cultured on 6% PVA, 0.4% chitosan (water soluble and insoluble) hydrogel membranes displayed excellent adhesion and spreading characteristics, in addition to negligible cell structural morphological changes in comparison to a polystyrene control. Similar vascular cell adhesion features were apparent on PVA membranes blended with water-soluble and -insoluble chitosan. Fluorescent activated cell sorter (FACS) analysis was used to determine BAEC and BASMC proliferation and cell viability. Apoptotic levels in BAEC after 7 days were 12.8% +/- 2.5% on the PVA- chitosan WS-1 membrane and 10.1% +/- 1.5% on the control well (n = 3) while comparable results were also noted for BASMC. Equivalent proliferative activity was apparent for BAEC on the control and PVA-chitosan membrane after 7 days, while BASMC showed increased proliferative activity on the membranes. These results indicate that the PVA-chitosan blended hydrogel membranes show promise for cell culture and tissue engineering applications.

An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC)-including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds.

Data from 305 non-proprietary compounds tested using the yeast RAD54-GFP (Green Fluorescent Protein) assay, GreenScreen GC, are presented, together with a detailed comparison with results from in vitro and in vivo genotoxicity tests and rodent carcinogenesis. In addition, observations on reproducibility and the performance of the test with autofluorescent and coloured compounds are described. Like the Ames test, the GreenScreen assay is shown to exhibit high specificity (82%), meaning that compounds with positive results are very likely to be genotoxic carcinogens. This is in contrast to mammalian cell tests established for use in regulatory testing that provide disappointingly low specificity and the inevitable generation of confounding false positive data. The analysis confirmed the observations of earlier studies, showing that a combination of an Ames test (or surrogate) with the yeast test provides high specificity as well as high sensitivity in the identification of rodent carcinogens.

Access of asylum seeker children to acute paediatric services.

To investigate the interface between primary care and paediatric services in the referral of asylum seekers. Over a 3 month period a questionnaire was administered, and clinical data gathered on every child attending the A&E department of UCHG whose parents were seeking asylum in this country. Control data was obtained for the next Irish child seen on-call. At the time of presentation to the paediatric service, an Irish child was 4 times more likely (32%) to have initially been seen and referred by a GP than an asylum seeker child (8%); 80% of asylum seeker families had registered with a GP, compared to 96% of controls. 24% of asylum seeker families had called and used an emergency response ambulance to get to hospital, compared to just 4% of Irish children. The rate of subsequent admission to the paediatric ward from A&E was nearly that in asylum seeker children (24%) compared to Irish controls (40%), get to hospital, compared to just 4% of Irish children. Asylum seeker children are less likely to have seen a GP prior to A&E presentation, more likely to go to hospital by ambulance and less likely to be subsequently admitted, suggesting an over-dependence on paediatric hospital services in this population.

An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening.

In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.