A Unification of Nanotopography and Extracellular Matrix in Electrospun Scaffolds for Bioengineered Hepatic Models.

Donor liver shortage is a crucial global public health problem as whole-organ transplantation is the only definitive cure for liver disease. Liver tissue engineering aims to reproduce or restore function through in vitro tissue constructs, which may lead to alternative treatments for active and chronic liver disease. The formulation of a multifunctional scaffold that has the potential to mimic the complex extracellular matrix (ECM) and their influence on cellular behavior, are essential for culturing cells on a construct. The separate employment of topographic or biological cues on a scaffold has both shown influences on hepatocyte survival and growth. In this study, we investigate both of these synergistic effects and developed a new procedure to directly blend whole-organ vascular perfusion-decellularized rat liver ECM (dECM) into electrospun fibers with tailored surface nanotopography. Water contact angle, tensile test, and degradation studies were conducted to analyze scaffold hydrophilicity, mechanical properties, and stability. The results show that our novel hybrid scaffolds have enhanced hydrophilicity, and the nanotopography retained its original form after hydrolytic degradation for 14 days. Human hepatocytes (HepG2) were seeded to analyze the scaffold biocompatibility. Cell viability and DNA quantification imply steady cell proliferation over the culture period, with the highest albumin secretion observed on the hybrid scaffold. Scanning electron microscopy shows that cell morphology was distinctly different on hybrid scaffolds compared to control groups, where HepG2 began to form a monolayer toward the end of the culture period; meanwhile, typical hepatic markers and ECM genes were also influenced, such as an increasing trend of albumin appearing on the hybrid scaffolds. Taken together, our findings provide a reproducible approach and utilization of animal tissue-derived ECM and emphasize the synergism of topographical stimuli and biochemical cues on electrospun scaffolds in liver tissue engineering.

Rat liver ECM incorporated into electrospun polycaprolactone scaffolds as a platform for hepatocyte culture.

Liver disease is expanding across the globe; however, health-care systems still lack approved pharmaceutical treatment strategies to mitigate potential liver failures. Organ transplantation is the only treatment for liver failure and with increasing cases of liver disease, transplant programs increasingly cannot provide timely transplant availability for all patients. The development of pharmaceutical mitigation strategies is clearly necessary and methods to improve drug development processes are considered vital for this purpose. Herein, we present a methodology for incorporating whole organ decellularised rat liver ECM (rLECM) into polycaprolactone (PCL) electrospun scaffolds with the aim of producing biologically relevant liver tissue models. rLECM PCL scaffolds have been produced with 5 w/w% and 10 w/w% rLECM:PCL and were analyzed by SEM imaging, tensile mechanical analyses and FTIR spectroscopy. The hepatocellular carcinoma cell line, HepG2, was cultured upon the scaffolds for 14 days and were analyzed through cell viability assay, DNA quantification, albumin quantification, immunohistochemistry, and RT-qPCR gene expression analysis. Results showed significant increases in proliferative activity of HepG2 on rLECM containing scaffolds alongside maintained key gene expression. This study confirms that rLECM can be utilized to modulate the bioactivity of electrospun PCL scaffolds and has the potential to produce electrospun scaffolds suitable for enhanced hepatocyte cultures and in-vitro liver tissue models.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

Response differences of HepG2 and Primary Mouse Hepatocytes to morphological changes in electrospun PCL scaffolds.

Liver disease cases are rapidly expanding across the globe and the only effective cure for end-stage disease is a transplant. Transplant procedures are costly and current supply of donor livers does not satisfy demand. Potential drug treatments and regenerative therapies that are being developed to tackle these pressing issues require effective in-vitro culture platforms. Electrospun scaffolds provide bio-mimetic structures upon which cells are cultured to regulate function in-vitro. This study aims to shed light on the effects of electrospun PCL morphology on the culture of an immortalised hepatic cell line and mouse primary hepatocytes. Each cell type was cultured on large 4-5 µm fibres and small 1-2 µm fibres with random, aligned and highly porous cryogenically spun configurations. Cell attachment, proliferation, morphology and functional protein and gene expression was analysed. Results show that fibre morphology has a measurable influence on cellular morphology and function, with the alteration of key functional markers such as CYP1A2 expression.

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 µm and 5 µm diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

Long-term outcome after anterolateral open reduction and Salter osteotomy for late presenting developmental dysplasia of the hip.

INTRODUCTION: Only a handful of studies report outcomes after open reduction for developmental hip dislocation beyond skeletal maturity. For successfully reduced hips it is the outcome into late adulthood on which the results of this intervention should be judged. These studies indicate clearly the importance of preservation of the acetabular growth centres during surgery. The acetabulum must also be addressed when insufficient growth remains reliably to remodel residual dysplasia even after stable, concentric reduction. SUMMARY: Comparing the longest-term outcome studies for open reduction to less invasive, but mainly historical, techniques of gradual traction reduction it is unsettling to note that the latter protocols are associated with the best results. Whereas open reduction and innominate osteotomy are practised as originally described by Salter, gradual traction reduction has largely been abandoned. CONCLUSIONS: There are probably aspects of the more time-consuming methods of gradual reduction that do not violate the hip joint capsule that expose the femoral head to a lower risk of femoral head osteonecrosis leading to better long-term outcomes.

MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation.

Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia.

A review of long-term outcomes for late presenting developmental hip dysplasia.

Successful management of late presenting hip dislocation in childhood is judged by the outcome not just at skeletal maturity but well beyond into adulthood and late middle age. This review considers different methods of treatment and looks critically at the handful of studies reporting long-term follow-up after successful reduction.

PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

The medial approach for the treatment of children with developmental dysplasia of the hip.

The medial approach for the treatment of children with developmental dysplasia of the hip (DDH) in whom closed reduction has failed requires minimal access with negligible blood loss. In the United Kingdom, there is a preference for these children to be treated using an anterolateral approach after the appearance of the ossific nucleus. In this study we compared these two protocols, primarily for the risk of osteonecrosis. Data were gathered prospectively for protocols involving the medial approach (26 hips in 22 children) and the anterolateral approach (22 hips in 21 children) in children aged < 24 months at the time of surgery. Osteonecrosis of the femoral head was assessed with validated scores. The acetabular index (AI) and centre-edge angle (CEA) were also measured. The mean age of the children at the time of surgery was 11 months (3 to 24) for the medial approach group and 18 months (12 to 24) for the anterolateral group, and the combined mean follow-up was 70 months (26 to 228). Osteonecrosis of the femoral head was evident or asphericity predicted in three of 26 hips (12%) in the medial approach group and four of 22 (18%) in the anterolateral group (p = 0.52). The mean improvement in AI was 8.8° (4° to 12°) and 7.9° (6° to 10°), respectively, at two years post-operatively (p = 0.18). There was no significant difference in CEA values of affected hips between the two groups. Children treated using an early medial approach did not have a higher risk of developing osteonecrosis at early to mid-term follow-up than those treated using a delayed anterolateral approach. The rates of acetabular remodelling were similar for both protocols.

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling.

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK MAPK pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified p21-activated kinase 1 (PAK1) as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 30--33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation.

Hypoxia regulates the production and activity of glucose transporter-1 and indoleamine 2,3-dioxygenase in monocyte-derived endothelial-like cells: possible relevance to infantile haemangioma pathogenesis.

BACKGROUND: Infantile haemangioma (IH) may present as a precursor area of pallor prior to the initial proliferative phase, which implies that the early lesion may be hypoxic. OBJECTIVES: To examine the effect of hypoxia on the expression and activity of two key molecular markers of IH, glucose transporter-1 (GLUT1) and indoleamine 2,3-dioxygenase (IDO). METHODS: IH endothelial cells express both haematopoietic and endothelial cell markers. CD14+ monocyte-derived endothelial-like cells have been employed in the study of IH and is the cell type used in this study. RESULTS: GLUT1 transcript, protein and activity levels were strongly induced by hypoxia and remained elevated following 2 days of normoxic recovery. IDO transcript levels were not affected by hypoxia, although IDO protein level was reduced fivefold and IDO activity >100-fold following 2 days of hypoxia. The protein and activity levels returned to normal following 2 days of normoxic recovery. CONCLUSIONS: The findings link the tissue hypoxia that precedes lesion development and the expression and/or activity of two key IH proteins. The early hypoxic insult may contribute to the elevated GLUT1 levels in IH lesions, while the very low IDO activity during the hypoxic phase may promote activation of immune cells in the lesion, which release cytokines that trigger IDO expression and activity and entry into the proliferative phase. Interestingly, IH lesion development shares some common features with ischaemia-reperfusion injury.

Use of the entero-test, a novel approach for the noninvasive capture of biliary metabolites in dogs.

Preclinical information on the biliary metabolites of a drug candidate is typically obtained through the collection of bile after surgical cannulation of the bile duct. In this study, we describe a novel approach using the Entero-Test, a simple device that facilitates the noninvasive sampling of duodenal bile. The Entero-Test was used to collect bile from six fasted dogs that had been dosed either orally with simvastatin (SV) or intravenously with simvastatin hydroxy acid (SVA), compounds that have been previously reported to undergo extensive metabolism and biliary secretion in the dog. The devices, consisting of a weighted gelatin capsule containing 90 cm of a highly absorbent nylon string, were swallowed by each dog with the proximal end of the string taped to the animal's face. Once the weighted string had reached the duodenum, gallbladder contraction was stimulated to release bile. Each bile-stained string was then retrieved via the mouth and, after solvent extraction, samples were analyzed for drug-related material by ultraperformance liquid chromatography-mass spectrometry and NMR spectroscopy. Numerous metabolites of SV and SVA were observed, and, in general, the major metabolites have been reported previously from studies with bile duct-cannulated animals dosed with [14C]SV or [14C]SVA. The results from this study demonstrate the utility of deploying the Entero-Test in absorption, distribution, metabolism, and elimination studies to provide information on the nature of biliary metabolites, which, on occasion, may be sufficient to negate the need for more invasive sampling techniques. The benefits and limitations of the technique are discussed.

The EuroPhysiome, STEP and a roadmap for the virtual physiological human.

Biomedical science and its allied disciplines are entering a new era in which computational methods and technologies are poised to play a prevalent role in supporting collaborative investigation of the human body. Within Europe, this has its focus in the virtual physiological human (VPH), which is an evolving entity that has emerged from the EuroPhysiome initiative and the strategy for the EuroPhysiome (STEP) consortium. The VPH is intended to be a solution to common infrastructure needs for physiome projects across the globe, providing a unifying architecture that facilitates integration and prediction, ultimately creating a framework capable of describing Homo sapiens in silico. The routine reliance of the biomedical industry, biomedical research and clinical practice on information technology (IT) highlights the importance of a tailor-made and robust IT infrastructure, but numerous challenges need to be addressed if the VPH is to become a mature technological reality. Appropriate investment will reap considerable rewards, since it is anticipated that the VPH will influence all sectors of society, with implications predominantly for improved healthcare, improved competitiveness in industry and greater understanding of (patho)physiological processes. This paper considers issues pertinent to the development of the VPH, highlighted by the work of the STEP consortium.

Are we overusing blood transfusing after elective joint replacement?--a simple method to reduce the use of a scarce resource.

OBJECTIVES: To determine the proportion of patients who received a blood transfusion after joint replacement, and to devise a simple method to ensure patients were transfused based on strict clinical and haematological need. DESIGN: Prospective audit over 2 years. PATIENTS AND METHODS: The study group was 151 patients who underwent total hip and knee arthroplasty in a typical district general hospital (Kettering) over a 2-year period. They were divided into three consecutive groups. Current practice was audited (producing the first group of 62 patients) and transfusion rates were compared to regional figures. Local guidelines were drawn up. A form was introduced on which the indications for any transfusion had to be documented prior to transfusion of the blood. This was designed to encourage transfusion only on strong clinical grounds or an haemoglobin (Hb) level < 8 g/dl. Transfusion practice was then re-audited (producing the second group of 44 patients) to assess whether practice had improved. A year later, all relevant staff were reminded by letter of the guidelines. The process was then re-audited (producing the third group of 45 patients) again to determine whether practice remained improved or not. RESULTS: In the first audit (current practice) of 62 patients, the overall transfusion rate was 71%, with a higher rate in the hip replacement group (84%) ordered mainly by anaesthetic staff. Ward staff were reluctant not to transfuse patients whose Hb level fell below 10 g/dl. In the second audit, the transfusion rate fell by nearly 50% to 37%, with almost identical figures for knee and hip replacement. In the third audit of 45 patients, a year later, the transfusion rate was 40% overall. CONCLUSIONS: Patients were being transfused routinely, generally without good clinical evidence of benefit to the patient. The audit process was successful in instituting change for the better in blood transfusion practice for elective joint replacement. The improved practice can be largely maintained provided staff are regularly reminded of appropriate guidelines and encouraged to transfuse for clinical need only. For absolute adherence to guidelines, we would recommend a compulsory form system be introduced for transfusion in the per-operative period, to ensure blood transfusion is only given when absolutely necessary.

Corrosion of cemented titanium femoral stems.

Cemented titanium stems in hip arthroplasty are associated with proximal cement-stem ebonding and early failure. This was well publicised with the 3M Capital hip. However, corrosion in this setting has been reported with only one stem design and is less widely accepted. We present a series of 12 cemented titanium Furlong Straight Stems which required revision at a mean of 78 months for thigh pain. At revision the stems were severely corroded in a pattern which was typical of crevice corrosion. Symptoms were eliminated after revision to an all-stainless steel femoral prosthesis of the same design. We discuss the likely causes for the corrosion. The combination of a titanium stem and cement appears to facilitate crevice corrosion.

Web-based tools for quantitative renal physiology.

We present the development strategy and present state of progress on an interactive website project for quantitative renal physiology: a) a quantitative kidney database (QKDB), and b) an interactive website presenting mathematical models covering the major aspects of renal physiology. QKDB will house data for quantitative evaluation of hypotheses of renal function, from the cellular, through the epithelial and tubular, to whole organ levels. It will thus facilitate comparisons among different species and under various experimental conditions. It will include especially: transport parameters, tubular concentrations and flow rates along the various nephron segments, and anatomical details, in human kidneys, in experimentally studied species, and in model epithelia, such as cultured cells and amphibian skin and urinary bladder. The modeling resource will provide an interactive user interface to a collection of published models at all levels of renal physiology, enabling non-modelers to exploit the models, altering key parameters according to hypotheses of their own and visualizing the simulation results, thus permitting quantitative exploration of new hypotheses. Implementation will be facilitated by translation of the models into a common markup language such as CellML (cell markup language) and SBML (systems biology markup language). There will thus be a modular separation of model descriptions from their numerical solution methods.

The pulmonary physician in critical care. Illustrative case 1: cystic fibrosis.

The case history of a patient with CF admitted to an ICU is presented and the appropriateness of intensive care management for patients with CF is discussed. Issues relevant to the ICU care of patients with CF are highlighted.