Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion)

The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine

High Hospital Volume Is Associated with Better Outcomes for Breast Cancer Surgery: Analysis of 233,247 Patients

Background:: The relationship between hospital volume and outcomes needs to be further elucidated for low-risk procedures such as surgical therapy of localized breast cancer. The objective of this investigation was to assess the relationship between hospital volume and outcomes for breast cancer surgery. Methods:: A total of 233,247 patients who underwent breast-conserving therapy (BCT) and breast-ablative therapy (BAT) for localized breast cancer were extracted from 13 years (1988-2000) of the Nationwide Inpatient Samples. Hospital volume was classified as low (<30 cases/year), intermediate (≥ 30 to <70cases/year), and high (≥ 70 cases/year). Multiple linear and logistic regression analyses were used to assess the risk-adjusted association between hospital volume and outcomes. Results:: In risk-adjusted analyses, patients operated on at low-volume hospitals were 3.04 (p = 0.03) times more likely to die after BCT compared with patients operated on at high-volume hospitals. Similarly, low-volume hospitals had a significantly higher likelihood of postoperative complications (odds ratio [OR] = 1.73, p = 0.01 for BCT; OR = 1.44, p < 0.001 for BAT) compared with high-volume hospitals. Compared with low-volume hospitals, length of hospital stay was significantly shorter and nonroutine patient discharge significantly lower for high-volume providers for both BCT and BAT (all p < 0.001). Patients were also significantly less likely to undergo BCT if operated on in a low- or intermediate-volume hospital compared with a high-volume provider (p < 0.001). Conclusions:: High-volume hospitals had significantly lower nonroutine patient discharge, postoperative morbidity and mortality, shorter length of hospital stay, and higher likelihood of performing BCT. Referral of patients with localized breast cancer to high-volume hospitals may be justifie

The relationship between hospital patients' ratings of quality of care and communication

Objective To assess the relationship between hospital patients' quality of care ratings and their experiences with health-related information exchanges and communication during hospitalization. Design Cross-sectional multivariate dimensional analysis of data from a quality of care experience questionnaire of hospital patients comparing scores across three levels of reported satisfaction. Setting and participants Five thousand nine hundred and fifty-two patients from a Swiss University Hospital responded to the questionnaire at discharge during 2010. Main outcome measures Survey questions measuring patients' evaluation of quality of care, patient loyalty and overall satisfaction. Results Different levels of reported satisfaction are associated with differing experiences of health-related information and communication during a hospital stay. Conclusions Patients who report lower satisfaction appear to attribute to the hospital staff enduring negative dispositions from behaviours that may be due to specific situational contexts. Negative experiences appear to influence scores on most other communication and information domains. Patients who report higher satisfaction, in contrast, appear to differentiate negative experiences and positive experiences and they appear to relativize and compartmentalize negative experiences associated with their hospital sta

Growth factors for clinical-scale expansion of human articular chondrocytes : Relevance for automated bioreactor systems

The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 celIS/cm(2) in medium containing 10 bovine serum or 10,000 cells/cm(2) with 1 chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF164 levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sproutin

Angiogenesis in tissue engineering : Breathing life into constructed tissue substitutes

Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ

Increased surface expression of CD18 and CD11b in leukocytes after tourniquet ischemia during elective hand surgery

The surface expression of β2-integrins was investigated in leukocytes from patients undergoing ischemia induced by tourniquet application for elective hand surgery. Blood samples were obtained before initiation, at the end of ischemia, and after 15 minutes of reperfusion from ischemic and contralateral arms of five patients. Comparable expression of CD18, CD11a, CD11b, and CD11c could be detected by immunofluorescence in leukocytes from samples drawn from either arm before tourniquet application. In contrast, a significant increase in the expression of CD18 was detectable in monocytes, granulocytes, and lymphocytes from the ischemic arm compared with that in the nonischemic contralateral control, at the end of the ischemia time (80 ± 16 minutes). A significantly increased expression of CD11b, but not CD11a or CD11c, determinants was also observed in granulocytes and monocytes. Concomitantly, a significant reduction in the percentages of granulocytes in samples from ischemic areas was detectable. After 15 minutes of reperfusion, differences in the expression of these adhesion molecules were no longer significant. The expression of the genes encoding interleukins IL-1α, IL-1β, and IL-6 and tumor necrosis factor alpha (TNFα) proinflammatory cytokines was also studied by reverse polymerase chain reaction (rPCR) in peripheral blood mononuclear cells (PBMCs) obtained from the same samples in three patients. IL-1β or IL-6 gene expression was never observed. Expression of IL-1α and TNFα genes, as detected in two patients, was not related with induction of ischemia. However, in these patients expression of one or both these genes was observed in samples derived from the ischemic but not the control arm after 15 minutes of reperfusion. These data document that overexpression of adhesion molecules and sequestration of leukocytes take place following short ischemia times, as routinely applied clinically for minor surgical procedure

Active Antigen-specific Immunotherapy of Melanoma: from Basic Science to Clinical Investigation

Advanced-stage melanoma here dismal prognosis, and novel therapeutic approaches are urgently required. The possibility of taking advantage of the immune response of patients for its treatment has been an appealing concept for almost a century. Only during the last decade, however, has the molecular identification of tumor-associated antigens (TAAs) offered the possibility of vaccinating patients (e.g., active induction of TAA-specific immune responses). Active antigen-specific immunotherapy (AASIT) is currently being investigated in a number of clinical centers as a treatment option for advanced-stage melanoma. A large number of melanoma TAAs have been molecularly characterized and are being used in vaccination trials in various molecular forms and according to various immunization protocols. Here we provide a short overview on melanoma TAAs, the technologies currently in use to induce specific cytotoxic T-lymphocyte (CTL) responses in vivo, and their monitoring. We also propose a tentative AASIT agenda for the next few years, aiming at improving the capacity to induce and monitor TAA-specific immune responses and to verify their clinical effectivenes

The impact of the Bohemian Spur on the cooling and exhumation pattern of the Eastern Alpine wedge

Fold and thrust belt dynamics and architecture may largely be impacted by the geometry of the overridden basement. The Bohemian Spur, the subcrop extension of the Bohemian massif, guided thrust propagation leading to the arcuate shape of the orogen and a narrowing of the Molasse Basin at the transition to the between the W-E trending Eastern Alps and the SW-NE trending Western Carpathians. Thermochronological studies in the Eastern Alps were mainly focused on the core of the collisional orogen, where deformation has been most prominent. Further to the east, some FT work is concentrated along fault zones but thermochronometers with lower closure temperatures have hardly been applied to higher elements of the nappe pile. Due to the scarcity of the dataset and preferential application of fission track dating uppermost crustal cooling below ca. 80 °C remains undetected. In this study we present new apatite (U-Th)/He and apatite fission track data from clastic units of the Rhenodanubian Flysch zone and the Northern Calcareous Alps. We find reset ages, that monitor a so far un(der)appreciated phase of prominent Late Oligocene to Miocene cooling. Thermal modeling of age data from the flysch samples reveals rapid Early Miocene cooling at rates of up to 40 °C/Ma between ca. 20 and 15 Ma. We propose a buttressing effect of the underlying tectonically structured eastern rim of the Bohemian Spur to be the driving mechanism for this phase of intensified exhumation. Our tectonic model (Fig. 1a) invokes contractional reactivation of pre-existing normal faults inherited from Penninic continental rifting. This positive inversion led to the shortening of the Jurassic half-graben infill and its extrusion as a major fold. Thermochronological data and thermal modeling of data from samples in the Lunz nappe of the Northern Calcareous Alps nappe pile indicate less punctuated cooling and exhumation. Modeling defines an increase of cooling rates at the latest at ca. 27 to 25 Ma, i.e., earlier than in the Flysch samples. Cooling occurred at a much lower rate of 3 to 6 °C/Ma and was synchronous with northward movement of the deformation front. In our tectonic model (Fig. 1b), we propose a staircase pattern that influences wedge dynamics: The topographically segmented downgoing plate leads to less localized and more distributed deformation invoking a broader area of uplift than the spatially focused uplift of the Flysch samples. Wedge propagation is initially inhibited or retarded by the relief of the basement. The ongoing northward movement of the propagating wedge is compensated through deep duplexing of the autochthonous foreland sequence. When calling upon deep-seated processes to explain the exhumation pattern the buttressing effect needs to be taken into account. Early Miocene drainage pattern reorganization in the Molasse Basin is proposed to be a consequence of uplift induced by the subcrop promontory