Hylaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues : in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system

Smart matrices are required in bone tissueengineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bioartificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.

O papel da angiogênese e da hemólise na úlcera de perna de pessoas com anemia falciforme

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72. h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20 compression, 1. Hz) over 3. days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24. h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24. h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.

The inflammatory role of angiogenic growth factors : a neutrophil perspective

De par sa présence dans tous les vaisseaux sanguins, l'endothélium joue un rôle clef dans le processus d’hémostase, tant par sa libération de facteurs anticoagulants que par ses changements protéiques qui permettent à l’organisme de déclencher la réparation tissulaire. La fonction anticoagulante de l’endothélium peut être mise en défaut en cas d’atteinte de son intégrité, entrainant la formation de thrombus, le rejet précoce de greffes ou encore l’induction de l’athérosclérose. L’intégrité de l’endothélium est donc capitale pour la prévention de nombreuses maladies cardiovasculaires. Chez l’adulte, les cellules endothéliales (CE), normalement quiescentes, sont rapidement activées en cas d’hypoxie ou d’inflammation, leur permettant ainsi d’amorcer le processus angiogénique comme suit: Tout d’abord, l’induction de l’hyperperméabilité vasculaire permet l’extravasation des protéines plasmatiques. Ensuite, la dégradation de la lame basale par des métalloprotéases permet aux CE de se détacher, de proliférer, de migrer et de s’organiser pour former l’ébauche du futur vaisseau. La dernière étape consiste en la maturation du vaisseau, c’est-à-dire son recouvrement par des cellules murales, telles que les cellules musculaires lisses et les péricytes. Ces processus sont régulés par de nombreux facteurs angiogéniques tels que les membres de la famille Notch, du vascular endothelial growth factor (VEGF), du fibroblast growth factor (FGF), des angiopoïétines, et des matrix metalloproteases (MMP). L’angiogenèse pathologique, soit une insuffisance ou un excès de vascularisation, est impliquée dans les blessures chroniques, les accidents cardiovasculaires, les pathologies coronariennes artérielles, les pathologies tumorales, l’arthrite rhumatoïde, la rétinopathie diabétique, l’athérosclérose, le psoriasis et l’asthme. Ces pathologies sont souvent issues d’une dérégulation de l’activité endothéliale, fréquemment observée conjointement à l’expression continue de molécules d’adhésion leucocytaires, à l’augmentation de la perméabilité vasculaire, et aux anomalies de la vasoréactivité. L’activation non-contrôlée de l’endothélium entraîne ainsi une inflammation chronique et la formation de structures vasculaires anarchiques. Les premiers leucocytes à répondre à l’appel inflammatoire sont les neutrophiles. Equippées d’une panoplie de produits antibactériens puissants mais aussi nocifs pour les tissus qui les entourent, ces cellules polylobées participent à chaque étape du processus inflammatoire, depuis l’induction de l’hyperperméabilité vasculaire jusqu’à la résolution. En effet, grâce à leurs récepteurs, les neutrophiles détectent et interprètent les signaux biochimiques présents dans la circulation et à la surface de l’endothélium, et libèrent aussi leurs propres médiateurs tels le VEGF, les MMP, et l’interleukine-8 (IL-8), dont les effets sont à la fois paracrines et autocrines. Existent-ils d’autres modulateurs typiques de la fonction endothéliale capables d’influencer le comportement des neutrophiles? En effet, notre laboratoire a démontré que chez l’humain, une stimulation directe aux angiopoïétines incitait les neutrophiles à adhérer aux CE, à migrer, à synthétiser et à relâcher l’IL-8, voire même à vivre plus longtemps. La présence du récepteur des angiopoïétines, Tie2, à la surface des neutrophiles laisse présager que la famille possèderait d’autres fonctions leucocytaires encore non-identifiées. Par ailleurs, dans un modèle classique de l’angiogenèse in vivo (matrigel), nous avons observé que sous l’effet du FGF1 et 2, les ébauches des nouveaux vaisseaux étaient parfois accompagnées d’une infiltration de cellules granulocytaires. Ainsi, en partant de ces observations, l’objectif de nos études (présentées ci-après) était d’approfondir nos connaissances sur la relation entre neutrophiles et facteurs angiogéniques, notamment les FGF et les angiopoïétines. Par tests in vitro, nous avons confirmé que les neutrophiles humains exprimaient plusieurs récepteurs du FGF (FGFR1-4) d’une façon hétérogène, et qu’ils migraient vers un gradient des ligands FGF1 et 2. Par ailleurs, nous nous sommes intéressés aux voies de signalisation inflammatoires activées par les ligands FGF1, FGF2, Ang1 et Ang2. Grâce à une stratégie génique ciblant 84 gènes inflammatoires, nous avons identifié plusieurs cibles d’intérêt touchées par Ang1, dont certains membres de la famille de l’IL-1, alors qu’aucun des gènes testés n’avait changé de façon significative sous l’effet des FGF ou d’Ang2. Suite à des cinétiques approfondies, nous avons démontré qu’Ang1 stimulait la transcription de l’ARN messager de l’IL-1β, et augmentait simultanément la quantité de protéine immature (pro-IL-1β; inactive) et clivée (IL-1β « mature »; active). En parallèle, Ang1 augmentait la sécrétion de l’antagoniste naturel de l’IL-1β, l’IL-1RA, sans pour autant stimuler la relâche de l’IL-1β. A l’instar des endotoxines bactériennes dont les effets liés à l’IL-1 dépendaient de la kinase p38, ceux d’Ang1 découlaient presque entièrement des voies de signalisation du p42/44.

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Spatial stimulus : Model making in the architectural design studio

This action research examines the enhancement of visual communication within the architectural design studio through physical model making. „It is through physical model making that designers explore their conceptual ideas and develop the creation and understanding of space,‟ (Salama & Wilkinson 2007:126). This research supplements Crowther‟s findings extending the understanding of visual dialogue to include physical models. „Architecture Design 8‟ is the final core design unit at QUT in the fourth year of the Bachelor of Design Architecture. At this stage it is essential that students have the ability to communicate their ideas in a comprehensive manner, relying on a combination of skill sets including drawing, physical model making, and computer modeling. Observations within this research indicates that students did not integrate the combination of the skill sets in the design process through the first half of the semester by focusing primarily on drawing and computer modeling. The challenge was to promote deeper learning through physical model making. This research addresses one of the primary reasons for the lack of physical model making, which was the limited assessment emphasis on the physical models. The unit was modified midway through the semester to better correlate the lecture theory with studio activities by incorporating a series of model making exercises conducted during the studio time. The outcome of each exercise was assessed. Tutors were surveyed regarding the model making activities and a focus group was conducted to obtain formal feedback from students. Students and tutors recognised the added value in communicating design ideas through physical forms and model making. The studio environment was invigorated by the enhanced learning outcomes of the students who participated in the model making exercises. The conclusions of this research will guide the structure of the upcoming iteration of the fourth year design unit.

Preparation, characterization and in vitro angiogenic capacity of cobalt substituted β-tricalcium phosphate ceramics

Divalent cobalt ions (Co2+) have been shown to possess the capacity to induce angiogenesis by activating hypoxia inducible factor-1α (HIF-1α) and subsequently inducing the production of vascular endothelial growth factor (VEGF). However, there are few reports about Co-containing biomaterials for inducing in vitro angiogenesis. The aim of the present work was to prepare Co-containing β-tricalcium phosphate (Co-TCP) ceramics with different contents of calcium substituted by cobalt (0, 2, 5 mol%) and to investigate the effect of Co substitution on their physicochemical and biological properties. Co-TCP powders were synthesized by a chemistry precipitation method and Co-TCP ceramics were prepared by sintering the powder compacts. The effect of Co substitution on phase transition and the sintering property of the β-TCP ceramics was investigated. The proliferation and VEGF expression of human bone marrow mesenchymal stem cells (HBMSCs) cultured with both powder extracts and ceramic discs of Co-TCP was further evaluated. The in vitro angiogenesis was evaluated by the tube-like structure formation of human umbilical vein endothelial cells (HUVECs) cultured on ECMatrix™ in the presence of powder extracts. The results showed that Co substitution suppressed the phase transition from β- to α-TCP. Both the powder extracts and ceramic discs of Co-TCP had generally good cytocompatibility to support HBMSC growth. Importantly, the incorporation of Co into β-TCP greatly stimulated VEGF expression of HBMSCs and Co-TCP showed a significant enhancement of network structure formation of HUVECs compared with pure TCP. Our results suggested that the incorporation of Co into bioceramics is a potential viable way to enhance angiogenic properties of biomaterials. Co-TCP bioceramics may be used for bone tissue regeneration with improved angiogenic capacity.

The effect of poser race on the happy categorization advantage depends on stimulus type, set size, and presentation duration

The question as to whether poser race affects the happy categorization advantage, the faster categorization of happy than of negative emotional expressions, has been answered inconsistently. Hugenberg (2005) found the happy categorization advantage only for own race faces whereas faster categorization of angry expressions was evident for other race faces. Kubota and Ito (2007) found a happy categorization advantage for both own race and other race faces. These results have vastly different implications for understanding the influence of race cues on the processing of emotional expressions. The current study replicates the results of both prior studies and indicates that face type (computer-generated vs. photographic), presentation duration, and especially stimulus set size influence the happy categorization advantage as well as the moderating effect of poser race.

Invading edge vs. inner (edvin) patterns of vascularization : an interplay between angiogenic and vascular survival factors defines the clinical behaviour of non-small cell lung cancer

Neo-angiogenesis during neoplastic growth involves endothelial mitogenic and migration stimuli produced by cancer or tumour stromal cells. Although this active angiogenesis takes place in the tumour periphery, the process of vessel growth and survival in inner areas and its clinical role remain largely unexplored. The present study compared the microvessel score (MS) as well as the single endothelial cell score (ECS) in the invading edge and in inner areas of non-small cell lung carcinomas (NSCLCs). Three different patterns of vascular growth were distinguished: the edvin (edge vs. inner) type 1, where a low MS was observed in both peripheral and inner tumour areas; the edvin type 2, where a high MS was noted in the invading front but a low MS in inner areas; and the edvin type 3, where both peripheral and inner tumour areas had a high MS. The ECS was high in the invading edge in edvin type 2 and 3 cases and was sharply decreased in both types in inner areas, suggesting that endothelial cell migration is unlikely to contribute to the angiogenic process in areas away from the tumour front. Expression of the vascular endothelial growth factor (VEGF) and of thymidine phosphorylase (TP) was associated with a high MS in the invading edge. VEGF was associated with a high MS in inner areas (edvin 3), while TP expression was associated with edvin type 2, showing that VEGF (and not TP) contributes to the preservation of the inner vasculature. Both edvin type 2 and 3 cases showed an increased incidence of node metastasis, but edvin type 3 cases had a poorer prognosis, even in the N1-stage group. The present study suggests that tumour factors regulating angiogenesis and vascular survival are not identical. A possible method is reported to quantify these two parameters by comparing the MS in the invading edge and inner areas (edvin types). This observation may contribute to the evaluation of the effectiveness of different therapeutic approaches, namely vascular targeting vs. anti-angiogenesis. Copyright (C) 2000 John Wiley and Sons, Ltd.

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non- small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-NO, 1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c- erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c- erbB-2-related immune response remains to be further investigated.

Lesions in the bed nucleus of the stria terminalis disrupt corticosterone and freezing responses elicited by a contextual but not by a specific cue-conditioned fear stimulus

The bed nucleus of the stria terminalis (BNST) is believed to be a critical relay between the central nucleus of the amygdala (CE) and the paraventricular nucleus of the hypothalamus in the control of hypothalamic–pituitary– adrenal (HPA) responses elicited by conditioned fear stimuli. If correct, lesions of CE or BNST should block expression of HPA responses elicited by either a specific conditioned fear cue or a conditioned context. To test this, rats were subjected to cued (tone) or contextual classical fear conditioning. Two days later, electrolytic or sham lesions were placed in CE or BNST. After 5 days, the rats were tested for both behavioral (freezing) and neuroendocrine (corticosterone) responses to tone or contextual cues. CE lesions attenuated conditioned freezing and corticosterone responses to both tone and con- text. In contrast, BNST lesions attenuated these responses to contextual but not tone stimuli. These results suggest CE is indeed an essential output of the amygdala for the expres- sion of conditioned fear responses, including HPA re- sponses, regardless of the nature of the conditioned stimu- lus. However, because lesions of BNST only affected behav- ioral and endocrine responses to contextual stimuli, the results do not support the notion that BNST is critical for HPA responses elicited by conditioned fear stimuli in general. Instead, the BNST may be essential specifically for contex- tual conditioned fear responses, including both behavioral and HPA responses, by virtue of its connections with the hippocampus, a structure essential to contextual condition- ing. The results are also not consistent with the hypothesis that BNST is only involved in unconditioned aspects of fear and anxiety.

Stimulation of osteogenic and angiogenic ability of cells on polymers by pulsed laser deposition of uniform akermanite-glass nanolayer

Polymer biomaterials have been widely used for bone replacement/regeneration because of their unique mechanical properties and workability. Their inherent low bioactivity makes them lack osseointegration with host bone tissue. For this reason, bioactive inorganic particles have been always incorporated into the matrix of polymers to improve their bioactivity. However, mixing inorganic particles with polymers always results in inhomogeneity of particle distribution in polymer matrix with limited bioactivity. This study sets out to apply the pulsed laser deposition (PLD) technique to prepare uniform akermanite (Ca2MgSi2O7, AKT) glass nanocoatings on the surface of two polymers (non-degradable polysulfone (PSU) and degradable polylactic acid (PDLLA)) in order to improve their surface osteogenic and angiogenic activity. The results show that a uniform nanolayer composed of amorphous AKT particles (∼30nm) of thickness 130nm forms on the surface of both PSU and PDLLA films with the PLD technique. The prepared AKT-PSU and AKT-PDLLA films significantly improved the surface roughness, hydrophilicity, hardness and apatite mineralization, compared with pure PSU and PDLLA, respectively. The prepared AKT nanocoatings distinctively enhance the alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, OPN and Col I) of bone-forming cells on both PSU and PDLLA films. Furthermore, AKT nanocoatings on two polymers improve the attachment, proliferation, VEGF secretion and expression of proangiogenic factors and their receptors of human umbilical vein endothelial cells (HUVEC). The results suggest that PLD-prepared bioceramic nanocoatings are very useful for enhancing the physicochemical, osteogenic and angiogenic properties of both degradable and non-degradable polymers for application in bone replacement/regeneration.