Detection of early osteogenic commitment in primary cells using Raman spectroscopy

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm−1) and B-type carbonate (1072 cm−1), DNA (782 cm−1) and collagen matrix (CH2 deformation at 1450 cm−1) and weaker phosphate bands (948 and 970 cm−1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945–952 cm−1) and carbonated apatite (957–962 cm−1) after only 3 days in culture and octacalcium phosphate (970 cm−1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells

Modulation of endoglin expression in islets of langerhans by VEGF reveals a novel regulator of islet endothelial cell function

BACKGROUND: Endoglin/CD105 is an auxiliary receptor for transforming growth factor-β with established roles in vascular remodelling. It has recently been shown that heterozygous endoglin deficiency in mice decreases insulin secretion in an animal model of obesity, highlighting a potential role for endoglin in the regulation of islet function. We have previously identified two different populations of endoglin expressing cells in human and mouse islets which are: (i) endothelial cells (ECs) and (ii) islet mesenchymal stromal cells. The contribution of islet EC endoglin expression to islet development and sensitivity to VEGF is unknown and is the focus of this study. RESULTS: In vitro culture of mouse islets with VEGF164 for 48 h increased endoglin mRNA levels above untreated controls but VEGF did not modulate VEGFR2, CD31 or CD34 mRNA expression or islet viability. Removal of EC-endoglin expression in vivo reduced islet EC area but had no apparent effect on islet size or architecture. CONCLUSION: EC-specific endoglin expression in islets is sensitive to VEGF and plays partial roles in driving islet vascular development, however such regulation appears to be distinct to mechanisms required to modulate islet viability and size

Local origins impart conserved bone type-related differences in human osteoblast behaviour

Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κβ ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro

MEPE is a novel regulator of growth plate cartilage mineralization

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and ?CT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide

Lucro líquido do franqueado: um sinal para a escolha de franquia em época de crise

Este artículo aborda desde la perspectiva de la Teoría de Señales la elección de una franquicia por un potencial franquiciado que elige este canal de distribución por primera vez. El objetivo es analizar la relación entre algunas señales enviadas por el franquiciador y la elección de una franquicia por el potencial franquiciado. Concluimos que los efectos de las variables macroeconómicas de España en el periodo 2006-2013 influyeron en los ingresos netos de los franquiciados para que se convirtiera en una señal empleada por éstos a la hora de elegir la franquicia donde abrir un establecimiento.This paper adopts the perspective of Theory of Signals to discuss how someone starting a business as a franchisee for the first time can choose a suitable franchise brand. The aim was to analyze the relationship between certain signals sent by the franchisor and the choice of a franchise brand by the prospective franchisee. Using panel data, we found that the effects of macroeconomic variables in Spain for the 2006-2013 crisis period influenced franchisees' net income, which thus became a relevant signal in their process of choosing a franchise brand to start a business.Sob a perspectiva da Teoria de Sinais, este artigo aborda a seleção de uma franquia por um franqueado que elege esse potencial canal de distribuição pela primeira vez. O objetivo é analisar a relação entre alguns sinais enviados pelo franqueador e a escolha de uma franquia pelo potencial franqueado. Usando a metodologia de dados em painel, os resultados obtidos nos permitiram concluir que os efeitos das variáveis macroeconômicas da Espanha no período de crise econômica (2006-2013) influenciaram as receitas líquidas dos franqueados, tornando-se um sinal para eles elegerem onde abrir um estabelecimento franqueado

Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs../Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals

Social interaction patterns, therapist responsiveness, and outcome in treatments for borderline personality disorder.

Inflexible social interaction patterns are defining features of borderline personality disorder (BPD). Specific beliefs about the self and others may be activated across interaction situations, often leading to instable relationships. It may be pivotal to address these difficulties in early treatment phases, through appropriate therapist responsiveness, which means an adaptation of therapist's activity to their client's behaviours using emerging information in the process (Stiles, 2009, Clinical Psychology: Science and Practice, 16, 86). In this process-outcome study, responsiveness is operationalized by the motive-oriented therapeutic relationship (Caspar, 2007, Handbook of psychotherapeutic case formulations, 2nd ed., 251-289, Guilford), based on the Plan analysis case formulation. The present study assesses the interplay between social interaction problems and therapist responsiveness, explaining symptoms at discharge and the therapeutic alliance. In total, N = 50 clients with BPD entered the study, and standard and responsive treatments were compared. Social interaction patterns were assessed by the newly developed Borderline Interaction Patterns Scale (BIPS), applied to recorded material of three sessions per therapy. Outcome was measured by general symptoms (OQ-45), borderline symptoms (BSL-23), interpersonal problems (IIP), and the therapeutic alliance (WAI). Results suggest that in standard treatment, social interaction patterns are neither related to outcome nor the therapeutic alliance. In responsive treatment, more activation of social interaction patterns predicted better outcome on IIP and lower therapist ratings of the alliance. The conclusions seem promising for specific effectiveness of responsive treatments in particular in the interpersonal problem area of BPD. Identifying social interaction patterns early in treatment may be a crucial pathway to change for BPD. Responsive therapy activating social interaction patterns may be crucial for better outcome. Future research should focus on mechanisms of change in early treatment phases for BPD. New scale for assessing social interaction patterns specific to borderline personality disorder

Outcome of crisis intervention for borderline personality disorder and post traumatic stress disorder: a model for modification of the mechanism of disorder in complex post traumatic syndromes

<p>Abstract</p> <p>Background</p> <p>This study investigates the outcome of crisis intervention for chronic post traumatic disorders with a model based on the theory that such crises manifest trauma in the present. The sufferer's behavior is in response to the current perception of dependency and entrapment in a mistrusted relationship. The mechanism of disorder is the sufferer's activity, which aims to either prove or disprove the perception of entrapment, but, instead, elicits more semblances of it in a circular manner. Patients have reasons to keep such activity private from therapy and are barely aware of it as the source of their symptoms.</p> <p>Methods</p> <p>The hypothesis is that the experimental intervention will reduce symptoms broadly within 8 to 24 h from initiation of treatment, compared to treatment as usual. The experimental intervention sidesteps other symptoms to engage patients in testing the trustworthiness of the troubled relationship with closure, thus ending the circularity of their own ways. The study compares 32 experimental subjects with 26 controls at similar crisis stabilization units.</p> <p>Results</p> <p>The results of the Brief Psychiatric Rating Scale (BPRS) supported the hypothesis (both in total score and for four of five subscales), as did results with Client Observation, a pilot instrument designed specifically for the circular behavior targeted by the experimental intervention. Results were mostly non-significant from two instruments of patient self-observation, which provided retrospective pretreatment scores.</p> <p>Conclusions</p> <p>The discussion envisions further steps to ascertain that this broad reduction of symptoms ensues from the singular correction that distinguishes the experimental intervention.</p> <p>Trial registration</p> <p>Protocol Registration System NCT00269139. The PRS URL is <url>https://register.clinicaltrials.gov</url></p