A novel bioreactor design for enhanced stem cells proliferation and differentiation in tissue engineered constructs

[Excerpt] Recent studies have shown that culturing undifferentiated stem cells in appropriate biochemical environments and under mechanical stimulation could provide the correct signals for cellular proliferation, differentiation and subsequent extracellular matrix production. This triggered a growing interest about in vitro biomechanically-stimulating culture environments. [...]info:eu-repo/semantics/publishedVersio

Autonomic modulation of heart rate of young and postmenopausal women undergoing estrogen therapy

The aim of the present study was to determine whether estrogen therapy (ET) reduces alterations of the autonomic control of heart rate (HR) due to hypoestrogenism and aging. Thirteen young (24 ± 2.6 years), 10 postmenopausal (53 ± 4.6 years) undergoing ET (PM-ET), and 14 postmenopausal (56 ± 2.6 years) women not undergoing ET (PM) were studied. ET consisted of 0.625 mg/day conjugated equine estrogen. HR was recorded continuously for 8 min at rest in the supine and sitting positions. HR variability (HRV) was analyzed by time (SDNN and rMSSD indices) and frequency domain methods. Power spectral components are reported as normalized units (nu) at low (LF) and high (HF) frequencies, and as LF/HF ratio. Intergroup comparisons: SDNN index was higher in young (median: supine, 47 ms; sitting, 42 ms) than in PM-ET (33; 29 ms) and PM (31; 29 ms) women (P < 0.05). PM showed lower HFnu, higher LFnu and higher LF/HF ratio (supine: 44, 56, 1.29; sitting: 38, 62, 1.60) than the young group in the supine position (61, 39, 0.63) and the PM-ET group in the sitting position (57, 43, 0.75; P < 0.05). Intragroup comparisons: HR was lower in the supine than in the sitting position for all groups (P < 0.05). The HRV decrease from the supine to the sitting position was significant only in the young group. These results suggest that HRV decreases during aging. ET seems to attenuate this process, promoting a reduction in sympathetic activity on the heart and contributing to the cardioprotective effect of estrogen hormones.49149

3-amino-1,2,4-triazole induces quick and strong fat loss in mice with high fat-induced metabolic syndrome

BACKGROUND: Obesity is a growing epidemic with limited effective treatments and an important risk factor for several diseases such as metabolic syndrome (MetS). In this study, we aimed to investigate the effect of 3-amino-1,2,4-triazole (ATZ), an inhibitor of catalase and heme synthesis, in a murine model for MetS. METHODS: Male C57BL/6 mice with high-fat diet-induced MetS received ATZ (500 mg·kg(-1)·24 h(-1)) for 12 weeks. RESULTS: The HFD group showed increased blood pressure and body weight, enhanced fat deposition accompanied by an increase in adipocyte diameter, and decreased lipolysis in white adipose tissue (WAT). The expression of genes related to inflammation was increased in WAT of the HFD group. Concurrently, these mice exhibited an increase in leptin, nonesterified fatty acid (NEFA), insulin, and glucose in plasma, coupled with glucose intolerance and insulin resistance. Strikingly, ATZ prevented the increase in blood pressure and the HFD-induced obesity as observed by lower body weight, WAT index, triglycerides, NEFA, and leptin in plasma. ATZ treatment also prevented the HFD-induced increase in adipocyte diameter and even induced marked atrophy and the accumulation of macrophages in this tissue. ATZ treatment also improved glucose metabolism by increasing glucose tolerance and insulin sensitivity, GLUT4 mRNA expression in WAT in parallel to decreased insulin levels. CONCLUSIONS: In the context of HFD-induced obesity and metabolic syndrome, the fat loss induced by ATZ is probably due to heme synthesis inhibition, which blocks adipogenesis by probably decreased RevErbα activity, leading to apoptosis of adipocytes and the recruitment of macrophages. As a consequence of fat loss, ATZ elicits a beneficial systemic antiobesity effect and improves the metabolic status

Effects of starch/polycaprolactone-based blends for spinal cord injury regeneration in neurons/glial cells viability and proliferation

Spinal cord injury (SCI) leads to drastic alterations on the quality of life of afflicted individuals. With the advent of Tissue Engineering and Regenerative Medicine where approaches combining biomaterials, cells and growth factors are used, one can envisage novel strategies that can adequately tackle this problem. The objective of this study was to evaluate a blend of starch with poly(ε-caprolactone) (SPCL) aimed to be used for the development of scaffolds spinal cord injury (SCI) repair. SPCL linear parallel filaments were deposited on polystyrene coverslips and assays were carried out using primary cultures of hippocampal neurons and glial cells. Light and fluorescence microscopy observations revealed that both cell populations were not negatively affected by the SPCL-based biomaterial. MTS and total protein quantification indicated that both cell viability and proliferation rates were similar to controls. Both neurons and astrocytes occasionally contacted the surface of SPCL filaments through their dendrites and cytoplasmatic processes, respectively, while microglial cells were unable to do so. Using single cell [Ca2+ ]i imaging, hippocampal neurons were observed growing within the patterned channels and were functional as assessed by the response to a 30 mM KCl stimulus. The present data demonstrated that SPCL-based blends are potentially suitable for the development of scaffolds in SCI regenerative medicine.Portuguese Foundation for Science and Technology through funds from POCTI and/or FEDER programs (Funding to ICVS, 3B's Research Group and post doctoral fellowship to A.J. Salgado-SFRH/BPD/17595/2004)

Reinforcement of poly-l-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engineering

Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (μ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1-3μm in width) with a homogeneous distribution of BBG-Sr microparticles (<45μm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membrane's mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration. STATEMENT OF SIGNIFICANCE: PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue

A novel enzymatically-mediated drug delivery carrier for bone tissue engineering applications: combining biodegradable starch-based microparticles and differentiation agents

In many biomedical applications, the performance of biomaterials depends largely on their degradation behavior. For instance, in drug delivery applications, the polymeric carrier should degrade under physiological conditions slowly releasing the encapsulated drug. The aim of this work was, therefore, to develop an enzymaticmediated degradation carrier system for the delivery of differentiation agents to be used in bone tissue engineering applications. For that, a polymeric blend of starch with polycaprolactone (SPCL) was used to produce a microparticle carrier for the controlled release of dexamethasone (DEX). In order to investigate the effect of enzymes on the degradation behavior of the developed system and release profile of the encapsulated osteogenic agent (DEX), the microparticles were incubated in phosphate buffer solution in the presence of a-amylase and/or lipase enzymes (at physiological concentrations), at 37 C for different periods of time. The degradation was followed by gravimetric measurements, scanning electron microscopy (SEM) and Fourier transformed infrared (FTIR) spectroscopy and the release of DEX was monitored by high performance liquid chromatography (HPLC). The developed microparticles were shown to be susceptible to enzymatic degradation, as observed by an increase in weight loss and porosity with degradation time when compared with control samples (incubation in buffer only). For longer degradation times, the diameter of the microparticles decreased significantly and a highly porous matrix was obtained. The in vitro release studies showed a sustained release pattern with 48% of the encapsulated drug being released for a period of 30 days. As the degradation proceeds, it is expected that the remaining encapsulated drug will be completely released as a consequence of an increasingly permeable matrix and faster diffusion of the drug. Cytocompatibility results indicated the possibility of the developed microparticles to be used as biomaterial due to their reduced cytotoxic effects

Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.M.L. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her grant (SFRH/BD/28708/2006), Marie Curie Actions-ALEA JACTA EST (MEST-CT-2004-008104), European NoE EXPERTISSUES (NMP3-CT-2004-500283), IP GENOSTEM (LSHB-CT-2003-503161) and CARTISCAFF (POCTI/SAUIBMA/58982

One naive T cell, multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets