Luís Barreiros Martins

Gellan gum: A new biomaterial for cartilage tissue engineering applications

Gellan gum is a polysaccharide manufactured by microbial fermentation of the Sphingomonas paucimobilis microorganism, being commonly used in the food and pharmaceutical industry. It can be dissolved in water, and when heated and mixed with mono or divalent cations, forms a gel upon lowering the temperature under mild conditions. In this work, gellan gum hydrogels were analyzed as cells supports in the context of cartilage regeneration. Gellan gum hydrogel discs were characterized in terms of mechanical and structural properties. Transmissionelectron microscopy revealed a quite homogeneous chain arrangement within the hydrogels matrix, and dynamic mechanical analysis allowed to characterize the hydrogels discs viscoelastic properties upon compression solicitation, being the compressive storage and loss modulus of approximately 40 kPa and 3 kPa, respectively, at a frequency of 1 Hz. Rheological measurements determined the sol-gel transition started to occur at approximately 36 degrees C, exhibiting a gelation time of approximately 11 s. Evaluation of the gellan gum hydrogels biological performance was performed using a standard MTS cytotoxicity test, which showed that the leachables released are not deleterious to the cells and hence were noncytotoxic. Gellan gum hydrogels were afterwards used to encapsulate human nasal chondrocytes (1 x 10(6) cells/mL) and culture them for total periods of 2 weeks. Cells viability was confirmed using confocal calcein AM staining. Histological observations revealed normal chondrocytes morphology and the obtained data supports the claim that this new biomaterial has the potential to serve as a cell support in the field of cartilage regeneration.

Injectable gellan gum hydrogels with autologous cells for the treatment of rabbit articular cartilage defects

In this work, the ability of gellan gum hydrogels coupled with autologous cells to regenerate rabbit full‐thickness articular cartilage defects was tested. Five study groups were defined: (a) gellan gum with encapsulated chondrogenic predifferentiated rabbit adipose stem cells (ASC + GF); (b) gellan gum with encapsulated nonchondrogenic predifferentiated rabbit adipose stem cells (ASC); (c) gellan gum with encapsulated rabbit articular chondrocytes (AC) (standard control); (d) gellan gum alone (control); (e) empty defect (control). Full‐thickness articular cartilage defects were created and the gellan gum constructs were injected and left for 8 weeks. The macroscopic aspect of the explants showed a progressive increase of similarity with the lateral native cartilage, stable integration at the defect site, more pronouncedly in the cell‐loaded constructs. Tissue scoring showed that ASC + GF exhibited the best results regarding tissue quality progression. Alcian blue retrieved similar results with a better outcome for the cell‐loaded constructs. Regarding real‐time PCR analyses, ASC + GF had the best progression with an upregulation of collagen type II and aggrecan, and a downregulation of collagen type I. Gellan gum hydrogels combined with autologous cells constitute a promising approach for the treatment of articular cartilage defects, and adipose derived cells may constitute a valid alternative to currently used articular chondrocytes.

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue‐engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real‐time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round‐shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real‐time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright

Motor uncoordination and neuropathology in a transgenic mouse model of Machado-Joseph disease lacking intranuclear inclusions and ataxin-3 cleavage products

Machado-Joseph disease (MJD) is a late-onset neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the ataxin-3 protein. We generated two transgenic mouse lineages expressing the expanded human ataxin-3 under the control of the CMV promoter: CMVMJD83 and CMVMJD94, carrying Q83 and Q94 stretches, respectively. Behavioral analysis revealed that the CMVMJD94 transgenic mice developed motor uncoordination, intergenerational instability of the CAG repeat and a tissue-specific increase in the somatic mosaicism of the repeat with aging. Histopathological analysis of MJD mice at early and late stages of the disease revealed neuronal atrophy and astrogliosis in several brain regions; however, we found no signs of microglial activation or neuroinflammatory response prior to the appearance of an overt phenotype. In our model, the appearance of MJD-like symptoms was also not associated with the presence of ataxin-3 cleavage products or intranuclear aggregates. We propose the transgenic CMVMJD94 mice as a useful model to study the early stages in the pathogenesis of MJD and to explore the molecular mechanisms involved in CAG repeat instability.

Lithium prevents stress-induced reduction of vascular endothelium growth factor levels

Understanding the mechanisms that regulate postnatal neurogenesis is becoming increasingly relevant since its modulation has been implicated in the pathogenesis of certain neuropsychiatric disorders. Lithium is a mood stabilizer known to increase hippocampal neurogenesis. Lithium also results in increased levels of the angiogenic factor vascular endothelial growth factor (VEGF). Since VEGF was recently shown to have neurogenic properties, we were interested to examine whether lithium administration might also be accompanied by alterations in VEGF expression in the hippocampus of normal and stressed rats; the latter treatment was introduced to reproduce some of the psychopathological signs for which lithium is used therapeutically. The expression of VEGF in the hippocampus in stressed animals was lower than that in controls, but the effect of stress was significantly attenuated in animals concomitantly receiving lithium. Double staining for VEGF and specific markers for immature neurons, mature neurons and astroglia revealed that immature neurons were most sensitive to the VEGF-inhibiting effects of stress. Confirming the involvement of a known regulatory pathway in these actions of lithium, we demonstrated that lithium co-administration prevented the stress-induced upregulation of glycogen synthase kinase-3beta (GSK-3beta) and down-regulation of beta-catenin expression; GSK-3beta is a known primary lithium target and its inhibition by this mood stabilizer leads to an upregulation of beta-catenin and subsequently, an increase of VEGF. Our results suggest that the actions of lithium, and possibly its therapeutic efficacy as a mood stabilizer also, are mediated by VEGF.

Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome

Rett syndrome is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 gene (MECP2). Several neural systems are affected in Rett, resulting in an autonomic dysfunction, a movement disorder with characteristic loss of locomotor abilities and profound cognitive impairments. A deregulation of monoamines has been detected in the brain and cerebrospinal fluid of both Rett patients and a Rett syndrome murine model, the Mecp2 knock-out mouse. Our goal was to characterize the onset and progression of motor dysfunction in Mecp2(tm1.1Bird) knock-out mice and the possible neurochemical alterations in different brain regions potentially playing a role in Rett-like pathophysiology, at two different time-points, at weaning (3 weeks old) and in young adults when overt symptoms are observed (8 weeks old). Our results revealed significant age- and region-dependent impairments in these modulatory neurotransmitter systems that correspond well with the motor phenotype observed in these mice. At 3 weeks of age, male Mecp2 knock-out mice exhibited ataxia and delayed motor initiation. At this stage, noradrenergic and serotonergic transmission was mainly altered in the prefrontal and motor cortices, whereas during disease progression the neurochemical changes were also observed in hippocampus and cerebellum. Our data suggest that the deregulation of norepinephrine and serotonin systems in brain regions that participate in motor control are involved in the pathophysiology of Rett syndrome motor phenotypes. Moreover, we highlight the contribution of cortical regions along with the brainstem to be in the origin of the pathology and the role of hippocampus and cerebellum in the progression of the disease rather than in its establishment.

Mapping cellular gains and losses in the postnatal dentate gyrus: Implications for psychiatric disorders

Neurogenesis and apoptosis occur contemporaneously in the postnatal hippocampal dentate gyrus and have been implicated in mood and cognitive disorders. Particularly, neurogenesis correlates with the manifestation of antidepressant effects, but its quantitative and topographical relationship with concomitant cell death has not been investigated. Accordingly, we applied stereological measurements to obtain synchronized topographical maps of these two events in rats aged 1 and 3 months under basal conditions; the two ages were chosen to represent neuro-developmental windows during which cell proliferation and death are occurring at peak and relatively steady levels, respectively. Our analysis shows that apoptotic cells are evenly distributed throughout the dentate gyrus, although the incidence of apoptosis decreased gradient-wise from the tip of the suprapyramidal layer and was highest in the external third of the granule cell layer. Interestingly, apoptosis was higher in the left hippocampus. In addition, we confirm previous less stringent studies demonstrating that neurogenesis occurs differentially in the dorsal-ventral axis of the hippocampus and in suprapyramidal-infrapyramidal blades of the dentate gyrus. These results raise intriguing new questions regarding the coordinated regulation of hippocampal neurogenesis and apoptosis since the two processes apparently share common regulatory factors. In addition, these findings open questions with respect to the functional significance of topographical gradients in neurogenesis and apoptosis in the context of the etiopathogenesis of neuropsychiatric diseases and the reported dependence on the efficacy of therapeutic agents on the generation of new hippocampal neurons.

Regenerative Braking Potential and Energy Simulations for a Plug-In Hybrid Electric Vehicle Under Real Driving Conditions

There are several possible configurations and technologies for the powertrains of electric and hybrid vehicles, but most of them will include advanced energy storage systems comprising batteries and ultra-capacitors. Thus, it will be of capital importance to evaluate the power and energy involved in braking and the fraction that has the possibility of being regenerated. The Series type Plug-in Hybrid Electric Vehicle (S-PHEV), with electric traction and a small Internal Combustion Engine ICE) powering a generator, is likely to become a configuration winner. The first part of this work describes the model used for the quantification of the energy flows of a vehicle, following a particular route. Normalised driving-cycles used in Europe and USA and real routes and traffic conditions were tested. The results show that, in severe urban driving-cycles, the braking energy can represent more than 70% of the required useful motor-energy. This figure is reduced to 40% in suburban routes and to a much lower 18% on motorway conditions. The second part of the work consists on the integration of the main energy components of an S-PHEV into the mathematical model. Their performance and capacity characteristics are described and some simulation results presented. In the case of suburban driving, 90% of the electrical motor-energy is supplied by the battery and ultra-capacitors and 10% by the auxiliary ICE generator, while on motorway these we got 65% and 35%, respectively. The simulations also indicate an electric consumption of 120 W.h/km for a small 1 ton car on a suburban route. This value increases by 11% in the absence of ultra-capacitors and a further 28% without regenerative braking.Copyright

On solving the profit maximization of small cogeneration systems

Cogeneration is a high-efficiency technology that has been adapted to small and micro scale applications. In this work, the development and test of a numerical optimization model is carried out in order to implement an analysis that will lead to the optimal design of a small cogeneration system. The main idea is the integration of technical and economic aspects in the design of decentralized energy production considering the requirements for energy consumption for the building sector. The nonlinear optimization model was solved in MatLab®environment using two local optimization methods: the Box and the SQP method. The optimal solution provided a positive annual worth and disclosed reasonable values for the decision variables of the thermo-economic model. Both methods converged for the same solution, demonstrating the validity of the implemented approach. This study confirmed that the use of numerical optimization models is of utmost importance in the assessment of energy systems sustainability.

Modeling a stirling engine for cogeneration applications

The interest on decentralized power generation technology has been drastically increasing over the last few years. This great interest is due to the necessity of achieving new ways for improving energy efficiency, the national security of energy supply and the reduction of carbon dioxide emissions. Combined heat and power generation (CHP) systems can be a good option to achieve those goals. In Europe and for the building sector, this fact can be translated in the development of low power systems (micro-CHP), designed to fulfill building equivalent loads. These systems will replace the usual boilers that satisfy the dwelling’s heat requirements and, additionally, generate electricity for own consumption or export back to the electricity grid. The most cited technologies in small and micro-scale are Fuel Cells, Internal Combustion Engines, and Stirling Engines. Stirling Engines are gaining some attention due to their advantages: high total efficiency, fuel flexibility, low emissions, low noise/vibration levels and good performance at partial load. Due to these characteristics, Stirling engines seem to be a good alternative for residential energy conversion, and thus, a pathway for more energy-efficient systems that rise to the challenges of increasing market competition. Many studies have been conducted in order to assess Stirling Engines performance, but the integration of technical and economic evaluation for micro-CHP systems applications is an issue that is not focused in literature, and is the final objective of this project.Copyright