RESUMO
The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.
RESUMO
In this work, dextran based membranes with potential to be used as implantable devices in Tissue Engineering and Regenerative Medicine (TERM) were prepared by a straightforward strategy. Briefly, two polymers approved by the Food and Drug Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with methacrylate moieties, and subjected to photocrosslinking. Employing different weight ratios of each polymer in the formulations allowed to obtain transparent membranes with tunable physicochemical properties and low adverse host tissue response. Independently of the material, all formulations have shown to be thermally stable up to 300⯰C whilst variations in the polymer ratio resulted in membranes with different glass transition temperatures (Tg) and flexibility. The swelling capacity ranged from 50% to 200%. On the other hand, in vitro hydrolytic degradation did not show to be material-dependent and all membranes maintained their structural integrity for more than 30 days, losing only 8-12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all membranes are capable of supporting cell adhesion and viability. The in vivo biocompatibility of membranes implanted subcutaneously in rats' dorsum indicate that M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as "slight-irritant" and "non-irritant", respectively. From the histological analysis performed on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis thereby excluding the presence of toxic degradation by-products deposited or accumulated in these tissues. In combination, these results suggest that the newly developed formulations hold great potential as engineered devices for biomedical applications, where the biological response of cells and tissues are greatly dependent on the physical and chemical cues provided by the substrate.
Assuntos
Materiais Biocompatíveis/química , Dextranos/química , Membranas Artificiais , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Polpa Dentária/citologia , Humanos , Masculino , Metacrilatos/química , Poliésteres/química , Próteses e Implantes , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/metabolismo , Tela Subcutânea/metabolismo , Tela Subcutânea/patologiaRESUMO
Development of synthetic bone substitutes has arisen as a major research interest in the need to find an alternative to autologous bone grafts. Using an ovine model, the present pre-clinical study presents a synthetic bone graft (Bonelike®) in combination with a cellular system as an alternative for the regeneration of non-critical defects. The association of biomaterials and cell-based therapies is a promising strategy for bone tissue engineering. Mesenchymal stem cells (MSCs) from human dental pulp have demonstrated both in vitro and in vivo to interact with diverse biomaterial systems and promote mineral deposition, aiming at the reconstruction of osseous defects. Moreover, these cells can be found and isolated from many species. Non-critical bone defects were treated with Bonelike® with or without MSCs obtained from the human dental pulp. Results showed that Bonelike® and MSCs treated defects showed improved bone regeneration compared with the defects treated with Bonelike® alone. Also, it was observed that the biomaterial matrix was reabsorbed and gradually replaced by new bone during the healing process. We therefore propose this combination as an efficient binomial strategy that promotes bone growth and vascularization in non-critical bone defects.
RESUMO
Mesenchymal Stromal cells (MSCs) have a potential role in cell-based therapies. Foetal bovine serum (FBS) is used to supplement the basal cell culture medium but presents several disadvantages and risks. Other alternatives have been studied, including human umbilical cord blood plasma (hUCBP), aiming at the development of xeno-free culturing protocols. A comparative characterization of multicomponent metabolic composition of hUCBP and commercial FBS based on Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical analysis was performed. The analysis of 1H-NMR spectra revealed both similarities and differences between the two proposed supplements. Similar metabolites (amino acids, glucose, lipids and nucleotides) were found in the hUCBP and FBS NMR spectra. The results show that the major difference between the metabolic profiles of the two proposed supplements are due to the significantly higher levels of glucose and lower levels of lactate, glutamate, alanine and branched chain amino acids in hUCBP. Similar or slightly different levels of important proteinogenic amino acids, as well as of nucleotides, lipids were found in the hUCBP and FBS. In order to validate it's suitability for cell culture, umbilical cord-MSCs (UC-MSCs) and dental pulp stem cells (DPSCs) were expanded using hUCBP. In both hMSCs, in vitro culture with hUCBP supplementation presented similar to improved metabolic performances when compared to FBS. The two cell types tested expressed different optimum hUCBP percentage content. For DPSCs, the optimum hUCBP content was 6% and for UC-MSCs, 4%. Cultured hMSCs displayed no changes in senescence indicators, as well as maintained characteristic surface marker's expression. FBS substitution was associated with an increase in early apoptosis events, in a dose dependent manner, as well as to slight up- and down-regulation of targeted gene's expression. Tri-lineage differentiation capacity was also influenced by the substitution of FBS by hUCBP.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Metabolômica/métodos , Soro/química , Cordão Umbilical/química , Animais , Diferenciação Celular , Células Cultivadas , Meios de Cultura/química , Polpa Dentária/citologia , Humanos , Técnicas In Vitro , Espectroscopia de Prótons por Ressonância Magnética , Células-Tronco/citologiaRESUMO
One of the main goals of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, six exercises have been organized. At the present edition (GHEP-MIX06), with 25 participant laboratories, the exercise main aim was to assess mixture profiles results by issuing a report, from the proposal of a complex mock case. One of the conclusions obtained from this exercise is the increasing tendency of participating laboratories to validate DNA mixture profiles analysis following international recommendations. However, the results have shown some differences among them regarding the edition and also the interpretation of mixture profiles. Besides, although the last revision of ISO/IEC 17025:2017 gives indications of how results should be reported, not all laboratories strictly follow their recommendations. Regarding the statistical aspect, all those laboratories that have performed statistical evaluation of the data have employed the likelihood ratio (LR) as a parameter to evaluate the statistical compatibility. However, LR values obtained show a wide range of variation. This fact could not be attributed to the software employed, since the vast majority of laboratories that performed LR calculation employed the same software (LRmixStudio). Thus, the final allelic composition of the edited mixture profile and the parameters employed in the software could explain this data dispersion. This highlights the need, for each laboratory, to define through internal validations its criteria for editing and interpreting mixtures, and to continuous train in software handling.
Assuntos
Impressões Digitais de DNA/normas , Genética Forense/normas , Laboratórios/estatística & dados numéricos , Repetições de Microssatélites , Sociedades Científicas , Humanos , Funções Verossimilhança , Relatório de Pesquisa/normas , SoftwareRESUMO
We have, in previous work developed, characterized and evaluated the biocompatibility of an engineered hyaluronic acid nanogel. Here we assess the targetability of a hyaluronic acid nanogel towards CD44 overexpressing cells, in vitro and in vivo. Results obtained by flow cytometry and confocal fluorescence microscopy shows that nanogel is greatly internalized by non-small cancer lung cells (A549 cells), that overexpress CD44 receptors. The biodistribution and tumor targetability of the nanogel labelled with a near-infrared (NIR) probe were performed, in mice, through a non-invasive imaging system. Results revealed nanogel high targetability towards an induced subcutaneous A549 tumor. Nanogels pharmacokinetics was evaluated also in healthy animals, and Alexa Fluor 680 labelled nanogel exhibited higher accumulation in liver, kidneys and skin. Also, a comparative biodistribution study was performed, using two NIR imaging probes, Cy5.5 and Alexa Fluor 680.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Géis , Ácido Hialurônico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanotecnologia , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Ácido Hialurônico/farmacocinética , Técnicas In Vitro , Rim/metabolismo , Fígado/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Pele/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição TecidualRESUMO
Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.
Assuntos
Cromossomos Humanos X , Cromossomos Humanos Y , Impressões Digitais de DNA , DNA Mitocondrial/genética , Laboratórios/normas , Repetições de Microssatélites , Amelogenina/genética , Análise Química do Sangue , Feminino , Genética Forense , Marcadores Genéticos , Haplótipos , Humanos , Masculino , Saliva/química , Sêmen/químicaRESUMO
One of the main objectives of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the area of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, three exercises have been organized (GHEP-MIX01, GHEP-MIX02 and GHEP-MIX03), with 32, 24 and 17 participant laboratories respectively. The exercise aims to give a general vision by addressing, through the proposal of mock cases, aspects related to the edition of mixture profiles and the statistical treatment. The main conclusions obtained from these exercises may be summarized as follows. Firstly, the data show an increased tendency of the laboratories toward validation of DNA mixture profiles analysis following international recommendations (ISO/IEC 17025:2005). Secondly, the majority of discrepancies are mainly encountered in stutters positions (53.4%, 96.0% and 74.9%, respectively for the three editions). On the other hand, the results submitted reveal the importance of performing duplicate analysis by using different kits in order to reduce errors as much as possible. Regarding the statistical aspect (GHEP-MIX02 and 03), all participants employed the likelihood ratio (LR) parameter to evaluate the statistical compatibility and the formulas employed were quite similar. When the hypotheses to evaluate the LR value were locked by the coordinators (GHEP-MIX02) the results revealed a minor number of discrepancies that were mainly due to clerical reasons. However, the GHEP-MIX03 exercise allowed the participants to freely come up with their own hypotheses to calculate the LR value. In this situation the laboratories reported several options to explain the mock cases proposed and therefore significant differences between the final LR values were obtained. Complete information concerning the background of the criminal case is a critical aspect in order to select the adequate hypotheses to calculate the LR value. Although this should be a task for the judicial court to decide, it is important for the expert to account for the different possibilities and scenarios, and also offer this expertise to the judge. In addition, continuing education in the analysis and interpretation of mixture DNA profiles may also be a priority for the vast majority of forensic laboratories.
Assuntos
Repetições de Microssatélites , Humanos , Inquéritos e QuestionáriosRESUMO
There has been very little work published on the variation of reporting practices of mixtures between laboratories, but it has been previously demonstrated that there is little consistency. This is because there is no current uniformity of practice, so different laboratories will operate using different rules. The interpretation of mixtures is not solely a matter of using some software to provide 'an answer'. An assessment of a case will usually begin with a consideration of the circumstances of a crime. Assumptions made about the numbers of contributors follow from an examination of the electropherogram(s)--and these may differ between the prosecution and the defence hypotheses. There may be a necessity to evaluate several sets of hypotheses for any given case if the circumstances are uncertain. Once the hypotheses are formulated, the mathematical analysis is complex and can only be accomplished by the use of specialist software. In order to obtain meaningful results, it is essential that scientists are trained, not only in the use of the software, but also in the methodology to understand the likelihood ratio concept that is used. The Euroforgen-NoE initiative has developed a training course that utilizes the LRmix program to carry out the calculations. This software encompasses the recommendations of the ISFG DNA commissions on mixture interpretation and is able to interpret samples that may come from two or more contributors and may also be partial profiles. Recently, eighteen different laboratories were trained in the methodology. Afterwards they were asked to independently analyze two different cases with partial mixture DNA evidence and to write a statement court-report. We show that by introducing a structured training programme, it is possible to demonstrate, for the first time, that a high degree of standardization, leading to uniformity of results can be achieved by participating laboratories.
Assuntos
Impressões Digitais de DNA/normas , Laboratórios/normas , Funções Verossimilhança , Software , Europa (Continente) , Humanos , Estatística como Assunto/educaçãoRESUMO
The GHEP-ISFG Working Group performed a collaborative exercise to monitor the current practice of mitochondrial (mt)DNA reporting. The participating laboratories were invited to evaluate a hypothetical case example and assess the statistical significance of a match between the haplotypes of a case (hair) sample and a suspect. A total of 31 forensic laboratories participated of which all but one used the EMPOP database. Nevertheless, we observed a tenfold range of reported LR values (32-333.4), which was mainly due to the selection of different reference datasets in EMPOP but also due to different applied formulae. The results suggest the need for more standardization as well as additional research to harmonize the reporting of mtDNA evidence.
Assuntos
DNA Mitocondrial/genética , Bases de Dados Genéticas , Haplótipos , HumanosRESUMO
Objetivo: La terapia asistida con animales se está utilizando de diversas formas para mejorar la calidad de vida de las personas con dolor crónico. El objetivo de este trabajo fue realizar una revisión sistemática de las publicaciones disponibles y resumir lo que se sabe sobre esta forma de terapia complementaria. Material y métodos: Se realizó una búsqueda en las bases de datos MEDLINE (vía PubMed), CINAHL, PsycINFO, Web of Science Core Collection, Psychology and Behavioral Sciences Collection desde sus inicios hasta enero de 2016. Resultados: Se identificaron un total de 179 artículos, de los que se revisaron 135 para su potencial inclusión en este trabajo. Finalmente, se han incluido 7 trabajos. Los resultados de estos estudios muestran que, en general, las personas que han participado de estas terapias informan de una menor intensidad de dolor, mejor estado de ánimo y, en general, de mejor calidad de vida. Los trabajos publicados no describen detalladamente el tipo de intervención planteada ni sus componentes específicos; así son difíciles de replicar. Conclusiones: Aunque los estudios revisados muestran resultados positivos, las investigaciones han utilizado diseños poco elaborados y de escasa exigencia metodológica. En futuras investigaciones es de esperar un mayor rigor y control. También una mejor descripción de las intervenciones, detalle fundamental para identificar qué variables son las responsables últimas de los efectos beneficiosos de los que se informan (AU)
Objective: Animal-assisted therapy is used in various ways to improve the quality of life of people with chronic pain. The aim of this work was to conduct a systematic review of the literature, and summarize what is known about this form of complementary therapy. Methods: A search was conducted in the following databases MEDLINE (via PubMed), CINAHL, PsycINFO, Web of Science Core Collection, Psychology and Behavioral Sciences Collection, since its inception until January 2016. Results: A total of 179 items were found, and 135 were reviewed for potential inclusion in this study. Finally, we have included 7 articles. The results of the studies reported in these articles show that, in general, people who have participated in animal-assisted therapy report lower pain intensity, improved mood and overall better quality of life. Published reports do not provide detailed information about the type of intervention being used, nor its specific components, thus offering little possibility of replication. Conclusions: Reviewed studies show positive results, however these are based on poor designs. Future research with greater rigor and control is warranted. Future publications need to provide better descriptions of the interventions that are used, this is critical to identify which variables are ultimately responsible for the beneficial effects that are being reported (AU)
Assuntos
Humanos , Animais , Masculino , Feminino , Terapia Assistida com Animais/métodos , Dor Crônica/terapia , Qualidade de Vida , Manejo da Dor/instrumentação , Manejo da Dor/métodos , Bibliometria , Cães , Manejo da Dor/normas , Manejo da Dor , Doença Crônica/epidemiologia , Doença Crônica/terapiaRESUMO
The present work quantifies, through principal components analysis (PCA) the relationships among the variability of breath-by-breath ventilatory parameters [minute-ventilation (VE), tidal volume (Vt), and respiratory rate (FR)] during a maximal progressive exercise test. The results show that the first and second eigenvalues of the covariant matrix contains almost 90% of the variables' variance possible to see through the PCA, which means that the problem can be reduced by a two-dimensional analysis. The results show a close similarity between the global variability in two groups test, athletes and sedentary (control). For the athletes group, the parameter Vt is responsible for the high VE variability values while in the sedentary group the FR is more relevant for VE variability. The result improves the knowledge about respiratory variability during exercise, showing that Vt's and FR's variabilities contribute in different ways to global ventilation variability during a maximal cardiopulmonary exercise test in athletes and sedentary men.
Assuntos
Mecânica Respiratória/fisiologia , Comportamento Sedentário , Esportes/fisiologia , Adulto , Antropometria/métodos , Teste de Esforço/métodos , Humanos , Masculino , Análise de Componente Principal , Adulto JovemRESUMO
A male ovine linkage map has been constructed on the basis of 11 half-sib families of a commercial population of Spanish Churra sheep as part of a genome scan for quantitative trait loci mapping. A total of 1421 daughters and their sires were genotyped for 182 microsatellite markers evenly distributed along the ovine autosomes. A total of 259,192 genotypes were obtained, generating an average of 669 informative meioses per marker. An autosomal genome length of 3262 cM was estimated for the Churra population with a mean marker interval of 17.86 cM. Our map represents an approximate 90% coverage of the autosomal ovine genome and constitutes a useful tool for the genetic dissection of complex traits in this breed. General agreement was found between the Churra map and other published maps for sheep, despite certain length discrepancies.
Assuntos
Ovinos/genética , Animais , Mapeamento Cromossômico , Masculino , Repetições de Microssatélites , Locos de Características Quantitativas , Ovinos/classificação , Espanha , Especificidade da EspécieRESUMO
The alpha-dispersion amplitude of suspensions of colloidal particles is usually calculated from the low-frequency asymptotic of the frequency-dependent solution of the electrodiffusion equations. Since these equations written in spheroidal coordinates do not separate, no theoretical results exist for the low-frequency dielectric properties of suspensions of spheroidal particles. In order to sidestep this problem, we used another method which relates the dielectric properties to the energy stored in the system (Grosse, C., Ferroelectrics 86, 171 (1988)) which, at low frequencies, mainly corresponds to the Gibbs free energy associated to the field-induced electrolyte concentration changes outside the double layer (Grosse, C. and Shilov, V. N., J. Colloid Interface Sci. 193, 178 (1997)). This method permits us to calculate the static permittivity by solving a purely static problem, which makes it possible to calculate analytically the alpha-dispersion amplitude of suspensions of spheroidal particles since the electrodiffusion equations do separate in the static case. We also calculate the characteristic time of the alpha-dispersion from the dispersion amplitude and the static and high-frequency values of the dipolar coefficient. The analytical results obtained are presented and discussed for both prolate and oblate geometries, and for parallel, perpendicular, and random orientations of the particles with respect to the applied field. Copyright 1999 Academic Press.