Dynamic Coculture of a Prevascularized Engineered Bone Construct

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect.

Hypoglyceamia in a Patient with a Solitary Fibrous Tumour

Objective: To illustrate an unusual mechanism causing hypoglycaemia. Material and methods: A 76-year-old man presented with episodes of agitation and confusion and was resuscitated with oral glucose gel when found to be hypoglycaemic. Results: A CT scan for an abdominal mass confirmed a solitary fibrous tumour (SFT). The sarcoma multidisciplinary team suggested conservative management. The patient's episodic hypoglycaemia was managed with diet modification including corn-based starch, scheduled snacks and dexamethasone. Glucose levels were within normal range at discharge from hospital. The patient was referred to the palliative care team for follow-up. Conclusion: SFTs causing non-islet cell tumour hypoglycaemia are difficult to treat. KEYWORDS

Dermatofibrosarcoma protuberans en pacientes pediátricos

Antecedentes y Objetivos. Presentamos 3 casos de dermatofibrosarcomaprotuberans (DFSP) en pacientes pediátricos para documentar la presentación clínica poco frecuente de esta patología. Pacientes y Método. Realizamos un análisis descriptivo retrospectivo de los 3 últimos casos de DFSP en nuestro centro durante los años 2010-2013. Resultados. Una niña de 12 años remitida desde otra institución por DFSP en parte distal del muslo, en la que ampliamos bordes quirúrgicos de resección en 3 cm y practicamos cobertura con dermis artificial e injerto autólogo; tras 1 año de observación sin recidiva, realizamos reconstrucción del defecto mediante injertos autólogos de grasa. Otra niña de 12 años diagnosticada de Síndrome de Hamartomatosis Múltiple asociado al gen PTEN, que presentó DFSP en labio mayor derecho y en mama derecha, recidivante en esta última localización. Por último, un DFSP congénito en una paciente remitida a los 3 meses de vida con tumoración gigante de pared abdominal. El diagnóstico orientativo inicial con resonancia magnética con gadolino, solo se confirmó finalmente por los hallazgos histopatológicos de la pieza de resección; ni la punción aspiración ni la biopsia cutánea lograron confirmar el diagnóstico. Conclusiones. El DFSP es un tipo de sarcoma raro de la piel del niño y del adulto, localmente agresivo y con una alta tasa de recidiva. La escisión local amplia puede provocar mutilaciones y desfigurar al paciente; por ello la cirugía micrográfica de Mohs permite reducir los márgenes quirúrgicos.

Uterine sarcomas: clinical presentation and MRI features

Uterine sarcomas are a rare heterogeneous group of tumors of mesenchymal origin, accounting for approximately 8% of uterine malignancies. They comprise leiomyosarcoma, endometrial stromal sarcoma, undifferentiated endometrial sarcoma, and adenosarcoma. Compared with the more common endometrial carcinomas, uterine sarcomas behave more aggressively and are associated with a poorer prognosis. Due to their distinct clinical and biological behavior, the International Federation of Gynecology and Obstetrics introduced a new staging system for uterine sarcomas in 2009, categorizing uterine carcinosarcoma as a variant of endometrial carcinoma, rather than a pure sarcoma. Magnetic resonance imaging (MRI) has a developing role in the assessment of these malignancies. Features such as tumor localization, irregular or nodular margins, necrosis, rapid growth, intense contrast enhancement, and restriction at diffusion-weighted imaging can suggest the diagnosis and help differentiate from more common leiomyomas and endometrial carcinoma. MRI is therefore extremely useful in preoperative detection and staging and, consequently, in determination of appropriate management. This pictorial review aims to discuss the clinical features of uterine sarcomas, as well as their most common appearances and distinct characteristics in MRI.

Efeitos ginecológicos do "tamoxifeno": contributo da radiologia

O “Tamoxifeno” (TAM) é a terapêutica anti-estrogénica de escolha nas doentes com cancro da mama. Os efeitos proliferativos do TAM em idade pós-menopausa têm sido associados a hiperplasia, pólipos, carcinoma e sarcoma do endométrio. As doentes tratadas com TAM têm também maior incidência de leiomiomas, adenomiose e endometriose, assim como maior risco de quistos do ovário. O método de primeira linha na vigilância das mulheres sob TAM é a ecografia transvaginal (US-TV). O endométrio apresenta-se frequentemente espessado e com áreas quísticas, aspectos passíveis de melhor caracterização por histerossonografia e ressonância magnética. Nas imagens ponderadas em T2, um endométrio espessado e heterogéneo com captação de aspecto reticulado e a opacificação da interface endométrio-miométrio associam-se a lesões de pior prognóstico (pólipos, hiperplasia atípica e neoplasia). O conhecimento dos efeitos ginecológicos do TAM e da sua tradução radiológica promove o diagnóstico precoce e adequado encaminhamento destas doentes.

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Inmunoterapia autóloga con células dendríticas en modelos caninos con tumor venéreo transmisible

La inmunoterapia autóloga utiliza células del mismo cuerpo para estimular y restaurar las defensas naturales del sistema inmunológico. Algunas de las células que han sido utilizado en años recientes son: linfocitos infiltrantes de tumor, linfocitos T citotóxicos, células asesinas activadas por linfocinas y células dendríticas (CD). Las CD son células especializadas y su función es el capturar, procesar y presentar antígenos a los linfocitos para iniciar una respuesta inmune. El tumor venéreo transmisible (TVT), también conocido como sarcoma infeccioso o tumor de Sticker, es un cáncer transmisible en perros que afecta mayormente los genitales externos y se transmite durante el coito. En este trabajo se implementó por primera vez un modelo de TVT en el órgano genital (vagina) de los pacientes y se les administró la inmunoterapia autóloga con CD específicas contra el tumor. Para estudiar esta terapia se utilizaron tres grupos experimentales: el tumor sin tratamiento, el tumor tratado con sangre completa autóloga, y el tumor tratado con CD autólogas específicas para el TVT. Por último se evaluó la capacidad inmunológica del extracto tumoral total (ETT) del tumor como método de prevención in vivo. Para el tratamiento autólogo con las CD, se esperó que el tumor midiese 3cm, se realizó un cultivo primario de las células de TVT y se les extrajo el 4% de su peso corporal de sangre a los pacientes para realizar una diferenciación de los monocitos a CD. Para evaluar el efecto de la inmunoterapia autóloga con CD se observaron los efectos secundarios, el tamaño tumoral, las poblaciones de linfocitos, los niveles de IFN-γ en sangre y los linfocitos infiltrantes de tumor por histopatología. Los monocitos se diferenciaron a CD y se les realizó un análisis fenotípico mediante citometría de flujo. Los monocitos mostraron una expresión de CD14+ de 80.1%, CD80+ de 15.6%, CD83+ de 0.4% y un DLA II de1.8%. En las CD se obtuvo una expresión de 8.7% para CD14+, 80.3% para CD80+, 76.4% para CD83+ y 86.5% para DLA II y 62% en la prueba de fagocitosis. La terapia no mostró ningún efecto secundario en nuestro grupo experimental y hubo una regresión tumoral del 100% para la semana doce. Se encontró un aumento de expresión celular en sangre de CD4+ de 29%, de CD8+ de 34% y de IFN-γ de 120 pg/mL. Nuestros resultados demuestran que la inmunoterapia autóloga con CD específicas inducen una regresión del TVT en caninos.

Evaluation of the antioxidant activity and antitumor activity of marine invertebrates extracts

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2014

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

Biomimetic topography in orthopaedic ceramic

The primary objective of this research was to perform an in vitro assessment of the ability of microscale topography to alter cell behaviour, with specific regard to producing favourable topography in an orthopaedic ceramic material suitable for implantation in the treatment of arthritis. Topography at microscale and nanoscale alters the bioactivity of the material. This has been used in orthopaedics for some time as seen with optimal pore size in uncemented hip and knee implants. This level of topography involves scale in hundreds of micrometres and allows for the ingrowth of tissue. Topography at smaller scale is possible thanks to progressive miniaturisation of technology. A topographic feature was created in a readily available clinically licensed polymer, Polycaprolcatone (PCL). The effect of this topography was assessed in vitro. The same topography was transferred to the latest generation composite orthopaedic ceramic, zirconia toughened alumina (ZTA). The fidelity of reproduction of the topography was examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). These investigations showed more accurate reproduction of the topography in PCL than ZTA with some material artefacts in the ZTA. Cell culture in vitro was performed on the patterned substrates. The response of osteoprogenitor cells was assessed using immunohistochemistry, real-time polymerase chain reaction and alizarin staining. These results showed a small effect on cell behaviour. Finally metabolic comparison was made of the effects created by the two different materials and the topography in each. The results have shown a reproducible topography in orthopaedic ceramics. This topography has demonstrated a positive osteogenic effect in both polycaprolactone and zirconia toughened alumina across multiple assessment modalities.

Controlled surface nanotopography and oxygen plasma treatment of PEEK to improve cellular response

Poly(aryl-ether-ether-ketone) (PEEK) is a semi crystalline polymer which exhibits properties that make it an attractive choice for use as an implant material. It displays natural radiolucency, and MRI compatibility, as well as good chemical and sterilization resistance, both of which make it of particular interest in orthopaedic implants. However, PEEK has demonstrated poor cellular adhesion both in vitro and in vivo. This is problematic as implant surfaces that do not develop a layer of adhesive cells are at risk of undergoing fibrous encapsulation, which in turn leads to lack of a strong interface between the implant device and the patient tissue, which can in turn lead to failure of the implant and revision surgery . As incorporating nanotopography into a polymer surface has been demonstrated to be able to direct the differentiation behaviour of stem cells, a possible solution to PEEKs underlying issues with poor cellular response would be to incorporate specific nanoscale topography into the material surface through injection moulding, and then analysing if this is a viable method for addressing PEEKs issues with cellular response. In addition to nanoscale topography, the experimental PEEK surfaces were treated with oxygen plasma to address the underlying cytophobicity of the material. As this type of treatment has been documented to be capable of etching the PEEK surface, experiments were carried out to quantify the effect of this treatment, both on the ability of cells to adhere to the PEEK surface, as well as the effect it has upon the nanotopography present at the PEEK surface. The results demonstrated that there were a range of plasma treatments which would significantly improve the ability of cells to adhere to the PEEK surface without causing unacceptable damage to the nanotopography. Three different types of cells with osteogenic capacity were tested with the PEEK surfaces to gauge the ability of the topography to alter their behaviour: SAOS-2, osteoprogenitors and 271+ MSCs. Due to PEEKs material properties (it is non transparent, exhibits birefringence and is strongly autofluorescent) a number of histological techniques were used to investigate a number of different stages that take place in osteogenesis. The different cell types did display slightly different responses to the topographies. The SAOS-2 cells cultured on surfaces that had been plasma treated for 2 minutes at 200W had statistically significantly higher levels of von Kossa staining on the NSQ surface compared to the planar surface, and the same experiment employing alizarin red staining, showed a statistically significantly lower level of staining on the SQ surface compared to the planar surface. Using primary osteoprogenitor cells designed to look into if whether or not the presence of nanotopography effected the osteogenic response of these cells, we saw a lack of statistically significant difference produced by the surfaces investigated. By utilising HRP based immunostaining, we were able to investigate, in a quantitative fashion, the production of the two osteogenic markers osteopontin and osteocalcin by cells. When stained for osteocalcin, the SQ nanotopography had total percentage of the surface with stained material, average area and average perimeter all statistically significantly lower than the planar surface. For the cells that were stained for osteopontin, the SQ nanotopgraphy had a total percentage of the surface with stained material, average area and average perimeter all highly statistically significantly lower than those of the planar surface. Additionally, for this marker the NSQ nanotopography had average areas and average perimeters that were highly significantly higher than those of the planar surface. There were no significant differences for any of the values investigated for the 271+ MSC’s When plasma treatment was varied, the SAOS-2 cells demonstrated an overall trend i.e. increasing the energy of plasma treatment in turn leads to an increase in the overall percentage of staining. A similar experiment employing stem cells isolated from human bone marrow instead of SAOS-2 cells showed that for polycarbonate surfaces , used as a control, mineralization is statistically significantly higher on the NSQ nanopattern compared to the planar surface, whereas on the PEEK surfaces we observe the opposite trend i.e. the NSQ nanotopography having a statistically significantly lower amount of mineralization compared to the planar surface at the 200W 2min and 30W 1min plasma treatments. The standout trend from the PEEK results in this experiment was that the statistically significant differences on the PEEK substrates were clustered around the lower energy plasma treatments, which could suggest that the plasma treatment disrupted a function of the nanotopograhy which is why, as the energy increases, there are less statistically significant differences between the NSQ nanotopography and the Planar surface This thesis documents the response of a number of different types of cells to specific nanoscale topographies incorporated into the PEEK surface which had been treated with oxygen plasma. It outlines the development of a number of histological methods which measure different aspects of osteogenesis, and were selected to both work with PEEK, and produce quantitative results through the use of Cell Profiler. The methods that have been employed in this body of work would be of interest to other researchers working with this material, as well as those working with similarly autofluorescent materials.

Establishment of antibody panels and histochemical techniques in routine tumor diagnosis in Veterinary Pathology

In Portugal, Veterinary Pathology is developing rapidly, and in recent years we assist to the emergence of private laboratories and the restructuring of universities,polytechnics and public laboratories.The Portuguese Society of Animal Pathology,through its actions and its associates has been keeping the discussion among its peers in order to standardizethe criteria of description,classification and evaluation of cases which are the subject of our daily work.One of the last challenges is associated with the use of routine histochemical techniques and immunohistochemistry, in an effort to establish standardized panels for tumour diagnosis, which could eventually reduce each analysis cost.For this purpose a simple survey was built, in which all collaborators answered questions about the markers used for carcinoma, sarcoma and round cell tumour diagnosis, as well as general questions related with the subject. We obtained twenty-one answered to the questions, from public and private laboratories.In general, in most cases immunohistochemical and histochemical methods are used for diagnosis.The wide spectrum cytokeratins are universally used to confirm carcinoma, and vimentin for sarcoma. The CD3 marker is used by all laboratories to identify T lymphocytes. For the diagnosis of B-cell lymphoma, the marker used is not consensual. In each laboratory there are different markers for more specific situations and only two labs perform PCR techniques for diagnosis. These data will be presented to promote extended discussion,namely to reach a consensus when different markers are used.

Microenvironment and prognostic factors in bone tumors: IDH mutations in chondrosarcoma

Microenvironment in bone tumors is a dynamic entity composed of cells from different origins (immune cells, stromal cells, mesenchymal stem cells, endothelial cells, pericytes) and vascular structures surrounded by a matrix of different nature (bone, cartilage, myxoid). Interactions between cancer cells and tumor microenvironment (TME) are complex and can change as tumor progress, but are also crucial in determining response to cancer therapies. Chondrosarcoma is the second most frequent bone cancer in adult age, but its treatment still represents a challenge, for the intrinsic resistance to conventional chemotherapy and radiation therapy. This resistance is mainly due to pathological features, as dense matrix, scarce mitoses and poor vascularization, sustained by biological mechanisms only partially delucidated. Somatic mutation in the Krebs cycle enzyme isocytrate dehydrogenase (IDH) have been described in gliomas, acute myeloid leukemia, cholangiocarcinoma, melanoma, colorectal, prostate cancer, thyroid carcinoma and other cancers. In mesenchymal tumors IDH mutations are present in about 50% of central chondrosarcoma. IDH mutations are an early event in chondrosarcoma-genesis, and contribute to the acquisition of malignancy through the block of cellular differentiation, hypoxia induction through HIF stabilization, DNA methylation and alteration of cellular red-ox balance. While in gliomas IDH mutations confers a good prognosis, in chondrosarcoma IDH prognostic role is controversial in different reported series. First aim of this project is to define the prevalence and the prognostic role of IDH mutation in high grade central conventional chondrosarcoma patients treated at Istituto Ortopedico Rizzoli. Second aim is the critical revision of scientific literature to understand better how a genomic event in cancer cell can trigger alteration in the TME, through immune infiltrate reshaping, angiogenesis induction, metabolic and methylation rewiring. Third aim is to screen other sarcoma histotypes for the presence of IDH mutation.

Development of novel multi-substituted apatite nanophases with advanced functionalities for bone regeneration

In the field of bone substitutes is highly researched an innovative material able to fill gaps with high mechanical performances and able to stimulate cell response, permitting the complete restoration of the bone portion. In this respect, the synthesis of new bioactive materials able to mimic the compositional, morphological and mechanical features of bone is considered as the elective approach for effective tissue regeneration. Hydroxyapatite (HA) is the main component of the inorganic part of bone. Additionally ionic substitution can be performed in the apatite lattice producing different effects, depending from the selected ions. Magnesium, in substitution of calcium, and carbonate, in substitution of phosphate, extensively present in the biological bones, are able to improve properties naturally present in the apatitic phase, (i.e. biomimicry, solubility e osteoinductive properties). Other ions can be used to give new useful properties, like antiresorptive or antimicrobial properties, to the apatitic phase. This thesis focused on the development of hydroxyapatite nanophases with multiple ionic substitutions including gallium, or zinc ions, in association with magnesium and carbonate, with the purpose to provide double synergistic functionality as osteogenic and antibacterial biomaterial. Were developed bioactive materials based on Sr-substituted hydroxyapatite in the form of sintered targets. The obtained targets were treated with Pulsed Plasma Deposition (PED) resulting in the deposition of thin film coatings able to improve the roughness and wettability of PEEK, enhancing its osteointegrability. Were investigated heterogeneous gas-solid reactions, addressed to the biomorphic transformations of natural 3D porous structures into bone scaffolds with biomimetic composition and hierarchical organization, for application in load-bearing sites. The kinetics of the different reactions of the process were optimized to achieve complete and controlled phase transformation, maintaining the original 3-D morphology. Massive porous scaffolds made of ion-substituted hydroxyapatite and bone-mimicking structure were developed and tested in 3-D cell culture models.

Methods for Biodata Analysis in different Omic Contexts

The world of Computational Biology and Bioinformatics presently integrates many different expertise, including computer science and electronic engineering. A major aim in Data Science is the development and tuning of specific computational approaches to interpret the complexity of Biology. Molecular biologists and medical doctors heavily rely on an interdisciplinary expert capable of understanding the biological background to apply algorithms for finding optimal solutions to their problems. With this problem-solving orientation, I was involved in two basic research fields: Cancer Genomics and Enzyme Proteomics. For this reason, what I developed and implemented can be considered a general effort to help data analysis both in Cancer Genomics and in Enzyme Proteomics, focusing on enzymes which catalyse all the biochemical reactions in cells. Specifically, as to Cancer Genomics I contributed to the characterization of intratumoral immune microenvironment in gastrointestinal stromal tumours (GISTs) correlating immune cell population levels with tumour subtypes. I was involved in the setup of strategies for the evaluation and standardization of different approaches for fusion transcript detection in sarcomas that can be applied in routine diagnostic. This was part of a coordinated effort of the Sarcoma working group of "Alleanza Contro il Cancro". As to Enzyme Proteomics, I generated a derived database collecting all the human proteins and enzymes which are known to be associated to genetic disease. I curated the data search in freely available databases such as PDB, UniProt, Humsavar, Clinvar and I was responsible of searching, updating, and handling the information content, and computing statistics. I also developed a web server, BENZ, which allows researchers to annotate an enzyme sequence with the corresponding Enzyme Commission number, the important feature fully describing the catalysed reaction. More to this, I greatly contributed to the characterization of the enzyme-genetic disease association, for a better classification of the metabolic genetic diseases.