Browsing by Author "Sousa, Susana R."
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- Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffoldPublication . Cruz-Neves, Susana; Ribeiro, Nilza; Pinho dos Santos Graça, Maria Inês; Jerónimo, Carmen; Sousa, Susana R.; Monteiro, Fernando J.Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies.
- A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regenerationPublication . Ribeiro, Nilza; Sousa, Susana R.; Van Blitterswijk, Clemens A.; Moroni, Lorenzo; Monteiro, Fernando J.This work aims to design a synthetic construct that mimics the natural bone extracellular matrix through innovative approaches based on simultaneous type I collagen electrospinning and nanophased hydroxyapatite (nanoHA) electrospraying using non-denaturating conditions and non-toxic reagents. The morphological results, assessed using scanning electron microscopy and atomic force microscopy (AFM), showed a mesh of collagen nanofibers embedded with crystals of HA with fiber diameters within the nanometer range (30 nm), thus significantly lower than those reported in the literature, over 200 nm. The mechanical properties, assessed by nanoindentation using AFM, exhibited elastic moduli between 0.3 and 2 GPa. Fourier transformed infrared spectrometry confirmed the collagenous integrity as well as the presence of nanoHA in the composite. The network architecture allows cell access to both collagen nanofibers and HA crystals as in the natural bone environment. The inclusion of nanoHA agglomerates by electrospraying in type I collagen nanofibers improved the adhesion and metabolic activity of MC3T3-E1 osteoblasts. This new nanostructured collagen–nanoHA composite holds great potential for healing bone defects or as a functional membrane for guided bone tissue regeneration and in treating bone diseases.
- Comprehensive Analysis of Secreted Protein, Acidic and Rich in Cysteine in Prostate Carcinogenesis: Development of a 3D Nanostructured Bone-Like ModelPublication . Ribeiro, Nilza; Costa-Pinheiro, Pedro; Henrique, Rui; Gomez-Lazaro, Maria; Pereira, Marisa P.; Mansur, Alexandra A. P.; Mansur, Herman S.; Jerónimo, Carmen; Sousa, Susana R.; Monteiro, Fernando J.Most aggressive prostate cancer (PCa) types tend to metastasize frequently to bone and SPARC, a matricellular protein, might participate in such biological processes. The objective of this study was to evaluate the effect of SPARC in prostate carcinogenesis and bone metastization. This was explored assessing the morphology, metabolic activity and SPARC expression of different PCa cell lines resembling different stages of carcinogenesis, using a 3D bone-biomimetic model (collagen nanofibers/nanohydroxyapatite) grafted with SPARC. Our findings highlight distinct cellular behavior depending on cell type and presence of exogenous SPARC. In fact, SPARC addition contributed to the survival and significant growth of a non-bone metastatic PCa cell line (LNCaP) on bone-like biomaterial. Moreover, SPARC expression levels were evaluated in a series of prostatic tissues, comparing normal prostate, pre-neoplastic prostate intraepithelial neoplasias and overtly malignant tumors, and also metastasis to its correspondent primary prostate tumors, ascertaining potential association between SPARC and clinicopathological data. Remarkably, SPARC was overexpressed in patients with higher Gleason Score, indicating tumors with poor prognosis, as well as in metastasis, particularly from bone sites, compared with their respective primary tumors. The results suggest a potential role of SPARC as a clinical target on PCa, due to its association with bone metastization.
- Ecotoxicological impact of two soil remediation treatments in Lactuca sativa seedsPublication . Rede, Diana; Santos, Lúcia H.M.L.M.; Ramos, Sandra; Oliva-Teles, Filipe; Antão, Cristina; Sousa, Susana R.; Delerue-Matos, CristinaPharmaceuticals have been identified as environmental emerging pollutants and are present in different compartments, including soils. Chemical remediation showed to be a good and suitable approach for soil remediation, though the knowledge in their impact for terrestrial organisms is still limited. Therefore, in this work, two different chemical remediation treatments (Fenton oxidation and nanoremediation) were applied to a soil contaminated with an environmental representative concentration of ibuprofen (3 ng g-1). The phytotoxic impact of a traditional soil remediation treatment (Fenton oxidation) and of a new and more sustainable approach for soil remediation (nanoremediation using green nano-scale zerovalent iron nanoparticles (nZVIs)) was evaluated in Lactuca sativa seeds. Percentage of seed germination, root elongation, shoot length and leaf length were considered as endpoints to assess the possible acute phytotoxicity of the soil remediation treatments as well as of the ibuprofen contaminated soil. Both chemical remediation treatments showed to have a negative impact in the germination and development of lettuce seeds, exhibiting a reduction up to 45% in the percentage of seed germination and a decrease around 80% in root elongation comparatively to the contaminated soil. These results indicate that chemical soil remediation treatments could be more prejudicial for terrestrial organisms than contaminated soils.
- Endothelialization of chitosan porous conduits via immobilization of a recombinant fibronectin fragment (rhFNIII7–10)Publication . Amaral, I. F.; Neiva, I.; Silva, F. Ferreira da; Sousa, Susana R.; Piloto, A. M.; Lopes, C. D. F.; Barbosa, Mário A.; Kirkpatrick, C. J.; Pêgo, A. P.The present study aimed to develop a pre-endothelialized chitosan (CH) porous hollowed scaffold for application in spinal cord regenerative therapies. CH conduits with different degrees of acetylation (DA; 4% and 15%) were prepared, characterized (microstructure, porosity and water uptake) and functionalized with a recombinant fragment of human fibronectin (rhFNIII7–10). Immobilized rhFNIII7–10 was characterized in terms of amount (125I-radiolabelling), exposure of cell-binding domains (immunofluorescence) and ability to mediate endothelial cell (EC) adhesion and cytoskeletal rearrangement. Functionalized conduits revealed a linear increase in immobilized rhFNIII7–10 with rhFNIII7–10 concentration, and, for the same concentration, higher amounts of rhFNIII7–10 on DA 4% compared with DA 15%. Moreover, rhFNIII7–10 concentrations as low as 5 and 20 lgml 1 in the coupling reaction were shown to provide DA 4% and 15% scaffolds, respectively, with levels of exposed cell-binding domains exceeding those observed on the control (DA 4% scaffolds incubated in a 20 lgml 1 human fibronectin solution). These grafting conditions proved to be effective in mediating EC adhesion/cytoskeletal organization on CH with DA 4% and 15%, without affecting the endothelial angiogenic potential. rhFNIII7–10 grafting to CH could be a strategy of particular interest in tissue engineering applications requiring the use of endothelialized porous matrices with tunable degradation rates.
- Heparinized nanohydroxyapatite/collagen granules for controlled release of vancomycinPublication . Coelho, Catarina C.; Sousa, Susana R.; Monteiro, Fernando J.The purpose of this study was to develop a bone substitute material capable of preventing or treating osteomyelitis through a sustainable release of vancomycin and simultaneously inducing bone regeneration. Porous heparinized nanohydroxyapatite (nanoHA)/collagen granules were characterized using scanning electron microscopy, micro-computed tomography and attenuated total reflectance Fourier transform infrared spectroscopy. After vancomycin adsorption onto the granules, its releasing profile was studied by UV molecular absorption spectroscopy. The heparinized granules presented a more sustainable release over time, in comparison with nonheparinized nanoHA and nanoHA/collagen granules. Vancomycin was released for 360 h and proved to be bioactive until 216 h. Staphylococcus aureus adhesion was higher on granules containing collagen, guiding the bacteria to the material with antibiotic, improving their eradication. Moreover, cytotoxicity of the released vancomycin was assessed using osteoblast cultures, and after 14 days of culture in the presence of vancomycin, cells were able to remain viable, increasing their metabolic activity and colonizing the granules, as observed by scanning electron microscopy and confocal laser scanning microscopy. These findings suggest that heparinized nanoHA/collagen granules are a promising material to improve the treatment of osteomyelitis, as they are capable of releasing vancomycin, eliminating the bacteria, and presented morphological and chemical characteristics to induce bone regeneration.
- Individual and mixture toxicity evaluation of three pharmaceuticals to the germination and growth of Lactuca sativa seedsPublication . Rede, Diana; Santos, Lúcia H.M.L.M.; Ramos, Sandra; Oliva-Teles, Filipe; Antão, Cristina; Sousa, Susana R.; Delerue-Matos, CristinaThis work aims to assess, individually and in mixtures, possible phytotoxic effects of three pharmaceuticals (paracetamol, ibuprofen and amoxicillin) on germination and early growth of Lactuca sativa seeds. Pharmaceuticals are an important group of emerging contaminants, whose presence has been described in several environmental compartments, including soils. However, knowledge on their possible impact in terrestrial organisms is still sparse and even more when mixtures are considered. Germination tests are important to evaluate the quality of soil and the toxic effects that contaminants can pose to plants. The acute effects of individual pharmaceuticals as well as binary and ternary mixtures were assessed using different endpoints, namely: percentage of seed germination, root elongation, shoot and leaf length, after an exposure time of five days. Overall, in the exposure of L. sativa seeds to individual pharmaceuticals there are indications of acute toxicity in the early plant growth. However, this inhibitory effect tends to be cancelled in the acute exposure to mixtures. This study shows the importance of evaluating the toxicity of mixtures of pharmaceuticals, since they might have distinct toxic effects when compared to the single compounds, and also because, probably, it is the closest scenario to the reality that can be found in the environment.
- Influence of crystallite size of nanophased hydroxyapatite on fibronectin and osteonectin adsorption and on MC3T3-E1 osteoblast adhesion and morphologyPublication . Ribeiro, N.; Sousa, Susana R.; Monteiro, F. J.The characteristic topographical features (crystallite dimensions, surface morphology and roughness) of bioceramics may influence the adsorption of proteins relevant to bone regeneration. This work aims at analyzing the influence of two distinct nanophased hydroxyapatite (HA) ceramics, HA725 and HA1000 on fibronectin (FN) and osteonectin (ON) adsorption and MC3T3-E1 osteoblast adhesion and morphology. Both substrates were obtained using the same hydroxyapatite nanocrystals aggregates and applying the sintering temperatures of 725ºC and 1000ºC, respectively. The two proteins used in this work, FN as an adhesive glycoprotein and ON as a counter-adhesive protein, are known to be involved in the early stages of osteogenesis (cell adhesion, mobility and proliferation). The properties of the nanoHA substrates had an important role in the adsorption behavior of the two studied proteins and clearly affected the MC3T3- E1 morphology, distribution and metabolic activity. HA1000 surfaces presenting slightly larger grain size, higher root-mean-square roughness (Rq), lower surface area and porosity, allowed for higher amounts of both proteins adsorbed. These substrates also revealed increased number of exposed FN cell-binding domains as well as higher affinity for osteonectin. Regarding the osteoblast adhesion results, improved viability and cell number were found for HA1000 surfaces as compared to HA725 ones, independently of the presence or type of adsorbed protein. Therefore the osteoblast adhesion and metabolic activity seemed to be more sensitive to surfaces morphology and roughness than to the type of adsorbed proteins.
- Melanoma in the Eyes of MechanobiologyPublication . Brás, M. Manuela; Radmacher, Manfred; Sousa, Susana R.; Granja, Pedro L.Skin is the largest organ of the human body with several important functions that can be impaired by injury, genetic or chronic diseases. Among all skin diseases, melanoma is one of the most severe, which can lead to death, due to metastization. Mechanotransduction has a crucial role for motility, invasion, adhesion and metastization processes, since it deals with the response of cells to physical forces. Signaling pathways are important to understand how physical cues produced or mediated by the Extracellular Matrix (ECM), affect healthy and tumor cells. During these processes, several molecules in the nucleus and cytoplasm are activated. Melanocytes, keratinocytes, fibroblasts and the ECM, play a crucial role in melanoma formation. This manuscript will address the synergy among melanocytes, keratinocytes, fibroblasts cells and the ECM considering their mechanical contribution and relevance in this disease. Mechanical properties of melanoma cells can also be influenced by pigmentation, which can be associated with changes in stiffness. Mechanical changes can be related with the adhesion, migration, or invasiveness potential of melanoma cells promoting a high metastization capacity of this cancer. Mechanosensing, mechanotransduction, and mechanoresponse will be highlighted with respect to the motility, invasion, adhesion and metastization in melanoma cancer.
- Role of SPARC in Bone Remodeling and Cancer‐Related Bone MetastasisPublication . Ribeiro, Nilza; Sousa, Susana R.; Brekken, Rolf A.; Monteiro, Fernando J.There is a growing socioeconomic recognition that clinical bone diseases such as bone infections, bone tumors and osteoporotic bone loss mainly associated with ageing, are major issues in today0s society. SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, may be a promising therapeutic target for preventing or treating bone‐related diseases. In fact, SPARC is associated with tissue remodeling, repair, development, cell turnover, bone mineralization and may also participate in growth and progression of tumors, namely cancer‐related bone metastasis. Yet, the function of SPARC in such biological processes is poorly understood and controversial. The main objective of this work is to review the current knowledge related to the activity of SPARC in bone remodeling, tumorigenesis, and bone metastasis. Progress in understanding SPARC biology may provide novel strategies for bone regeneration and the development of anti‐angiogenic, anti‐proliferative, or counter‐adhesive treatments specifically against bone metastasis.