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- 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.
- 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.
- 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.
- 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.
- 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.
- 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.