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- Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering: In vitro and in vivo assessment of biological performancePublication . Yan, Le-Ping; Silva-Correia, Joana; Oliveira, Mariana B.; Vilela, Carlos; Pereira, Hélder; Sousa, Rui A.; Mano, João F.; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Novel porous bilayered scaffolds, fully integrating a silk fibroin (SF) layer and a silk-nano calcium phosphate (silk-nanoCaP) layer for osteochondral defect (OCD) regeneration, were developed. Homogeneous porosity distribution was achieved in the scaffolds, with calcium phosphate phase only retained in the silk-nanoCaP layer. The scaffold presented compressive moduli of 0.4 MPa in the wet state. Rabbit bone marrow mesenchymal stromal cells (RBMSCs) were cultured on the scaffolds, and good adhesion and proliferation were observed. The silk-nanoCaP layer showed a higher alkaline phosphatase level than the silk layer in osteogenic conditions. Subcutaneous implantation in rabbits demonstrated weak inflammation. In a rabbit knee critical size OCD model, the scaffolds firmly integrated into the host tissue. Histological and immunohistochemical analysis showed that collagen II positive cartilage and glycosaminoglycan regeneration presented in the silk layer, and de novo bone ingrowths and vessel formation were observed in the silk-nanoCaP layer. These bilayered scaffolds can therefore be promising candidates for OCD regeneration.
- Tumor growth suppression induced by biomimetic silk fibroin hydrogelsPublication . Yan, Le-Ping; Silva-Correia, Joana; Ribeiro, Viviana P.; Miranda-Gonçalves, Vera; Correia, Cristina; Morais, Alain da Silva; Sousa, Rui A.; Reis, Rui M.; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to β-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become β-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent β-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.
- Silk-based anisotropical 3D biotextiles for bone regenerationPublication . Ribeiro, Viviana P.; Silva-Correia, Joana; Nascimento, Ana I.; Morais, Alain da Silva; Marques, Alexandra P.; Ribeiro, Ana S.; Silva, Carla J.; Bonifácio, Grata; Sousa, Rui A.; Oliveira, Joaquim M.; Oliveira, Ana L.; Reis, Rui L.Bone loss in the craniofacial complex can been treated using several conventional therapeutic strategies that face many obstacles and limitations. In this work, novel three-dimensional (3D) biotextile architectures were developed as a possible strategy for flat bone regeneration applications. As a fully automated processing route, this strategy as potential to be easily industrialized. Silk fibroin (SF) yarns were processed into weft-knitted fabrics spaced by a monofilament of polyethylene terephthalate (PET). A comparative study with a similar 3D structure made entirely of PET was established. Highly porous scaffolds with homogeneous pore distribution were observed using micro-computed tomography analysis. The wet state dynamic mechanical analysis revealed a storage modulus In the frequency range tested, the storage modulus values obtained for SF-PET scaffolds were higher than for the PET scaffolds. Human adipose-derived stem cells (hASCs) cultured on the SF-PET spacer structures showed the typical pattern for ALP activity under osteogenic culture conditions. Osteogenic differentiation of hASCs on SF PET and PET constructs was also observed by extracellular matrix mineralization and expression of osteogenic-related markers (osteocalcin, osteopontin and collagen type I) after 28 days of osteogenic culture, in comparison to the control basal medium. The quantification of convergent macroscopic blood vessels toward the scaffolds by a chick chorioallantoic membrane assay, showed higher angiogenic response induced by the SF-PET textile scaffolds than PET structures and gelatin sponge controls. Subcutaneous implantation in CD-1 mice revealed tissue ingrowth's accompanied by blood vessels infiltration in both spacer constructs. The structural adaptability of textile structures combined to the structural similarities of the 3D knitted spacer fabrics to craniofacial bone tissue and achieved biological performance, make these scaffolds a possible solution for tissue engineering approaches in this area.
- Modulating cell adhesion to polybutylene succinate biotextile constructs for tissue engineering applicationsPublication . Ribeiro, Viviana P.; Almeida, Lília R.; Martins, Ana R.; Pashkuleva, Iva; Marques, Alexandra P.; Ribeiro, Ana S.; Silva, Carla J.; Bonifácio, Graça; Sousa, Rui A.; Oliveira, Ana L.; Reis, Rui L.Textile-based technologies are powerful routes for the production of three-dimensional porous architectures for tissue engineering applications because of their feasibility and possibility for scaling-up. Herein, the use of knitting technology to produce polybutylene succinate fibre-based porous architectures is described. Furthermore, different treatments have been applied to functionalize the surface of the scaffolds developed: sodium hydroxide etching, ultraviolet radiation exposure in an ozone atmosphere and grafting (acrylic acid, vinyl phosphonic acid and vinyl sulphonic acid) after oxygen plasma activation as a way to tailor cell adhesion. A possible effect of the applied treatments on the bulk properties of the textile scaffolds has been considered and thus tensile tests in dry and hydrated states were also carried out. The microscopy results indicated that the surface morphology and roughness were affected by the applied treatments. The X-ray photoelectron spectroscopy and contact angle measurements showed the incorporation of oxygen-containing groups and higher surface free energy as result of the surface treatments applied. The DNA quantification and scanning electron microscopy analysis revealed that these modifications enhanced cell adhesion and altered cell morphology. Generally, sodium hydroxide treatment altered most significantly the surface properties, which in turn resulted in a high number of cells adherent to these surfaces. Based on the results obtained, the proposed surface treatments are appropriate to modify polybutylene succinate knitting scaffolds, influencing cell adhesion and its potential for use in tissue engineering applications.
- Supercritical CO2 technology: the next standard sterilization technique?Publication . Soares, Gonçalo C.; Learmonth, David A.; Vallejo, Mariana C.; Davila, Sara Perez; González, Pío; Sousa, Rui A.; Oliveira, Ana L.Sterilization of implantable medical devices is of most importance to avoid surgery related complications such as infection and rejection. Advances in biotechnology fields, such as tissue engineering, have led to the development of more sophisticated and complex biomedical devices that are often composed of natural biomaterials. This complexity poses a challenge to current sterilization techniques which frequently damage materials upon sterilization. The need for an effective alternative has driven research on supercritical carbon dioxide (scCO(2)) technology. This technology is characterized by using low temperatures and for being inert and non-toxic. The herein presented paper reviews the most relevant studies over the last 15 years which cover the use of scCO(2) for sterilization and in which effective terminal sterilization is reported. The major topics discussed here are: microorganisms effectively sterilized by scCO(2), inactivation mechanisms, operating parameters, materials sterilized by scCO(2) and major requirements for validation of such technique according to medical devices' standards.
- Influence of different surface modification treatments on silk biotextiles for tissue engineering applicationsPublication . Ribeiro, Viviana P.; Almeida, Lília R.; Martins, Ana R.; Pashkuleva, Iva; Marques, Alexandra P.; Ribeiro, Ana S.; Silva, Carla J.; Bonifácio, Graça; Sousa, Rui A.; Reis, Rui L.; Oliveira, Ana L.Biotextile structures from silk fibroin have demonstrated to be particularly interesting for tissue engineering (TE) applications due to their high mechanical strength, interconnectivity, porosity, and ability to degrade under physiological conditions. In this work, we described several surface treatments of knitted silk fibroin (SF) scaffolds, namely sodium hydroxide (NaOH) solution, ultraviolet radiation exposure in an ozone atmosphere (UV/O3) and oxygen (O2) plasma treatment followed by acrylic acid (AAc), vinyl phosphonic acid (VPA), and vinyl sulfonic acid (VSA) immersion. The effect of these treatments on the mechanical properties of the textile constructs was evaluated by tensile tests in dry and hydrated states. Surface properties such as morphology, topography, wettability and elemental composition were also affected by the applied treatments. The in vitro biological behavior of L929 fibroblasts revealed that cells were able to adhere and spread both on the untreated and surface-modified textile constructs. The applied treatments had different effects on the scaffolds' surface properties, confirming that these modifications can be considered as useful techniques to modulate the surface of biomaterials according to the targeted application.