Percorrer por autor "Morais, Alain da Silva"
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- Combinatory approach for developing silk fibroin scaffolds for cartilage regenerationPublication . Ribeiro, Viviana P.; Morais, Alain da Silva; Maia, F. Raquel; Canadas, R. F.; Costa, João B.; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. Statement of Significance In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.
- Combinatory approach for developing silk fibroin-based scaffolds seeded with human adipose-derived stem cells for cartilage tissue engineering applicationsPublication . Ribeiro, Viviana; Morais, Alain da Silva; Maia, Fátima; Oliveira, Ana; Oliveira, Joaquim; Reis, RuiSeveral processing technologies have been combined to create scaffolds for different tissue engineering (TE) applications. Hydrogels have been extensively used for cartilage TE applications, presenting several structural similarities to the natural extracellular matrix of cartilage tissue environment[1]. From the different biodegradable materials proposed as matrices for cartilage scaffolding[2], silk fibroin (SF) presents high versatility, processability and tailored mechanical properties, which make this protein attractive for the development of innovative matrices for cartilage TE purposes[3]. In a previous study, we proposed fast formed SF hydrogels produced through a horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) crosslinking reaction, taking advantage of the presence of tyrosine groups[4]. In this work, macro-/micro-porous SF scaffolds derived from enzymatically cross- linked SF hydrogels by a HRP/H2O2 complex were produced in combination with salt-leaching and freeze-drying methodologies. The scaffolds morphology, mechanical properties and chemical characterization were assessed by mean of different characterization techniques (SEM, micro-CT, Instron, FTIR and XRD). The scaffolds structural integrity was evaluated by swelling ratio and degradation profile studies. The in vitro ability to support the adhesion, proliferation and differentiation into the chondrogenic lineage was tested using human adipose-derived stem cells (hASCs) cultured over 28 days in basal and chondrogenic conditions. Cell behaviour in the presence of the SF scaffolds was evaluated through different quantitative (GAGs/DNA and RT-PCR) and qualitative (live/dead, SEM, histology and immunocytochemistry) assays. The in vivo biocompatibility of the SF-based scaffolds was also assessed by subcutaneous implantation in mice for 2 and 4 weeks and analysed by means of hematoxylin & eosin (H&E) staining and immunohistochemical analysis of CD31 angiogenic marker. The results showed highly porous and interconnected SF structures that allowed cell adhesion and infiltration into the scaffolds. In vitro cell viability and proliferation were also observed over the 28 days of culturing in basal conditions and a significant increase of GAGs content was detected on constructs cultured in chondrogenic differentiation medium. In vivo results showed that the implanted scaffolds allowed tissue ingrowth’s and blood vessels formation/infiltration. The obtained results demonstrated that the innovative approach of combining enzymatically cross-linked SF hydrogels with the salt- leaching and freeze-drying methodologies allowed to produce more versatile scaffold architectures with appropriate mechanical properties and large swelling ability. The positive influence over in vitro chondrogenic differentiation and in vivo response, revealed by the new tissue formation and angiogenesis within the porous scaffolds, validates the proposed macro-/micro-porous SF scaffolds for being used in cartilage TE applications. Moreover, the versatility of these combinatory approach can allow for further applications in other musculoskeletal TE strategies.
- 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.
- 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.