Loading...
10 results
Search Results
Now showing 1 - 10 of 10
- 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.
- In vitro evaluation of the biological performance of macro/ micro-porous silk fibroin and silk-nano calcium phosphate scaffoldsPublication . Yan, L.-P.; Oliveira, J. M.; Oliveira, A. L.; Reis, R. L.This study evaluates the biological performance of salt-leached macro/microporous silk scaffolds (S16) and silk-nano calcium phosphate scaffolds (SC16), both deriving from a 16 wt % aqueous SF solution. Enzymatic degradation results showed that the silk-based scaffolds presented desirable biostability, and the incorporation of calcium phosphate further improved the scaffolds' biostability. Human adipose tissue derived stromal cells (hASCs) were cultured onto the scaffolds in vitro. The Alamar blue assay and DNA content revealed that both scaffolds were non-cytotoxic and can support the viability and proliferation of the hASCs. Scanning electron microscopy observation demonstrated that the microporous structure was beneficial for the cell adhesion while the macroporous structure favored the cell migration and proliferation. The histological analysis displayed abundant extracellular matrix formed inside the scaffolds, leading to the significant increase of scaffolds' modulus. These results revealed that S16 and SC16 could be promising alternatives for cartilage and bone tissue engineering scaffolding applications, respectively.
- 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.
- Rapidly responsive silk fibroin hydrogels as an artificial matrix for the programmed tumor cells deathPublication . Ribeiro, Viviana P.; Silva-Correia, Joana; Gonçalves, Cristiana; Pina, Sandra; Radhouani, Hajer; Montonen, Toni; Oliveira, Joaquim M.; Oliveira, Ana L.Timely and spatially-regulated injectable hydrogels, able to suppress growing tumors in response to conformational transitions of proteins, are of great interest in cancer research and treatment. Herein, we report rapidly responsive silk fibroin (SF) hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, and demonstrate their use as an artificial biomimetic three-dimensional (3D) matrix. The proposed SF hydrogels presented a viscoelastic nature of injectable hydrogels and spontaneous conformational changes from random coil to β-sheet conformation under physiological conditions. A human neuronal glioblastoma (U251) cell line was used for screening cell encapsulation and in vitro evaluation within the SF hydrogels. The transparent random coil SF hydrogels promoted cell viability and proliferation up to 10 days of culturing, while the crystalline SF hydrogels converted into β-sheet structure induced the formation of TUNEL-positive apoptotic cells. Therefore, this work provides a powerful tool for the investigation of the microenvironment on the programed tumor cells death, by using rapidly responsive SF hydrogels as 3D in vitro tumor models.
- Enzymatically cross-linked silk fibroin-based hierarchical scaffolds for osteochondral regenerationPublication . Ribeiro, Viviana P.; Pina, Sandra; Costa, João B.; Cengiz, Ibrahim Fatih; García-Fernández, Luis; Fernández-Gutiérrez, Maria del Mar; Paiva, Olga C.; Oliveira, Ana L.; San-Román, Julio; Oliveira, Joaquim M.; Reis, Rui L.Osteochondral (OC) regeneration faces several limitations in orthopedic surgery, owing to the complexity of the OC tissue that simultaneously entails the restoration of articular cartilage and subchondral bone diseases. In this study, novel biofunctional hierarchical scaffolds composed of a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like layer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped β-tricalcium phosphate (β-TCP) subchondral bone-like layer (HRP-SF/dTCP layer) were proposed as a promising strategy for OC tissue regeneration. For comparative purposes, a similar bilayered structure produced with no ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity distribution was achieved throughout the scaffolds, as shown by micro-computed tomography analysis. The ion-doped bilayered scaffolds presented a wet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical properties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90 kPa) superior to that of the control bilayered scaffolds (189.18 ± 90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37 kPa, respectively). Apatite crystal formation, after immersion in simulated body fluid (SBF), was observed in the subchondral bone-like layers for the scaffolds incorporating TCP powders. Human osteoblasts (hOBs) and human articular chondrocytes (hACs) were co-cultured onto the bilayered structures and monocultured in the respective cartilage and subchondral bone half of the partitioned scaffolds. Both cell types showed good adhesion and proliferation in the scaffold compartments, as well as adequate integration of the interface regions. Osteoblasts produced a mineralized extracellular matrix (ECM) in the subchondral bone-like layers, and chondrocytes showed GAG deposition. The gene expression profile was different in the distinct zones of the bilayered constructs, and the intermediate regions showed pre-hypertrophic chondrocyte gene expression, especially on the BdTCP constructs. Immunofluorescence analysis supported these observations. This study showed that the proposed bilayered scaffolds allowed a specific stimulation of the chondrogenic and osteogenic cells in the co-culture system together with the formation of an osteochondral-like tissue interface. Hence, the structural adaptability, suitable mechanical properties, and biological performance of the hierarchical scaffolds make these constructs a desired strategy for OC defect regeneration.
- Core-shell silk hydrogels with spatially tuned conformations as drug-delivery systemPublication . Le-Ping, Yan; Oliveira, Joaquim M.; Oliveira, Ana L.; Reis, Rui L.Hydrogels of spatially controlled physicochemical properties are appealing platforms for tissue engineering and drug delivery. In this study, core-shell silk fibroin (SF) hydrogels of spatially controlled conformation were developed. The core-shell structure in the hydrogels was formed by means of soaking the preformed (enzymatically crosslinked) random coil SF hydrogels in methanol. When increasing the methanol treatment time from 1 to 10 min, the thickness of the shell layer can be tuned from about 200 to about 850 μm as measured in wet status. After lyophilization of the rehydrated core-shell hydrogels, the shell layer displayed compact morphology and the core layer presented porous structure, when observed by scanning electron microscopy. The conformation of the hydrogels was evaluated by Fourier transform infrared spectroscopy in wet status. The results revealed that the shell layer possessed dominant β-sheet conformation and the core layer maintained mainly random coil conformation. Enzymatic degradation data showed that the shell layers presented superior stability to the core layer. The mechanical analysis displayed that the compressive modulus of the core-shell hydrogels ranged from about 25 kPa to about 1.1 MPa by increasing the immersion time in methanol. When incorporated with albumin, the core-shell SF hydrogels demonstrated slower and more controllable release profiles compared with the non-treated hydrogel. These core-shell SF hydrogels of highly tuned properties are useful systems as drug-delivery system and may be applied as cartilage substitute
- 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.
- Current concepts and challenges in osteochondral tissue engineering and regenerative medicinePublication . Yan, Le Ping; Oliveira, Joaquim M.; Oliveira, Ana L.; Reis, Rui L.In the past few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate toward osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cell implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant, and reprogrammed cells for osteochondral regeneration.
- Thermal annealed silk fibroin membranes for periodontal guided tissue regenerationPublication . Geão, Catarina; Costa-Pinto, Ana R.; Cunha-Reis, Cassilda; Ribeiro, Viviana P.; Vieira, Sílvia; Oliveira, Joaquim M.; Reis, Rui L.; Oliveira, A. L.Guided tissue regeneration (GTR) is a surgical procedure applied in the reconstruction of periodontal defects, where an occlusive membrane is used to prevent the fast-growing connective tissue from migrating into the defect. In this work, silk fibroin (SF) membranes were developed for periodontal guided tissue regeneration. Solutions of SF with glycerol (GLY) or polyvinyl alcohol (PVA) where prepared at several weight ratios up to 30%, followed by solvent casting and thermal annealing at 85 °C for periods of 6 and 12 h to produce high flexible and stable membranes. These were characterized in terms of their morphology, physical integrity, chemical structure, mechanical and thermal properties, swelling capability and in vitro degradation behavior. The developed blended membranes exhibited high ductility, which is particular relevant considering the need for physical handling and adaptability to the defect. Moreover, the membranes were cultured with human periodontal ligament fibroblast cells (hPDLs) up to 7 days. Also, the higher hydrophilicity and consequent in vitro proteolytic degradability of these blends was superior to pure silk fibroin membranes. In particular SF/GLY blends demonstrated to support high cell adhesion and viability with an adequate hPDLs’ morphology, make them excellent candidates for applications in periodontal regeneration.