TISSUE2TISSUE-Using Textile Technologies to Develop Innovative 3D Architectures to Be Applied in Bone Tissue Engineering

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Modulating cell adhesion to polybutylene succinate biotextile constructs for tissue engineering applications

Publication . 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.

2017Journal article

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Core-shell silk hydrogels with spatially tuned conformations as drug-delivery system

Publication . 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

2017Journal article

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Silk-based anisotropical 3D biotextiles for bone regeneration

Publication . 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.

2017Journal article

Open access

In vitro evaluation of the biological performance of macro/ micro-porous silk fibroin and silk-nano calcium phosphate scaffolds

Publication . 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.

2015Journal article

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Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering: In vitro and in vivo assessment of biological performance

Publication . 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.

2015Journal article

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