Browsing by Author "Reis, Rui L."
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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.
- 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 with hierarchical porosity and enhanced performance for cartilage tissue engineering applicationsPublication . Ribeiro, Viviana P.; Yan, Le-Ping; Oliveira, Ana; Oliveira, Joaquim M.; Reis, Rui L.Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from highconcentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications.Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically crosslinked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding.
- Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applicationsPublication . Ribeiro, Viviana; Yan, Le-Ping; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from high-concentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications.
- Continuous-flow precipitation as a route to prepare highly controlled nanohydroxyapatite: In vitro mineralization and biological evaluationPublication . Castro, Filipa; Ribeiro, Viviana. P.; Ferreira, António; Oliveira, Ana L.; Reis, Rui L.; Teixeira, José A.; Rocha, FernandoThis work reports the biological evaluation of nanosized hydroxyapatite (HAp) previously synthesized by continuous-flow precipitation in a scaled-up meso oscillatory flow reactor (meso-OFR). Physicochemical characterization of the synthesized HApsuggests high surface reactivity namely because of its high specific surface area and low crystallinity. On the other hand, in vitro biomineralization assays demonstrated the apatite-forming activity of the prepared HAp and their higher surface reactivity when compared to a commercial HAp. Furthermore, human osteoblastic-like (Saos-2) cells culture evidenced that the synthesized HAp stimulated cell proliferation, especially when applied at lower concentrations (30 and 50 μg ml−1), although its cellular uptake behavior. Therefore, the prepared HApshows immense potential as biomedical material, as well as drug and gene delivery vehicle. The results are also very promising regarding further scaling up of the process, as the designed methodology allow for the preparation in a continuous mode of nanosized HAp with controlled physico-chemical properties.
- 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
- 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.
- 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.
- Finely tuned fiber-based porous structures for bone tissue engineering applicationsPublication . Ribeiro, Viviana; Silva-Correia, Joana; Morais, Alain; Correlo, Vitor M.; Marques, Alexandra P.; Ribeiro, Ana; Silva, Carla; Durães, Nelson; Bonifácio, Graça; Sousa, Rui A.; Oliveira, Joaquim M.; Oliveira, Ana L.; Reis, Rui L.
- In vivo skin hydrating efficacy of fish collagen from greenland halibut as a high-value active ingredient for cosmetic applicationsPublication . Martins, Eva; Reis, Rui L.; Silva, Tiago H.The industrial processing of fish for food purposes also generates a considerable number of by-products such as viscera, bones, scales, and skin. From a value-added perspective, fish by-products can act also as raw materials, especially because of their collagen content (particularly in fish skin). Interestingly, the potential of marine collagen for cosmetic applications is enormous and, remarkably, the extraction of this protein from fish skins has been established for different species. Using this approach, we investigated the integration of marine collagen (COLRp_I) extracted from the skin of the Greenland halibut as an active ingredient in a cosmetic hydrogel formulation. In this study, extracts of marine collagen at concentrations up to 10 mg/mL showed a non-cytotoxic effect when cultured with fibroblast cells for 3 days. In addition, marine collagen extract, when incorporated into a cosmetic hydrogel formulation, met criterion A of ISO 11930:2019 regarding the efficacy of the preservative system (challenge test). In addition, the cosmetic formulations based on marine collagen at dosages of 0.1, 0.25 and 0.5% were tested in a clinical study on the skin of the forearms of 23 healthy volunteers, showing a sightly hydration effect, suggesting its potential for beauty applications. Moreover, this work illustrates that the circular economy concept applied to the fish processing industry can represent important benefits, at innovation, environmental and economic levels.