Browsing by Author "Ferreira, Frederico Castelo"
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- Additive manufactured Poly("-caprolactone)-graphene scaffolds: Lamellar crystal orientation, mechanical properties and biological performancePublication . Biscaia, Sara; Silva, João C.; Moura, Carla; Viana, Tânia; Tojeira, Ana; Mitchell, Geoffrey R.; Pascoal-Faria, Paula; Ferreira, Frederico Castelo; Alves, NunoUnderstanding the mechano–biological coupling mechanisms of biomaterials for tissue engineering is of major importance to assure proper scaffold performance in situ. Therefore, it is of paramount importance to establish correlations between biomaterials, their processing conditions, and their mechanical behaviour, as well as their biological performance. With this work, it was possible to infer a correlation between the addition of graphene nanoparticles (GPN) in a concentration of 0.25, 0.5, and 0.75% (w/w) (GPN0.25, GPN0.5, and GPN0.75, respectively) in three-dimensional poly("-caprolactone) (PCL)-based scaffolds, the extrusion-based processing parameters, and the lamellar crystal orientation through small-angle X-ray scattering experiments of extruded samples of PCL and PCL/GPN. Results revealed a significant impact on the scaffold’s mechanical properties to a maximum of 0.5% of GPN content, with a significant improvement in the compressive modulus of 59 MPa to 93 MPa. In vitro cell culture experiments showed the scaffold’s ability to support the adhesion and proliferation of L929 fibroblasts (fold increase of 28, 22, 23, and 13 at day 13 (in relation to day 1) for PCL, GPN0.25, GPN0.5, and GPN0.75, respectively) and bone marrow mesenchymal stem/stromal cells (seven-fold increase for all sample groups at day 21 in relation to day 1). Moreover, the cells maintained high viability, regular morphology, and migration capacity in all the different experimental groups, assuring the potential of PCL/GPN scaffolds for tissue engineering (TE) applications.
- Chondrogenic differentiation of mesenchymal stem/stromal cells on 3D porous poly (ε-caprolactone) scaffolds: Effects of material alkaline treatment and chondroitin sulfate supplementationPublication . Moura, Carla; Silva, João Carlos; Faria, Sofia; Fernandes, Paulo Rui; Silva, Cláudia Lobato da; Cabral, Joaquim Manuel Sampaio; Linhardt, Robert; Bártolo, Paulo Jorge; Ferreira, Frederico CasteloCartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BMMSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D extruded PCL scaffolds, characterized by mCT, featured a 21 mmL1 surface area to volume ratio, 390 mm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CSsupplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.
- Comparison of Three-dimensional Extruded Poly (ɛ-Caprolactone) and Polylactic acid Scaffolds with Pore size VariationPublication . Monteiro de Moura, Carla Sofia; Ferreira, Frederico Castelo; Bártolo, Paulo JorgeAdditive manufacturing (AM) has become a prominent approach among the scientific community for the production of three-dimensional (3D) matrices able to support tissue engineering approaches, promoting cell adhesion, proliferation and organization aiming to repair different tissues, such as bone or cartilage. In this study we used an extrusion-based technique for the production of poly (ɛ-caprolactone) (PCL) and polylactic acid (PLA) scaffolds and performed a side-by-side scaffold characteristics comparison. Using this technique we were able to create fully 3D interconnected porous scaffolds with pore size variations ranging from 190 μm to 390 μm with both materials. These scaffolds were assessed for stiffness, wettability and cell adhesion using mesenchymal stem/stromal cells (MSC). Comparisons between these two materials were made. The compressive modulus obtained is on the same order of magnitude for both materials. However, PCL presents a statistically significant higher compressive modulus. Results confirmed that PCL is a more hydrophobic material, so it presents a lower wettability when compared to PLA. Interestingly cell adhesion is similar for PLA and PCL, therefore selection between these two materials for the use of this versatile platform can be defined according with biodegradability aimed.
- Effects of glycosaminoglycan supplementation in the chondrogenic differentiation of bone marrow- and synovial- derived mesenchymal stem/stromal cells on 3D-extruded poly (ε-caprolactone) scaffoldsPublication . Silva, João C.; Moura, Carla; Matos, António P. Alves de; Cabral, Joaquim M. S.; Linhardt, Robert J.; Ferreira, Frederico CasteloThe lack of effective and long-term treatments for articular cartilage defects has increased the interest for innovative tissue engineering strategies. Such approaches, combining cells, biomaterial matrices and external biochemical/physical cues, hold promise for generating fully functional cartilage tissue. Herein, this study aims at exploring the use of the major cartilage glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronic acid (HA), as external biochemical cues to promote the chondrogenic differentiation of human bone marrow- and synovium-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) on custom-made 3 D porous poly (ε-caprolactone) (PCL) scaffolds. The culture conditions, namely the chondrogenic medium and hypoxic environment (5% O2 tension), were firstly optimized by culturing hBMSCs on PCL scaffolds without GAG supplementation. For both MSC sources, GAG supplemented media, particularly with HA, promoted significantly cartilage-like extracellular matrix (ECM) production (higher sulfated GAG amounts) and chondrogenic gene expression. Remarkably, in contrast to tissues generated using hBMSCs, the hSMSC-based constructs showed decreased expression of hypertrophic marker COL X. Histological, immunohistochemical and transmission electron microscopy (TEM) analysis confirmed the presence of typical articular cartilage ECM components (GAGs, aggrecan, collagen fibers) in all the tissue constructs produced. Overall, our results highlight the potential of integrating GAG supplementation, hSMSCs and customizable 3 D scaffolds toward the fabrication of bioengineered cartilage tissue substitutes with reduced hypertrophy.
- Extracellular matrix decorated polycaprolactone scaffolds for improved mesenchymal stem/stromal cell osteogenesis towards a patient-tailored bone tissue engineering approachPublication . Silva, João C.; Carvalho, Marta S.; Udangawa, Ranodhi N.; Moura, Carla; Cabral, Joaquim M. S.; Silva, Cláudia L. da; Ferreira, Frederico Castelo; Vashishth, Deepak; Linhardt, Robert J.The clinical demand for tissue-engineered bone is growing due to the increase of non-union fractures and delayed healing in an aging population. Herein, we present a method combining additive manufacturing (AM) techniques with cell-derived extracellular matrix (ECM) to generate structurally well-defined bioactive scaffolds for bone tissue engineering (BTE). In this work, highly porous three-dimensional polycaprolactone (PCL) scaffolds with desired size and architecture were fabricated by fused deposition modeling and subsequently decorated with human mesenchymal stem/stromal cell (MSC)-derived ECM produced in situ. The successful deposition of MSC-derived ECM onto PCL scaffolds (PCL-MSC ECM) was confirmed after decellularization using scanning electron microscopy, elemental analysis, and immunofluorescence. The presence of cell derived ECM within the PCL scaffolds significantly enhanced MSC attachment and proliferation, with and without osteogenic supplementation. Additionally, under osteogenic induction, PCL-MSC ECM scaffolds promoted significantly higher calcium deposition and elevated relative expression of bone-specific genes, particularly the gene encoding osteopontin, when compared to pristine scaffolds. Overall, our results demonstrated the favorable effects of combining MSC-derived ECM and AM-based scaffolds on the osteogenic differentiation of MSC, resulting from a closer mimicry of the native bone niche. This strategy is highly promising for the development of novel personalized BTE approaches enabling the fabrication of patient defect-tailored scaffolds with enhanced biological performance and osteoinductive properties.
- Extruded bioreactor perfusion culture supports the chondrogenic differentiation of human mesenchymal stem/stromal cells in 3D porous poly(ɛ-caprolactone) scaffoldsPublication . Silva, João C.; Moura, Carla; Borrecho, Gonçalo; Matos, António P. Alves de; Silva, Cláudia L. da; Cabral, Joaquim M. S.; Bártolo, Paulo J.; Linhardt, Robert J.; Ferreira, Frederico CasteloNovel bioengineering strategies for the ex vivo fabrication of native-like tissue-engineered cartilage are crucial for the translation of these approaches to clinically manage highly prevalent and debilitating joint diseases. Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, expensive, and not very versatile. In the current study, a novel, cost-effective, and customizable perfusion bioreactor totally fabricated by additive manufacturing (AM) is proposed for the study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly(ɛ-caprolactone) (PCL) scaffolds. hBMSCs are first seeded and grown on PCL scaffolds and hBMSC–PCL constructs are then transferred to 3D-extruded bioreactors for continuous perfusion culture under chondrogenic inductive conditions. Perfused constructs show similar cell metabolic activity and significantly higher sulfated glycosaminoglycan production (≈1.8-fold) in comparison to their non-perfused counterparts. Importantly, perfusion bioreactor culture significantly promoted the expression of chondrogenic marker genes while downregulating hypertrophy. This work highlights the potential of customizable AM platforms for the development of novel personalized repair strategies and more reliable in vitro models with a wide range of applications.
- A multimodal stimulation cell culture bioreactor for tissue engineering: A numerical modelling approachPublication . Meneses, João; Silva, João C.; Fernandes, Sofia R.; Datta, Abhishek; Ferreira, Frederico Castelo; Moura, Carla; Amado, Sandra; Alves, Nuno; Pascoal-Faria, PaulaThe use of digital twins in tissue engineering (TE) applications is of paramount importance to reduce the number of in vitro and in vivo tests. To pursue this aim, a novel multimodal bioreactor is developed, combining 3D design with numerical stimulation. This approach will facilitate the reproducibility between studies and the platforms optimisation (physical and digital) to enhance TE. The new bioreactor was specifically designed to be additive manufactured, which could not be reproduced with conventional techniques. Specifically, the design suggested allows the application of dual stimulation (electrical and mechanical) of a scaffold cell culture. For the selection of the most appropriate material for bioreactor manufacturing several materials were assessed for their cytotoxicity. Numerical modelling methods were then applied to the new bioreactor using one of the most appropriate material (Polyethylene Terephthalate Glycol-modified (PETG)) to find the optimal stimulation input parameters for bone TE based on two reported in vitro studies.
- PEDOT:PSS-coated polybenzimidazole electroconductive nanofibers for biomedical applicationsPublication . Sordini, Laura; Silva, João C.; Garrudo, Fábio F. F.; Rodrigues, Carlos A. V.; Marques, Ana C.; Linhardt, Robert J.; Cabral, Joaquim S. M.; Morgado, Jorge; Ferreira, Frederico CasteloBioelectricity drives several processes in the human body. The development of new materials that can deliver electrical stimuli is gaining increasing attention in the field of tissue engineering. In this work, novel, highly electrically conductive nanofibers made of poly [2,20 - m-(phenylene)-5,50 -bibenzimidazole] (PBI) have been manufactured by electrospinning and then coated with cross-linked poly (3,4-ethylenedioxythiophene) doped with poly (styrene sulfonic acid) (PEDOT:PSS) by spin coating or dip coating. These scaffolds have been characterized by scanning electron microscopy (SEM) imaging and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The electrical conductivity was measured by the four-probe method at values of 28.3 S·m−1 for spin coated fibers and 147 S·m−1 for dip coated samples, which correspond, respectively, to an increase of about 105 and 106 times in relation to the electrical conductivity of PBI fibers. Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) cultured on the produced scaffolds for one week showed high viability, typical morphology and proliferative capacity, as demonstrated by calcein fluorescence staining, 40 ,6-diamidino-2-phenylindole (DAPI)/Phalloidin staining and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay. Therefore, all fiber samples demonstrated biocompatibility. Overall, our findings highlight the great potential of PEDOT:PSS-coated PBI electrospun scaffolds for a wide variety of biomedical applications, including their use as reliable in vitro models to study pathologies and the development of strategies for the regeneration of electroactive tissues or in the design of new electrodes for in vivo electrical stimulation protocols.
- Strategy to improve the mechanical properties of bioabsorbable materials based on chitosan for orthopedic fixation applicationsPublication . Figueiredo, Lígia; Fonseca, Rita; Pinto, Luís F. V.; Ferreira, Frederico Castelo; Almeida, Amélia; Rodrigues, AlexandraBioabsorbable polymeric fixation devices have been used as an alternative to metallic implants in orthopedics,preventing the stress shielding effect and avoiding a second surgery for implant removal. However,several problems are still associated with current bioabsorbable implants, including the limited mechanical stiffness and strength, and the adverse tissue reactions generated. To minimize or even eliminate the problems associated with these implants, strategies have been developed to synthesize new implant materials based on chitosan. To overcome the brittle behavior of most 3D chitosan-based structures, glycerol and sorbitol were blended to chitosan and the effect of these plasticizers in the produced specimens was analyzed by flexural tests, Berkovich tests, scanning electron microscopy (SEM) and micro-CT analyzes. The improvement of the mechanical properties was also tested by adding ceramics, namely hydroxyapatite powder and biphasic mixtures of hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP). In the plasticizers group, the best combination of the measured properties was obtained for chitosan with 10% glycerol (flexural strength of 53.8 MPa and indentation hardness of 19.4 kgf/mm2), while in the ceramics group the best mechanical behavior was obtained for chitosan with 10% HAþβ-TCP powder (flexural strength of 67.5 MPa and indentation hardness 28.2 kgf/mm2). All the tested material compositions were dense and homogeneous, fundamental condition for a good implant performance. These are encouraging results, which support the continued development of chitosan-based materials for orthopedic fixation applications.