Browsing by Author "Linhardt, Robert J."
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- 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.
- 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.