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- A Brief Review on Processes for Cartilage RepairPublication . Moura, Carla; Santos-Rocha, Rita; Franco, Susana; Malça, Cândida; Galhano, Cristina; Henriques, Marta; Morouço, PedroThe aim of the present review was to highlight some of the available processes for cartilage repair and regeneration. Considering the high impact that cartilage degeneration has in the quality of life, in an aging society, efforts to promote better treatments are crucial. The current available processes have advantages and drawbacks, that should be further investigated, aiming to obtain tailored and successful repair. Finally, some suggestions for tissue engineering strategies are presented, so that the scientific community can debate pros and cons to be investigated.
- Comprehensive review on full bone regeneration through 3D printing approachesPublication . Fernandes, Cristiana; Moura, Carla; Ascenso, Rita M.T.; Amado, Sandra; Alves, Nuno; Pascoal-Faria, PaulaOver the last decades, the number of work accidents associated with bone fractures has increased leading to a growing concern worldwide. Currently, autografts, allografts, and xenografts are used for bone regeneration. However, their application has associated risks. Tissue engineering (TE) has brought solutions to address these problems, through the production of temporary supports, providing mechanical support to the formation of new bone tissue and biocompatible and biodegradable scaffolds, which allow cell adhesion and proliferation to ensure bone formation. The combination of materials and structure with the technique to be used will directly influence their physical and chemical properties and, consequently, their action in contributing to bone regeneration. Thus, the focus of this chapter is to perform an exhaustive literature review and a critical analysis of the state of the art in bone TE and present a proposal of an optimized temporary support geometry for bone regeneration in case of large bone defects. For this, it was listed and identified the best choice of biomaterials, fabrication method, cell type and their culture conditions (static vs. dynamic), and/or the inclusion of growth factors for the repair of large bone defects.
- 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.
- 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.
- Characterization of Biocompatible Poly(Ethylene Glycol)-Dimethacrylate Hydrogels for Tissue EngineeringPublication . Lopes, João; Fonseca, Rita; Viana, Tânia; Fernandes, Cristiana; Morouço, Pedro; Moura, Carla; Biscaia, SaraTissue Engineering depends on broadly techniques to regenerate tissues and/or organ functions. To do so, tailored polymeric and/or hydrogel scaffolds may be used to ensure the appropriate regeneration. Hydrogels are suitable materials for constructing cell-laden matrices as they can be produced with incorporation of cells and rapidly cross-linked in situ through photopolymerisation reactions. Measurement of the polymerization degree, as well as resistance to compression and water retention are fundamental tests to evaluate the characteristics of hydrogels. In this work, free-radical polymerisation of poly(ethylene glycol)-dimethacrylate (PEGDMA) in UV light was assessed. Several hydrogels with different photoinitiator and water contents were produced to evaluate their influence on hydrogels behaviour. Experiments showed that variations on water and photoinitiator content induce changes in the physical and chemical behaviour of hydrogels. As it was found, water content prevents polymerisation to occur and reduces the mechanical properties of hydrogels weakening them. Furthermore, differences were found in varying water content from 15 to 30%, since this increase turned hydrogels more fragile and increase their stabilization time for water retention.
- Biofísica - Caso ClínicoPublication . Carrão, Luis Miguel Costa; Moura, Carla
- From animal to human: (Re)using acellular extracellular matrices for temporomandibular disc substitutionPublication . Trindade, Daniela; Alves, Nuno; Moura, CarlaCurrent treatments for temporomandibular joint (TMJ) disc dysfunctions are not fully effective and lack regenerative capacity. Therefore, the search for tissue-engineered materials for TMJ disc substitution is critical to fill this gap. Decellularization presents tremendous potential, as it is possible to obtain an extracellular matrix with an adequate biomechanical structure and biochemical components. However, its application to the TMJ disc is still in progress, since there are few studies in the literature, and those that exist have many gaps in terms of characterisation, which is decisive to ensure its success. Ultimately, we intend to emphasize the importance of the decellularization technique for the development of an engineered TMJ disc.
- Preclinical randomized controlled trial of bilateral discectomy versus bilateral discopexy in Black Merino sheep temporomandibular joint: TEMPOJIMS e Phase 1- histologic, imaging and body weight resultsPublication . Ângelo, David Faustino; Morouço, Pedro; Gil, Florêncio Monje; Mónico, Lisete; González-Garcia, Raúl; Sousa, Rita; Neto, Lia; Caldeira, Inês; Smith, Margaret; Smith, Susan; Sanz, David; Santos, Fábio Abade dos; Pinho, Mário; Carrapiço, Belmira; Cavaco, Sandra; Moura, Carla; Alves, Nuno; Salvado, Francisco; Little, ChristopherIntroduction: The role of temporomandibular joint (TMJ) surgery is not well defined due to a lack of quality randomized controlled clinical trials, comparing different TMJ surgical treatments with medical and placebo interventions. The temporomandibular joint interposal study (TEMPOJIMS) is a rigorous preclinical trial divided in 2 phases. In phase 1 the authors investigated the role of the TMJ disc and in phase 2 the authors evaluated 3 different interposal materials. The present work of TEMPOJIMS e phase 1, aims to evaluate histopathologic and imaging changes of bilateral discectomy and discopexy in Black Merino sheep TMJ, using a high-quality trial following the ARRIVE guidelines. Material and methods: This randomized, blinded and controlled preclinical trial was conducted in 9 Black Merino sheep to investigate histopathologic (primary outcome), imaging and body weight (secondary outcomes) changes after bilateral discectomy, discopexy and sham surgery. Results: Significant changes were noticed in discectomy group, both in imaging and histopathologic analyses. Body weight changes were most pronounced in the discectomy group in the first 4 months after surgery with recovery to baseline weight 6 months after surgery. Discopexy induced nonsignificant changes in histopathologic, imaging and body weight analyses. Conclusions: This study reinforces the importance of developing an effective interposal material to substitute the TMJ disc and the need to explore the molecular mechanisms that underlie TMJ cartilage degeneration. The study design proposed in TEMPOJIMS represents an important progress towards future rigorous TMJ investigations.
- Multi-material implants for temporomandibular joint disc repair: Tailored additive manufacturing productionPublication . Moura, Carla; Trindade, Daniela; Vieira, Milena; Francisco, Luís; Ângelo, David Faustino; Alves, NunoTemporomandibular disorders (TMD) affect a substantial percentage of the population, and the resources spent on their treatment are considerable. Despite the worldwide efforts around Tissue Engineering of the temporomandibular joint (TMJ) disc, a proper implant offering a long-term solution for TMD was not yet developed. To contribute to these efforts, this work is focused on the research and development of implants for TMJ disc regeneration. Scaffolds and hydrogels mimicking the TMJ disc of black Merino sheep were produced using different materials, poly(+-caprolactone) (PCL) and poly(ethylene glycol) diacrylate (PEGDA), and as a multi-material structure. Different parameters of the scaffold manufacturing were assessed: the influence of processing temperatures, filament diameter, and biological environment. Moreover, two multimaterial approaches were also assessed, scaffold with a hydrogel shell and scaffold with a hydrogel core. It was found that increasing temperature, the scaffolds’ porosity decreases, increasing their compressive modulus. Decreasing the filament size (300 to 200 mm) decreases the compressive modulus to almost half of the initial value. Scaffolds with 200 mm filaments are the ones with a closer modulus to the native disc and their properties are maintained under hydrated conditions. The introduction of a hydrogel core in these scaffolds presented better mechanical properties to TMJ disc substitution.
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