19 results on '"Canadas RF"'
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2. Vancomycin-Loaded, Nanohydroxyapatite-Based Scaffold for Osteomyelitis Treatment: In Vivo Rabbit Toxicological Tests and In Vivo Efficacy Tests in a Sheep Model.
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Alegrete N, Sousa SR, Padrão T, Carvalho Â, Lucas R, Canadas RF, Lavrador C, Alexandre N, Gärtner F, Monteiro FJ, and Gutierres M
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The treatment for osteomyelitis consists of surgical debridement, filling of the dead space, soft tissue coverage, and intravenous administration of antimicrobial (AM) agents for long periods. Biomaterials for local delivery of AM agents, while providing controllable antibiotic release rates and simultaneously acting as a bone scaffold, may be a valuable alternative; thus, avoiding systemic AM side effects. V-HEPHAPC is a heparinized nanohydroxyapatite (nHA)/collagen biocomposite loaded with vancomycin that has been previously studied and tested in vitro. It enables a vancomycin-releasing profile with an intense initial burst, followed by a sustained release with concentrations above the Minimum Inhibitory Concentration (MIC) for MRSA. In vitro results have also shown that cellular viability is not compromised, suggesting that V-HEPHAPC granules may be a promising alternative device for the treatment of osteomyelitis. In the present study, V-HEPHAPC (HEPHAPC with vancomycin) granules were used as a vancomycin carrier to treat MRSA osteomyelitis. First, in vivo Good Laboratory Practice (GLP) toxicological tests were performed in a rabbit model, assuring that HEPHAPC and V-HEPHAPC have no relevant side effects. Second, V-HEPHAPC proved to be an efficient drug carrier and bone substitute to control MRSA infection and simultaneously reconstruct the bone cavity in a sheep model.
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- 2023
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3. Pharmacological and Non-Pharmacological Agents versus Bovine Colostrum Supplementation for the Management of Bone Health Using an Osteoporosis-Induced Rat Model.
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Kydonaki EK, Freitas L, Reguengo H, Simón CR, Bastos AR, Fernandes EM, Canadas RF, Oliveira JM, Correlo VM, Reis RL, Vliora M, Gkiata P, Koutedakis Y, Ntina G, Pinto R, Carrillo AE, Marques F, and Amorim T
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- Alendronate pharmacology, Animals, Body Weight, Cattle, Colostrum metabolism, Dietary Supplements, Female, Humans, Ovariectomy, Pregnancy, Rats, Rats, Sprague-Dawley, Bone Density, Osteoporosis drug therapy
- Abstract
Osteoporosis is defined by loss of bone mass and deteriorated bone microarchitecture. The present study compared the effects of available pharmacological and non-pharmacological agents for osteoporosis [alendronate (ALE) and concomitant supplementation of vitamin D (VD) and calcium (Ca)] with the effects of bovine colostrum (BC) supplementation in ovariectomized (OVX) and orchidectomized (ORX) rats. Seven-month-old rats were randomly allocated to: (1) placebo-control, (2) ALE group (7.5 μg/kg of body weight/day/5 times per week), (3) VD/Ca group (VD: 35 μg/kg of body weight/day/5 times per week; Ca: 13 mg/kg of body weight/day/3 times per week), and (4) BC supplementation (OVX: 1.5 g/day/5 times per week; ORX: 2 g/day/5 times per week). Following four months of supplementation, bone microarchitecture, strength and bone markers were evaluated. ALE group demonstrated significantly higher Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC and significantly lower Ct.Pr, Tb.Pr, Tb.Sp, Ct.BMD and Tb.BMD, compared to placebo (p < 0.05). BC presented significantly higher Ct.Pr, Ct.BMD, Tb.Pr, Tb.Sp, and Tb.BMD and significantly lower Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC compared to ALE in OVX rats (p < 0.05). OVX rats receiving BC experienced a significant increase in serum ALP and OC levels post-supplementation (p < 0.05). BC supplementation may induce positive effects on bone metabolism by stimulating bone formation, but appear not to be as effective as ALE.
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- 2022
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4. Numerical and experimental simulation of a dynamic-rotational 3D cell culture for stratified living tissue models.
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Canadas RF, Liu Z, Gasperini L, Fernandes DC, Maia FR, Reis RL, Marques AP, Liu C, and Oliveira JM
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- Bioreactors, Computer Simulation, Humans, Perfusion, Cell Culture Techniques, Three Dimensional, Tissue Engineering methods
- Abstract
Human tissues and organs are inherently heterogeneous, and their functionality is determined by the interplay between different cell types, their secondary architecture, and gradients of signalling molecules and metabolites. To mimic the dynamics of native tissues, perfusion bioreactors and microfluidic devices are widely used in tissue engineering (TE) applications for enhancing cell culture viability in the core of 3D constructs. Still, most in vitro screening methods for compound efficacy and toxicity assessment include cell or tissue exposure to constant and homogeneous compound concentrations over a defined testing period. Moreover, a prevalent issue inhibiting the large-scale adoption of microfluidics and bioreactor is the tubing dependence to induce a perfusion regime. Here, we propose a compartmentalized rotational (CR) 3D cell culture platform for a stable control over gradient tissue culture conditions. Using the CR bioreactor, adjacent lanes of constructs are patterned by controlled flow dynamics to enable tissue stratification. Numerical and experimental simulations demonstrate cell seeding dynamics, as well as culture media rotational perfusion and gradient formations. Additionally, the developed system induces vertical and horizontal rotations, which increase medium exchange and homogeneous construct maturation, allowing both perfused tubing-based and tubing-free approaches. As a proof-of-concept, experiments and accompanying simulation of cellular inoculation and growth in 3D scaffold and hydrogel were performed, before the examination of a blood-brain-barrier model, demonstrating the impact of a heterotypic culture on molecular permeability under mimetic dynamic conditions. Briefly, the present work discloses the simulation of 3D dynamic cultures, and a semi-automated platform for heterotypic tissues in vitro modelling, for broad TE and drug discovery/screening applications., (© 2022 IOP Publishing Ltd.)
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- 2022
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5. Porous aligned ZnSr-doped β-TCP/silk fibroin scaffolds using ice-templating method for bone tissue engineering applications.
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Bicho D, Canadas RF, Gonçalves C, Pina S, Reis RL, and Oliveira JM
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- Bone and Bones, Calcium Phosphates, Ice, Porosity, Tissue Scaffolds, Fibroins, Tissue Engineering
- Abstract
The bone is a complex and dynamic structure subjected to constant stress and remodeling. Due to the worldwide incidence of bone disorders, tissue scaffolds and engineered bone tissues have emerged as solutions for bone grafting, which require sophisticated scaffolding architectures while keeping high mechanical performance. However, the conjugation of a bone-like scaffold architecture with efficient mechanical properties is still a critical challenge for biomedical applications. In this sense, the present study focused on the modulating the architecture of silk fibroin (SF) scaffolds crosslinked with horseradish peroxidase and mixed with zinc (Zn) and strontium (Sr)-doped β-tricalcium phosphate (ZnSr.TCP) to mimic bone structures. The ZnSr.TCP-SF hydrogels were tuned by programmable ice-templating parameters, and further freeze-dried, in order to obtain 3D scaffolds with controlled pore orientation. The results showed interconnected channels in the ZnSr.TCP-SF scaffolds that mimic the porous network of the native subchondral bone matrix. The architecture of the scaffolds was characterized by microCT, showing tunable pore size according to freezing temperatures (-196 °C: ∼80.2 ± 20.5 µm; -80 °C: ∼73.1 ± 20.5 µm; -20 °C: ∼104.7 ± 33.7 µm). The swelling ratio, weight loss, and rheological properties were also assessed, revealing efficient scaffold integrity and morphology after aqueous immersion. Thus, the ZnSr.TCP-SF scaffolds made of aligned porous structure were developed as affordable candidates for future applications in clinical osteoregeneration and in vitro bone tissue modelling.
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- 2021
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6. 3DICE coding matrix multidirectional macro-architecture modulates cell organization, shape, and co-cultures endothelization network.
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Canadas RF, Costa JB, Mao Z, Gao C, Demirci U, Reis RL, Marques AP, and Oliveira JM
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- Coculture Techniques, Porosity, Regenerative Medicine, Tissue Scaffolds, Cryogels, Tissue Engineering
- Abstract
Natural extracellular matrix governs cells providing biomechanical and biofunctional outstanding properties, despite being porous and mostly made of soft materials. Among organs, specific tissues present specialized macro-architectures. For instance, hepatic lobules present radial organization, while vascular sinusoids are branched from vertical veins, providing specific biofunctional features. Therefore, it is imperative to mimic such structures while modeling tissues. So far, there is limited capability of coupling oriented macro-structures with interconnected micro-channels in programmable long-range vertical and radial sequential orientations. Herein, a three-directional ice crystal elongation (3DICE) system is presented to code geometries in cryogels. Using 3DICE, guided ice crystals growth templates vertical and radial pores through bulky cryogels. Translucent isotropic and anisotropic architectures of radial or vertical pores are fabricated with tunable mechanical response. Furthermore, 3D combinations of vertical and radial pore orientations are coded at the centimeter scale. Cell morphological response to macro-architectures is demonstrated. The formation of endothelial segments, CYP450 activity, and osteopontin expression, as liver fibrosis biomarkers, present direct response and specific cellular organization within radial, linear, and random architectures. These results unlock the potential of ice-templating demonstrating the relevance of macro-architectures to model tissues, and broad possibilities for drug testing, tissue engineering, and regenerative medicine., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
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- 2021
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7. Bovine Colostrum Supplementation Improves Bone Metabolism in an Osteoporosis-Induced Animal Model.
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Kydonaki EK, Freitas L, Fonseca BM, Reguengo H, Raposo Simón C, Bastos AR, Fernandes EM, Canadas RF, Oliveira JM, Correlo VM, Reis RL, Vliora M, Gkiata P, Koutedakis Y, Ntina G, Pinto R, Carrillo AE, Marques F, and Amorim T
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- Animals, Bone Density, Bone and Bones drug effects, Bone and Bones metabolism, Cattle, Dietary Supplements, Disease Models, Animal, Female, Ovariectomy, Rats, Rats, Wistar, Colostrum metabolism, Osteoporosis drug therapy, Osteoporosis metabolism
- Abstract
Osteoporosis is characterized by bone loss. The present study aims to investigate the effects of bovine colostrum (BC) on bone metabolism using ovariectomized (OVX) and orchidectomized (ORX) rat models. Twenty-seven-week-old Wistar Han rats were randomly assigned as: (1) placebo control, (2) BC supplementation dose 1 (BC1: 0.5 g/day/OVX, 1 g/day/ORX), (3) BC supplementation dose 2 (BC2: 1 g/day/OVX, 1.5 g/day/ORX) and (4) BC supplementation dose 3 (BC3: 1.5 g/day/OVX, 2 g/day/ORX). Bone microarchitecture, strength, gene expression of VEGFA, FGF2, RANKL, RANK and OPG, and bone resorption/formation markers were assessed after four months of BC supplementation. Compared to the placebo, OVX rats in the BC1 group exhibited significantly higher cortical bone mineral content and trabecular bone mineral content ( p < 0.01), while OVX rats in the BC3 group showed significantly higher trabecular bone mineral content ( p < 0.05). ORX rats receiving BC dose 2 demonstrated significantly higher levels of trabecular bone mineral content ( p < 0.05). Serum osteocalcin in the ORX was pointedly higher in all BC supplementation groups than the placebo (BC1: p < 0.05; BC2, BC3: p < 0.001). Higher doses of BC induced significantly higher relative mRNA expression of OPG, VEGFA, FGF2 and RANKL ( p < 0.05). BC supplementation improves bone metabolism of OVX and ORX rats, which might be associated with the activation of the VEGFA, FGF2 and RANKL/RANK/OPG pathways.
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- 2021
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8. Bioengineered Nanoparticles Loaded-Hydrogels to Target TNF Alpha in Inflammatory Diseases.
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Oliveira IM, Fernandes DC, Maia FR, Canadas RF, Reis RL, and Oliveira JM
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Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients' mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.
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- 2021
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9. Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone and porous silk fibroin for meniscus tissue engineering.
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Cengiz IF, Maia FR, da Silva Morais A, Silva-Correia J, Pereira H, Canadas RF, Espregueira-Mendes J, Kwon IK, Reis RL, and Oliveira JM
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- Animals, Biocompatible Materials chemistry, Biocompatible Materials pharmacology, Cell Adhesion drug effects, Cell Survival drug effects, Humans, Male, Meniscus cytology, Meniscus metabolism, Meniscus transplantation, Mice, Mice, Nude, Porosity, Stem Cell Transplantation, Stem Cells cytology, Stem Cells metabolism, Fibroins chemistry, Polyesters chemistry, Printing, Three-Dimensional, Tissue Engineering methods, Tissue Scaffolds chemistry
- Abstract
The meniscus has critical functions in the knee joint kinematics and homeostasis. Injuries of the meniscus are frequent, and the lack of a functional meniscus between the femur and tibial plateau can cause articular cartilage degeneration leading to osteoarthritis development and progression. Regeneration of meniscus tissue has outstanding challenges to be addressed. In the current study, novel Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone (PCL) and porous silk fibroin were proposed for meniscus tissue engineering. As confirmed by micro-structural analysis the entrapment of silk fibroin was successful, and all scaffolds had excellent interconnectivity (≥99%). The EiC scaffolds had more favorable micro-structure compared with the PCL cage scaffolds by improving the pore size while keeping the interconnectivity almost the same. When compared with the PCL cage, the entrapment of porous silk fibroin into the PCL cage decreased the high compressive modulus in a favorable matter in the wet state thanks to the silk fibroin's high swelling properties. The in vitro studies with human stem cells or meniscocytes seeded constructs, demonstrated that the EiC scaffolds had superior cell adhesion, metabolic activity, and proliferation compared to the PCL cage scaffolds. Upon subcutaneous implantation of scaffolds in nude mice, all groups were free of adverse incidents, and mildly invaded by inflammatory cells with neovascularization, while the EiC scaffolds showed better tissue infiltration. The results of this work indicated that the EiC scaffolds of PCL and silk fibroin are favorable for meniscus tissue engineering, and the findings are encouraging for further studies using a larger animal model.
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- 2020
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10. Dynamic Culture Systems and 3D Interfaces Models for Cancer Drugs Testing.
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Fernandes DC, Canadas RF, Reis RL, and Oliveira JM
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- Animals, Bioreactors, Humans, Microfluidics, Tissue Engineering, Antineoplastic Agents pharmacology, Cell Culture Techniques methods, Drug Screening Assays, Antitumor methods, Models, Biological, Neoplasms drug therapy, Neoplasms pathology
- Abstract
The mass use of biological agents for pharmaceutical purposes started with the development and distribution of vaccines, followed by the industrial production of antibiotics. The use of dynamic systems, such as bioreactors, had been already applied in the food industry in fermentation processes and started being used for the development of pharmaceutical agents from this point on. In the last decades, the use of bioreactors and microfluidic systems has been expanded in different fields. The emergence of the tissue engineering led to the development of in vitro models cultured in dynamic systems. This is particularly relevant considering the urgent reduction of the total dependence on animal disease models that is undermining the development of novel drugs, using alternatively human-based models to make the drug discovery process more reliable. The failure out coming from animal models has been more prevalent in certain types of cancer, such as glioblastoma multiform and in high-grade metastatic cancers like bone metastasis of breast or prostatic cancer. The difficulty in obtaining novel drugs for these purposes is mostly linked to the barriers around the tumors, which these bioactive molecules have to overcome to become effective. For that reason, the individualized study of each interface is paramount and is only realistic once applying human-based samples (e.g. cells or tissues) in three-dimensions for in vitro modeling under dynamic conditions. In this chapter, the most recent approaches to model these interfaces in 3D systems will be explored, highlighting their major contributions to the field. In this section, these systems' impact on increased knowledge in relevant aspects of cancer aggressiveness as invasive or motile cellular capacity, or even resistance to chemotherapeutic agents will have particular focus. The last section of this chapter will focus on the integration of the tumor interfaces in dynamic systems, particularly its application on high-throughput drug screening. The industrial translation of such platforms will be discussed, as well as the main upcoming challenges and future perspectives.
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- 2020
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11. A soft 3D polyacrylate hydrogel recapitulates the cartilage niche and allows growth-factor free tissue engineering of human articular cartilage.
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Jiménez G, Venkateswaran S, López-Ruiz E, Perán M, Pernagallo S, Díaz-Monchón JJ, Canadas RF, Antich C, Oliveira JM, Callanan A, Walllace R, Reis RL, Montañez E, Carrillo E, Bradley M, and Marchal JA
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- Animals, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Acrylic Resins chemistry, Cartilage, Articular metabolism, Chondrocytes metabolism, Extracellular Matrix chemistry, Hydrogels chemistry, Tissue Engineering, Tissue Scaffolds chemistry
- Abstract
Cartilage degeneration or damage treatment is still a challenge, but, tissue engineering strategies, which combine cell therapy strategies, which combine cell therapy and scaffolds, and have emerged as a promising new approach. In this regard, polyurethanes and polyacrylates polymers have been shown to have clinical potential to treat osteochondral injuries. Here, we have used polymer microarrays technology to screen 380 different polyurethanes and polyacrylates polymers. The top polymers with potential to maintain chondrocyte viability were selected, with scale-up studies performed to evaluate their ability to support chondrocyte proliferation during long-term culture, while maintaining their characteristic phenotype. Among the selected polymers, poly (methylmethacrylate-co-methacrylic acid), showed the highest level of chondrogenic potential and was used to create a 3D hydrogel. Ultrastructural morphology, microstructure and mechanical testing of this novel hydrogel revealed robust characteristics to support chondrocyte growth. Furthermore, in vitro and in vivo biological assays demonstrated that chondrocytes cultured on the hydrogel had the capacity to produce extracellular matrix similar to hyaline cartilage, as shown by increased expression of collagen type II, aggrecan and Sox9, and the reduced expression of the fibrotic marker's collagen type I. In conclusion, hydrogels generated from poly (methylmethacrylate-co-methacrylic acid) created the appropriate niche for chondrocyte growth and phenotype maintenance and might be an optimal candidate for cartilage tissue-engineering applications. SIGNIFICANCE STATEMENT: Articular cartilage has limited self-repair ability due to its avascular nature, therefore tissue engineering strategies have emerged as a promising new approach. Synthetic polymers displaygreat potential and are widely used in the clinical setting. In our study, using the polymer microarray technique a novel type of synthetic polyacrylate was identified, that was converted into hydrogels for articular cartilage regeneration studies. The hydrogel based on poly (methylmethacrylate-co-methacrylic acid-co-PEG-diacrylate) had a controlable ultrastructural morphology, microstructure (porosity) and mechanical properties (stiffness) appropriate for cartilage engineering. Our hydrogel created the optimal niche for chondrocyte growth and phenotype maintenance for long-term culture, producing a hyaline-like cartilage extracellular matrix. We propose that this novel polyacrylate hydrogel could be an appropriate support to help in the treatment efficient cartilage regeneration., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2019
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12. Tunable anisotropic networks for 3-D oriented neural tissue models.
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Canadas RF, Ren T, Tocchio A, Marques AP, Oliveira JM, Reis RL, and Demirci U
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- Animals, Cell Survival physiology, Cerebral Cortex cytology, Human Umbilical Vein Endothelial Cells, Humans, Mice, Inbred C57BL, Microfluidics methods, Tissue Engineering methods, Anisotropy, Nerve Tissue cytology, Tissue Scaffolds chemistry
- Abstract
Organized networks are common in nature showing specific tissue micro-architecture, where cells can be found isotropically or anisotropically distributed in characteristic arrangements and tissue stiffness. However, when addressing an in vitro tissue model, it is challenging to grant control over mechanical properties while achieving anisotropic porosity of polymeric networks, especially in three-dimensional systems (3-D). While progress was achieved organizing cells in two-dimension (2-D), fabrication methods for aligned networks in 3-D are limited. Here, we describe the use of a biomimetic extra-cellular matrix system allowing programming of anisotropic structures into precisely advancing pore diameters in 3-D. Using control over polymeric composition, crosslinking directionality and freezing gradient dynamics, we revealed a mechanism to top-down biofabricate 3-D structures with tunable micro-porosity capable of directing cellular responses at millimeter scale such as axonal anisotropic outgrowth that is a unique characteristic of the brain cortex. Further, we showed the unique integration of this method with a microfluidic system establishing a neural-endothelial heterotypic conjugation, which can potentially be broadly applied to multiple organ systems., (Copyright © 2018. Published by Elsevier Ltd.)
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- 2018
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13. Marine Collagen/Apatite Composite Scaffolds Envisaging Hard Tissue Applications.
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Diogo GS, Senra EL, Pirraco RP, Canadas RF, Fernandes EM, Serra J, Pérez-Martín RI, Sotelo CG, Marques AP, González P, Moreira-Silva J, Silva TH, and Reis RL
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- Animals, Apatites isolation & purification, Biocompatible Materials chemistry, Biocompatible Materials isolation & purification, Bone and Bones injuries, Collagen isolation & purification, Cross-Linking Reagents chemistry, Guided Tissue Regeneration methods, Materials Testing, Tissue Engineering methods, Apatites chemistry, Collagen chemistry, Sharks, Tissue Scaffolds chemistry
- Abstract
The high prevalence of bone defects has become a worldwide problem. Despite the significant amount of research on the subject, the available therapeutic solutions lack efficiency. Autografts, the most commonly used approaches to treat bone defects, have limitations such as donor site morbidity, pain and lack of donor site. Marine resources emerge as an attractive alternative to extract bioactive compounds for further use in bone tissue-engineering approaches. On one hand they can be isolated from by-products, at low cost, creating value from products that are considered waste for the fish transformation industry. One the other hand, religious constraints will be avoided. We isolated two marine origin materials, collagen from shark skin ( Prionace glauca ) and calcium phosphates from the teeth of two different shark species ( Prionace glauca and Isurus oxyrinchus ), and further proposed to mix them to produce 3D composite structures for hard tissue applications. Two crosslinking agents, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) and hexamethylene diisocyanate (HMDI), were tested to enhance the scaffolds' properties, with EDC/NHS resulting in better properties. The characterization of the structures showed that the developed composites could support attachment and proliferation of osteoblast-like cells. A promising scaffold for the engineering of bone tissue is thus proposed, based on a strategy of marine by-products valorisation.
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- 2018
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14. Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration.
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Ribeiro VP, da Silva Morais A, Maia FR, Canadas RF, Costa JB, Oliveira AL, Oliveira JM, and Reis RL
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- Animals, Fibroins, Humans, Materials Testing, Mice, Mice, Inbred ICR, Stem Cells cytology, Subcutaneous Fat cytology, Tissue Engineering, Cartilage physiology, Chondrogenesis, Regeneration, Stem Cells metabolism, Subcutaneous Fat metabolism, Tissue Scaffolds chemistry
- Abstract
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., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2018
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15. Stem Cells for Osteochondral Regeneration.
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Canadas RF, Pirraco RP, Oliveira JM, Reis RL, and Marques AP
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- Adult Stem Cells transplantation, Bone Marrow Transplantation, Cells, Cultured transplantation, Chondrocytes transplantation, Chondrogenesis, Embryonic Stem Cells cytology, Forecasting, Humans, Induced Pluripotent Stem Cells transplantation, Mesenchymal Stem Cell Transplantation methods, Mesenchymal Stem Cells cytology, Osteogenesis, Regenerative Medicine trends, Tissue Engineering methods, Bone Diseases therapy, Cartilage Diseases therapy, Regenerative Medicine methods, Stem Cell Transplantation methods
- Abstract
Stem cell research plays a central role in the future of medicine, which is mainly dependent on the advances on regenerative medicine (RM), specifically in the disciplines of tissue engineering (TE) and cellular therapeutics. All RM strategies depend upon the harnessing, stimulation, or guidance of endogenous developmental or repair processes in which cells have an important role. Among the most clinically challenging disorders, cartilage degeneration, which also affects subchondral bone becoming an osteochondral (OC) defect, is one of the most demanding. Although primary cells have been clinically applied, stem cells are currently seen as the promising tool of RM-related research because of its availability, in vitro proliferation ability, pluri- or multipotency, and immunosuppressive features. Being the OC unit, a transition from the bone to cartilage, mesenchymal stem cells (MSCs) are the main focus for OC regeneration. Promising alternatives, which can also be obtained from the patient or at banks and have great differentiation potential toward a wide range of specific cell types, have been reported. Still, ethical concerns and tumorigenic risk are currently under discussion and assessment. In this book chapter, we revise the existing stem cell-based approaches for engineering bone and cartilage, focusing on cell therapy and TE. Furthermore, 3D OC composites based on cell co-cultures are described. Finally, future directions and challenges still to be faced are critically discussed.
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- 2018
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16. Bioreactors and Microfluidics for Osteochondral Interface Maturation.
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Canadas RF, Marques AP, Reis RL, and Oliveira JM
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- Animals, Biological Transport, Bone and Bones cytology, Cell Communication, Cell Culture Techniques instrumentation, Cell Culture Techniques methods, Cells, Cultured, Chondrocytes cytology, Equipment Design, Forecasting, Humans, Implants, Experimental, Lab-On-A-Chip Devices, Rheology, Bioreactors, Chondrogenesis physiology, Microfluidics methods, Osteogenesis physiology, Tissue Engineering methods
- Abstract
The cell culture techniques are in the base of any biology-based science. The standard techniques are commonly static platforms as Petri dishes, tissue culture well plates, T-flasks, or well plates designed for spheroids formation. These systems faced a paradigm change from 2D to 3D over the current decade driven by the tissue engineering (TE) field. However, 3D static culture approaches usually suffer from several issues as poor homogenization of the formed tissues and development of a necrotic center which limits the size of in vitro tissues to hundreds of micrometers. Furthermore, for complex tissues as osteochondral (OC), more than recovering a 3D environment, an interface needs to be replicated. Although 3D cell culture is already the reality adopted by a newborn market, a technological revolution on cell culture devices needs a further step from static to dynamic already considering 3D interfaces with dramatic importance for broad fields such as biomedical, TE, and drug development. In this book chapter, we revised the existing approaches for dynamic 3D cell culture, focusing on bioreactors and microfluidic systems, and the future directions and challenges to be faced were discussed. Basic principles, advantages, and challenges of each technology were described. The reported systems for OC 3D TE were focused herein.
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- 2018
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17. Posterior talar process as a suitable cell source for treatment of cartilage and osteochondral defects of the talus.
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Correia SI, Silva-Correia J, Pereira H, Canadas RF, da Silva Morais A, Frias AM, Sousa RA, van Dijk CN, Espregueira-Mendes J, Reis RL, and Oliveira JM
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- Humans, Cartilage, Cell Proliferation, Talus cytology, Talus metabolism, Tissue Engineering methods
- Abstract
Osteochondral defects of the ankle are common lesions affecting the talar cartilage and subchondral bone. Current treatments include cell-based therapies but are frequently associated with donor-site morbidity. Our objective is to characterize the posterior process of the talus (SP) and the os trigonum (OT) tissues and investigate their potential as a new source of viable cells for application in tissue engineering and regenerative medicine. SP and OT tissues obtained from six patients were characterized by micro-computed tomography and histological, histomorphometric and immunohistochemical analyses. Proliferation and viability of isolated cells were evaluated by MTS assay, DNA quantification and live/dead staining. The TUNEL assay was performed to evaluate cell death by apoptosis. Moreover, the production of extracellular matrix was evaluated by toluidine blue staining, whereas cells phenotype was investigated by flow cytometry. Characterization of ankle explants showed the presence of a cartilage tissue layer in both SP and OT tissues, which represented at least 20%, on average, of the explant. The presence of type II collagen was detected in the extracellular matrix. Isolated cells presented a round morphology typical of chondrocytes. In in vitro studies, cells were viable and proliferating for up to 21 days of culture. No signs of apoptosis were detected. Flow-cytometry analysis revealed that isolated cells maintained the expression of several chondrocytic markers during culture. The results indicated that the SP and OT tissues were a reliable source of viable chondrocytes, which could find promising applications in ACI/MACI strategies with minimal concerns regarding donor zone complications. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)
- Published
- 2017
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- View/download PDF
18. Biofunctional Ionic-Doped Calcium Phosphates: Silk Fibroin Composites for Bone Tissue Engineering Scaffolding.
- Author
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Pina S, Canadas RF, Jiménez G, Perán M, Marchal JA, Reis RL, and Oliveira JM
- Subjects
- Biocompatible Materials pharmacology, Biomechanical Phenomena, Bone and Bones cytology, Bone and Bones physiology, Cell Differentiation, Fibroins pharmacology, Humans, Tissue Engineering, Tissue Scaffolds, Biocompatible Materials chemistry, Bone and Bones drug effects, Calcium Phosphates chemistry, Fibroins chemistry
- Abstract
The treatment and regeneration of bone defects caused by traumatism or diseases have not been completely addressed by current therapies. Lately, advanced tools and technologies have been successfully developed for bone tissue regeneration. Functional scaffolding materials such as biopolymers and bioresorbable fillers have gained particular attention, owing to their ability to promote cell adhesion, proliferation, and extracellular matrix production, which promote new bone growth. Here, we present novel biofunctional scaffolds for bone regeneration composed of silk fibroin (SF) and β-tricalcium phosphate (β-TCP) and incorporating Sr, Zn, and Mn, which were successfully developed using salt-leaching followed by a freeze-drying technique. The scaffolds presented a suitable pore size, porosity, and high interconnectivity, adequate for promoting cell attachment and proliferation. The degradation behavior and compressive mechanical strengths showed that SF/ionic-doped TCP scaffolds exhibit improved characteristics for bone tissue engineering when compared with SF scaffolds alone. The in vitro bioactivity assays using a simulated body fluid showed the growth of an apatite layer. Furthermore, in vitro assays using human adipose-derived stem cells presented different effects on cell proliferation/differentiation when varying the doping agents in the biofunctional scaffolds. The incorporation of Zn into the scaffolds led to improved proliferation, while the Sr- and Mn-doped scaffolds presented higher osteogenic potential as demonstrated by DNA quantification and alkaline phosphatase activity. The combination of Sr with Zn led to an influence on cell proliferation and osteogenesis when compared with single ions. Our results indicate that biofunctional ionic-doped composite scaffolds are good candidates for further in vivo studies on bone tissue regeneration., (© 2017 S. Karger AG, Basel.)
- Published
- 2017
- Full Text
- View/download PDF
19. Polyhydroxyalkanoates: waste glycerol upgrade into electrospun fibrous scaffolds for stem cells culture.
- Author
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Canadas RF, Cavalheiro JM, Guerreiro JD, de Almeida MC, Pollet E, da Silva CL, da Fonseca MM, and Ferreira FC
- Subjects
- Biomarkers metabolism, Cell Adhesion, Cell Culture Techniques, Cell Differentiation, Cell Survival, Humans, Hydrophobic and Hydrophilic Interactions, Mechanical Phenomena, Nanofibers chemistry, Nanofibers ultrastructure, Polyhydroxyalkanoates biosynthesis, Stem Cells cytology, Stem Cells metabolism, Waste Products, Glycerol chemistry, Polyhydroxyalkanoates chemistry, Tissue Scaffolds chemistry
- Abstract
This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic the structure of the native extracellular matrix, the natural environment where cells inhabit. Following a biorefinery approach, crude glycerol directly recovered from a biodiesel post-reaction stream was fed as major C source to Cupriavidus necator DSM 545 to produce polyhydroxyalkanoates at polymer titers of 9-25g/L. Two of the P(3HB-4HB-3HV) terpolymers produced, one containing 11.4% 4HB and 3.5% 3HV and the other containing 35.6% 4HB and 3.4% 3HV, were electrospun into fibers of average diameters of 600 and 1400nm, respectively. hMSC were cultured for 7 days in both fiber meshes, showing their ability to support stem cell growth at acceptable proliferation levels. Comparative results clearly demonstrate that scaffold topology is critical, with electrospun PHA fibers succeeding on the support of significant cell adhesion and proliferation, where planar PHA films failed., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
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