RESUMO
One of the challenges in substituting dairy products by alternative proteins is that the properties of mixed protein gels cannot necessarily be predicted by those of single protein gels, whereas the need of mixing is often driven by nutritional aspects. However, mixing plant proteins could also open a door to new textures. The main goal of this study was to investigate the impact of binary mixing of hemp (H), yellow pea (P), and brown lentil (L) protein concentrates/isolates on their gel and water-holding properties. Dispersions of reconstituted proteins and mixtures thereof were gelled using glucono-δ-lactone (GDL), transglutaminase (TG), and temperature (T) at a protein content of 12% (w/w). Mixtures of pea and lentil proteins showed gel strengths for TG- and T-induced gels that are proportional to the ratio of the mixture constituents (linear mixing behavior), whereas synergistic effects were observed for GDL-induced gelation. In contrast, all mixtures containing hemp exhibited a non-linear mixing behavior for the three gelation methods, usually resulting in lower gel strengths compared to theoretically expected values. The study showed that mixing plant-based proteins of different protein sources can lead to very different mixing behaviors in terms of gel properties, showing either a reinforcing, an indifferent or a weakening effect compared to the theoretically expected properties. The results can help developing more targeted plant protein-based soft gel products such as yogurt alternatives with specific techno-functional properties, while adjusting the nutritional characteristics.
Assuntos
Cannabis , Lens (Planta) , Géis , Gluconatos , Lactonas , Pisum sativum , Proteínas de Plantas , Transglutaminases , ÁguaRESUMO
Intra-articular administration of glucocorticoids such as dexamethasone is a common treatment for osteoarthritic inflammation and pain. Despite its potent anti-inflammatory properties, multiple barriers hinder the drug's effectiveness in the articular space. In particular, the high turnover rate of the synovial fluid and the dense cartilage extracellular matrix (ECM) lead to poor drug penetration into cartilage. In order to increase the infiltration and retention time, two dexamethasone prodrugs were developed. Firstly, dexamethasone was conjugated to polycationic chitosan, which led to deep and sustained infiltration of the drug into full thickness cartilage, due to its strong electrostatic interactions with the high negative fixed charges of the cartilage ECM. Secondly, dexamethasone was conjugated to a collagen type II-binding peptide, WYRGRL, and this prodrug was shown to be retained in the deep zones of cartilage through specific interactions with cartilage-specific collagen type II bundles. In both cases, active dexamethasone was released from the carrier by ester linkage hydrolysis. Complexing dexamethasone with either chitosan or collagen type II-affinity carriers increased its binding and therapeutic efficacy inside cartilage, compared to the free drug. Both dexamethasone conjugates significantly reduced levels of inflammatory markers and slowed the loss of glycosaminoglycans in an ex vivo model. A single dose of a cartilage-targeting dexamethasone prodrug represents a promising alternative to the repetitive glucocorticoid injections needed to compensate for its rapid clearance from the joint cavity.
Assuntos
Cartilagem Articular/metabolismo , Dexametasona/administração & dosagem , Sistemas de Liberação de Medicamentos , Glucocorticoides/administração & dosagem , Pró-Fármacos/administração & dosagem , Adulto , Animais , Bovinos , Células Cultivadas , Quitosana/metabolismo , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Dexametasona/farmacocinética , Dexametasona/farmacologia , Portadores de Fármacos/metabolismo , Feminino , Glucocorticoides/farmacocinética , Glucocorticoides/farmacologia , Humanos , Masculino , Peptídeos/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Adulto JovemRESUMO
Cellular processes involve dynamic rearrangement of the cytoskeleton. The GTPase RhoA plays a fundamental role in controlling cytoskeletal architecture. The phenotypic stability of chondrocytes is enhanced through inhibition of RhoA, whereas RhoA activation leads to dedifferentiation. We hypothesized that local inhibition of this pathway could induce chondrogenesis and cartilage regeneration. In this study, a novel alginate-derived hydrogel system was developed for sustained RhoA targeting. Specifically, an engineered variant of C. botulinum C3 transferase, a potent RhoA inhibitor, was immobilized onto a hydrogel to achieve sustained release and enzymatic activity. Chondrocytes encapsulated within this fully biocompatible, mechanically stable scaffold produced a stable collagen type II-rich matrix in vitro which matured over a six-week period. Samples were implanted subcutaneously in mice, and similar production of a collagen type II-rich matrix was observed. The intrinsically versatile system has the potential to treat a number of clinical disorders, including osteoarthritis, linked with RhoA dysregulation.
Assuntos
Alginatos/química , Hidrogéis/química , Proteína rhoA de Ligação ao GTP/química , ADP Ribose Transferases/farmacologia , Animais , Materiais Biocompatíveis , Biomarcadores , Toxinas Botulínicas/farmacologia , Desdiferenciação Celular , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Enzimas Imobilizadas/farmacologia , Camundongos , Alicerces Teciduais , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
The bacterial ligase Sortase A (SA) and its mutated variants have become increasingly popular over the last years for post-translational protein modifications due to their unparalleled specificity and efficiency. The aim of this work was to study SA as a cross-linking enzyme for hydrogel-based tissue engineering. For this, we optimized SA pentamutant production and purification from E. coli to achieve high yields and purity. Then using hyaluronan (HA) as a model biopolymer and modifying it with SA-substrate peptides, we studied the cross-linking kinetics obtained with SA, the enzyme stability, cytocompatibility, and immunogenicity, and compared those to state-of-the-art standards. The transglutaminase activated factor XIII (FXIIIa) was used as the reference cross-linking enzyme, and the clinical collagen scaffold Chondro-Gide (CG) was used as a reference biocompatible material for in vivo studies. We found SA could be produced in large amounts in the lab without special equipment, whereas the only viable source of FXIIIa is currently a prescription medicine purified from donated blood. SA was also remarkably more stable in solution than FXIIIa, and it could provide even much faster gelation, making it possible to achieve nearly-instantaneous gel formation upon delivery with a double-barrel syringe. This is an interesting improvement for in vivo work, to allow in situ gel formation in a wet environment, and could also be useful for applications like bioprinting where very fast gelation is needed. The cytocompatibility and lack of immunogenicity were still uncompromised. These results support the use of SA as a versatile enzymatic cross-linking strategy for 3D culture and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Enzymatic crosslinking has immense appeal for tissue engineers as one of the most biocompatible methods of hydrogel crosslinking. Sortase A has a number of unique advantages over previous systems. We show an impressive and tunable range of crosslinking kinetics, from almost instantaneous gelation to several minutes. We also demonstrate that Sortase A crosslinked hydrogels have good cytocompatibility and cause no immune reaction when implanted in vivo. With its additional benefits of excellent stability in solution and easy large-scale synthesis available to any lab, we believe this novel crosslinking modality will find multiple applications in high throughput screening, tissue engineering, and biofabrication.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Reagentes de Ligações Cruzadas/química , Cisteína Endopeptidases/química , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Biopolímeros/química , Linhagem Celular , Condrócitos/citologia , Endotoxinas/química , Escherichia coli/metabolismo , Fator XIII/química , Fibroblastos/citologia , Células HEK293 , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Inflamação , Cinética , Peptídeos/química , Processamento de Proteína Pós-Traducional , Reologia , Células-Tronco/citologia , Tensoativos/química , Transglutaminases/químicaRESUMO
The goal of this study was to evaluate human epiphyseal chondroprogenitor cells (ECPs) as a potential new cell source for cartilage regeneration. ECPs were compared to human bone marrow stromal cells (MSCs) and human adult articular chondrocytes (ACs) for their chondrogenic potential and phenotypic stability in vitro and in vivo. The cells were seeded in Optimaix-3D scaffolds at 5 × 104 cells/mm3 and gene expression, matrix production and mechanical properties were analysed up to 6 weeks. In vitro, ECPs synthesized consistently high collagen 2 and low collagen 10. AC-seeded constructs exhibited high donor variability in GAG/DNA values as well as in collagen 2 staining, but showed low collagen 10 production. MSCs, on the other hand, expressed high levels of collagen 2 but also of collagens 1 and 10, and were therefore not considered further. In vivo, there was considerable loss of matrix proteins in ECPs compared to in vitro cultured samples. To overcome this, a second implantation study investigated the effect of mixing cells with alginate prior to seeding in the scaffold. ECPs in alginate maintained their cartilage matrix and resisted mineralization and vessel infiltration better 6 weeks after subcutaneous implantation, whereas ACs lost their chondrogenic matrix completely. This study shows the great potential of ECPs as an off-the-shelf, highly chondrogenic cell type that produces stable cartilage in vivo. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Alginatos , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno , Células-Tronco/metabolismo , Alicerces Teciduais/química , Adulto , Alginatos/química , Alginatos/farmacologia , Cartilagem/citologia , Condrócitos/citologia , Colágeno/química , Colágeno/farmacologia , Feminino , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Engenharia TecidualRESUMO
A true biomimetic of the cartilage extracellular matrix (ECM) could greatly contribute to our ability to regenerate this tissue in a mechanically demanding, often inflamed environment. Articular cartilage is a composite tissue made of cells and fibrillar proteins embedded in a hydrophilic polymeric meshwork. Here, a polyanionic functionalized alginate is used to mimic the glycosaminoglycan component of the native ECM. To create the fibrillar component, cryoelectrospinning of poly(ε-caprolactone) on a -78 °C mandrel, subsequently treated by O2 plasma, is used to create a stable, ultraporous and hydrophillic nanofiber network. In this study, cell-laden, fiber-reinforced composite scaffolds thicker than 1.5 mm can be created by infiltrating a chondrocyte/alginate solution into the fiber mesh, which is then physically cross-linked. The fibrillar component significantly reinforces the chondroinductive, but mechanically weak sulfated alginate hydrogels. This allows the production of a glycosaminoglycan- and collagen type II-rich matrix by the chondrocytes as well as survival of the composite in vivo. To further enhance the system, the electrospun component is loaded with dexamethasone, which protected the cells from an IL-1ß-mediated inflammatory insult.