Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis

Abstract Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine

El avance de la agenda legislativa de género en las provincias argentinas: ¿cuánto importa el apoyo de los/as gobernadores/as?

Fil: Chicatun, Lucía. Universidad de San Andrés. Departamento de Ciencias Sociales; Argentina

In vitro generation of a bilayered dense collagen / chitosan hydrogel scaffold as an osteochondral model

Success of osteochondral tissue engineering (TE) requires stratified scaffolds that mimic the biophysicochemical composition of the cellular environment of both the cartilage and subchondral bone. In vitro reconstituted collagen type I (Coll) hydrogels are widely used as biomimetic scaffolds for TE, however due to their highly-hydrated nature they collapse due to gravitational forces (self-compression; SC). Plastic compression (PC) is a method that rapidly enables the generation of dense scaffolds with solid weight percent approaching native tissues values.The aim of this doctoral research was to develop and characterize a bilayered model system for osteochondral TE applications based on the incorporation of a GAG-analog (i.e. chitosan; CTS), within a dense Coll hydrogel, to closely mimic the native extracellular matrix (ECM) of the osteochondral interface. The first objective was to develop and optimize a co-gelling system for the generation of highly-hydrated Coll/CTS hybrid gels with different CTS proportions. PC was shown to be an effective and rapid process able to generate, within minutes, dense Coll/CTS hybrid gels with increased solid weight percent, compressive modulus and resistance to enzymatic degradation, as dictated by CTS content. As a second objective, the effect of CTS incorporation on modulating MC3T3-E1 pre-osteoblast seeded-cell function within dense Coll gels was investigated. Dense Coll/CTS hydrogels supported MC3T3-E1 cell viability, proliferation, and differentiation under osteogenic-inducing conditions. These findings demonstrated that dense Coll/CTS hybrids provide an osteoid-like structure as an in vitro model for bone TE.As a third objective, the effect of CTS incorporation into dense Coll gel discs was investigated to support RCJ3.1C5.18 chondroprogenitors (RCJ) differentiation. Immunohistochemistry for collagen type II, in combination with Safranin O staining and GAG quantification, indicated greater chondroprogenitor differentiation within Coll/CTS scaffolds, compared to Coll alone. The results demonstrated the suitability of dense Coll/CTS scaffolds to be used as in vitro models for cartilage repair. The fourth objective was to develop a bilayered dense Coll/CTS hydrogel with ratios approaching those of Coll/GAGs found in the ECM at the osteochondral interface. In addition, the optimization of the co-culturing conditions to maintain the simultaneous chondro- and osteogenesis was investigated. The results demonstrated the potential of bilayered dense Coll/CTS hydrogels to be used as effective in vitro osteochondral models. As the fifth objective, CTS effect on Coll gel consolidation was investigated by monitoring the spatiotemporal distribution of fluorescent beads using confocal microscopy during Coll/CTS hydrogels consolidation. The Happel model was used to predict the hydraulic permeability of the hydrogels. In addition, the effect of CTS fixed charge on Coll hydrogels was investigated through structural, mechanical and swelling characterizations under isotonic and hypertonic conditions. The results indicate the ability of a charged GAG-analog to tailor the biophysicochemical properties of Coll hydrogels, thus providing a reliable 3D in vitro tissue model for various TE applications. In conclusion, the integrated bilayered dense Coll/CTS construct developed and characterized in this doctoral research effectively provided a tailored in vitro cell culture milieu that closely mimics a complex physiologic ECM to be used as a three-dimensional model and with the potential for clinical use as a biomimetic implant with osteochondral regenerative capacity.Le succès de régénération du tissu ostéochondral requiert le développement des matrices stratifiées afin d'imiter la composition biophysiquechimiques du cartilage et l'os sous-chondral. Les hydrogels de collagène de type I (Coll) reconstitués in vitro sont grandement utilisés en tant que matrices biomimétiques pour le génie tissulaire (GT). En raison de leur nature hautement hydratée s'affaissent à cause des forces gravitationnelles (auto-compression; SC). La compression plastique (CP) est un procédé rapide qui génère des matrices denses avec un pourcentage massique de solide que se rapprochant du taux de solide des tissus naturels. Cette recherche de doctorat a pour but de développer et caractériser un modèle à deux couches pour des applications en GT ostéochondral basés sur l'incorporation de GAGs analogiques (i.e. chitosan; CTS) dans un hydrogel Coll dense afin de reproduire de près la matrice extra-cellulaire (MEC) naturelle de l'interface ostéochondrale. Le premier objectif était de développer et d'optimiser un système co-gélifiant pour la génération de gels hybrides de Coll/CTS hautement hydraté avec diverses proportions de CTS. In a été démontré que la CP est un procédé rapide capable de générer des gels hybrides de Coll/CTS dense avec un taux de solide accru, un module de compression et une résistance à la dégradation enzimatique, le tout dicté par la teneur en CTS.Comme second objectif l'effet de l'incorporation de CTS sur la modulation de la fonction de cellules ensemencées pré-ostéoblastes MC3T3-E1 à l'intérieur de gels Coll denses a été étudié. Les hydrogels de Coll/CTS dense ont permis la viabilité et la prolifération de cellules ainsi que leur différentiation dans des conditions ostéogéniques. Ces résultats démontrent que les hybrides de Coll/CTS denses sont une approche pour l'assemblage de structures de type ostéoïdes en tant que modèle in vitro pour GT. En tant que troisième objectif l'effet de l'incorporation de CTS à des disques de gel Coll dense afin de supporter la différentiation de chondro-progéniteurs RCJ3.1C5.18 (RCJ) a été étudié. L'immunohistochimie du colagène de type II, combiné avec la coloration avec du Safranin O et la quantification des GAGs, ont indiqué que la différentiation des chondro-progéniteurs est meilleures avec les matrices de Coll/CTS. Les résultats ont démontré la pertinence de les hybrides de Coll/CTS denses pour être utilisé comme modèles in vitro pour la réparation du cartilageLe quatrième objectif était de développer une structure à deux couches d'hydrogel de Coll/CTS denses avec des ratios se rapprochant celui de Coll/GAGs se retrouvant dans l'interface ostéochondrale. De plus l'optimisation des conditions de co-culture permettant de supportent les réactions concurrentes de chondrogénèse et d'ostéogénèse. Les résultats démontrent la possibilité d'utiliser les hydrogels de Coll/CTS denses à deux couches en tant que modèles ostéochondraux in vitro. En tant que cinquièmes objectif l'effet des CTS sur la consolidation du gel de colagène a été étudié en surveillant la distribution spatiotemporelle de billes fluorescentes par microscopie confocale. Le modèle de Happel a été utilisé afin de prédire la perméabilité hydraulique des hydrogels. Aussi l'effet de la charge fixe des CTS sur les hydrogels Coll/CTS a été étudié par leur caractérisation structurelle, mécanique et de gonflement dans des conditions isotoniques et hypertoniques. Les résultats ont indiqué la capacité d'un analogue de GAG chargé à s'adapter aux propriétés biophysicochimiques des hydrogels Coll, offrant un modèle de tissus in vitro pour diverses applications de GT. En conclusion la structure à deux couches de Coll/CTS dense développée et caractérisée dans le cadre de ce doctorat a procuré un milieu de culture de cellules in vitro reproduisant la MEC complexe. Cette structure pourrait potentiellement être utilisé cliniquement en tant qu'implant biomimétique avec des capacités régénératrices ostéochondrales

Carbon nanotube deposits and CNT/SiO2 composite coatings by electrophoretic deposition

Multiwalled carbon nanotube (CNT) films have been successfully fabricated by electrophoretic deposition (EPD) on stainless steel substrates. Electrophoretic deposition was performed using optimised aqueous suspensions under constant voltage conditions. Triton X-100 was used as a surfactant to disperse CNT bundles, and iodine was added as a particle charger. CNT/SiO2 composite coatings were prepared by electrophoretic co-deposition. Experimental results show that the CNTs were efficiently mixed with SiO2 nanoparticles to form a network structure. Layered CNT/SiO2 porous composites were obtained by sequential EPD experiments alternating the deposition of CNT and SiO2 nanoparticles. The structure of all films deposited was studied in detail by scanning electron microscopy. Possible applications of CNT and CNT/SiO2 films are as porous coatings in the biomedical field, thermal management devices, biomedical sensors and other functional applications where the properties of CNTs are required

Osteoblastic differentiation under controlled bioactive ion release by silica and titania doped sodium-free calcium phosphate-based glass

Sodium-free phosphate-based glasses (PGs) doped with both SiO2 and TiO2 (50P2O5-40CaO-xSiO2-(10-x)TiO2, where x=10, 7, 5, 3, and 0mol%) were developed and characterised for controlled ion release applications in bone tissue engineering. Substituting SiO2 with TiO2 directly increased PG density and glass transition temperature, indicating a cross-linking effect of Ti on the glass network which was reflected by significantly reduced degradation rates in an aqueous environment. X-ray diffraction confirmed the presence of Ti(P2O7) in crystallised TiO2-containing PGs, and nuclear magnetic resonance showed an increase in Q1 phosphate species with increasing TiO2 content. Substitution of SiO2 with TiO2 also reduced hydrophilicity and surface energy. In biological assays, MC3T3-E1 pre-osteoblasts effectively adhered to the surface of PG discs and the incorporation of TiO2, and hence higher stability of the PG network, significantly increased cell viability and metabolic activity indicating the biocompatibility of the PGs. Addition of SiO2 increased ionic release from the PG, which stimulated alkaline phosphatase (ALP) activity in MC3T3-E1 cells upon ion exposure. The incorporation of 3mol% TiO2 was required to stabilise the PG network against unfavourable rapid degradation in aqueous environments. However, ALP activity was greatest in PGs doped with 5-7mol% SiO2 due to up-regulation of ionic concentrations. Thus, the properties of PGs can be readily controlled by modifying the extent of Si and Ti doping in order to optimise ion release and osteoblastic differentiation for bone tissue engineering applications.Peer reviewed: YesNRC publication: Ye