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Disruption of retinal vasculature is linked to various diseases, including diabetic retinopathy and macular degeneration, leading to vision loss. We present here a novel algorithmic approach that generates highly realistic digital models of human retinal blood vessels, based on established biophysical principles, including fully-connected arterial and venous trees with a single inlet and outlet. This approach, using physics-informed generative adversarial networks (PI-GAN), enables the segmentation and reconstruction of blood vessel networks with no human input and which out-performs human labelling. Segmentation of DRIVE and STARE retina photograph datasets provided near state-of-the-art vessel segmentation, with training on only a small (n = 100) simulated dataset. Our findings highlight the potential of PI-GAN for accurate retinal vasculature characterization, with implications for improving early disease detection, monitoring disease progression, and improving patient care.
Assuntos
Algoritmos , Aprendizado Profundo , Retina , Vasos Retinianos , Humanos , Vasos Retinianos/diagnóstico por imagem , Retina/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Retinopatia Diabética/diagnóstico , Degeneração Macular/patologiaRESUMO
There is a significant need across multiple indications for an off-the-shelf bioengineered tubular graft which fulfils the mechanical and biological requirements for implantation and function but does not necessarily require cells for manufacture or deployment. Herein, we present a tissue-like tubular construct using a cell-free, materials-based method of manufacture, utilizing densified collagen hydrogel. Our tubular grafts are seamless, mechanically strong, customizable in terms of lumen diameter and wall thickness, and display a uniform fibril density across the wall thickness and along the tube length. While the method enables acellular grafts to be generated rapidly, inexpensively, and to a wide range of specifications, the cell-compatible densification process also enables a high density of cells to be incorporated uniformly into the walls of the tubes, which we show can be maintained under perfusion culture. Additionally, the method enables tubes consisting of distinct cell domains with cellular configurations at the boundaries which may be useful for modelling aortic disease. Further, we demonstrate additional steps which allow for luminal surface patterning. These results highlight the universality of this approach and its potential for developing the next generation of bioengineered grafts.
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Colágeno , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Engenharia Biomédica , HidrogéisRESUMO
Glioblastoma (GBM) angiogenesis is critical for tumor growth and recurrence, making it a compelling therapeutic target. Here, a disease-relevant, vascularized tumoroid in vitro model with stem-like features and stromal surrounds is reported. The model is used to recapitulate how individual components of the GBM's complex brain microenvironment such as hypoxia, vasculature-related stromal cells and growth factors support GBM angiogenesis. It is scalable, tractable, cost-effective and can be used with biologically-derived or biomimetic matrices. Patient-derived primary GBM cells are found to closely participate in blood vessel formation in contrast to a GBM cell line containing differentiated cells. Exogenous growth factors amplify this effect under normoxia but not at hypoxia suggesting that a significant amount of growth factors is already being produced under hypoxic conditions. Under hypoxia, primary GBM cells strongly co-localize with umbilical vein endothelial cells to form sprouting vascular networks, which has been reported to occur in vivo. These findings demonstrate that our 3D tumoroid in vitro model exhibits biomimetic attributes that may permit its use as a preclinical model in studying microenvironment cues of tumor angiogenesis.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Neovascularização Patológica , Técnicas de Cultura de Tecidos , Microambiente Tumoral , Biomarcadores , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Imunofluorescência , Glioblastoma/etiologia , Glioblastoma/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imuno-Histoquímica , Esferoides CelularesRESUMO
BACKGROUND: Glioblastoma (GBM) is a highly aggressive incurable brain tumor. The main cause of mortality in GBM patients is the invasive rim of cells migrating away from the main tumor mass and invading healthy parts of the brain. Although the motion is driven by forces, our current understanding of the physical factors involved in glioma infiltration remains limited. This study aims to investigate the adhesion properties within and between patients' tumors on a cellular level and test whether these properties correlate with cell migration. METHODS: Six tissue samples were taken from spatially separated sections during 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. Navigated biopsy samples were collected from strongly fluorescent tumor cores, a weak fluorescent tumor rim, and nonfluorescent tumor margins. A microfluidics device was built to induce controlled shear forces to detach cells from monolayer cultures. Cells were cultured on low modulus polydimethylsiloxane representative of the stiffness of brain tissue. Cell migration and morphology were then obtained using time-lapse microscopy. RESULTS: GBM cell populations from different tumor fractions of the same patient exhibited different migratory and adhesive behaviors. These differences were associated with sampling location and amount of 5-ALA fluorescence. Cells derived from weak- and nonfluorescent tumor tissue were smaller, adhered less well, and migrated quicker than cells derived from strongly fluorescent tumor mass. CONCLUSIONS: GBM tumors are biomechanically heterogeneous. Selecting multiple populations and broad location sampling are therefore important to consider for drug testing.
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Albumin-based hydrogels are increasingly attractive in tissue engineering because they provide a xeno-free, biocompatible and potentially patient-specific platform for tissue engineering and drug delivery. The majority of research on albumin hydrogels has focused on bovine serum albumin (BSA), leaving human serum albumin (HSA) comparatively understudied. Different gelation methods are usually employed for HSA and BSA, and variations in the amino acid sequences of HSA and BSA exist; these account for differences in the hydrogel properties. Heat-induced gelation of aqueous HSA is the easiest method of synthesizing HSA hydrogels however hydrogel opacity and poor cell attachment limit their usefulness in downstream applications. Here, a solution to this problem is presented. Stable and translucent HSA hydrogels were created by controlled thermal gelation and the addition of sodium chloride. The resulting bio-inert hydrogel was then subjected to air plasma treatment which functionalised its surface, enabling the attachment of basement membrane matrix (Geltrex). In vitro survival and proliferation studies of foetal human osteoblasts subsequently demonstrated good biocompatibility of functionalised albumin hydrogels compared to untreated samples. Thus, air plasma treatment enables functionalisation of inert heat-derived HSA hydrogels with extracellular matrix proteins and these may be used as a xeno-free platform for biomedical research or cell therapy.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Gases em Plasma , Albumina Sérica Humana/química , Engenharia Tecidual/métodos , Materiais Biocompatíveis/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/toxicidade , Proteínas da Matriz Extracelular/ultraestrutura , Temperatura Alta , Humanos , Hidrogéis/toxicidade , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteoblastos , Albumina Sérica Humana/toxicidade , Albumina Sérica Humana/ultraestrutura , Cloreto de Sódio/química , Propriedades de SuperfícieRESUMO
One of the greatest challenges in fabricating artificial tissues and organs is the incorporation of vascular networks to support the biological requirements of the embedded cells, encouraging tissue formation and maturation. With the advent of 3D printing technology, significant progress has been made with respect to generating vascularized artificial tissues. Current algorithms to generate arterial/venous trees are computationally expensive and offer limited freedom to optimize the resulting structures. Furthermore, there is no method for algorithmic generation of vascular networks that can recapitulate the complexity of the native vasculature for more than two trees, and export directly to a 3D printing format. Here, we report such a method, using an accelerated constructive constrained optimization approach, by decomposing the process into construction, optimization, and collision resolution stages. The new approach reduces computation time to minutes at problem sizes where previous implementations have reported days. With the optimality criterion of maximizing the volume of useful tissue which could be grown around such a network, an approach of alternating stages of construction and batch optimization of all node positions is introduced and shown to yield consistently more optimal networks. The approach does not place a limit on the number of interpenetrating networks that can be constructed in a given space; indeed we demonstrate a biomimetic, liver-like tissue model. Methods to account for the limitations of 3D printing are provided, notably the minimum feature size and infill at sharp angles, through padding and angle reduction, respectively.
Assuntos
Impressão Tridimensional , Engenharia Tecidual , Algoritmos , Artérias , BiomiméticaRESUMO
Pursuing long-term self-healing infrastructures has gained popularity in the construction field. Vascular networks have the potential to achieve long-term self-healing in cementitious infrastructures. To avoid further monitoring of non-cementitious tubes, sacrificial material can be used as a way of creating hollow channels. In this research, we report a new method for fabrication of complex 3D internal hollow tunnels using 3D printing of polyvinyl alcohol (PVA). The behaviour of 3D printed PVA structures in cement pastes was investigated using computed-tomography (CT) combined with X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDX). Results showed that (i) 1300 min were needed to fully dissolve 1 g of a 3D printed PVA structure, and different pH solutions did not significantly change the PVA dissolving process compared with a neutral environment; (ii) a low water/cement ratio can minimize early stage cracking resulting from PVA expansion; (iii) and PVA-cement interaction products were mainly calcite and a Ca-polymer compound. In conclusion, controlling the PVA expansion by decreasing the water/cement (w/c) ratio provides a promising approach to achieve 3D hollow channels in cement and, therefore, makes it possible to create complex tunnels within self-healing cementitious materials.
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Porous coatings on prosthetic implants encourage implant fixation. Enhanced fixation may be achieved using a magneto-active porous coating that can deform elastically in vivo on the application of an external magnetic field, straining in-growing bone. Such a coating, made of 444 ferritic stainless steel fibres, was previously characterised in terms of its mechanical and cellular responses. In this work, co-cultures of human osteoblasts and endothelial cells were seeded into a novel fibrin-based hydrogel embedded in a 444 ferritic stainless steel fibre network. Albumin was successfully incorporated into fibrin hydrogels improving the specific permeability and the diffusion of fluorescently tagged dextrans without affecting their Young's modulus. The beneficial effect of albumin was demonstrated by the upregulation of osteogenic and angiogenic gene expression. Furthermore, mineralisation, extracellular matrix production, and formation of vessel-like structures were enhanced in albumin-enriched fibrin hydrogels compared to fibrin hydrogels. Collectively, the results indicate that the albumin-enriched fibrin hydrogel is a promising bio-matrix for bone tissue engineering and orthopaedic applications.
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Albumin, the most abundant plasma protein in mammals, is a versatile and easily obtainable biomaterial. It is pH and temperature responsive, dissolvable in high concentrations and gels readily in defined conditions. This versatility, together with its inexpensiveness and biocompatibility, makes albumin an attractive biomaterial for biomedical research and therapeutics. So far, clinical research in albumin has centered mainly on its use as a carrier molecule or nanoparticle to improve drug pharmacokinetics and delivery to target sites. In contrast, research in albumin-based hydrogels is less established albeit growing in interest over recent years. In this minireview, we report current literature and critically discuss the synthesis, mechanical properties, biological effects and uses, biodegradability and cost of albumin hydrogels as a xeno-free, customizable, and transplantable construct for tissue engineering and regenerative medicine.
Assuntos
Albuminas , Transplante de Células , Portadores de Fármacos , Hidrogéis , Nanopartículas , Medicina Regenerativa , Albuminas/química , Albuminas/uso terapêutico , Animais , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Humanos , Hidrogéis/química , Hidrogéis/uso terapêutico , Nanopartículas/química , Nanopartículas/uso terapêuticoRESUMO
There is currently an interest in "active" implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibers that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1α1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fiber spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.
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Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.
Assuntos
Ductos Biliares/citologia , Ductos Biliares/fisiologia , Organoides/citologia , Organoides/fisiologia , Regeneração , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Separação Celular/métodos , Células Cultivadas , Desenho de Equipamento , Humanos , Camundongos , Engenharia Tecidual/instrumentação , Alicerces Teciduais/químicaRESUMO
The generation of bioengineered biliary tissue could contribute to the management of some of the most impactful cholangiopathies associated with liver transplantation, such as biliary atresia or ischemic cholangiopathy. Recent advances in tissue engineering and in vitro cholangiocyte culture have made the achievement of this goal possible. Here we provide an overview of these developments and review the progress towards the generation and transplantation of bioengineered bile ducts. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.
Assuntos
Doenças dos Ductos Biliares/cirurgia , Ductos Biliares/transplante , Órgãos Bioartificiais , Engenharia Tecidual/métodos , Animais , Ductos Biliares/citologia , Engenharia Biomédica/métodos , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Células Epiteliais , Humanos , Transplante de Fígado/efeitos adversos , Transplante de Fígado/métodos , Técnicas de Cultura de Órgãos/métodosRESUMO
The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.
Assuntos
Ductos Biliares Extra-Hepáticos/fisiologia , Células Epiteliais/citologia , Vesícula Biliar/fisiologia , Organoides/fisiologia , Regeneração/fisiologia , Engenharia Tecidual/métodos , Animais , Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Extra-Hepáticos/lesões , Sistema Biliar/citologia , Sistema Biliar/lesões , Sistema Biliar/fisiologia , Transplante de Células , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Vesícula Biliar/lesões , Humanos , Técnicas In Vitro , Queratina-19/metabolismo , Queratina-7/metabolismo , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Secretina/farmacologia , Somatostatina/farmacologia , Alicerces Teciduais , gama-Glutamiltransferase/metabolismoRESUMO
Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of â¼14 mm s-1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Osteoblastos/metabolismo , Rotação , Alicerces Teciduais/química , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Osteoblastos/citologiaRESUMO
Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. We report a new method for producing a hierarchical, three-dimensional (3D) and perfusable vasculature in a large, cellularized fibrin hydrogel. Bifurcating channels, varying in size from 1 mm to 200-250 µm, are formed using a novel process in which we convert a 3D printed thermoplastic material into a gelatin network template, by way of an intermediate alginate hydrogel. This enables a CAD-based model design, which is highly customizable, reproducible, and which can yield highly complex architectures, to be made into a removable material, which can be used in cellular environments. Our approach yields constructs with a uniform and high density of cells in the bulk, made from bioactive collagen and fibrin hydrogels. Using standard cell staining and immuno-histochemistry techniques, we showed good cell seeding and the presence of tight junctions between channel endothelial cells, and high cell viability and cell spreading in the bulk hydrogel.
Assuntos
Alginatos/química , Gelatina/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hidrogéis/química , Alicerces Teciduais/química , Sobrevivência Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Células Endoteliais da Veia Umbilical Humana/citologia , HumanosRESUMO
Substrate grain structure and topography play major roles in mediating cell and bacteria activities. Severe plastic deformation techniques, known as efficient metal-forming and grain refining processes, provide the treated material with novel mechanical properties and can be adopted to modify nanoscale surface characteristics, possibly affecting interactions with the biological environment. This in vitro study evaluates the capability of severe shot peening, based on severe plastic deformation, to modulate the interactions of nanocrystallized metallic biomaterials with cells and bacteria. The treated 316L stainless steel surfaces were first investigated in terms of surface topography, grain size, hardness, wettability and residual stresses. The effects of the induced surface modifications were then separately studied in terms of cell morphology, adhesion and proliferation of primary human osteoblasts (bone forming cells) as well as the adhesion of multiple bacteria strains, specifically Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and ampicillin-resistant Escherichia coli. The results indicated a significant enhancement in surface work hardening and compressive residual stresses, maintenance of osteoblast adhesion and proliferation as well as a remarkable decrease in the adhesion and growth of gram-positive bacteria (S. aureus and S. epidermidis) compared to non-treated and conventionally shot peened samples. Impressively, the decrease in bacteria adhesion and growth was achieved without the use of antibiotics, for which bacteria can develop a resistance towards anyway. By slightly grinding the surface of severe shot peened samples to remove differences in nanoscale surface roughness, the effects of varying substrate grain size were separated from those of varying surface roughness. The expression of vinculin focal adhesions from osteoblasts was found to be singularly and inversely related to grain size, whereas the attachment of gram-positive bacteria (S. aureus and S. epidermidis) decreased with increasing nanoscale surface roughness, and was not affected by grain refinement. Ultimately, this study demonstrated the advantages of the proposed shot peening treatment to produce multifunctional 316L stainless steel materials for improved implant functions without necessitating the use of drugs.
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Aderência Bacteriana , Nanoestruturas/química , Osteoblastos/citologia , Aço Inoxidável/química , Materiais Biocompatíveis , Adesão Celular , Proliferação de Células , Força Compressiva , Escherichia coli , Humanos , Metais/química , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Osseointegração , Osteoblastos/metabolismo , Osteoblastos/microbiologia , Pseudomonas aeruginosa , Staphylococcus aureus , Staphylococcus epidermidis , Propriedades de Superfície , Molhabilidade , Difração de Raios XRESUMO
Microgrooved surfaces have been used extensively to influence cell contact guidance. Guiding cell growth, extracellular matrix deposition, and mineralization is important for bone implant longevity. In this study, we investigated the osteoblast response to microgrooved metallic surfaces in serum-supplemented medium. Groove spacing was comparable with the spread osteoblast size. Focal adhesions were observed to confine to the intervening ridge/groove boundaries. Osteoblasts bridged over the grooves and were unable to conform to the concave shape of the underlying grooves. Microgrooved surfaces induced higher osteoblast proliferation and metabolic activity after 14 days in osteogenic medium compared with as-received surfaces, resulting in higher mineralization and alignment of cell-secreted collagen after 28 days. To establish whether preferential cell attachment at the ridge/groove boundaries was influenced by the adhesion proteins contained in the serum-supplemented media, fluorescently labeled fibronectin was adsorbed onto the microgrooved substrates at low concentrations, mimicking the concentrations found in blood serum. Fibronectin was found to selectively adsorb onto the ridge/groove boundaries, the osteoblast focal adhesion sites, suggesting that protein adsorption may have influenced the cell attachment pattern.
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Osteoblastos/citologia , Proteínas/metabolismo , Adsorção , Osteoblastos/metabolismo , Propriedades de SuperfícieRESUMO
Ferromagnetic fiber networks have the potential to deform in vivo imparting therapeutic levels of strain on in-growing periprosthetic bone tissue. 444 Ferritic stainless steel provides a suitable material for this application due to its ability to support cultures of human osteoblasts (HObs) without eliciting undue inflammatory responses from monocytes in vitro. In the present article, a 444 fiber network, containing 17 vol% fibers, has been investigated. The network architecture was obtained by applying a skeletonization algorithm to three-dimensional tomographic reconstructions of the fiber networks. Elastic properties were measured using low-frequency vibration testing, providing globally averaged properties as opposed to mechanical methods that yield only local properties. The optimal region for transduction of strain to cells lies between the ferromagnetic fibers. However, cell attachment, at early time points, occurs primarily on fiber surfaces. Deposition of fibrin, a fibrous protein involved in acute inflammatory responses, can facilitate cell attachment within this optimal region at early time points. The current work compared physiological (3 and 5 g·L(-1)) and supraphysiological fibrinogen concentrations (10 g·L(-1)), using static in vitro seeding of HObs, to determine the effect of fibrin deposition on cell responses during the first week of cell culture. Early cell attachment within the interfiber spaces was observed in all fibrin-containing samples, supported by fibrin nanofibers. Fibrin deposition influenced the seeding, metabolic activity, and early stage differentiation of HObs cultured in the fibrin-containing fiber networks in a concentration-dependant manner. While initial cell attachment for networks with fibrin deposited from low physiological concentrations was similar to control samples without fibrin deposition, significantly higher HObs attached onto high physiological and supraphysiological concentrations. Despite higher cell numbers with supraphysiological concentrations, cell metabolic activities were similar for all fibrinogen concentrations. Further, cells cultured on supraphysiological concentrations exhibited lower cell differentiation as measured by alkaline phosphatase activity at early time points. Overall, the current study suggests that physiological fibrinogen concentrations would be more suitable than supraphysiological concentrations for supporting early cell activity in porous implant coatings.
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Fibrina/química , Fibrina/farmacologia , Mecanotransdução Celular/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Módulo de Elasticidade , Humanos , Campos Magnéticos , Teste de Materiais , Mecanotransdução Celular/efeitos dos fármacos , Nanofibras/administração & dosagem , Nanofibras/química , Nanofibras/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Porosidade , Estresse MecânicoRESUMO
Beneficial effects on bone-implant bonding may accrue from ferromagnetic fiber networks on implants which can deform in vivo inducing controlled levels of mechanical strain directly in growing bone. This approach requires ferromagnetic fibers that can be implanted in vivo without stimulating undue inflammatory cell responses or cytotoxicity. This study examines the short-term in vitro responses, including attachment, viability, and inflammatory stimulation, of human peripheral blood monocytes to 444 ferritic stainless steel fiber networks. Two types of 444 networks, differing in fiber cross section and thus surface area, were considered alongside austenitic stainless steel fiber networks, made of 316L, a widely established implant material. Similar high percent seeding efficiencies were measured by CyQuant® on all fiber networks after 48 h of cell culture. Extensive cell attachment was confirmed by fluorescence and scanning electron microscopy, which showed round monocytes attached at various depths into the fiber networks. Medium concentrations of lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) were determined as indicators of viability and inflammatory responses, respectively. Percent LDH concentrations were similar for both 444 fiber networks at all time points, whereas significantly lower than those of 316L control networks at 24 h. All networks elicited low-level secretions of TNF-α, which were significantly lower than that of the positive control wells containing zymosan. Collectively, the results indicate that 444 networks produce comparable responses to medical implant grade 316L networks and are able to support human peripheral blood monocytes in short-term in vitro cultures without inducing significant inflammatory or cytotoxic effects.
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Imãs/química , Imãs/toxicidade , Monócitos/citologia , Monócitos/efeitos dos fármacos , Aço Inoxidável/química , Aço Inoxidável/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Monócitos/imunologia , Próteses e Implantes , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs.