RESUMEN
Thermoplastic polyurethane (TPU) is a polymer used in a variety of fields, including medical applications. Here, we aimed to verify if the brush and bar coater deposition techniques did not alter TPU properties. The topography of the TPU-modified surfaces was studied via AFM demonstrating no significant differences between brush and bar coater-modified surfaces, compared to the un-modified TPU (TPU Film). The effect of the surfaces on planktonic bacteria, evaluated by MTT assay, demonstrated their anti-adhesive effect on E. coli, while the bar coater significantly reduced staphylococcal planktonic adhesion and both bacterial biofilms compared to other samples. Interestingly, Pearson's R coefficient analysis showed that Ra roughness and Haralick's correlation feature were trend predictors for planktonic bacterial cells adhesion. The surface adhesion property was evaluated against NIH-3T3 murine fibroblasts by MTT and against human fibrinogen and human platelet-rich plasma by ELISA and LDH assay, respectively. An indirect cytotoxicity experiment against NIH-3T3 confirmed the biocompatibility of the TPUs. Overall, the results indicated that the deposition techniques did not alter the antibacterial and anti-adhesive surface properties of modified TPU compared to un-modified TPU, nor its bio- and hemocompatibility, confirming the suitability of TPU brush and bar coater films in the biomedical and pharmaceutical fields.
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We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.
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The brain is the most complex organ of our body. Such a complexity spans from the single-cell morphology up to the intricate connections that hundreds of thousands of neurons establish to create dense neuronal networks. All these components are involved in the genesis of the rich patterns of electrophysiological activity that characterize the brain. Over the years, researchers coming from different disciplines developedin vitrosimplified experimental models to investigate in a more controllable and observable way how neuronal ensembles generate peculiar firing rhythms, code external stimulations, or respond to chemical drugs. Nowadays, suchin vitromodels are namedbrain-on-a-chippointing out the relevance of the technological counterpart as artificial tool to interact with the brain: multi-electrode arrays are well-used devices to record and stimulate large-scale developing neuronal networks originated from dissociated cultures, brain slices, up to brain organoids. In this review, we will discuss the state of the art of the brain-on-a-chip, highlighting which structural and biological features a realisticin vitrobrain should embed (and how to achieve them). In particular, we identified two topological features, namely modular and three-dimensional connectivity, and a biological one (heterogeneity) that takes into account the huge number of neuronal types existing in the brain. At the end of this travel, we will show how 'far' we are from the goal and how interconnected-brain-regions-on-a-chip is the most appropriate wording to indicate the current state of the art.
Asunto(s)
Dispositivos Laboratorio en un Chip , Neuronas , Encéfalo , Fenómenos Electrofisiológicos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
OBJECTIVE: The goal of this work is to develop and characterize an innovative experimental framework to design interconnected (i.e. modular) heterogeneous (cortical-hippocampal) neuronal cultures with a three-dimensional (3D) connectivity and to record their electrophysiological activity using micro-electrode arrays (MEAs). APPROACH: A two-compartment polymeric mask for the segregation of different neuronal populations (cortex and hippocampus) was coupled to the MEA surface. Glass microbeads were used as a scaffold to mimic the 3D brain micro-architecture. MAIN RESULTS: We built a fully functional heterogeneous 3D neuronal network. From an electrophysiological point of view, we found that the heterogeneity induces a global increase of the activity rate, while the 3D connectivity modulates the duration and the organization of the bursting activity. SIGNIFICANCE: In vivo, studies of network dynamics and interactions between neuronal populations are often time-consuming, low-throughput, complex, and suffer from reproducibility. On the other hand, most of the commonly used in vitro brain models are too simplified and thus far from the in vivo situation. The achieved results demonstrate the feasibility to build a more realistic and controllable experimental in vitro model of interconnected brain regions on-a-chip whose applications may have impacts on the study of neurological disorders that impair the connectivity between brain areas (e.g. Parkinson disease).
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Hipocampo , Red Nerviosa , Fenómenos Electrofisiológicos , Neuronas , Reproducibilidad de los ResultadosRESUMEN
Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)-sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. Graphical abstract.
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Antineoplásicos/administración & dosificación , Endocitosis , Liposomas , Oxaliplatino/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Espectroscopía Dieléctrica , Humanos , Oxaliplatino/farmacologíaRESUMEN
The high sensitivity of silicon microcantilever sensors has expanded their use in areas ranging from gas sensing to bio-medical applications. Photochromic molecules also represent promising candidates for a large variety of sensing applications. In this work, the operating principles of these two sensing methods are combined in order to detect the reversible conformational change of a molecular switch, spiropyran. Thus, arrays of silicon microcantilever sensors were functionalized with spiropyran on the gold covered side and used as test microcantilevers. The microcantilever deflection response was observed, in five sequential cycles, as the transition from the spiropyran (SP) (CLOSED) to the merocyanine (MC) (OPEN) state and vice-versa when induced by UV and white light LED sources, respectively, proving the reversibility capabilities of this type of sensor. The microcantilever deflection direction was observed to be in one direction when changing to the MC state and in the opposite direction when changing back to the SP state. A tensile stress was induced in the microcantilever when the SP to MC transition took place, while a compressive stress was observed for the reverse transition. These different type of stresses are believed to be related to the spatial conformational changes induced in the photochromic molecule upon photo-isomerisation.
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Benzopiranos/química , Técnicas Biosensibles , Indoles/química , Conformación Molecular , Nitrocompuestos/química , Silicio/química , Benzopiranos/síntesis química , Indoles/síntesis química , Nitrocompuestos/síntesis química , Estrés Mecánico , Propiedades de Superficie , Rayos UltravioletaAsunto(s)
Leiomioma/patología , Leiomiosarcoma/patología , Microscopía de Fuerza Atómica , Miometrio/ultraestructura , Neoplasias Uterinas/patología , Adulto , Anciano , Diagnóstico Diferencial , Módulo de Elasticidad , Femenino , Humanos , Leiomioma/diagnóstico , Leiomioma/fisiopatología , Leiomiosarcoma/diagnóstico , Leiomiosarcoma/fisiopatología , Persona de Mediana Edad , Miometrio/anatomía & histología , Miometrio/fisiología , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/fisiopatologíaRESUMEN
The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
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Fenómenos Electrofisiológicos , Acoplamiento Excitación-Contracción , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Señalización del Calcio , Análisis de Datos , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Imagen MolecularRESUMEN
Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs' drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca2+ was studied. The differential evaluation explained the observed effects despite variability of biological samples. The ß-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.
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Técnicas Biosensibles , Células Madre Pluripotentes Inducidas/ultraestructura , Distrofia Muscular de Duchenne/fisiopatología , Miocitos Cardíacos/citología , Diferenciación Celular/genética , Distrofina/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Isoproterenol/farmacología , Microelectrodos , Microscopía de Fuerza Atómica , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Verapamilo/farmacologíaRESUMEN
The availability of 3D biomimetic in vitro neuronal networks of mammalian neurons represents a pivotal step for the development of brain-on-a-chip experimental models to study neuronal (dys)functions and particularly neuronal connectivity. The use of hydrogel-based scaffolds for 3D cell cultures has been extensively studied in the last years. However, limited work on biomimetic 3D neuronal cultures has been carried out to date. In this respect, here we investigated the use of a widely popular polysaccharide, chitosan (CHI), for the fabrication of a microbead based 3D scaffold to be coupled to primary neuronal cells. CHI microbeads were characterized by optical and atomic force microscopies. The cell/scaffold interaction was deeply characterized by transmission electron microscopy and by immunocytochemistry using confocal microscopy. Finally, a preliminary electrophysiological characterization by micro-electrode arrays was carried out.
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Quitosano/farmacología , Microesferas , Red Nerviosa/fisiología , Neuronas/fisiología , Andamios del Tejido/química , Animales , Red Nerviosa/ultraestructura , Neuronas/ultraestructura , Imagen Óptica , Ratas Sprague-DawleyRESUMEN
Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 µm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.
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Dermis/patología , Epidermis/patología , Microscopía de Fuerza Atómica , Fenómenos Biomecánicos , Biopsia , Dermis/diagnóstico por imagen , Dermis/efectos de la radiación , Elasticidad , Humanos , Obesidad/complicaciones , Obesidad/metabolismo , Piel/patología , Estrés Mecánico , Quemadura Solar/complicacionesRESUMEN
OBJECTIVE: We aim to develop a novel non-invasive or minimally invasive method for neural stimulation to be applied in the study and treatment of brain (dys)functions and neurological disorders. APPROACH: We investigate the electrophysiological response of in vitro neuronal networks when subjected to low-intensity pulsed acoustic stimulation, mediated by piezoelectric nanoparticles adsorbed on the neuronal membrane. MAIN RESULTS: We show that the presence of piezoelectric barium titanate nanoparticles induces, in a reproducible way, an increase in network activity when excited by stationary ultrasound waves in the MHz regime. Such a response can be fully recovered when switching the ultrasound pulse off, depending on the generated pressure field amplitude, whilst it is insensitive to the duration of the ultrasound pulse in the range 0.5 s-1.5 s. We demonstrate that the presence of piezoelectric nanoparticles is necessary, and when applying the same acoustic stimulation to neuronal cultures without nanoparticles or with non-piezoelectric nanoparticles with the same size distribution, no network response is observed. SIGNIFICANCE: We believe that our results open up an extremely interesting approach when coupled with suitable functionalization strategies of the nanoparticles in order to address specific neurons and/or brain areas and applied in vivo, thus enabling remote, non-invasive, and highly selective modulation of the activity of neuronal subpopulations of the central nervous system of mammalians.
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Estimulación Acústica/métodos , Potenciales de Acción/fisiología , Corteza Cerebral/fisiología , Hipocampo/fisiología , Nanopartículas/administración & dosificación , Potenciales de Acción/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Hipocampo/efectos de los fármacos , Nanopartículas/química , RatasRESUMEN
Studies in vitro have demonstrated that ß3-adrenergic receptors (ß3-ARs) regulate protein metabolism in skeletal muscle by promoting protein synthesis and inhibiting protein degradation. In this study, we evaluated whether activation of ß3-ARs by the selective agonist CL316,243 modifies the functional and structural properties of skeletal muscles of healthy mice. Daily injections of CL316,243 for 15 days resulted in a significant improvement in muscle force production, assessed by grip strength and weight tests, and an increased myofiber cross-sectional area, indicative of muscle hypertrophy. In addition, atomic force microscopy revealed a significant effect of CL316,243 on the transversal stiffness of isolated muscle fibers. Interestingly, the expression level of mammalian target of rapamycin (mTOR) downstream targets and neuronal nitric oxide synthase (NOS) was also found to be enhanced in tibialis anterior and soleus muscles of CL316,243 treated mice, in accordance with previous data linking ß3-ARs to mTOR and NOS signaling pathways. In conclusion, our data suggest that CL316,243 systemic administration might be a novel therapeutic strategy worthy of further investigations in conditions of muscle wasting and weakness associated with aging and muscular diseases.
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Agonistas de Receptores Adrenérgicos beta 3/farmacología , Dioxoles/farmacología , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 3/administración & dosificación , Animales , Dioxoles/administración & dosificación , Regulación de la Expresión Génica , Hipertrofia , Masculino , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.
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Cartílago Articular/ultraestructura , Colágenos Fibrilares/química , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patología , Cartílago Articular/patología , Articulación de la Cadera/metabolismo , Articulación de la Cadera/patología , Humanos , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Microscopía Electrónica de Rastreo , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Rodilla/metabolismoRESUMEN
Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150-4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150-200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.
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Adenocarcinoma/patología , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Microambiente Tumoral , Alginatos/química , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Análisis por Conglomerados , Módulo de Elasticidad , Elasticidad , Femenino , Citometría de Flujo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Hidrogeles/química , Técnicas In Vitro , Células MCF-7 , Microscopía de Fuerza Atómica , Presión , Ingeniería de Tejidos/métodosRESUMEN
Nuclear aggregates of polyamines (NAPs) are supramolecular compounds generated by the self-assembly of protonated nuclear polyamines (spermine, spermidine and putrescine) and phosphate ions. In the presence of genomic DNA, the hierarchical process of self-structuring ultimately produces nanotube-like polymers that envelop the double helix. Because of their modular nature and their aggregation-disaggregation dynamics, NAPs confer plasticity and flexibility to DNA. Through the disposition of charges, NAPs also enable a bidirectional stream of information between the genome and interacting moieties. High mobility group (HMG) B1 is a non-histone chromosomal protein that binds to DNA and that influences multiple nuclear processes. Because genomic DNA binds to either NAPs or HMGB1 protein, we explored the ability of in vitro self-assembled NAPs (ivNAPs) to mediate the DNA-HMGB1 interaction. To this end, we structured DNA-NAPs-HMGB1 and DNA-HMGB1-NAPs ternary complexes in vitro through opportune sequential incubations. Mobility shift electrophoresis and atomic force microscopy showed that the DNA-ivNAPs-HGMB1 complex had conformational assets supposedly more suitable those of the DNA-HGMB1-ivNAPs to comply with the physiological and functional requirements of DNA. Our findings indicated that ivNAPs act as mediators of the DNA-HMGB1 interaction.
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Núcleo Celular/metabolismo , ADN/metabolismo , Proteína HMGB1/metabolismo , Poliaminas/metabolismo , Agregado de Proteínas/fisiología , Genoma/genética , Humanos , Microscopía de Fuerza Atómica/métodos , Conformación de Ácido Nucleico , Espermidina/metabolismo , Espermina/metabolismoRESUMEN
The ability to sense mechanical stimuli and elaborate a response to them is a fundamental process in all organisms, driving crucial mechanisms ranging from cell volume regulation up to organ development or regeneration. Nevertheless, only in few cases the underlying molecular players are known. In particular, mammals possess a large variety of mechanoreceptors, providing highly specialized functions in sensory cells, but also several housekeeping molecular systems are involved in the complex mechanism of mechanotransduction. Recently, a new class of almost ubiquitous membrane channels has been identified in mammalians, namely piezo1 and piezo2, that is thought to play a crucial role in the mechanobiology of mammals. This review focuses on recent findings on these novel channels, and highlights open biophysical questions that largely remain to be addressed.
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Canales Iónicos/metabolismo , Animales , Humanos , Canales Iónicos/química , Canales Iónicos/genéticaRESUMEN
In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduced by the microtubule network elicits reactive oxygen species. X-ROS tunes Ca(2+) signalling in healthy muscle, but in diseases such as Duchenne muscular dystrophy (DMD), microtubule alterations drive elevated X-ROS, disrupting Ca(2+) homeostasis and impairing function. Here we show that detyrosination, a post-translational modification of α-tubulin, influences X-ROS signalling, contraction speed and cytoskeletal mechanics. In the mdx mouse model of DMD, the pharmacological reduction of detyrosination in vitro ablates aberrant X-ROS and Ca(2+) signalling, and in vivo it protects against hallmarks of DMD, including workload-induced arrhythmias and contraction-induced injury in skeletal muscle. We conclude that detyrosinated microtubules increase cytoskeletal stiffness and mechanotransduction in striated muscle and that targeting this post-translational modification may have broad therapeutic potential in muscular dystrophies.
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Microtúbulos/fisiología , Fibras Musculares Esqueléticas/fisiología , Miocitos Cardíacos/fisiología , Animales , Fenómenos Biomecánicos , Calcio , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Microscopía de Fuerza Atómica , Ratas , Ratas Sprague-DawleyRESUMEN
In this paper, a quantitative interpretation for atomic force microscopy-based dynamic nanoindentation (AFM-DN) tests on the superficial layers of bovine articular cartilage (AC) is provided. The relevant constitutive parameters of the tissue are estimated by fitting experimental results with a finite element model in the frequency domain. Such model comprises a poroelastic stress-strain relationship for a fibril reinforced tissue constitution, assuming a continuous distribution of the collagen network orientations. The identification procedure was first validated using a simplified transversely isotropic constitutive relationship; then, the experimental data were manually fitted by using the continuous distribution fibril model. Tissue permeability is derived from the maximum value of the phase shift between the input harmonic loading and the harmonic tissue response. Tissue parameters related to the stiffness are obtained from the frequency response of the experimental storage modulus and phase shift. With this procedure, an axial to transverse stiffness ratio (anisotropy ratio) of about 0.15 is estimated.