RESUMEN
Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 µm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 µm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 µm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.
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Mecanotransducción Celular , Línea Celular Tumoral , Elasticidad , Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Seudópodos/metabolismo , Análisis de la Célula Individual , Quinasas Asociadas a rho/metabolismoRESUMEN
Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.
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Compuestos de Bencilideno/química , Materiales Biocompatibles/química , Ácido Hialurónico/química , Hidrogeles/química , Fotólisis , Polietilenglicoles/química , Línea Celular , Química Clic , Humanos , Células Madre Mesenquimatosas/citología , Nitrobencenos/química , Fotones , Fármacos Fotosensibilizantes/química , Compuestos de Sulfhidrilo/química , Ingeniería de TejidosRESUMEN
Graphical Abstract.
RESUMEN
Here, an artificial intelligence (AI)-based approach was employed to optimize the production of electrospun scaffolds for in vivo wound healing applications. By combining polycaprolactone (PCL) and poly(ethylene glycol) (PEG) in various concentration ratios, dissolved in chloroform (CHCl3) and dimethylformamide (DMF), 125 different polymer combinations were created. From these polymer combinations, electrospun nanofiber meshes were produced and characterized structurally and mechanically via microscopic techniques, including chemical composition and fiber diameter determination. Subsequently, these data were used to train a neural network, creating an AI model to predict the optimal scaffold production solution. Guided by the predictions and experimental outcomes of the AI model, the most promising scaffold for further in vitro analyses was identified. Moreover, we enriched this selected polymer combination by incorporating antibiotics, aiming to develop electrospun nanofiber scaffolds tailored for in vivo wound healing applications. Our study underscores three noteworthy conclusions: (i) the application of AI is pivotal in the fields of material and biomedical sciences, (ii) our methodology provides an effective blueprint for the initial screening of biomedical materials, and (iii) electrospun PCL/PEG antibiotic-bearing scaffolds exhibit outstanding results in promoting neoangiogenesis and facilitating in vivo wound treatment.
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Collagen fibrils are the fundamental structural elements in vertebrate animals and compose a structural framework that provides mechanical support to load-bearing tissues. Understanding how these fibrils initially form and mechanically function has been the focus of a myriad of detailed investigations over the last few decades. From these studies a great amount of knowledge has been acquired as well as a number of new questions to consider. In this review, we examine the current state of our knowledge of the mechanical properties of extant fibrils. We emphasize on the mechanical response and related deformation of collagen fibrils upon tension, which is the predominant load imposed in most collagen-rich tissues. We also illuminate the gaps in knowledge originating from the intriguing results that the field is still trying to interpret. STATEMENT OF SIGNIFICANCE: Collagen is the result of millions of years of biological evolution and is a unique family of proteins, the majority of which provide mechanical support to biological tissues. Cells produce collagen molecules that self-assemble into larger structures, known as collagen fibrils. As simple as they appear under an optical microscope, collagen fibrils display a complex ultrastructural architecture tuned to the external forces that are imposed upon them. Even more complex is the way collagen fibrils deform under loading, and the nature of the mechanisms that drive their formation in the first place. Here, we present a cogent synthesis of the state-of-knowledge of collagen fibril mechanics. We focus on the information we have from in vitro experiments on individual, isolated from tissues, collagen fibrils and the knowledge available from in silico tests.
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Colágeno , Matriz Extracelular , Animales , Fenómenos Biomecánicos , Colágeno/química , Matriz Extracelular/metabolismo , Soporte de Peso , MicroscopíaRESUMEN
Collagen fibrils are the basic structural building blocks that provide mechanical properties such as stiffness, toughness, and strength to tissues from the nano- to the macroscale. Collagen fibrils are highly hydrated and transient deformation mechanisms contribute to their mechanical behavior. One approach to describe and quantify the apparent viscoelastic behavior of collagen fibrils is to find rheological models and fit the resulting empirical equations to experimental data. In this study, we consider a nonlinear rheological Maxwell model for this purpose. The model was fitted to tensile stress-time data from experiments conducted in a previous study on hydrated and partially dehydrated individual collagen fibrils via AFM. The derivative tensile modulus, estimated from the empirical equation, increased for decreasing hydration of the collagen fibril. The viscosity is only marginally affected by hydration but shows a dependency with strain rate, suggesting thixotropic behavior for low strain rates.
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Colágeno , Estrés Mecánico , Fenómenos Biomecánicos , Viscosidad , Resistencia a la TracciónRESUMEN
Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates ("2D") or embedded in fibrin hydrogels ("3D") with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels' fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7-30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.
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Bone is a biological tissue with unique mechanical properties, owing to a complex hierarchical structure ranging from the nanoscale up to the macroscale. To better understand bone mechanics, investigation of mechanical properties of all structural elements at every hierarchical level and how they interact is necessary. Testing of bone structures at the lower microscale, e.g. bone lamellae, has been least performed and remains a challenge. Focused ion beam (FIB) milling is an attractive technique for machining microscopic samples from bone material and performing mechanical testing at the microscale using atomic force microscopy (AFM) and nanoindentation setups. So far, reported studies at this length scale have been performed on bone samples of animal origin, mostly in a dehydrated state, except for one study. Here we present an AFM-based microbeam bending method for performing bending measurements in both dehydrated and rehydrated conditions at the microscale. Single lamella bone microbeams of four human donors, aged 65-94 yrs, were machined via FIB and tested both in air and fully submerged in Hank's Balanced Salt Solution (HBSS) to investigate the effect of (de)hydration and to a certain extent, of age, on bone mechanics. Bending moduli were found to reduce up to 5 times after 2 h of rehydration and no trend of change in bending moduli with respect to age could be observed. Mechanical behavior changed from almost purely elastic to viscoelastic upon rehydration and a trend of lower dissipated energy in samples from older donors could be observed in the rehydrated state. These results confirm directly the importance of water for the mechanical properties of bone tissue at the microscale. Moreover, the trend of lowered capability of energy dissipation in older donors may contribute to a decrease of fracture toughness and thus an increase in bone fragility with age.
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Huesos , Fémur , Anciano , Hueso Cortical , HumanosRESUMEN
Collagen is the major structural protein in human bodies constituting about 30% of the entire protein mass. Through a self-assembly process, triple helical collagen molecules assemble into high aspect-ratio fibers of tens to hundreds of nanometer diameter, known as collagen fibrils (CFs). In the last decade, several methods for tensile testing these CFs emerged. However, these methods are either overly time-consuming or offer low data acquisition bandwidth, rendering dynamic investigation of tensile properties impossible. Here, we describe a novel instrument for tensile testing of individual CFs. CFs are furnished with magnetic beads using a custom magnetic tweezer. Subsequently, CFs are lifted by magnetic force, allowing them to be picked-up by a microgripper structure, which is mounted on a cantilever-based interferometric force probe. A piezo-lever actuator is used to apply tensile displacements and to perform tensile tests of tethered CFs, after alignment. Once the mechanical tests are finished, CFs are removed from the microgripper by application of a magnetic field. Our novel instrument enables tensile tests with at least 25-fold increased throughput compared to tensile testing with an atomic force microscope while achieving force resolution (p-p) of 10 nN at a strain resolution better than 0.1%.
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Colágeno , Humanos , Microscopía de Fuerza Atómica/métodos , Piel , Resistencia a la TracciónRESUMEN
Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.
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Colágeno/fisiología , Fibrosis Pulmonar/metabolismo , Biomarcadores , Células Cultivadas , Colágeno/química , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Factor 1 Inducible por Hipoxia , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Estrés Oxidativo/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s-1 ) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies.
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Bioimpresión , Gelatina , Rayos Láser , Norbornanos , Impresión Tridimensional , Compuestos de Sulfhidrilo , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.
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Tissue hydration is well known to influence tissue mechanics and can be tuned via osmotic pressure. Collagen fibrils are nature's nanoscale building blocks to achieve biomechanical function in a broad range of biological tissues and across many species. Intrafibrillar covalent cross-links have long been thought to play a pivotal role in collagen fibril elasticity, but predominantly at large, far from physiological, strains. Performing nanotensile experiments of collagen fibrils at varying hydration levels by adjusting osmotic pressure in situ during atomic force microscopy experiments, we show the power the intrafibrillar noncovalent interactions have for defining collagen fibril tensile elasticity at low fibril strains. Nanomechanical tensile tests reveal that osmotic pressure increases collagen fibril stiffness up to 24-fold in transverse (nanoindentation) and up to 6-fold in the longitudinal direction (tension), compared to physiological saline in a reversible fashion. We attribute the stiffening to the density and strength of weak intermolecular forces tuned by hydration and hence collagen packing density. This reversible mechanism may be employed by cells to alter their mechanical microenvironment in a reversible manner. The mechanism could also be translated to tissue engineering approaches for customizing scaffold mechanics in spatially resolved fashion, and it may help explain local mechanical changes during development of diseases and inflammation.
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Colágeno/química , Estrés Mecánico , Animales , Masculino , Ratones , Microscopía de Fuerza AtómicaRESUMEN
Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale "cables" acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the non-enzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.
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Colágenos Fibrilares/química , Electricidad Estática , Animales , Reactivos de Enlaces Cruzados/química , Femenino , Glutaral/química , RatonesRESUMEN
Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.
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Fenómenos Mecánicos , Microscopía de Fuerza Atómica/métodos , Calibración , Coloides , Módulo de ElasticidadRESUMEN
Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.
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Aminoácido Oxidorreductasas/antagonistas & inhibidores , Colágeno/química , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/química , Fibrosis Pulmonar/tratamiento farmacológico , Reticulina/química , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos/química , Animales , Fenómenos Biomecánicos , Estudios de Casos y Controles , Colágeno/metabolismo , Colágeno/ultraestructura , Reactivos de Enlaces Cruzados/química , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Homeostasis/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Mecanotransducción Celular , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteína-Lisina 6-Oxidasa , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Reticulina/metabolismo , Reticulina/ultraestructura , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a 'guiding' structure to support the body's self-healing process.
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Implantes Absorbibles , Cartílago Articular/crecimiento & desarrollo , Condrocitos/fisiología , Regeneración Tisular Dirigida/instrumentación , Poliésteres/química , Andamios del Tejido , Materiales Biocompatibles/química , Cartílago Articular/citología , Células Cultivadas , Condrocitos/citología , Condrogénesis/fisiología , Módulo de Elasticidad/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Ingeniería de Tejidos/instrumentaciónRESUMEN
Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects.
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Reactores Biológicos , Cartílago/citología , Condrocitos/citología , Técnicas Analíticas Microfluídicas/instrumentación , Perfusión/instrumentación , Ingeniería de Tejidos/instrumentación , Anciano de 80 o más Años , Cartílago/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Diseño de Equipo , Femenino , Cabeza Femoral/lesiones , Humanos , MasculinoRESUMEN
The nanomechanical assessment of collagen fibrils via atomic force microscopy (AFM) is of increasing interest within the biomedical research community. In contrast to conventional nanoindentation there exists no common standard for conducting experiments and analysis of data. Currently used analysis approaches vary between studies and validation of quantitative results is usually not performed, which makes comparison of data from different studies difficult. Also there are no recommendations with regards to the maximum indentation depth that should not be exceeded to avoid substrate effects. Here we present a methodology and analysis approach for AFM cantilever-based nanoindentation experiments that allows efficient use of captured data and relying on a reference sample for determination of tip shape. Further we show experimental evidence that maximum indentation depth on collagen fibrils should be lower than 10-15% of the height of the fibril to avoid substrate effects and we show comparisons between our and other approaches used in previous works. While our analysis approach yields similar values for indentation modulus compared to the Oliver-Pharr method we found that Hertzian analysis yielded significantly lower values. Applying our approach we successfully and efficiently indented collagen fibrils from human bronchi, which were about 30 nm in size, considerably smaller compared to collagen fibrils obtained from murine tail-tendon. In addition, derived mechanical parameters of collagen fibrils are in agreement with data previously published. To establish a quantitative validation we compared indentation results from conventional and AFM cantilever-based nanoindentation on polymeric samples with known mechanical properties. Importantly we can show that our approach yields similar results when compared to conventional nanoindentation on polymer samples. Introducing an approach that is reliable, efficient and taking into account the AFM tip shape, we anticipate that the present work may act as a guideline for conducting AFM cantilever-based nanoindentation of collagen fibrils. This may aid understanding of collagen-related diseases such as asthma, lung fibrosis or bone disease with potential alterations of collagen fibril mechanics.
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Materiales Biocompatibles/química , Colágeno/química , Microscopía de Fuerza Atómica , Nanoestructuras/química , Nanotecnología/métodos , Animales , Fenómenos Biomecánicos , Bronquios/patología , Elasticidad , Humanos , Ensayo de Materiales , Ratones , Distribución de Poisson , Polímeros/química , Ratas , Reproducibilidad de los Resultados , Estrés MecánicoRESUMEN
An improved understanding of bone mechanics is vital in the development of evaluation strategies for patients at risk of bone fracture. The current evaluation approach based on bone mineral density (BMD) measurements lacks sensitivity, and it has become clear that as well as bone mass, bone quality should also be evaluated. The latter includes, among other parameters, the bone matrix material properties, which in turn depend on the hierarchical structural features that make up bone as well as their composition. Optimal load transfer, energy dissipation and toughening mechanisms have, to some extent, been uncovered in bone. Yet, the origin of these properties and their dependence upon the hierarchical structure and composition of bone are largely unknown. Here we investigate load transfer in the osteonal and sub-osteonal levels and the mechanical behaviour of osteonal lamellae and interlamellar areas during loading. Using cantilever-based nanoindentation, in situ microtensile testing during atomic force microscopy (AFM) and digital image correlation (DIC), we report evidence for a previously unknown mechanism. This mechanism transfers load and movement in a manner analogous to the engineered "elastomeric bearing pads" used in large engineering structures. µ-RAMAN microscopy investigations showed compositional differences between lamellae and interlamellar areas. The latter have lower collagen content but an increased concentration of noncollagenous proteins (NCPs). Hence, NC-enriched areas on the microscale might be similarly important for bone failure as ones on the nanoscale. Finally, we managed to capture stable crack propagation within the interlamellar areas in a time-lapsed fashion, proving their significant contribution towards fracture toughness.