Where shall I go? The mechanosensing adventures of a computational single cell

Cell adhesion and migration play an essential role in processes within the human body such as embryogenesis, tissue regeneration or cancer. Thus, to fully understand the behaviour of the mechanisms that regulate them would be a big step in biomedicine. In recent years, computational models have been postulated as firm candidates in terms of cellular research, since they constitute a very powerful tool complementary to traditional in vitro research: thanks to them, we are able to analyze what is happening within the cell even at subcellular level. In this work, we used a computational model to approach such cellular processes by studying the mechanical stimuli that govern the interaction of a cell with its environment. In particular, our interest resided in analyzing how the cell exerts traction through its actomyosin stress fibers by sensing the substrate stiffness, which is known as mechanosensing; as a consequence, the cell is deformed and this allows cell migration. To mimic some biological functions regarding cell-matrix adhesions, Bell's model and fiber maturation were implemented in the computational model. From the results we obtained after running some simulations, it is shown that there are many factors that influence cell traction. For instance, the total amount of focal adhesions at a certain time determines the number of fibers exerting force at the same time, which is translated as a higher force. Also, if those focal adhesions are able to live longer, there are more fibers coexisting. Substrate stiffness also plays an important role: as stiffness increases, stress fibers mature further and thus exert higher forces on the substrate; in addition, it also determines the size of the contact interface between the cell and the substrate. All in all, computational methods give quantitative and qualitative data with a lot of detail; hence, further research in this line is indeed a big step forward

Bone regeneration in patient-specific scaffolds from microfluidics to computational simulation

Los trastornos musculoesqueléticos y sus correspondientes enfermedades óseas son una de las principales causas de dolor y discapacidad, así como una carga social y económica para nuestra sociedad. Cuando la función articular se ve afectada o los defectos óseos son demasiado grandes para los injertos óseos, los implantes protésicos son el método estándar para tratar los trastornos musculoesqueléticos graves, aunque existe la necesidad clínica de que los implantes permanezcan activos durante un período de tiempo más largo y reduzcan las tasas de revisión. Para abordar la mayor durabilidad de los implantes ortopédicos, recientemente han surgido implantes impresos en tres dimensiones (3D) para fabricar superficies porosas específicas del paciente en la superficie del hueso-implante, mejorando así la fijación biológica del implante. La traslación de los principios de la medicina regenerativa a la ortopedia permitiría definir una nueva generación de implantes que completen la transición de materiales inertes a andamios bioactivos que guíen el proceso de regeneración ósea. A corto plazo, es probable que los andamios ortopédicos regenerativos impresos en 3D aumenten la vida útil del implante, mientras que a largo plazo puedan degradarse una vez que el tejido huésped esté completamente reparado. El objetivo global de esta tesis es evaluar el potencial regenerativo asociado a los andamiajes óseos impresos en 3D para aplicaciones ortopédicas específicas del paciente.Para ello, el primer estudio tuvo como objetivo determinar el papel del entorno mecánico del huésped en el proceso de regeneración ósea guiado por andamios óseos impresos en 3D en aplicaciones de carga. Se desarrolló un modelo computacional de regeneración ósea impulsada por un mecanismo en andamios porosos y se basó en la especificidad del sujeto, el sitio de implantación y la sensibilidad al entorno mecánico. A continuación, se simuló el crecimiento óseo en el interior de andamiajes porosos de titanio implantados en el fémur distal y la tibia proximal de tres cabras y se comparó con los resultados experimentales. Los resultados mostraron que el crecimiento óseo en el interior cambió de un patrón de distribución homogéneo, cuando los andamios estaban en contacto con el hueso trabecular, a un crecimiento óseo localizado cuando los andamios se implantaron en una ubicación diafisaria. En general, la dependencia de la respuesta osteogénica de la biomecánica del huésped sugirió que, desde una perspectiva mecánica, el potencial regenerativo dependía tanto del andamio como del entorno del huésped.El segundo estudio de esta tesis tuvo como objetivo evaluar la actividad osteogénica específica del paciente en un entorno controlado in vitro donde las células óseas humanas, aisladas de sujetos individuales, imitan los rasgos esenciales del proceso de formación ósea. Los sistemas in vitro tradicionales ya permitieron demostrar que los osteoblastos humanos primarios embebidos en una matriz fibrada de colágeno se diferencian en osteocitos en condiciones específicas. Por lo tanto, se planteó la hipótesis de que la traslación de este entorno a la escala de órgano en un chip crea una unidad funcional mínima para recapitular la maduración de los osteoblastos hacia los osteocitos y la mineralización de la matriz. Con este propósito, se sembraron osteoblastos humanos primarios en un hidrogel de colágeno de tipo I, para conocer mejor el papel de la densidad de siembra de células en su diferenciación a osteocitos. Los resultados muestran que las células cultivadas a mayor densidad aumentan la longitud de la dendrita con el tiempo, dejan de proliferar, exhiben morfología dendrítica, regulan positivamente la actividad de la fosfatasa alcalina y expresan marcadores de osteocitos. Este estudio reveló que los sistemas de microfluídica son una estrategia funcional que permite crear un modelo de tejido óseo específico del paciente e investigar el potencial osteogénico individual de las células óseas del paciente.En conjunto, los resultados de esta tesis enfatizan la importancia de utilizar un sistema de modelado múltiple al investigar el proceso de regeneración in vivo guiado por armazones óseos específicos adecuados al paciente. Ambos actores de una estrategia regenerativa libre de células in situ, a saber, el andamio y el paciente, tienen un efecto significativo en el resultado regenerativo final y necesitan ser modelados. Las técnicas avanzadas de in vitro e in silico, combinadas con datos de in vivo, evalúan aspectos distintivos del proceso de regeneración ósea para aplicaciones específicas del paciente. Las futuras estrategias personalizadas de ingeniería de tejidos podrían depender de la integración de esos modelos para mitigar en última instancia la variabilidad en el proceso de regeneración ósea guiado por un andamio específico para el paciente.<br /

Use of micro-CT-based finite element analysis to accurately quantify peri-implant bone strains: a validation in rat tibiae

Although research has been addressed at investigating the effect of specific loading regimes on bone response around the implant, a precise quantitative understanding of the local mechanical response close to the implant site is still lacking. This study was aimed at validating micro-CT-based finite element (μFE) models to assess tissue strains after implant placement in a rat tibia. Small implants were inserted at the medio-proximal site of 8 rat tibiae. The limbs were subjected to axial compression loading; strain close to the implant was measured by means of strain gauges. Specimen-specificμFE models were created and analyzed. For each specimen, 4 different models were created corresponding to different representations of the bone-implant interface: bone and implant were assumed fully osseointegrated (A); a low stiffness interface zone was assumed with thickness of 40μm (B), 80μm (C), and 160μm (D). In all cases, measured and computational strains correlated highly (R 2= 0.95, 0.92, 0.93, and 0.95 in A, B, C, and D, respectively). The averaged calculated strains were 1.69, 1.34, and 1.15 times higher than the measured strains for A, B, and C, respectively, and lower than the experimental strains for D (factor = 0.91). In conclusion, we demonstrated that specimen-specific FE analyses provide accurate estimates of peri-implant bone strains in the rat tibia loading model. Further investigations of the bone-implant interface are needed to quantify implant osseointegratio

Inverse method based on 3D nonlinear physically constrained minimisation in the framework of traction force microscopy

Traction force microscopy is a methodology that enables to estimate cellular forces from the measurement of the displacement field of an extracellular matrix (ECM)-mimicking hydrogel that a cell is mechanically interacting with. In this paper, a new inverse and physically-consistent methodology is developed and implemented in the context of 3D nonlinear elasticity. The proposed method searches for a displacement field that approximates the measured one, through the imposition of fulfillment of equilibrium with real and known forces acting in the hydrogel. The overall mathematical formulation leads to a constrained optimisation problem that is treated through a Lagrange operator and that is solved numerically by means of a nonlinear finite element framework. In order to illustrate the potential and enhanced accuracy of the proposed inverse method, it is applied to a total of 5 different real cases of cells cultured in a 3D hydrogel that is considered to behave as a nonlinear elastic material. Different error indicators are defined in order to compare ground truth simulated displacements and tractions to the ones recovered by the new inverse as well as by the forward method. Results indicate that the evaluation of displacement gradients leads to errors, in terms of recovered tractions, that are more than three times lower (on average) for the inverse method compared to the forward method. They highlight the enhanced accuracy of the developed methodology and the importance of appropriate inverse methods that impose physical constraints to traction and stress recovery in the context of traction force microscopyMinisterio de Economía y Competitividad (MINECO). PGC2018-097257-B-C31Consejo Europeo de Resucitación 308223Consejo Europeo de Resucitación G087018NMinisterio de Educación, Cultura y Deporte (MECD). España CAS17/00096Hércules G0H6316NFonds Wetenschappelijk Onderzoek (FWO) 12ZR120NFonds Wetenschappelijk Onderzoek (FWO) V413019

TFMLAB: A MATLAB toolbox for 4D traction force microscopy

Article number 100723We present TFMLAB, a MATLAB software package for 4D (x;y;z;t) Traction Force Microscopy (TFM). While various TFM computational workflows are available in the literature, open-source programs that are easy to use by researchers with limited technical experience and that can analyze 4D in vitro systems do not exist. TFMLAB integrates all the computational steps to compute active cellular forces from confocal microscopy images, including image processing, cell segmentation, image alignment, matrix displacement measurement and force recovery. Moreover, TFMLAB eases usability by means of interactive graphical user interfaces. This work describes the package's functionalities and analyzes its performance on a real TFM caseKU Leuven 1S68818NFonds Wetenschappelijk Onderzoek (FWO) V413019NFonds Wetenschappelijk Onderzoek (FWO) G085018NCambridge Conservation Initiative (CCI) 2014TC16RFCB046Ministerio de Educación, Cultura y Deporte de España CAS17/00096Ministerio de Economía y Competitividad de España (MINECO) PGC2018-097257-B-C31Consejo Europeo de Investigación 308223KU Leuven C14/17/111Fonds Wetenschappelijk Onderzoek (FWO)/Hercules G0H6316

Effect of ultrasound on bone fracture healing:A computational bioregulatory model

peer reviewedBone healing is a complex biological procedure in which several cellular actions, directed by biochemical and mechanical signals, take place. Experimental studies have shown that ultrasound accelerates bone ossification and has a multiple influence on angiogenesis. In this study a mathematical model predicting bone healing under the presence of ultrasound is demonstrated. The primary objective is to account for the ultrasound effect on angiogenesis and more specifically on the transport of the Vascular Endothelial Growth Factor (VEGF). Partial differential equations describing the spatiotemporal evolution of cells, growth factors, tissues and ultrasound acoustic pressure and velocity equations determining the development of the blood vessel network constitute the present model. The effect of the ultrasound characteristics on angiogenesis and bone healing is investigated by applying different boundary conditions of acoustic pressure at the periosteal region of the bone model, which correspond to different intensity values. The results made clear that ultrasound enhances angiogenesis mechanisms during bone healing. The proposed model could be regarded as a step towards the monitoring of the effect of ultrasound on bone regeneration. © 2018Action “Supporting Postdoctoral Researchers” of the Operational Program “Education and Lifelong Learning” (Action’s Beneficiary: General Secretariat for Research and Technology); Greek State (PE8-3347

Effect of ultrasound on bone fracture healing:A computational mechanobioregulatory model

Bone healing process is a complicated phenomenon regulated by biochemical and mechanical signals. Experimental studies have shown that ultrasound (US) accelerates bone ossification and has a multiple influence on cell differentiation and angiogenesis. In a recent work of the authors, a bioregulatory model for providing bone-healing predictions was addressed, taking into account for the first time the salutary effect of US on the involved angiogenesis. In the present work, a mechanobioregulatory model of bone solidification under the US presence incorporating also the mechanical environment on the regeneration process, which is known to affect cellular processes, is presented. An iterative procedure is adopted, where the finite element method is employed to compute the mechanical stimuli at the linear elastic phases of the poroelastic callus region and a coupled system of partial differential equations to simulate the enhancement by the US cell angiogenesis process and thus the oxygen concentration in the fractured area. Numerical simulations with and without the presence of US that illustrate the influence of progenitor cells' origin in the healing pattern and the healing rate and simultaneously demonstrate the salutary effect of US on bone repair are presented and discussed

L1-regularized reconstruction for traction force microscopy

Proceeding of: 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI). IEEE, Prague, 13-16 April, 2016.Traction Force Microscopy (TFM) is a technique widely used to recover cellular tractions from the deformation they cause in their surrounding substrate. Traction recovery is an ill-posed inverse problem that benefits of a regularization scheme constraining the solution. Typically, Tikhonov regularization is used but it is well known that L1-regularization is a superior alternative to solve this type of problems. For that, recent approaches have started to explore what could be their contribution to increase the sensitivity and resolution in the estimation of the exerted tractions. In this manuscript, we adapt the L1-regularization of the curl and divergence to 2D TFM and compare the recovered tractions on simulated and real data with those obtained using Tikhonov and L1-norm regularization.This work was partially supported by the European Research Council (ERC) under the EU-FP7/2007-2013 through ERC Grant Agreement nº 308223, and the Spanish Ministry of Economy and Competitiveness (TEC2013-48552-C2-1-R). European Community's Seventh Framework ProgramEuropean Community's Seventh Framework Progra

Functional and biomechanical evaluation of a completely recellularized stentless pulmonary bioprosthesis in sheep

ObjectiveIn a previous study we showed that recellularization of a stentless bioprosthetic valve is stimulated 1 month after implantation in the pulmonary position, when its matrix (acellular photo-oxidized bovine pericardium) was preseeded by intraperitoneal implantation during a 3-day period.MethodsThe present study reports on the functional and biomechanical properties of such valves (n = 19) in sheep up to 5 months after implantation. Similar valves (n = 20) that were not intraperitoneally preseeded served as controls.ResultsRecellularization was partial in control valves and excessive in preseeded valves: 66% versus 223% of cellularity of native valves, respectively (P < .05). The valves were endothelialized and contained interstitial cells depositing new matrix (collagens and elastin). However, phenotyping revealed an increased proportion of cells with contractile properties (30%–40% alpha smooth muscle actin+) in both groups. Intraperitoneally seeded valves had thicker and shorter leaflets that were associated with mildly increased peak gradients and regurgitation. Characterization of the matrix properties revealed a gradually degrading matrix (±25% loss of collagen organization at 5 months) and a concomitant alteration of its biomechanical properties, that is, decreased strength, stiffness, and maximum force. However, overall valve function remained intact, and the biomechanical properties of the whole valves were superior to that of the native valves.ConclusionThe ectopic in vivo seeding paradigm provides full recellularization. However, the volume fraction of the cellular phenotypes is not optimal, resulting in inadequate remodeling of the valves