A Novel Flexible and Steerable Probe for Minimally Invasive Soft Tissue Intervention

Current trends in surgical intervention favour a minimally invasive (MI) approach, in which complex procedures are performed through increasingly small incisions. Specifically, in neurosurgery, there is a need for minimally invasive keyhole access, which conflicts with the lack of maneuverability of conventional rigid instruments. In an attempt to address this fundamental shortcoming, this thesis describes the concept design, implementation and experimental validation of a novel flexible and steerable probe, named “STING” (Soft Tissue Intervention and Neurosurgical Guide), which is able to steer along curvilinear trajectories within a compliant medium. The underlying mechanism of motion of the flexible probe, based on the reciprocal movement of interlocked probe segments, is biologically inspired and was designed around the unique features of the ovipositor of certain parasitic wasps. Such insects are able to lay eggs by penetrating different kinds of “host” (e.g. wood, larva) with a very thin and flexible multi-part channel, thanks to a micro-toothed surface topography, coupled with a reciprocating “push and pull” motion of each segment. This thesis starts by exploring these foundations, where the “microtexturing” of the surface of a rigid probe prototype is shown to facilitate probe insertion into soft tissue (porcine brain), while gaining tissue purchase when the probe is tensioned outwards. Based on these findings, forward motion into soft tissue via a reciprocating mechanism is then demonstrated through a focused set of experimental trials in gelatine and agar gel. A flexible probe prototype (10 mm diameter), composed of four interconnected segments, is then presented and shown to be able to steer in a brain-like material along multiple curvilinear trajectories on a plane. The geometry and certain key features of the probe are optimised through finite element models, and a suitable actuation strategy is proposed, where the approach vector of the tip is found to be a function of the offset between interlocked segments. This concept of a “programmable bevel”, which enables the steering angle to be chosen with virtually infinite resolution, represents a world-first in percutaneous soft tissue surgery. The thesis concludes with a description of the integration and validation of a fully functional prototype within a larger neurosurgical robotic suite (EU FP7 ROBOCAST), which is followed by a summary of the corresponding implications for future work

Biomechanics of a parasitic wasp ovipositor : Probing for answers

Insects such as mosquitoes, true bugs, and parasitic wasps, probe for resources hidden in various substrates. The resources are often, located deep within the substrate and can only be reached with long and thin (slender) probes. Such probes can, however, easily bend or break (buckle) when pushed inside the substrate, which makes probing a challenging task. Nevertheless, the mentioned insects use their probes repeatedly throughout their lifetime without apparent damage. Furthermore, the probes are also used for sensing the targets, can be steered during insertion, and can transport both fluids (e.g. blood, phloem sap) and eggs. Insect probes seem highly versatile structures that satisfy many functional requirements, including buckling avoidance, steering, sensing, and transport. Similar requirements also hold for minimally invasive medical procedures, where slender tools are used to minimize damage to the patient. Understanding the probing process in insects can bring insights in the insect ecology and evolution and it may also help in the development of novel surgical tools. In this thesis, I focus on the mechanical and motor adaptations of insect probing, while other aspects are only briefly discussed. In chapter 2, we review the literature on the probing structures and their operating principles across mosquitoes, parasitic wasps, and hemipterans. Probes are either modified mouthparts (mosquitoes, true bugs) or special tubular outgrowths of the abdomen (parasitic wasps). Despite having different developmental origins, the probes share three major morphological characteristics, which may reflect the shared functional requirements of buckling avoidance and steering: (i) the probes consist of multiple, interconnected elements that can slide along each other, (ii) the probe diameters are very small, which leaves no space for internal musculature, and (iii) the distal ends (tips) of the probe elements are asymmetric and often bear various serrations, hooks, bulges, or notches. How such slender multi-element probes avoid buckling during insertion has been hypothesized in the so-called push–pull mechanism. According to this mechanism, the probe is inserted into the substrate by reciprocal movements of the elements. The insects therefore simultaneously push on some of the probe elements, while pulling on the others. The tip serrations are directed such, that they primarily increase the friction upon pulling of the elements. This puts the pulled elements under tension and makes them effectively stiffer in bending (like when pulling a rope). The elements under tension can serve as guides along which the other elements are pushed inside the substrate without the risk of buckling. The insect alternates the pushing and pulling between the elements to incrementally insert the probe in the substrate. This mechanism has, however, never been quantified in insects and it was hitherto unknown whether the animals rely on it during probing. The probe tip asymmetry presumably facilitates steering. The asymmetric tip geometry leads to asymmetric reaction forces from the substrate on the tip during insertion, which push the probe tip sideways into a curved path. Controlling the tip geometry therefore allows for control of probing direction. Although offsetting the elements by sliding already changes the shape of the probe tip, these changes might be too small to induce the necessary change of probing direction. A number of mechanisms that enhance the tip asymmetry during the sliding of the elements have been suggested. However, few mechanisms have been observed or studied in vivo, so it is not completely clear how insects steer with their probes. Additionally, the effect of the substrate on both the steering and insertion mechanisms is unknown. To understand the biomechanics of insect probing, we investigated the probing behaviour of the braconid parasitic wasp Diachasmimorpha longicaudata. This is an ideal species for studying the buckling avoidance and steering, because it: (i) possess a slender ovipositor several millimetres in length, (ii) probes into solid material (e.g. citrus fruits), and (iii) attack fruit-fly larvae that are freely moving within the substrate (i.e. steering can be expected). The ovipositor of D. longicaudata is similar to other hymenopterans and consists of three interconnected elements (valves), one dorsal and two ventral ones. The interconnection is a tongue-and-groove mechanism, which allows for sliding of the valves, but prevents their separation. The ovipositor has an asymmetric tip—the distal end of the dorsal valve is enlarged (bulge), while the ventral valve tips have harpoon-like serrations. Additionally, just proximal to the bulge of the dorsal valve, the ovipositor is characteristically bent in an S-shape. This seems to be a feature present only in D. longicaudata and closely related species. The wasps also possess a pair of sheaths that envelop the ovipositor at rest and throughout most of the probing process, but do not penetrate into the substrate. In chapter 3, we studied the kinematics of ovipositor insertion into translucent, artificial substrates of various stiffnesses. Ovipositor insertion was filmed in a three camera setup, which allowed us to reconstruct the ovipositor insertion in 3D, while also monitoring the orientation of the insect’s body. We discovered that the wasps can explore a wide range of the substrate by probing in any direction with respect to their body orientation from a single puncture point. Probing range and speed decreased with increasing substrate stiffness. Wasps used two strategies of ovipositor insertion. In soft substrates, all ovipositor valves were pushed inside the substrate at the same time. In stiff substrates, wasps always moved the valves alternatively, presumably employing the hypothesized push–pull mechanism. We observed that ovipositors can follow curved trajectories inside the substrate. Detailed kinematic analysis revealed that the ovipositors followed a curved path during probing with protracted ventral valve(s). In contrast, probing with protracted dorsal valve resulted in straight trajectories. We linked the changes in the probing direction to the shape changes in the ovipositor tip. When the ventral valves were protracted, they curved towards the dorsal valve, resulting in an enhanced bevel which presumably caused a change in insertion direction. In chapter 4, we investigated the above described steering mechanism by quantifying the bending stiffness (three point bend test) and the geometry (high-resolution computer tomography) of the ovipositor in D. longicaudata. Additionally, we qualitatively assessed the material composition of the valves using fluorescence imaging. The thick dorsal valve bulge might be stiff and could straighten the S-shaped region of the ovipositor during the valve offset, causing bending of the tip. We discovered that the S-shaped region of the ovipositor is significantly softer than its neighbouring regions, which is mostly due to the presence of resilin in the S-shaped region of the ventral valve. Resilin is a rubber-like protein and reduces the stiffness of the otherwise heavily sclerotized valves. Additionally, we showed that the ventral valves have a higher bending stiffness than the dorsal valve along most of their length. The exception is presumably the bulge on the dorsal valve—although we could not directly measure its bending stiffness, its geometrical properties show that it is the thickest (and therefore stiffest) region in the distal end of the ovipositor. Outside the substrate, offsetting of the valves in any direction (i.e. pro- or retraction of the ventral valves) caused a straightening of the S-shaped region of the ovipositor and a curving towards the dorsal side. However, during probing in a substrate, such curving was only observed upon protraction of the ventral valves. We hypothesize this is due to the interaction of the ovipositor with the substrate. Namely, the bevelled ventral valve tips generate substrate reaction forces that promote dorsal curving, while the bevelled tip of the dorsal valve generates substrate forces that promote ventral bending. The interaction between the ventral and dorsal valves straightens the S-shaped region of the ovipositor and enhances dorsal curving. This therefore facilitates strong shape changes of the tip only upon protraction of the ventral valves, while counteracting the ventral curving of the dorsal valve. These opposing mechanisms presumably result in an approximately straight protraction of the dorsal valve. In chapters 2 and 3 we describe how the wasps use the reciprocal valve movements when probing in stiff substrates. As such substrates presumably require strong forces during insertion, the reciprocal valve movements may indeed serve to avoid buckling. However, how the valves are actuated or the forces generated during probing have never been quantified. In chapter 5, we therefore investigated the ovipositor base and the muscles driving the movements of the valves. At the base, the valves attach to plate-like structures that are interconnected with a series of linkages. The muscles attach to these plates and can move them with respect to each other. Such movements also result in the movements of the valves. To analyse the mechanics of this linked system, we performed high-resolution computer tomography scans of wasps in different stages of the probing cycle. This allowed us to compare the configurational changes of the basal plates to the valve offset, and measure the muscle cross-sections and attachment sites. We also calculated the muscle moment arms and estimated the forces and moments of the most relevant musculature actuating the ovipositor movements, by assuming a tensile muscle stress previously reported for insect muscles. For the ventral valves only, we also calculated the forces the valves can exert onto the substrate. The dorsal valve can only be moved by moving the base that is linked inside the abdomen, and therefore force estimation could not be made. The displacement magnitude of the basal plates corresponded to the valve offset, indicating that the valves are indeed moved due to the changes in the arrangement of the basal plates. We also showed that the ventral valve plates move most during the probing cycle, while the magnitude of the dorsal valve plate movements is much smaller. This suggests that the ventral valves move along the dorsal valve, while the dorsal valve moves together with the abdomen during probing. Additionally, in the situation where the animal keeps its abdomen stationary, we estimated the maximal forces actuating the ventral valves. The estimated maximal pushing forces can be higher than the estimated buckling load of the unsupported ovipositor outside the substrate. Assuming the maximal pushing forces are required during probing, antibuckling mechanisms are needed to avoid damaging the ovipositor. Buckling can be limited (prevented) by either supporting the ovipositor outside the substrate with additional sheaths, employing the push–pull mechanism, or both. Subtracting the maximal estimated pushing and pulling forces on the ventral valves, results in a net pushing force that is very close to the buckling threshold of the ovipositor, albeit still slightly higher. The sheaths, although being flexible, might provide the additional support if needed. In this thesis, I show that multi-element probes are inserted into the substrate using reciprocal movements of the individual elements. These movements appear to be necessary in stiff substrates, which presumably require high pushing forces on a single element during probing. This is in accordance with the hypothesis that reciprocal valve movements serve as an anti-buckling mechanism. Additionally, such valve movements are also important for steering of the probe during insertion. The valve offset controls the shape of the probe tip and therefore the net substrate reaction forces that result in bending of the probe. Wasps evolved special structures that enhance the shape changes of their ovipositor tips and facilitate steering. Our findings may be interesting for a broad range of audiences. Entomologists, evolutionary biologists, and ecologists may find them useful when studying the diversification of probing insects, their evolutionary success, or their ecological interactions (e.g. insect–plant, parasite–host). The anti-buckling and steering mechanisms may be helpful when developing novel, man-made probes. These mechanisms allow for minimization of the probe thickness and accurate steering control, which minimizes substrate damage during probing. Our findings may be particularly useful in the development of slender, steerable needles for minimally invasive surgery.</p

A Novel Bio-Inspired Insertion Method for Application to Next Generation Percutaneous Surgical Tools

The use of minimally invasive techniques can dramatically improve patient outcome from neurosurgery, with less risk, faster recovery, and better cost effectiveness when compared to conventional surgical intervention. To achieve this, innovative surgical techniques and new surgical instruments have been developed. Nevertheless, the simplest and most common interventional technique for brain surgery is needle insertion for either diagnostic or therapeutic purposes. The work presented in this thesis shows a new approach to needle insertion into soft tissue, focussing on soft tissue-needle interaction by exploiting microtextured topography and the unique mechanism of a reciprocating motion inspired by the ovipositor of certain parasitic wasps. This thesis starts by developing a brain-like phantom which I was shown to have mechanical properties similar to those of neurological tissue during needle insertion. Secondly, a proof-of-concept of the bio-inspired insertion method was undertaken. Based on this finding, the novel method of a multi-part probe able to penetrate a soft substrate by reciprocal motion of each segment is derived. The advantages of the new insertion method were investigated and compared with a conventional needle insertion in terms of needle-tissue interaction. The soft tissue deformation and damage were also measured by exploiting the method of particle image velocimetry. Finally, the thesis proposes the possible clinical application of a biologically-inspired surface topography for deep brain electrode implantation. As an adjunct to this work, the reciprocal insertion method described here fuelled the research into a novel flexible soft tissue probe for percutaneous intervention, which is able to steer along curvilinear trajectories within a compliant medium. Aspects of this multi-disciplinary research effort on steerable robotic surgery are presented, followed by a discussion of the implications of these findings within the context of future work

Cyclic motion control for programmable bevel-tip needles 3D steering: a simulation study

Flexible, steerable, soft needles are desirable in Minimally Invasive Surgery to achieve complex trajectories while maintaining the benefits of percutaneous intervention compared to open surgery. One such needle is the multi-segment Programmable Bevel-tip Needle (PBN), which is inspired by the mechanical design of the ovipositor of certain wasps. PBNs can steer in 3D whilst minimizing the force applied to the surrounding substrate, due to the cyclic motion of the segments. Taking inspiration also from the control strategy of the wasp to perform insertions and lay their eggs, this paper presents the design of a cyclic controller that can steer a PBN to produce a desired trajectory in 3D. The performance of the controller is demonstrated in simulation in comparison to that of a direct controller without cyclic motion. It is shown that, while the same steering curvatures can be attained by both controllers, the time taken to achieve the configuration is longer for the cyclic controller, leading to issues of potential under-steering and longer insertion times

Biomimicry - medical design concepts inspired by nature

Biomimicry is the application of existing features in nature to human technologies, such as the invention of aircraft inspired by bird flight. In the development of medical solutions, biomimicry is a growing field of research, where a holistic understanding of nature can inspire cutting-edge design. The purpose of this study was to create an educational, visual resource exemplifying up-and-coming medical applications of biomimicry. A website was created to present 2D motion graphics (animations) and illustrations. Animation is an established and useful method of communicating health information to the public. This presents an accessible interface for the public to interact with and learn about this area of research, bridging the gap between the two. Increasing public knowledge, engagement, and interest can expand the reach and thereby influence future research. A survey was conducted to assess public engagement and opinions on both the resource and the topic of biomimicry and medical design. The results suggested that participants positively engaged with the resource; 95.7% strongly agreed/agreed that the animations were beneficial for learning. All responding participants agreed that biomimicry could provide useful solutions in medical design. This study suggests that graphic motions are effective at communicating complex ideas for public outreach.</p

Development of an online progressive mathematical model of needle deflection for application to robotic-assisted percutaneous interventions

A highly flexible multipart needle is under development in the Mechatronics in Medicine Laboratory at Imperial College, with the aim to achieve multi-curvature trajectories inside biological soft tissue, such as to avoid obstacles during surgery. Currently, there is no dedicated software or analytical methodology for the analysis of the needle’s behaviour during the insertion process, which is instead described empirically on the basis of experimental trials on synthetic tissue phantoms. This analysis is crucial for needle and insertion trajectory design purposes. It is proposed that a real-time, progressive, mathematical model of the needle deflection during insertion be developed. This model can serve three purposes, namely, offline needle and trajectory design in a forward solution of the model, when the loads acting on needle from the substrate are known; online, real-time identification of the loads that act on the needle in a reverse solution, when the deflections at discrete points along the needle length are known; and the development of a sensitivity matrix, which enables the calculation of the corrective loads that are required to drive the needle back on track, if any deviations occur away from a predefined trajectory. Previously developed mathematical models of needle deflection inside soft tissue are limited to small deflection and linear strain. In some cases, identical tip path and body shape after full insertion of the needle are assumed. Also, the axial load acting on the needle is either ignored or is calculated from empirical formulae, while its inclusion would render the model nonlinear even for small deflection cases. These nonlinearities are a result of the effects of the axial and transverse forces at the tip being co-dependent, restricting the calculation of the independent effects of each on the needle’s deflection. As such, a model with small deflection assumptions incorporating tip axial forces can be called “quasi-nonlinear” and a methodology is proposed here to tackle the identification of such axial force in the linear range. During large deflection of the needle, discrepancies between the shape of the needle after the insertion and its tip path, computed during the insertion, also significantly increase, causing errors in a model based on the assumption that they are the same. Some of the models developed to date have also been dependent on existing or experimentally derived material models of soft tissue developed offline, which is inefficient for surgical applications, where the biological soft tissue can change radically and experimentation on the patient is limited. Conversely, a model is proposed in this thesis which, when solved inversely, provides an estimate for the contact stiffness of the substrate in a real-time manner. The study and the proposed model and techniques involved are limited to two dimensional projections of the needle movements, but can be easily extended to the 3-dimensional case. Results which demonstrate the accuracy and validity of the models developed are provided on the basis of simulations and via experimental trials of a multi-part 2D steering needle in gelatine.Open Acces

Protrusion-retraction switches and traction forces in spontaneous cell polarization

Cell migration is a fundamental property of all animal cells which is involved in processes such as embryogenesis, immune response, tissue regeneration and cancer metastasis. Directional migration requires cell polarization which could happen in response to external signals but also spontaneously. Polarization involves the change in the cell edge activities from random distribution of protrusion and retraction to their separation into two large regions corresponding to the leading and trailing edges of the cell. Many studies investigated cytoskeletal mechanisms of protrusions and retractions but it is still not well understood how the cell orchestrates protrusion and retraction along its edge and what triggers transitions between these two types of activity. These questions are essential to understand both cell polarization and cycles of protrusion and retraction which characterize exploratory edge dynamics before polarization. This thesis focuses on the analysis of cell edge dynamics during the initiation of motion and on the mechanisms of transition (switches) between protrusion and retraction. We considered the cell polarization and protrusion-retraction cycles as two related phenomena and aimed to identify common mechanisms. Live cell imaging, cytoskeletal inhibitors, traction force microscopy, patterned substrates, micromanipulation, and computational analysis and modeling were used to examine cell polarization in the experimental model of fish epithelial keratocytes that are characterized by simple and regular shape and robust polarization and motion. We found that protrusion-retraction switches happened at maximal distance from the cell center both during cell edge fluctuation and directional motion and did not depend on the edge orientation with respect to the cell motion direction. Computational model demonstrated that switches at a threshold distance were sufficient for self-organization of edge activity leading to spontaneous polarization and stable cell shape and motion suggesting that distance-sensing is a fundamental mechanism of cell symmetry breaking. Next we investigated the mechanisms of distance-sensing focusing on two hypotheses: traction forces and tridimensional cell shape. Traction force may increase with the distance from the cell center (e.g., due to a build-up of actomyosin network) leading to detachment of the edge and initiation of retraction. We discovered that traction forces indeed increased with the distance and that protrusion-retraction switches occurred near maximal forces. Local external force also induced protrusion-retraction switch. However, inhibition of contractility reduced traction forces and abolished the dependence of force on the distance, but did not prevent cell polarization. Taken together, these results suggest that traction forces are sufficient, but not necessary mediators of distance-dependent switch and cell polarization. In an alternative mechanism, tridimensional shape of the cell edge may depend on the distance from the cell center and affect the balance of forces at the edge, inducing switches. We have locally modified this tridimensional force balance by using substrates with topographic features and showed that switches preferentially happen near these features. These results provide a novel framework to understand cell edge dynamics and symmetry breaking in terms of protrusion-retraction switches and physical force balance at the cell edge

Dynamics of epithelial gap closure using microfabrication and micromechanical approaches

Les cellules peuvent migrer sous différentes conditions qui dépendent de l environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d une blessure. Lorsque l épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d un anneau contractile d acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface libre .Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite molle qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d un processus collectif au travers de la formation d un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l embryogenèse.Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. 11Abstract (English)Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. Next, we also addressed the mechanical component of epithelial gap closure. In this study, we took advantage of a laser-ablation system to disrupt some cells within an epithelial monolayer, and study how the remaining cells sealed that gap. By measuring the traction forces that cells exert on the substrate along the closure, we observed that cells first pulled on the substrate to propel themselves. By the last steps of closure, there is a transition in the direction of the force, so that cells are pulled to the center of the gap due to the assembly of a supracellular actin cable. Altogether, this work provides valuable knowledge on the current understanding of the mechanisms accounting for epithelial gap closure. We believe that a better comprehension of these mechanisms can help to shed light in clinically relevant situations where epithelial gap closure is impaired.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF