Attuatori bio-ibridi

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1 XXXII Scuola Annuale di Bioingegneria (Approccio integrato per la medicina rigenerativa) Bressanone, Settembre 2013 Attuatori bio-ibridi Arianna Menciassi, Leonardo Ricotti The BioRobotics Institute Scuola Superiore Sant Anna (Pisa)

2 Come si colloca questa lezione nell ambito della Scuola Well established technologies in a field which can be disruptive in a different field Christensen, "The Innovator's Dilemma (1997): new technology are separated into two categories: sustaining and disruptive. Sustaining technology relies on incremental improvements to an already established technology. Disruptive technology lacks refinement, often has performance problems because it is new, appeals to a limited audience, and may not yet have a proven practical application. CMOS cameras and high power LEDs + = Endoscopic pills Traditional endoscope

3 Come si colloca questa lezione nell ambito della Scuola... The link between this lecture and the GNB School topics Technologies for tissue engineering, related to cell culturing, scaffolding, differentiation etc. can be applied in fields different from regenerative medicine with a dramatic impact.?

4 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

5 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

6 Il problema dell attuazione nella progettazione e sviluppo di macchine Future machines: the bottleneck of actuation Actuation is an essential function of any artificial or living machine, allowing its movement and its interaction with the surrounding environment Large machines are characterized by lack of flexibility and of life-like movements A hot topic already in 2005 (GNB School by Giusti and Cigada) Micro-machines are limited by scalability issues

7 Il problema dell attuazione nella progettazione e sviluppo di macchine Large machines: lack of flexibility and life-like movements NAO robot Roberto Bolle Rigid constitutive elements and joints Non-compliant actuators Centralized intelligence Flexible constitutive elements and joints Compliant actuators (muscles) Distributed intelligence

8 Il problema dell attuazione nella progettazione e sviluppo di macchine... Small machines: scalability issues At small scales, the relative importance of the physical laws changes L = characteristic length Volumetric quantities: inertia, weight, heat capacity, body forces Scale as ~ L 3 Surface quantities: friction, heat transfer, surface forces Scale as ~ L 2 Intermolecular van der Waals force: F vdw = Hr 8πx 2 com/watch?v=hjivvimus4 H = material-dependent Hamaker constant r = radius of a sphere x = separation distance (sphere infinite halfspace) Scales as ~ L, if only r scales Scales as ~ L -1, if both r and x scale J.J. Abbott et al. Robotics in the small. IEEE Rob Autom Mag. 14: (2007)

9 Il problema dell attuazione nella progettazione e sviluppo di macchine Force vs Size - Comparative analysis (Scuola GNB 2005 Attuatori e Sensori, Dario, Menciassi, Stefanini)

10 Il problema dell attuazione nella progettazione e sviluppo di macchine Comparison between actuator technologies M. Zupan et al. Actuator classification and selection the development of a database. Adv. Eng. Mat. 4(12): (2002)

11 Il problema dell attuazione nella progettazione e sviluppo di macchine Comparison between actuator technologies Piezoelectric motors Converse piezoelectric effect: change in shape when an electrical field is applied Acoustic or ultrasonic vibrations Linear or rotary motion CYcVT1yFv7A Delta-3 demonstrator, by Noliac om/watch?v=ojj18 mqo1ym High-precision positioning Fast actuation (high frequency) Extremely limited stroke

12 Il problema dell attuazione nella progettazione e sviluppo di macchine Comparison between actuator technologies M. Zupan et al. Actuator classification and selection the development of a database. Adv. Eng. Mat. 4(12): (2002)

13 Il problema dell attuazione nella progettazione e sviluppo di macchine Comparison between actuator technologies Pneumatic actuators Conversion of energy (tipically in the form of compressed air) into mechanical (linear or rotary) motion McKibben surgical robot, by KU Leuven bd_nyfs =xl5binuumbm Good compromise between force output and stroke Mimicking of few muscle features Need of an extremely bulky energy source

14 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

15 I vantaggi del muscolo naturale The unique properties of muscles Natural muscles rely on a finely regulated acto-myosin contractile machinery, optimized by millions of years of natural evolution. Benz Patent Motor Car B. Trimmer. A journal of soft robotics: why now? Soft Robotics. 1: 1-4 (2013)

16 I vantaggi del muscolo naturale Non-key skeletal muscle features J.M. Hollerbach et al. Comparative analysis of actuator technologies for robotics. The Rob. Rev (1992)

17 I vantaggi del muscolo naturale Key skeletal muscle features Backdrivability Stiffness control High transduction efficiency Self-sensing / self-healing properties Chemically fueling High aerobic transduction efficiency (1000 J per gram of glucose) D.G. Caldwell. Natural and artificial muscle elements as robot actuators. Mechatronics. 3: (1993) No artificial actuators are able to reproduce the behavior of muscle integrated series-elastic components, which are thought to give rise to many of the life-like characteristics of animal movements. S. Lv et al. Designed biomaterials to mimic the mechanical properties of muscles. Nature. 465: (2010)

18 I vantaggi del muscolo naturale Key skeletal muscle features Natural muscle has a modular architecture based on microscopic contractile units (the acto-myosin molecular machinery) Performance invariance at both small and large scales

19 Meccanismi o materiali smart per l attuazione How obtaining muscle-like behaviour Design of smart mechanisms Development of a bio-inspired robotic hand: Natural hand: ~ 40 muscles 40 artificial actuators: non feasible, due to: System bulkyness Unavailability of 40 different control signals Underactuated mechanisms require few control signals but can still endow the hand with many degrees of freedom (DOFs). Underactuated mechanisms: # Actuators < # DOFs 2 motors, 9 DOFs E. Mattar. A survey of bio-inspired robotics hand implementation: new directions in dexterous manipulation. Rob. Auton. Syst. 61: (2013)

20 Real OCTOPUS arm Meccanismi o materiali smart per l attuazione How obtaining muscle-like behaviour Design of smart mechanisms Artificial OCTOPUS arm Local processing, wires Transverse actuation system Longitudinal Muscles Transverse Muscles Oblique Muscles Longitudinal actuation system Embedding material Mechanical interface / containment (nylon) C. Laschi et al. Design of a biomimetic robotic octopus arm. Bioinspir. Biomim. 4(1): (2009) M. Cianchetti et al. Design concept and validation of a robotic arm inspired by the octopus. Mat. Sci. Eng.: C. 31: (2011)

21 Meccanismi o materiali smart per l attuazione How obtaining muscle-like behaviour Design of smart mechanisms C. Laschi et al. Design of a biomimetic robotic octopus arm. Bioinspir. Biomim. 4(1): (2009) M. Cianchetti et al. Design concept and validation of a robotic arm inspired by the octopus. Mat. Sci. Eng.: C. 31: (2011)

22 Meccanismi o materiali smart per l attuazione How obtaining muscle-like behaviour Biomimetic actuators based on smart materials Electroactive polymers (EAPs) Polymers that exhibit a change in size or shape when stimulated by an electric field Dielectric elastomers Conductive polymers Ionic polymer metal composites Polyelectrolyte mechano-chemical gels Piezoelectric polymers Flex robot, by SRI International be.com/watch?v =nl4-s-ddo-m Spring Roll actuator, by SRI International atch?v=eih8l59sd30 Robot blimp, by Swiss Federal Lab for Materials Testing and Research

23 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

24 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali Explanted living muscles-based devices Muscle-powered swimming robot: Autonomy: 4 h Speed: 1/3 body length / s Main drawbacks: 12 cm H. Herr et al. A swimming robot actuated by living muscle tissue. J. Neuroeng. Rehab. 1: 1-6 (2004) need of sacrificing vertebrate animals; extremely limited lifetime (explanted muscles are characterized by functional structures, such as the vascular network, which rapidly degenerate out of their native environment); possible actuator architectures are limited by the those available in nature.

25 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali... Self-beating cardiomyocyte-based devices Speed: 38 µm/s = 2.3 mm/min (1/4 body length / s) 100 µm J. Xi et al. Self-assembled microdevices driven by muscle. Nature. 4: (2005)

26 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali... Self-beating cardiomyocyte-based devices (meso scale device with scalability potentialities) A.W. Feinberg et al. Muscular thin films for building actuators and powering devices. Science. 317: (2007) 1 mm Speed: 3 mm/min [24 mm/min if paced at 1 Hz (10 V stimulation)]

27 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali Self-beating cardiomyocyte-based devices F = Eab3 4L 3 x F = bending force E = hydrogel elastic modulus x = micropillar displacement F ~ 80 nn (larger than forces generated by molecular motors or laser tweezers: typically, few pn) K. Morishima et al. Demonstration of a bio-microactuator powered by cultured cardiomyocytes coupled to hydrogel micropillars. Sens Act B: Chem. 119: (2006) Cardiomyocyte-actuated micropump Microchannel flow rate: 2 nl/min (typical flow rate for a microchamber: 100 nl/min) Y. Tanaka et al. An actuated pump on-chip powered by cultured cardiomyocytes. Lab Chip. 6: (2006)

28 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali... Self-beating insect cell-based devices Mammals ~ 37 C / precise ph values Larvae of Ctenoplusia agnate 4 40 C / wide range of ph Operability at room temperature (25 C) Long-term stability, even without medium replacement (~ 30 days) Speed: 3.5 µm/s (almost in straight line) Y. Akiyama et al. Room temperature operable autonomously moving bio-microrobot powered by insect dorsal vessel tissue. PLoS ONE. 7: e38274 (2012)

29 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali... Skeletal muscle-based devices K. Nagamine et al. Spatiotemporally controlled contraction of micropatterned skeletal muscle cells on a hydrogel sheet. Lab Chip. 11: (2011) M.S. Sakar et al. Formation and optogenetic control of engineered 3D skeletal muscle bioactuators. Lab Chip. 12: (2012) Limited myotube maturation / low contractility Short-term stability

30 La soluzione: crescere muscoli naturali interfacciandoli con meccanismi artificiali Comparison between actuator technologies Cardiomyocytebased devices Skeletal musclebased devices

31 Modeling of bio-hybrid systems For optimizing the device behaviur, modelling can help Multi-scale and multi-purpose modeling Aspect to model Type of modeling Instruments Cell-substrate interaction and device contraction Electrical stimulation, fluidic renewal, etc. Cell/protein engineering (e.g. due to cellnanoparticle interactions) Non-linear hyperelastic and cell-based models Modeling of combined physical effects Modeling of micro/nanoscale phenomena and quantum effects Finite element tools (e.g. Abaqus) Multiphysics analysis tools (e.g. COMSOL) Molecular Dynamics (MD) simulations (e.g. NAMD2 software)

32 Modeling of bio-hybrid systems Cell-substrate interaction and device contraction: an example of modeling strategy Modeling of muscle thin films: Deformation gradient: F = x X X = reference position x = current position J. Shim et al. Modeling of cardiac muscle thin films: pre-stretch, passive and active behavior. J. Biomech. 45: (2012)

33 Modeling of bio-hybrid systems Cell-substrate interaction and device contraction: an example of modeling strategy Elastomeric substrate Cauchy stress (for an elastomeric substrate) Neo-Hookean model: T = T v + Tisot Muscle cells Cauchy stress (for beating cardiomyocytes) Phenomenological model: T = T v + Tisot + Tani pas + Tani(att) T = k(j 1) + E 3J dev(bi) J = det (F) k = bulk modulus of the material E = Elastomer initial elastic modulus B i = left Cauchy-Green tensor q(t) = t/t 2 e 1 t T 2 k E c λ, E f λ, P, q k = bulk modulus of the cells E c = initial elastic modulus of the intercellular part λ = muscle cell pretensioning E f = elastic modulus of muscle fibers P = Max contraction force per cell cross section area q = activation level T = time characteristic of muscle cell contraction

34 Modeling of bio-hybrid systems Cell-substrate interaction and device contraction: an example of modeling strategy

35 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

36 Problemi da risolvere per costruire attuatori bioibridi usabili The pieces of a bio-hybrid puzzle Embedded microelectrodes and control systems Development of engineered substrates Miniaturized flexible bioreactors Functional interfaces to transmit force/power Choice of suitable cell lines Maximization of cell contractility Living / non-living interface Assembly of single contractile units L. Ricotti and A. Menciassi. Bio-hybrid muscle cell-based actuators. Biomed. Microdev. 14(6): (2012)

37 Problemi da risolvere per costruire attuatori bioibridi usabili Choice of suitable cell lines Primary mammalian cardiomyocytes (high self-beating ability) Primary insect dorsal vessel cells (operating at low temperatures) Immortalized skeletal muscle cell lines (C2C12, low differentiation) Primary skeletal muscle cells (need of sacrificing animals) Insect embryos-deriving myotubes (environmentally robust) Human ESCs or ipscs (high-impact translation in regenerative medicine) In any case: Co-culture of different cell types is better!

38 Problemi da risolvere per costruire attuatori bioibridi usabili Maximization of cell contractility Combination of chemical (growth factors) and physical stimuli Low-serum media Fibroblast-deriving growth factors Anisotropic micro/nano topographical cues Electrical stimulation Exogenous genetic materials insertion (mirna) mirna-1 promotes myogenesis mirna-133 enhances myoblast proliferation (thus hampering differentiation) J.F. Chen et al. The role of microrna-1 and microrna-133 in skeletal muscle proliferation and differentiation. Nature Gen. 38: (2005) Indirect nanoparticle-based stimuli Responsive nanoparticles internalized by muscle cells and stimulated by means of outer sources

39 Problemi da risolvere per costruire attuatori bioibridi usabili Embedded microelectrodes and control systems Electrode integrability, biocompatibility and long-term stability Ability to avoid undesired effects (e.g. cytotoxycity, water electrolysis, etc.) Control system: low level (activation pattern) and high-level (possible muscle synergies) Functional interfaces to transmit force/power Tendon-like structures able to connect the actuator with the external world Design of active and passive mechanical features

40 Problemi da risolvere per costruire attuatori bioibridi usabili Miniaturized flexible bioreactors Ability to keep cells in a controlled environment Tuned mechanical features to not hamper actuator contraction F. Vozzi et al. A flexible bioreactor system for constructing in vitro tissue and organ models. Biotechnol Bioeng. 108(9): (2011) Miniaturized or large scale actuators Assembly of single contractile units Possibility of outperforming natural solutions, in terms of shapes and architectures, in correspondence to specific tasks

41 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

42 Alcuni casi di studio sugli attuatori bioibridi Choice of suitable cell lines Development of engineered substrates Maximization of cell contractility Muscle cells Co-cultures Indirect nanoparticle-based stimuli 2D Matrices 3D Matrices

43 Alcuni casi di studio sugli attuatori bioibridi Development of compliant substrates Free-standing robust polymeric substrates with sub-micrometric thickness Poly(L-lactic acid) T. Fujie et al. Evaluation of substrata effect on cell adhesion properties using freestanding poly(l-lactic acid) nanosheets. Langmuir. 27: (2011)

44 Alcuni casi di studio sugli attuatori bioibridi Development of compliant substrates Low controllability Use of a murine skeletal muscle cell line (problems of stability and low differentiation) 100 µm L. Ricotti et al. Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) uòtra-thin films. Biomed. Microdev. 12: (2010)

45 Alcuni casi di studio sugli attuatori bioibridi Choice of suitable cell lines Development of engineered substrates Maximization of cell contractility Muscle cells Co-cultures Indirect nanoparticlebased stimuli 2D Matrices 3D Matrices

46 Alcuni casi di studio sugli attuatori bioibridi Development of compliant substrates Thin free-standing polyacrylamide gels were obtained by: O 2 plasma on glass coverslips; 10% acrylamide/bis-acrylamide; Flat or micro-grooved Si molds; Thickness: 12.2 ± 5.2 µm Young s modulus: 14.7 ± 1.5 kpa Detachment of molds and rinsing; Covalent binding of fibronectin.

47 Alcuni casi di studio sugli attuatori bioibridi Anisotropic co-culture of fibroblasts and muscle cells 100 µm Strong cell anisotropy on µg substrates 24 h after seeding 100 µm

48 Alcuni casi di studio sugli attuatori bioibridi Maximization of cell contractility Indirect nanoparticlebased stimuli Nanoparticles as intracellular vectors The use of nanoparticles as drug/gene vectors is a hot topic in nanomedicine However, nanoparticles can be used also to indirectly convey physical stimuli (electrical, mechanical, etc.) within cells

49 Alcuni casi di studio sugli attuatori bioibridi Piezoelectric nanoparticles as intracellular vectors BNNTs were coated with glycol chitosan and dispersed in the culture medium (10 µg/ml) Direct piezoelectric effect: internal generation of electrical charge resulting from an applied mechanical force US Stimulation: 20 W, 40 khz

50 Alcuni casi di studio sugli attuatori bioibridi Synergistic effects on co-cultures Differentiation: Day 7 25 µm 100 µm

51 Alcuni casi di studio sugli attuatori bioibridi Synergistic effects on co-cultures Electrical activity (calcium imaging) Day 7 F F+BNNT+US µg µg+bnnt+us L. Ricotti et al. Boron nitride nanotube-mediated stimulation of cell coculture on micro-engineered hydrogels. PLoS ONE. 8(8): e71707 (2013)

52 Alcuni casi di studio sugli attuatori bioibridi... From 2D to 3D Controlled assembly of several 2D layers Positioning and chemical issues Can be approached by means of robot-assisted micromanipulation and surface functionalization strategies Development of actual 3D constructs Need of 3D geometry and vascularization Use of advanced methods for 3D scaffold fabrication and vascularization of engineered constructs

53 Alcuni casi di studio sugli attuatori bioibridi Choice of suitable cell lines Development of engineered substrates Maximization of cell contractility Muscle cells Co-cultures Indirect nanoparticlebased stimuli 2D Matrices 3D Matrices

54 Alcuni casi di studio sugli attuatori bioibridi Three dimensional self-assembling matrices Polydimethylsiloxane (PDMS) Thin films by spin-assisted deposition Different monomer / curing agent ratio (different elastic moduli) Stress-induced rolling membrane technique

55 Alcuni casi di studio sugli attuatori bioibridi Three dimensional self-assembling matrices Polydimethylsiloxane (PDMS) Thin films by spin-assisted deposition Different monomer / curing agent ratio (different elastic moduli) Stress-induced rolling membrane technique

56 Alcuni casi di studio sugli attuatori bioibridi Characterization of morphology Characterization of top layer Characterization of bottom layer

57 Alcuni casi di studio sugli attuatori bioibridi Surface chemical stability PDMS is characterized by hydrophobic recovery Need of a strategy for assuring long-term cell adhesion Genipin: natural cross-linker, which assures covalent binding of proteins to PDMS substrates G. Genchi et al. Bio/non-bio interfaces: a straightforward method for obtaining long term PDMS/muscle cell biohybrid constructs. Coll. Surf. B: Biointerf. 105: (2013) Stable fibronectin coating (> 3 weeks)

58 Alcuni casi di studio sugli attuatori bioibridi Three dimensional self-assembling matrices Rolled structure (SEM imaging):

59 Alcuni casi di studio sugli attuatori bioibridi Actuator contraction modeling Finite Element Method (FEM) simulations: Hollow cylindrical structure (3 concentric layers); Yeoh s model, 3rd order From literature data: Cell dimensions and distribution Forces exerted by cells: ~ 10 µn for each cardiomyocyte ~ 1.2 µn for each myotube

60 Alcuni casi di studio sugli attuatori bioibridi Actuator contraction modeling (Initial actuator length: 10 mm) Achievable contractions: ~ 40% Achievable contractions: ~ 3.5% L. Ricotti et al. Three-dimensional tubular self-assembling structure for bio-hybrid actuation. Proc. Conf. Liv. Mach. (2013)

61 Sommario Outline Il problema dell attuazione nella progettazione e sviluppo di macchine I vantaggi del muscolo naturale Approccio ibrido all attuazione Problemi da risolvere per costruire attuatori bioibridi usabili Esempi di lavori in corso Conclusioni e contributi da e per il tissue engineering

62 Conclusioni e contributi da e per il tissue engineering Contributions FROM tissue engineering Advanced scaffolds for skeletal muscle tissue engineering Development of engineered substrates Nanomaterials and methods for cell engineering: maximization of contractility, long-term stability and environmental robustness Choice of suitable cell lines Maximization of cell contractility

63 Conclusioni e contributi da e per il tissue engineering Contributions TO tissue engineering Embedded microelectrodes and control systems Miniaturized flexible bioreactors Advanced tools for stimulation, monitoring and maintenance of cell constructs Robot-assisted micromanipulation systems and advanced assembly strategies to build complex 3D structures Assembly of single contractile units

64 Thank you for your attention

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