Mechanical Engineering | Physics |

Preservation Of The Architectural

Heritage | Structural, Seismic
And Geotechnical Engineering |
Urban Planning, Design And
Policy | Aerospace Engineering
| architectural composition |
Architecture, Built Environment
And Construction Engineering |
Architectural, Urban And Interior
Design | Bioengineering | Design |
Electrical Engineering | Energy And
Nuclear Science And Technology |
Environmental And Infrastructure
Engineering|IndustrialChemistryAnd
Chemical Engineering | Information
Technology|ManagementEngineering
| MATERIALs ENGINEERING | Mathematical
Models And Methods In Engineering PhD Yearbook | 2019
 research programs within experimental laboratories located at the Politecnico di Milano or outside it, typically

PhD Yearbook | 2019

biomedical research centers, hospitals or industries. When the research is performed within the Politecnico, PhD
Doctoral Program candidates are usually assigned to one of the following laboratories belonging to the DEIB and CMIC: Laboratory
of Biological Structure Mechanics (LaBS, CMIC), Laboratory of movement analysis “Luigi Divieti” (DEIB), Medical
in bioengineering Informatics laboratory (DEIB), Neuroengineering and medical robotics Laboratory (NearLab, DEIB), Biosignals,
Bioimaging and Bioinformatics Lab (B3 lab, DEIB), Biomaterials
Composition of the PhD Board laboratory (CMIC), Biomedical Technology Lab (TBMLab, DEIB),
Experimental Micro and Biofluid dynamics (µBS Lab, DEIB),
Aliverti Andrea (Coordinator) DEIB
Computational Biomechanics Lab (DEIB), Biocompatibility 117
Bianchi Anna Maria DEIB and Cell culture Lab (BioCell, CMIC), Bioreactors Laboratory

bioENGINEERING
Candiani Gabriele CMIC (CMIC). The Istituto di Elettronica, Ingegneria dell’Informazione
e delle Telecomunicazioni (IEIIT) of the Consiglio Nazionale
Chair: The main objective of the PhD Programme in Bioengineering is to prepare Cerveri Pietro DEIB delle Ricerche (CNR), which is located at DEIB, represents
Prof. Andrea Aliverti the PhD candidates to develop high level engineering problem-solving Chiesa Roberto CMIC another possible option. Stage periods in distinguished research
abilities in biomedical, healthcare and life sciences, inside research groups institutes in Italy and abroad are an essential feature of the PhD
Cimolin Veronica DEIB
or in private/public industrial contexts, through a strong interdisciplinary candidate training. The candidates are encouraged to carry out
training bridging engineering and medical/biological knowledge. Costantino Maria Laura CMIC part of their research activities in contact with other research
During the PhD, the candidates develop a scientific research project groups, preferably abroad through periods of at least three
Dellacà Raffaele DEIB
dealing with a complex problem which can be at different scales, from months spent in laboratories where the candidate can acquire
the molecular and the cellular levels to living organisms up to biomedical De Momi Elena DEIB further skills to develop his/her research work and thesis.
systems. They investigate original methods, devices, and systems with Farè Silvia CMIC Collaborations that may involve the PhD students are presently
different purposes: increasing knowledge, proposing innovative methods active with several national and international research and
for diagnosis and therapy as well as improving healthcare and daily life Ferrante Simona DEIB
academic Institutions. Very often, the involvement of companies
structures and services. At the end of the PhD programme, the candidate Fiore Gianfranco Beniamino DEIB and clinical partners facilitates the technological transfer of
are expected to be able to carry out innovative projects and research applied research into industry and clinical applications.
Gastaldi Dario CMIC
and development in the field of Bioengineering, by proposing new The educational offer includes ad hoc advanced courses
methodological and technological solutions and properly evaluating the Guazzoni Chiara DEIB specifically designed for the PhD in Bioengineering. The offer
technology impact in healthcare, life science and biomedical industry. Mainardi Luca DEIB includes also the school of the National Bioengineering Group,
During the three years of the program, PhD candidates perform which is held yearly for one week in Bressanone (Bz). Every
their research through theoretical and experimental activities in four Mantero Sara CMIC year, the School is focused on different topics. As examples,
major areas: biomimetic engineering and micro-nano technologies; Pattini Linda DEIB the themes of the last few years have been: Neuro-informatics
rehabilitation engineering and technology; technologies for therapy; (2011), Biomedical devices from research to market (2012),
Pennati Giancarlo CMIC
physiological modelling and non-invasive diagnostics. Regenerative medicine (2013), From functional recovery to
More specific areas include but are not limited to: molecular and cellular Pozzi Giuseppe DEIB artificial organs (2014), Experimental models for development
engineering, biomaterials, tissue engineering, bio-artificial interfaces methods for 3R (2015), Bioengineering for Active ageing (2016),
Raimondi Manuela Teresa CMIC
and devices, neuro-prostheses, movement analysis, cardiovascular and E-Health and digital medicine (2017), Biomedical Images (2018).
respiratory system bioengineering, central nervous system signal and Ravazzani Paolo CNR The PhD Board of professors (‘PhD Board’) is composed by
image processing for rehabilitation, biomechanics, computational fluid- Redaelli Alberto DEIB highly qualified and active researchers in Bioengineering,
dynamics, computer assisted surgery and radiotherapy, robotics, artificial belonging to DEIB and CMIC. The PhD Board is responsible
organs, implantable devices, biomedical signal and image processing, Rodriguez Matas José Felix CMIC
of all the candidate’s activities. The competencies of Faculty
E-Health, bioinformatics, functional genomics and molecular medicine. Signorini Maria Gabriella DEIB members cover a wide spectrum of research fields. This allows
The PhD Program in Bioengineering is organized with an inter- a continuous updating of the PhD program and ensures that the
Soncini Monica DEIB
departmental structure. Faculty members of the PhD Advisory Board PhD candidates are involved in innovative work.
belong to two Departments of the Politecnico di Milano, namely DEIB Villa Tomaso CMIC
(Department of Electronics, Information and Bioengineering) and CMIC
(Department of Chemistry, Materials and Chemical Engineering “G. The PhD Programme in Bioengineering relies also on an Advisory Board Member, formed by distinguished experts
Natta”). coming from R&D industries, research and clinical centers, in order to ensure that that the goals of the PhD
PhD candidates (who are, in average, 20 per year) develop their PhD Program are in line also with the needs of non-academic world.
 118

Computational cerebellar models and

PhD Yearbook | 2019

their embodiment in behavioral loops to
understand neural bases of motor learning
Alberto Antonietti - Advisors: Prof. Alessandra Pedrocchi, Claudia Casellato
119
Computational neural models such as the confirmation of view that cerebellar plasticity is a always noisier than the worst-case potentiality. The network size was project can lead to accelerated

bioENGINEERING
are fundamental to explain how the role of multiple plasticity dynamic and distributed process, simulation can accomplish, and increased, including geometrical computational brain research
ensemble brain functions might sites in cerebellar learning. The in which cortical plasticity is more learning is actually a long-lasting information and plausible since we have demonstrated the
emerge from elementary neuronal simulations demonstrated that the rapidly activated and drives a set experience-dependent change in convergence/divergence ratios use of an effective tool-flow in
components. This project has addition of nuclear plasticity sites of changes that reverberate onto behavior, which can realistically and plastic mechanisms. We have the research of complex brain
been focused on the generation improved model performance and the more slowly adapting nuclei. be observed only from an exploited a new SNN simulator, models. This platform showcases
and tuning of cerebellar showed the different timescales of Changes or deficiencies occurring embodied system. This approach the NEST simulator, a popular and a paradigm for large-scale
computational models, which had learning. We validated the model in one site are compensated by has the challenging potential more advanced tool, improving simulations that can be adapted
been gradually refined to increase robustness in learning associative the others suggesting possible to represent the initial building the realism of the model, including to any other neuron modeling
their biological realism. Eventually, responses with different inter interventional sites for therapy block of a more complex brain- new neural populations and problems, greatly accelerating
these computational models have stimuli intervals and we have shed and repair. The model predictions inspired controller, into which synapses. Leveraging High- the scientific process and the
been embedded within simulated light on acquisition, extinction are warranted future experimental other realistic SNNs emulating Performance Computing systems, development of brain research.
and real robotic platforms and and consolidation mechanisms, investigations, e.g. performing in different brain structures involved we succeed in generating a full
challenged in various closed loop associable to the different active vivo multi-electrode recordings in sensorimotor integration could reconstruction of the mouse
protocols. plasticity sites. of the plastic evolution of neural be embedded. flocculus (almost 1 million neurons
The models were developed as As a second step, we used the discharges in cortical and deep During the project development, and 360 million synapses) and
realistic Spiking Neural Networks cerebellar model to generate cerebellar nuclei neurons. multiple features have been simulating it in two learning tasks
(SNN) and have been tested in expectations from potential As a third step, the neural implemented in the model, taking (Figure 2).
different sensorimotor tasks: ad-hoc experiments and to network model was challenged inspiration from physiology, in The technological platform that
Eye Blink Classical Conditioning generate hypotheses about the in different learning paradigms, order to increase its realism and has been developed in this
(EBCC), Vestibulo-Ocular Reflex mechanisms underlying behavioral integrated with robotic platforms
(VOR), movements perturbed components and modifications. (Figure 1). These provided bodies
by force fields, and motor By manipulating the model to the simulated “little” brain,
correction. Eventually, fitting real parameters, it was possible to closing the loop between action
experimental data coming from tune the model to fit the behavior and perception. As a result, we
human or animal subjects. of either healthy subjects or have efficiently linked low-level
A mechanistic quantitative subjects perturbed by Transcranial cerebellar circuits with high-level
interpretation of the dynamic Magnetic Stimulation (TMS). We functions integrating a SNN into
evolution of cerebellar plasticity have shown that a macroscopic a neurorobot operating in real-
during skill acquisition is still measurement during a behavioral time. The main added value of
lacking, and computational models task can be successfully explained learning in our neurorobot is
can be very effective to provide by using an appropriate model that it succeeded in reproducing
new insights. Thus, an extensive constructed at the microscopic how biological systems acquire,
analysis of the relations between level. In particular, we have been extinguish and express knowledge Fig. 2 - The 2D projection on
model parameters and behavioral able to tune the cerebellar model of a noisy and changing world, the Coronal Plane of the mouse
Fig. 1 - (Left) The architecture of the cerebellar SNN, the different neural brain volume (in grey) and the 3D
outputs have been exploited. against human EBCC data before in multiple cerebellum-driven populations are connected by synapses, the three plasticity sites are positions of the neurons used
As a first step, a realistic and after perturbation with TMS, learning tasks performed by a highlighted in orange: the cortical plasticity (PF-PC) and the nuclear plasticities to reconstruct the SNN circuit of
model was developed to test in two different protocols. The real robot moving in perturbed (MF-DCN and PC-DCN). (Right) The robotic set-up of the Pavlovian task the mouse flocculus, the olivary
reproduced with NAO robot, which had to protect himself with the provided complex and the deep cerebellar
neurophysiological theories, results supported the emerging environments. The real world is shield from the laser beam. nuclei.
 120

Fluorescent transcription factors as tools

PhD Yearbook | 2019

to evaluate the influence of mechanical
stimuli on nuclear import
Lucia Boeri
Advisors: Manuela Teresa Raimondi, Diego Albani, Emanuela Jacchetti 121
Mesenchymal stem cell (MSCs)- nuclear import rate assessment real-time PCR results highlighted of MyoD in comparison to PAGFP.

bioENGINEERING
based therapy is a promising in different MSC stretching an inhibitory effect on MyoD Furthermore, structural analyses
strategy to regenerate injured conditions. functionality. showed that the interactions
tissues and regulate immune To reach the goal, five fluorescent Key experimental evidence of the between (-30)GFP and MyoD were
responses. The challenges transcription factor variants fluorescent transcription factors mainly mediated by negative and
concerning the clinical application were designed, purified and was deepened with computational positive residues, respectively.
of MSCs injection are mainly validated. The transcription investigations. In particular, the In conclusion, this work developed
related to the reduced post- factor experimentally selected (-30)GFP inhibition effect observed and validated several potential
transplantation cell survival and was a promoter of myogenesis, during the transcription promoting tools for nucleocytoplasmic
the low control of MSC fate. MyoD (Myoblast determination activity analysis was assessed by transport measurement. Among
Recent advancements focused protein). Five variants of MyoD molecular dynamics (MD). MyoD- the fluorescent variants of MyoD,
on MSC response by studying were purified, each fused to a PAGFP was used as negative the photoactivatable MyoD-
their mechanotransduction, different fluorescent protein control in MD simulation. (-30) PAGFP is the tool that best
that is the mechanism by which (probe): Tat-GFP, (-30)GFP, PAGFP, GFP and PAGFP 3D models were resembles the properties of the
MSCs transduce environmental PAmCherry, mEOS3.2. Each variant generated by homology modeling, native transcription factor and
mechanical inputs into chemical of MyoD presented a peculiar while in the case of MyoD protein, it is suitable for further nuclear
signals. The NICHOID project characteristic: Tat-GFP-MyoD the 3D structure of the basic helix import measurements. Otherwise,
started in 2015 with the goal and (-30)GFP-MyoD followed loop helix (BHLH) DNA binding (-30)GFP-MyoD showed great
to verify the hypothesis that specific protein transduction- domain was the only available. The properties in terms of protein
MSC key mechanisms, such as based delivery procedures while goal of MD analyses was to verify delivery and nucleo-cytoplasmic
differentiation, are regulated by MyoD-PAGFP, MyoD-PAmCherry if the presence of the peculiar shuttling. In this variant, MD
stretched-dependent nuclear and MyoD-mEos3.2 were major negative surface charge of simulations suggested an
import of specific transcription sensible to photoactivation or (-30)GFP could interact with BHLH interference mechanism based
factors. According to this photoconversion. The validation of domain of MyoD, thus altering its on a strong interaction between
hypothesis the nuclear pores are the fluorescent molecules required functionality. Considering the bias MyoD and (-30)GFP that could
mechanically sensitive elements analysis of protein localization by of the lacking part of the MyoD explain the inhibition of MyoD
and respond to cell stretching fluorescence confocal microscopy, structure, they were generated activity.
with nuclear pore enlarging nucleocytoplasmic transport four different configurations by Overall, this work demonstrated
and increased nuclear import measurement by fluorescence positioning the BHLH domain of that, the design and the
of transcription factors. The recovery after photobleaching MyoD in different positions in development of the fluorescent
experimental validation of the (FRAP) method and transcription respect to the fluorescent protein, probes in an intracellular dynamics
NICHOID hypothesis is based promoting activity assay by real- so that the interface between the study are fundamental steps to
on the nuclear import rate time PCR. The results showed that two molecules was different in deeply understand their possible
measurement of transcription the fluorescent variant of MyoD every simulation. When analyzing applications.
factors able to promote MSC best representing the physiology root mean square deviation
differentiation. The aim of the of the native MyoD is the MyoD- (RMSD) data and non-bonded
Ph.D. thesis was to engineer PAGFP. (-30)GFP-MyoD resulted a interaction energies, results
transcription factors to achieve suitable tool for nucleocytoplasmic showed that (-30)GFP interacted
fluorescent tools to allow the transport measurement but more strongly with the monomer
 122

RespirHó: an inertial sensor-based system

PhD Yearbook | 2019

for daily life breathing monitoring in
neuromuscular patients
Ambra Cesareo - Advisor: Prof. Andrea Aliverti
123
The main function of the that could answers to the account the two-degree-of- selection of a single quaternion approved by the Italian Minister and analysis method; however,

bioENGINEERING
respiratory system is to guarantee abovementioned needs is based freedom model of the chest wall. component. The algorithm has of Health. The main aims of the some conclusions can already
appropriate gas exchange which on the measurement of chest wall The third MIMU unit is used to been subsequently adapted to study were to validate the device be drawn. Preliminary data
involves the uptake of oxygen movements related to breath, have a local reference system, dynamic conditions (e.g. walking), in static conditions, to assess collected from the first patients
and release of carbon dioxide. using inertial sensors. Assessment recording body (non-breathing) allowing automatic breath-by- its feasibility of autonomous enrolled, provided encouraging
Ventilatory control system of breathing pattern by measuring motion, and works as logging breath extraction of breathing use during daily activities and results considering the device
attempts to correct deficiencies chest wall displacement, or node. The device is integrated pattern temporal parameters. ultimately to collect information comfortable, tolerable and
of oxygen (hypoxemia) and/ derived tissue changes, it is into an acquisition platform, in Finally, the device and method about the need of further design easy to use. Validation in static
or excess of carbon dioxide acknowledged as preferred which data recorded by the units developed have been applied improvements. Data of this pilot conditions gave good results in
(hypercarbia) by increasing both monitoring method because does are sent to a smartphone and and tested on clinical target study are reported in my thesis, terms of reliability and agreement
tidal volume and respiratory not interfere with the airways, stored into a remote server, for population, namely Muscular representing the first available with the reference method. Data
rate, that are the main variables being minimally invasive. Inertial telemonitoring service. Figure 1 Dystrophy (MD). It is characterized results in the literature about obtained during daily activities
of breathing pattern. Breathing sensors open the door to new shows the concept of the platform, by progressive muscle-weakening breathing rate assessment, demonstrated the feasibility of
frequency, therefore, is per se an opportunities to develop this describing the three sensor units. and wasting conditions, that obtained by using inertial-based monitoring breathing rate for
important informative parameter approach in a low-cost, wearable A new position-independent affect also respiratory muscles, devices in patients with respiratory long periods using the device
of respiratory function that may be and easy to use manner. The algorithm, to extract breathing leading to respiratory failure, muscular weakness. The pilot developed. The research activity
a key predictor of adverse events. present thesis wants to offer new information from orientation the main cause of death in study on clinical population is described in this thesis represents
Monitoring breathing frequency, insights into breathing monitoring changes recorded with the three these patients. A pilot study to ongoing and, when it will be a step forward the implementation
could early identify patients at by using inertial sensors, trying units has been developed and assess usability, acceptance and finalized, it will provide information of at home continuous breathing
risk of developing respiratory and to overcome limitations of this tested on healthy subjects in static wearability of the device in 15 about necessary adjustments for frequency monitoring in patients
cardiac dysfunction, with high fields, such as high sensitivity and semi-static conditions, using patients with Duchenne Muscular what concerns the design of the at high risk of developing
specificity. The need for accurate, to motion artefacts and limited Optoelectronic Plethysmography Dystrophy and Limb-girdle device (e.g. dimension, shape of respiratory dysfunction and
objective methods to assess and validation in clinical population. as reference method. A study Muscular Dystrophy has been the housing, fixation methods...) failure.
monitor breathing frequency is A new wearable, modular comparing different approaches
thus evident, inside and outside and wireless device for long- has been conducted, to identify
the clinic. Monitoring tools should term monitoring of breathing the best method of dimension
be less invasive and obtrusive frequency has been designed, reduction allowing to move
as possible, easy to use and low developed, implemented, tested from 4-dimension problem,
cost to foster the autonomous and optimized. It is based on characterized by the four
use by patients and promote sensor fusion of data collected components of the orientation
the dissemination and transfer from accelerometer, gyroscope quaternion to a single-dimension
of the technology to the clinical and magnetometer (Magnetic problem, with a single signal
and healthcare practice. Systems and inertial sensor Unit, MIMU) to representing the breathing signal.
proposed in the literature for compute complete quaternion- This study underlined as the
breathing pattern monitoring based orientation, and its variation approach based on the fusion of
during daily life activities do not over time. The device is composed the four quaternion components
address these requirements by three MIMU units. Two units by using Principal Component
and are confined to research are placed on the thorax and Analysis provided best estimation
Fig.1 - Proof of concept of a wearable, wireless, modular respiratory Holter based on inertial measurement units (IMUS) for
application. An emerging approach on the abdomen, to take into than approaches based on the the continuous breathing pattern monitoring through the detection of chest wall breathing related movements.
 TC
124

PhD Yearbook | 2019

100 µm 100 µm (a)

i ii
Nicola Contessi Negrini - Advisor: Prof. Silvia Farè

GEL_bulk
125
Adipose tissue (AT, commonly regeneration. in vitro adhesion, proliferation macroscopic shape with a can be fabricated by different

bioENGINEERING
named as fat) is a loose connective Gelatin, a collagen derivative, was and adipogenic differentiation defined porous structure, the techniques that allow producing
tissue dedicated to energy selected for its solubility in water, of preadipocytes on the gelatin crosslinked gelatin hydrogel was scaffolds successfully matching AT
storage, organs protection, lower antigenic and immunogenic hydrogels were demonstrated. also used as ink and 3D printed engineering requirements.
and contributing to organism response compared to collagen, A strategy based on the by an optimized procedure to
homeostasis. AT can be affected versatility, readily availability, use of sacrificial hydrogel 100 µm
obtain 3D printed scaffolds for 100 µm (b)
by severe pathologies, including and exposure of cell-binding microbeads and 3D printed AT engineering. The rheological
lipodystrophies, congenital
defects, trauma or resection after
motives and metalloproteinase
target-sites for degradation. The
structures was subsequently
engineered to obtain a porous
properties of the gelatin solution
were investigated during the
i ii
pathological cases (e.g., breast crosslinking reaction, necessary to crosslinked gelatin hydrogel crosslinking and a printability

GEL_porous
tumor removal) that urgently produce gelatin hydrogels stable with a controlled vasculature, time window (i.e., G’ < G”) was
require AT restoration. Current at 37 °C (i.e., in vivo applications) respectively. Microbeads and 3D identified. The gelatin solution
clinical treatments mainly aim at and investigated for scaffolds printed strands with controlled was printed on a substrate
AT volume restoration with no production, is based on a Michael- dimension and geometry were by keeping the cartridge
functional regain (i.e., synthetic type addition between gelatin simultaneously embedded in temperature at 35 °C, after
prosthesis mainly in silicone), and methylenebisacrylamide, the the gelatin hydrogel during its printing parameters optimization
or are affected by donor-site crosslinker. Gelatin concentration preparation and, at complete (figure 2). The printed hydrogels
morbidity (i.e., tissue flaps) and and reaction stoichiometry were gelatin crosslinking, the sacrificial were crosslinked at an optimized
unpredictable long-term results varied to tune the hydrogels structures were removed by temperature and the obtained 100 µm 100 µm
(i.e., lipofilling). AT engineering biomimetics towards different AT an optimized procedure. The structures showed patterned (c)
offers a unique alternative to depots. All the produced hydrogels so-produced scaffolds showed geometry and macroscopic shape
target at both tissue volume were stable in water at 37 °C, ideal properties in terms of reproducing the CAD design
replacement and functional regain; thus proving the efficiency of the porosity (200 – 400 m pore size), used for the printing. The printed Fig. 1 - Oil Red O staining of human mesenchymal stem cells cultured on
porous gelatin hydrogels
however, strict requirements crosslinking reaction. Different mechanical compressive response hydrogels were stable at 37 °C
must be met to obtain an ideal AT crosslinking degrees were (E = 3 kPa), and enzymatic and biomimetic AT mechanical
engineering scaffold, including (i) measured by varying either gelatin degradability. Moreover, the properties were achieved;
3D printing
adequate porosity, (ii) biochemical concentration and/or reaction obtained hollow channels allowed moreover, printed hydrogels
cartridge (35 °C)
cues to allow cells adhesion, (iii) stoichiometry; the hydrogels fluids flow (i.e., water and blood, maintained their shape during in
biomimetic structural properties, showed different weight variations tested ex vivo) and cells adhesion vitro cells cultures and sustained Needle
(iv) biodegradability, and (v) after immersion in physiological- to the hollow channels walls, preadipocytes in vitro adhesion,
a promoted vascularization. like environment, which in turn promising aspects for a promoted proliferation and adipogenic 3D printed
The aim of this PhD thesis is influenced their mechanical vascularisation. Finally, in vitro differentiation. gelatin hydrogel
to produce, by innovative and and rheological properties. The tests proved the ability of the In conclusion, this thesis
advanced fabrication technologies, variation of synthesis parameters produced scaffolds in supporting describes, for the first time, the 3D printing platform
AT scaffolds matching the allowed controlling the properties human mesenchymal stem cells use of a chemically crosslinked surface (4 °C)
requirements previously stated, of the produced hydrogels so to adhesion, proliferation, and hydrogels processed by advanced
by using a chemically crosslinked achieve biomimetic properties of adipogenic differentiation (figure fabrication technologies useful
gelatin hydrogel investigated different AT depots (i.e., breast 1). for AT engineering. This hydrogel
Fig. 2 - Optimized 3D printing process of chemically crosslinked gelatin
here for the first time for AT and heel pad AT). Moreover, To achieve a controlled is a versatile material that hydrogels
 126

PhD Yearbook | 2019

Beatrice De Maria
Advisors: Prof. Sergio Cerutti, Prof. Alberto Porta 127
Introduction over both cBR and sBR branches. affected by postural challenge rate in response to AP decreases asymmetry given that, while the

bioENGINEERING
The baroreflex is a complex Consequently, the first aim of this and ALS; v) when assessed from than to AP increases. It can be HP variability asymmetry was
physiological mechanism aimed doctoral thesis is to adapt the SEQ spontaneous variations the concluded that when the cBR and progressive reduced with age, the
at the maintenance of the arterial and bivariate PRSA methods for asymmetric behaviour of sBR sBR characterization is performed same trend was much weaker
pressure (AP) homeostasis, the assessment of cBR and sBR seems to be more evident than on the basis of the spontaneous when the BR asymmetry markers
exploiting different branches. The asymmetries. that of the cBR in young healthy variability, the asymmetry seemed were considered.
cardiac BR (cBR) branch reacts to subjects and this conclusion does to be a peculiar aspect of the As to the characterization of ALS
AP changes modulating the heart Experimental protocols not hold in the ALS patients; sBR, but not of the cBR. From a this thesis confirmed that cBRS
period (HP), while the sympathetic Three different experimental vi) the asymmetry of the cBR is methodological point of view, was lower in ALS patients but the
BR (sBR) one regulating the protocols were analysed: i) 12 linked to the HP asymmetry; vii) while the SEQ method seems response to TILT was preserved.
sympathetic activity. The cBR young healthy subjects performing in ALS patients the sBR is more to be more powerful for the Conversely, sBR appeared to be
is commonly characterized by incremental tilt, to verify the effect impaired than the cBR, but the cBR quantification of the asymmetry much more impaired than cBR
computing the cBR sensitivity of an orthostatic challenge (TILT) and sBR asymmetry indexes are of the BR branches compared to given that sBR was not affected
(cBRS) defined as the amplitude of on cBR and sBR asymmetries; ii) uncorrelated with patients’ clinical PRSA, the opposite conclusion by TILT and sBR asymmetry was
the HP changes in response to a 100 healthy subjects characterized features; viii) the HP asymmetry is holds when the characterization lost. However, these results need
unit variation of AP, while the sBR by different age (20 subjects for linked to the functional status in of BRS, regardless of the sign of a check after increasing the size of
by computing the sBR sensitivity each decade, from 21 to 7o yrs) ALS patients. the AP variations, is the focus. the group of healthy controls. The
(sBRS) defined as the amplitude performing active standing, to Therefore, this thesis recommends few significant correlations of the
of the MSNA changes in response assess the effect of aging on cBR Conclusions the use of SEQ method for BR BR parameters and asymmetry
to a unit variation of DAP. As a asymmetry; iii) 107 amyotrophic In this doctoral thesis, the PRSA asymmetry evaluation and the makers with the clinical status of
result of its inner functioning, lateral sclerosis (ALS) patients and method was successfully adapted PRSA method for the traditional the ALS patients stresses the need
BR reacts both to AP increases 44 age-matched healthy controls to the separated computation computation of BRS especially in to reduce the heterogeneity of the
and decreases and the eventual performing tilt, to assess the effect of cBRS by considering positive pathological populations. ALS population by classifying more
different response of the BR to AP of a pathological condition, here and negative AP variations, Remarkably, this thesis suggests homogenous subgroups.
positive and negative variations is ALS, on sBR and cBR asymmetries. both in healthy subjects and in that the cBR asymmetry plays a
referred to as the BR asymmetry. ALS patients, and successfully role in producing the HP variability
Results extended for the characterization asymmetry. Indeed, although
Aim The most important findings of of the sBR. As to cBR functioning, no significant difference was
The purpose of this thesis is to this doctoral thesis are: i) the in all the considered populations, detected between contribution
propose the assessment of the possibility to exploit the bivariate no significantly relevant given by positive and negative
asymmetry of BR by avoiding PRSA method to evaluate both the differences between cBRS AP variations to cBRS, the
the use of pharmacological cBR and sBR asymmetries based estimates derived by positive cBR asymmetry indexes were
intervention but exploiting two on the spontaneous variability; and negative AP variations were correlated to HP asymmetry ones,
analytical methods based on ii) the SEQ and PRSA methods detected. Conversely, in young suggesting that the small non-
the spontaneous variability: the cannot be considered equivalent healthy subjects the sBR exhibited significant difference between
sequence (SEQ) method and the in the assessment of the BR a significant asymmetry with them contribute nevertheless
bivariate phase rectified signal asymmetry; iii) cBRS markers are the same absolute changes of to the HP asymmetry. However,
averaging (PRSA) one. The BR modified by postural challenges AP producing more important the BR asymmetry explained
asymmetry will be evaluated and ALS; iv) sBRS markers are not changes of sympathetic burst only partially the HP variability
 128

Development, characterization and feasibility

PhD Yearbook | 2019

of industrialization of a novel hybrid semi-
degradable vascular access for haemodialysis
Sebastiao N. Dentinho van Uden
Advisors: Prof. Alberto C. L. Redaelli, Stefania A. Riboldi 129

bioENGINEERING
Early-stage renal disease patients access approach that behaves as optimal immediate recovery showing sub-micrometric factor for the success of a vascular manufacturing, while increasing
desperately need haemodialysis a graft in the short-term and as from haemodialysis punctures fibrous morphologies, tuneable access (e.g., vein-graft compliance quality, was developed for clinical
®
to compensate the loss of natural a fistula in the long-run. This way and continuously intermediating mechanical properties, matching). The Silkothane graft validation and initial commercial
blood filtration and maintain providing the benefits of a readily haemodynamic divergences permeability before and after demonstrated early cannulation application. Results from directly
system homeostasis. However, available and predictable graft for between artery and vein. puncture in the same order of potential in line with self-sealing comparing the current and the
for decades vascular accesses, early cannulation, which slowly Silk fibroin and polyurethane magnitude as commercial grafts commercial synthetic grafts, and a upgraded electrospinning systems
required for haemodialysis, converts into native vascular were the chosen materials currently used in the clinics. degradability driven by enzymatic revealed a 5-fold drop in cost per
®
have been the bottleneck of this tissue; adapting and evolving to develop this hybrid semi- The development of the activity. Moreover, the fibroin-only Silkothane graft, placing it well
®
procedure, limiting the kidney according to local haemodynamics degradable vascular graft due Silkothane graft followed as a layers and extracellular matrix- below average production cost
transplant (3.7% chance a year) in a similar fashion as the artery to their respective bioactivity subsequent milestone to reach. like morphology, presented by per commercial synthetic grafts,
wait time to an average of 1000 and vein connected by the and elasticity. In this sense, the Considering the demonstrated the graft, revealed to be crucial at a rate of six 30 cm-long grafts
days. vascular access. first milestone was to achieve an haemocompatible and cell in providing a non-haemolytic fabricated per day.
When possible, arteriovenous In this work, principles of in situ electrospun blend of these two adhesive properties of silk fibroin, character, long clotting time, and In summary, the promising
®
fistula is the choice to create tissue engineering (Figure 1) are materials – Silkothane material the graft design concerned three favourable adhesion of human findings achieved in this thesis
a vascular access for long- followed, using electrospinning, – to serve as the core structure electrospun concentric layers umbilical vein endothelial cells provide the means to fabricate
term haemodialysis. Although, to achieve a vascular graft that of the graft. Physicochemical (Figure 2). Thus, composed of a with increasing viability after 3 and a pioneering in situ tissue
®
functional fistulae have the highest performs as a bioactive scaffold, analyses demonstrated the thick core layer of Silkothane 7 days. Accordingly, the proposed engineering vascular access,
success rates in the long-term hosting the colonisation of maintenance of the characteristic material in between two thin approach represents a promising addressing all needs from the
due to its purely native tissue vascular cells that trigger tissue features of fibroin and layers of pure electrospun fibroin, step forward towards an in situ commercial, industrial and –
composition, two thirds never development. Enzymes released polyurethane upon solubilisation, providing a continuous presence tissue engineering vascular potentially – clinical perspectives,
reach the maturation point to during this process degrade blending, electrospinning and of this cell adherent material access and its potential for vein- while aiming to finally eliminate
®
allow haemodialysis – failing in the most of the scaffold, allowing post-processing with ethanol across the Silkothane graft’s graft anastomosis stability, early the half-century old haemodialysis
short-term. At this point synthetic progressive and controlled or methanol. Envisioning their entire structure. cannulation, and biointegration. bottleneck, which is failing vascular
grafts fill the gap, providing remodelling from scaffold to possible application as semi- The full three-layered graft, Finally, to ensure industrial accesses.
®
immediate access (24-72h after vascular tissue. However, an elastic degradable substrates for influenced by the polyurethane feasibility of the Silkothane
minor surgery) – early cannulation. artificial bioinert mesh remains haemodialysis, tubular meshes presence, ensured mechanical graft, a pilot plant electrospinning
However, being fully composed embedded in the tissue, ensuring were further characterized, properties that are a determinant system able to scale up
of synthetic bioinert material they
are not able to biointegrate or
match the compliance of adjacent
anastomosed vessels, eventually
failing in the long-term.
Following on what fails with
fistulae and synthetic grafts,
it is clear how both have
complementary advantages (and
disadvantages). Therefore, this
Fig. 1 - Schematic representation of in situ tissue engineering approach upon Fig. 2 - Silkothane® graft and respective cross-section depicting the three-
work envisioned an ideal vascular implantation of a vascular graft. layered structure.
 130

Design and prototyping of a novel of the device, once injected biomaterial for the design of analysis of the device performance

PhD Yearbook | 2019

in the vitreous cavity. Two drug delivery systems in the and functioning in terms of
biodegradable magnesium-based device for stressful simulated conditions
were chosen as representative
ocular field and, in addition
to the implemented method,
corrosion times and anti-VEGF
release. Finally, a Mg-Nd-Zn-Zr
intravitreal release of anti-VEGF of the fluid-dynamic impact of
the ophthalmological milieu on
represented a novelty of relevance
in the research field and for the
(denoted as JDBM) alloy up-scaled
prototypes were manufactured for
the device for the analysis of clinical application. Noted the the future in-vitro experimental
Marco Ferroni - Advisor: Federica Boschetti
biocorrosion phenomena and corrosion behavior of magnesium campaign to prove the concept,
compared to a static control. in the vitreous cavity, a numerical focusing on the anti-VEGF drug 131
Nowadays, fully biocompatible, show the influence of analytical saccades in the vitreous cavity. The main body of the project campaign was developed by stability in presence of magnesium

bioENGINEERING
biodegradable and intravitreal configurations of the ocular led to the characterization of using numerical analysis based and on the effective kinetics of
drug delivery system, with saccadic movements and the magnesium, chosen as very on finite element method in order drug release from the prototype.
sustained-release of anti- permeation characterization of Moreover, the implementation promising biomaterial to be to define the peculiar corrosion The presented and developed
VEGF drugs, would be the ideal surrounding tissues on fluid flow of such a computational used as ideal platform for drug of the magnesium layer and, project was registered as patent
alternative to the current gold- inside the vitreous body. The model posed the bases for the release in the ocular environment, consequently, the final design application.
standard intravitreal injections for computational fluid-dynamic comprehension of the fluid- focusing on its corrosion of the device. Then, a numerical
the treatment of exudative age- model was extended to the study dynamic conditions for the design mechanism and rate, induced by investigation of anti-VEGF release
related macular degeneration. of drug delivery mechanisms of the drug delivery device. An the shear stress action. Firstly, was performed. Bevacizumab
In fact, several negative aspects when a gold-standard amount injection of a hypothetical device a further computational fluid- (Avastin®) was used as clinical
are associated to this treatment of inhibitors of the vascular was simulated, and the fluid- dynamic model based on a sparse reference drug. After the monthly
strategy, such as systemic/ endothelial growth factors is induced shear stress field on the non-linear optimizer optimization layer corrosion, bevacizumab was
intraocular complications, injected intravitreally. The novelty device itself in contact with the method was used to design considered as 95% uptaken after
infection risks, and particularly low in the research field in combining liquefied vitreous was studied, the experimental conditions 236 seconds of continuous ocular
patient compliance and expensive analytical saccadic movements comparing computational fluid- in order to recreate the shear motions. The proposed corrosion
therapy on National Health and permeation characterization dynamic with fluid-structure stress fields computed previously model was able to simulate more
Systems. In this project a novel of surrounding tissues, in addition interaction approaches. The on manufactured magnesium complex corrosion conditions,
fully biodegradable and injectable to the experimental evaluation computed fluid-induced shear samples. After performing ad hoc related to an ocular non-
magnesium-based device was of scleral hydraulic conductivity, stress distribution was evaluated corrosion tests, morphological and homogeneous shear stress field
designed and a first prototype led to computational results that and clearly suggested the profilometric maps of the free- acting on the device; moreover,
was developed. Considering the confirmed the rapid enhancement possibility to have a consequent eroded surfaces of magnesium its coupling with the bevacizumab
absence of drug delivery systems to anti-VEGF distribution due to uniform corrosion in the majority samples were acquired by using release model represented a
for the treatment of the age- scanning electron microscopy perspective pre-clinical tool for the
related macular degeneration and confocal laser scanning
on the market, the design of microscopy to evaluate the
such a device has the main corrosion mechanisms. The
clinical relevance to overcome magnesium corrosion rates were
the limitations associated to the evaluated (1.9, 2.7 and 3.435 μm/
current clinical practice. In order day) in three fluid-induced shear
to develop a strong and novel stress conditions and coupled to
method that aims to the design the current clinical timing between
and development of an ideal two consecutive injections,
drug release device, a classic obtaining the prospective
engineering design process magnesium thickness of the final
was proposed and developed. layer (57, 81 and 103 μm).
The implementation of a novel
combined computational fluid- The reported results highlighted Figure 2. 24 hour time-scale analysis of the sample morphology: localizations
dynamic model of the ocular Fig. 1 - In-plane velocity magnitude in a 3D view at two time instants (A) and B) the great potentialities in are present before testing them (t = 0). After 24 hours the fluid flow oxidized the
of the combined CFD model in presence of saccades. Zoom of the equatorial magnesium localizations and then erode the corrosion products, making the
posterior chamber was able to (C-F) and vertical (D-E) planes of both the 3D views. using magnesium as reference eroded surface as flat.
 132

Innovative strategies to control cell fate and

PhD Yearbook | 2019

gene delivery behaviour

Elisa Giupponi - Advisors: Gabriele Candiani, Lina Altomare

133
It is common knowledge that of surfaces, but the chemical the higher was the thiol content surfaces. SDG.

bioENGINEERING
cells are naturally exposed to functionalization has attracted onto the surfaces, the higher was The grail of gene delivery is Overall, we present: i) the
a multitude of stimuli in their more and more attention over the number of cells retained. the development of safe and validation of thiol-functionalized
surroundings that actively the last decades, since it can drive This result ascertained that effective gene delivery vectors, surfaces as platforms for
participate in determining the and regulate the first interfacial EPTs actively participated in cell and, in this scenario, there is the selective cell adhesion and the
in vivo cell behaviour and fate. interaction between the material adhesion and they bound cells to urgent need for the assessment control of cell behaviour and
The control of cell behaviour and and the cells. In this context, thiol-presenting surfaces by means of transfection efficiency and differentiation; ii) the validation
fate in vitro is possible through novel thiol-enriched surfaces of disulphide bond formation. cytotoxicity in a fast and reliable and characterization of a
the design of biomaterials and have been developed by using Based on these preliminary but manner. In this scenario, an easy- microfluidic device for cell culture
tools with specific properties the well-known sol-gel chemistry appealing results, thiols were to-use microfluidic platform for and the screening of transfectants
able to sustain and regulate cell (Chapter 2), to investigate explored more in-depth as the quantitative assessment performances. These issues are
adhesion, proliferation and even the possibility to immobilize regulators of cell behaviour and of cell trapping, culture and aimed at designing a future device
differentiation. Science is making cells exclusively by disulphide functions (Chapter 3). Sol-gel spin- transfection, and for the high- where thiol-functionalized surfaces
great efforts to recapitulate the bond formation between the coating was used for the synthesis throughput screening of gene are adopted as substrates of the
complex scenario of the cellular exofacial protein thiols (EPTs) of thin and homogeneous delivery vectors, has been microfluidic platform, in order to
microenvironment to study and thiols immobilized onto the coatings. We focused on the study validated and characterized. The combine the fine control of cell
cell behaviour and fate, and to surfaces. Two different alkoxides, of thiols role in mediating the device was endowed with a serial adhesion and behaviour, with
this aim, several approaches tetraethyl orthosilicate (TEOS) cell response to the material by dilution generator (SDG) for the gene delivery studies.
such as surface modifications and n-propyl trimethoxysilane using MC3T3-E1 pre-osteoblasts. generation of a linear dilution
with topographical cues and/or (CH3), each with a variable Thiol-functionalized surfaces of gene delivery particles and
chemical functionalities, and gene amount of (3-mercaptopropyl) turned out to affect cell adhesion, a downstream culture area for
delivery, can be adopted. trimethoxysilane (MSH), have cytoskeleton organization, focal the trapping and culture of small
The main objective of the present been used for the synthesis of adhesions (FAs) formation and cell groups of cells. The device allowed
thesis is the development of sols that have been deposited differentiation. Summarizing, we trapping roughly 10 cells per
innovative strategies to control onto glass surfaces by means of demonstrated that thiols activated chamber and displayed enough
cell behaviour and fate: i) sol-gel dip-coating process. A physico- the Rho GTPases pathways which room for exponential cell growth
chemistry has been applied for chemical characterization was are responsible for cytoskeleton during the whole culture period.
the functionalization of glass carried out, followed by the and FAs assembly, as well as Moreover, the platform proved
surfaces with different chemistries preliminary biological validation. for lamellipodia and filopodia to be a reliable, very useful tool
and variable amounts of thiols to The resulting surfaces showed formation. By increasing the thiol for the high-throughput analysis
study these groups as mediators variable wettability, roughness content onto the surfaces, such of transfection in a miniaturized
of cell adhesion and functions; and coating thickness according to effects raised, confirming that fashion. The developed device
ii) a microfluidic platform was their chemistry and thiol content, thiols were fundamental players in allowed the screening and the
validated to trap, culture and that depended on the recipe of cell adhesion mechanisms. optimization of the performances
transfect small cell populations, the starting solutions. Moreover, Finally, TEOS-based surfaces of gene delivery vectors through
and for the fast screening of the surfaces have been tested as demonstrated to affect cell the selection of the best
transfectants performances. platforms for the selective thiol- differentiation, by promoting transfection conditions among
Several strategies have been mediated cell adhesion, by using osteogenesis, while a very light five different transfectants doses
adopted for the modification HeLa cell line. We observed that effect was observed for CH3-based generated simultaneously by the
 134

Multiscale and Multiphysics Methodologies for to be considered, we could mention • the handling of water transport modelling will be presented.

PhD Yearbook | 2019

the integration of the oxygenating condensation on the gas side (one Chapter IV moves from the geometry
Blood Oxygenators Design bundle and heat exchanger, or the of the most common issues during analysis to the fluid characterization,
configuration of feed and discharge oxygenators clinical use): how focusing on the description of
sections and the limitations imposed is it generated, and how can we blood as a multiphase suspension
by progressive volume reductions; prevent it? of RBC in plasma. For this, the first
scaling from the red blood cells Work structure part of the chapter will describe
Ricardo Gómez Bardón
(RBC, of a few microns) to the The three arguments listed above will experimental work with microfluidic
Advisors: Gabriele Dubini, Giancarlo Pennati overall device dimensions (dozens be developed in the following chapters channels inspired in the oxygenating 135
of centimeters); coupling of blood following a multiscale/multiphysics bundles. Blood flow (in particular,

bioENGINEERING
Introduction with optimized efficiency. oxygenating devices has been flow, hemorheological response structure, organized attending to the RBC patterns) will be analyzed in
Blood oxygenators act as an This thesis is organized on a multiscale achieved thanks to the combined and biocompatibility; temperature effects of each physical phenomenom setups replicating the fluid dynamic
extracorporeal artificial lung during and multiphysics perspective, efforts of medical institutions, gradients and O and CO diffusion; at different geometric scales. This conditions equivalent to real clinical
2 2
certain types of cardiac surgery and combining computational fluid engineering companies and research counterbalance of global performance, approach aims to reveal their use. Again, the effects at different
intensive care therapies, where it is dynamics (CFD) and experimental centers. Over the last decade, survival production cost and usability... As will interconnections towards an overall characteristic lengths (single fibre
1
required to support or completely microfluidics. After an introduction to rates above 99% have been reached , be seen in Chapter II, the design of view of the performance of the and entire microchannel) will receive
replace the functioning of the patient’s the research problem and our goals with an estimated use above 650,000 blood oxygenators integrates a wide oxygenating device. special attention. Complementary,
2
lungs. Their operating principle (chapter I) and a historical background patients worldwide per year . range of engineering and medical Chapter II will present a historical the second part will describe a CFD
consists in oxygen diffusion to blood (chapter II), we will focus on different Although different technologies factors, many of which still demand a background on the evolution of multiphase blood approach, replicating
through a semipermeable membrane, physical phenomena relevant to blood are available in the market for finer analysis and improved solutions. blood oxygenators, together with a the experimental results. This method
typically arranged in a set of hollow oxygenators. Chapters III and IV cover blood oxygenation, here we will In this sense, this work is focused on brief outline of the most common will be then coupled with the strategy
fibres. These fibres carry gas inside fluid dynamics (blood flowing around focus on the most widely used. This the development of efficient methods engineering tools used in the design of for describing fibre bundles from
(intraluminal flow), while blood flows the fibers), multiphase effects of blood consists on a set (often reffered to for the analysis of blood oxygenators, modern devices. Chapter III, exploring its usefulness
externally around them (extraluminal (cells suspended in plasma) and mass as bundle) of hollow fibres made of aiming to introduce useful tools with Chapter III starts by describing during the analysis of new design
flow). Several engineering tools transport (O diffusion through blood). a microporous membrane. A gas an industrial application during their in greater detail the traditional configurations. Finally, the implications
2
are used by the medical industry Moreover, chapter V investigates mixture flows inside the fibres, while design process. For this, a number computational approaches for of the multiphase nature of blood in
during oxygenators design, including heat exchange (blood temperature blood circulates outside them. Thanks of simplifications frequently adopted representing the blood fluid dynamics a more accurate modelling of oxygen
experimental and computational regulation and water condensation on to the submicron-sized pores inside to describe oxygenators behavior inside oxygenating bundles. Their transport will be described.
techniques. Broadly speaking, the the gas phase). Each of them will be the fibre membrane, O and CO will be reconsidered, in pursuit of a motivations and the needs for their Chapter V presents a detailed
2 2
information gathered from these tools considered at different levels, from the can be exchanged across both sides further insight into the mechanisms simplifications will be discussed, and description of water condensation
characterizes the blood flow field device as a whole (tens of centimeters) (a detailed description will be given underlying their performance. A a novel strategy for setting up more on the gas side of blood oxygenators
and temperature across the device, to the red blood cells (RBC, a few in Chapter II). This way, the gaseous combined approach of numerical accurate models in an efficient way during their use, considering
the oxygen transport through the microns). Finally, general conclusions composition of blood is regulated techniques (Computational Fluid will be described. The entire process of the various mechanisms (air
blood stream, the structural integrity and possible applications for future in an equivalent way to what occurs Dynamics, or CFD simulations) and model generation and analysis will be humidification, heat transfer and
of the different components and designs will be discussed in chapter VI. inside the human lungs, with the main experimental work (microfluidic presented, illustrating the advantages change of phase) governing the
the associated risk of blood damage The main contributions of this work difference that the oxygenation occurs devices) will be applied to the following of its application for the design of new process through different sections of
during use. The scope of this research to the state-of-the-art in blood outside the patient’s body (being blood topics: devices. Limitations of classical models the device. Finally, the influence of the
is to develop advanced methodologies oxygenators are an innovative CFD oxygenators non-implantable devices). • the CFD modelling of the will be assessed by comparison with suggested method for fibre bundle
for an optimized design of blood modelling approach for an accurate Typically, a small heat exchanger is oxygenating fibre bundle: to what the suggested alternative, considering numerical modelling from Chapter
oxygenators. Some of the traditional description of the fibre bundle; an included in the device to control the extent is the traditional approach the most common arrangements in III will be discussed, evaluating its
assumptions adopted during their experimental investigation of the blood temperature during its use. (porous media model) accurate modern oxygenating bundles. Special usefulness for design purposes in
analysis will be revised, applying a heterogeneous distribution of RBC Despite this great success, some under certain configurations of attention will be given to the different comparison with traditional methods.
different perspective to handle some around the oxygenating fibres, and considerable challenges are still modern devices? effects at the fibre level (flow micro- Chapter VI will conclude by
of the problems associated with their a predictive model for the risk of posed by the oxygenators complex • the treatment of blood as a patterns) and along the different summarizing the findings from
development. New approaches will condensation during use. structure, the wide range of length homogeneous fluid: does its regions of the device (macro-level the previous sections, trying to
provide a better understanding of their Research goals scales involved or the different multiphase nature (as suspension perfusion of the oxygenating bundle). give a general perspective of their
functioning, being the ultimate goal to During the last 70 years, a substantial physical phenomena governing their of RBC in plasma), have any Finally, an extension of the benefits implications in possible optimizations
apply these methods in future designs evolution of extracorporeal blood efficiency. Among the various issues influence? from the strategy proposed to oxygen of future desings.
 136

Investigation of heart valve transcatheter sample under pulsatile flow impact on the hemodynamics was adapted for hands-on clinicians’

PhD Yearbook | 2019

conditions. unknown. In the experimental training in transcatheter therapies.
therapies – from a platform to a new therapy Novel treatments assessment
The tricuspid regurgitation is
models of mitral and tricuspid
regurgitation, the long-arm clip
The training-oriented platform
was employed for various
concept conventionally treated at valve-
level only. However, papillary
treatment improved overall
hemodynamic conditions without
training purposes including (i)
facilitating the definition of training
muscle displacement is, next inducing significant increase strategies in transcatheter edge-
Michal Jaworek
to annular dilation, the main of transvalvular pressure drop to-edge treatment of tricuspid
Advisors: Riccardo Vismara, Alberto Redaelli determinant of the tricuspid across mitral and tricuspid valve. regurgitation and providing visual 137
regurgitation. In this work, We demonstrated that tricuspid data for training materials, (ii)

bioENGINEERING
Background The aim of this dissertation was to of novel treatments under right ventricular papillary valve clip-based therapy was serving as demonstrative and
The aging of society and facilitate current transformations repeatable and controllable muscle approximation was successful when septal leaflet was training tool during cardiological
associated comorbidities are in valvular therapies by setting, which is not possible to be comprehensively assessed in grasped along with either anterior congresses across Europe in
major stimulators for the spread development of novel passive obtained in real clinical scenario. an ex-vivo model of functional or posterior one. It confirmed transcatheter therapies for mitral,
of valvular transcatheter therapies. beating heart platforms featuring An experimental approach tricuspid regurgitation under initial clinical observations and tricuspid and aortic valve. This
This sets new challenges for models of valvular pathologies to induce functional mitral tight control of the direction and experimental observations with work demonstrated a possible
clinicians, researchers and device and their application in studying regurgitation conditions in a level of the approximation with standard-length clip. Moreover, it spin-off application of platforms
developers. new transcatheter therapies and cyclically actuated healthy porcine the support of computational 3D was found that left atrial pressure initially intended for research
The need for new valvular clinicians’ hand-on training. left ventricle was explored. The morphology reconstructions from monitoring could be used as an purposes.
treatments or improvement of Experimental toolbox tailored proposed method was based volumetric echocardiographic additional indicator of procedure
existing ones is related to poor for transcatheter therapies on mechanical dilation of mitral data. The treatment caused success.
clinical outcomes of current state- Experimental models of functional annulus and displacement of a significant reduction of Hands-on training
of-the-art therapies, especially tricuspid regurgitation and left ventricular papillary muscles the tricuspid regurgitation, The experimental platform was
in case of functional mitral and functional mitral regurgitation using ad-hoc designed and tricuspid valve tenting volume
tricuspid regurgitation, which were developed and incorporated 3D-printed devices. This strategy and dimensions of tricuspid
showed high pathology recurrence into passive beating heart provided a realistic, repeatable annulus (Fig. 2). Moreover, low
rate in long-term. Moreover, platforms. and reversible ex-vivo pathological baseline cardiac output and
tricuspid regurgitation treatment A mock circulation loop of model, featuring lesions clinically low baseline dimensions of the
specifically has been neglected pulmonary circulation was associated with the pathology in tricuspid annulus were identified
surgically until recent years designed, manufactured and terms of hemodynamics, valve as the predictors of treatment
and the available interventional assessed. The platform (Fig. morphology and kinematics. It is success. In a follow-up study we
options are limited. 1) housed whole right porcine the first experimental approach demonstrated that the greatest
The quest for new, more durable heart passively actuated under which reproduced actual main reductions in regurgitation and
and less invasive therapies pulsatile flow conditions. The determinants of functional mitral morphological anomalies of the
can be assisted with realistic incorporated experimental regurgitation in whole heart tricuspid valve were obtained
preclinical evaluation. Reliable model of functional tricuspid after the concomitant treatment
experimental platforms tailored regurgitation was reversible, (at valvular and subvalvular
for transcatheter therapies and easily manageable and yielded level) addressing directly both
featuring models of valvular stable hemodynamic conditions determinants of the pathology.
pathologies could help in over simulated typical procedure Next, we investigated the
accelerating the development time. We tackled the challenges hemodynamic effects of a new
of new therapies. Moreover, related with the management of design of a transcatheter edge-
a possibility for hand-on high- cyclically pressurized compliant- to-edge device for mitral and
fidelity training in transcatheter wall right ventricle and control tricuspid insufficiency, featuring a
device implantation could help to of the tricuspid regurgitation long-arms clip. A standard-length Fig. 2 - Assessment of right ventricular papillary muscle approximation concept
potentially reduce the occurrence state in an experimental setup. clip is widely used commercially. for treatment of functional tricuspid regurgitation in a beating heart platform. A:
of procedural errors and improve The developed platform enables The long-arm clip could ease hemodynamic and morphological changes following the treatment, B: images
Fig. 1 - Passive beating platform of tricuspid valve in peak systole from fiberscope and 3D computational
the learning curve. systematic preclinical assessment scheme. grasping procedure, however the reconstruction from echocardiographic data.
 138

Characterization of Autonomic and cover both intrinsic and extrinsic effect of this position, changing the while the exposed group instead

PhD Yearbook | 2019

risk factors, as proposed by the sympatho-vagal balance. showed a blunted response.
Cardiorespiratory Control in Newborn triple risk model, as well as their Effect of prematurity: Results In conclusion, we showed
Populations at Risk for Sudden Infant Death interaction. showed increasing mean RR how complex parameters

Syndrome Effect of sleep states: Our results

indicate that, while Quiet Sleep
intervals, short-term HRV,
HR complexity and linear
and techniques for bivariate
analysis of breathing and HR
(QS) generally presents lower cardiorespiratory coupling as provide additional information
Maristella Lucchini - Advisor: Prof. Maria Gabriella Signorini
global HRV, higher complexity a function of Gestational Age with respect to traditional
Co-Advisor: Prof. William P. Fifer values were observed probably (GA), indicating a significant techniques in the description 139
Sudden Infant Death Syndrome peripheral resistance of the techniques, given previous due to increased interactions increasing cardiorespiratory of how physiological systems

bioENGINEERING
(SIDS) is defined as a sudden vascular tree, effects of circulating positive applications in adults, among physiological systems, coupling and autonomic control. dynamically interact to maintain
and unexplained death of an hormones and metabolic newborns and fetuses. Our second as higher TE values indicate. GA at birth influenced significantly an optimal health status. The
infant younger than 1 year of supply to the heart generates a objective was to characterize and A difference in directionality directionality of phase interaction. proposed novel techniques are
age. Although infrequent, SIDS is range of HRV patterns. For this quantify the mutual influence of balance based on sleep state In QS all the three GA groups advantageous for addressing
still the most common cause of reason, HRV has been for long cardiovascular and respiratory was observed, which could showed the dominant influence specific time scales and different
infant death between 1 month time considered a powerful rhythms. Many contributions be driven by differences of breathing on HR, and this modes of interaction for data
and 1 year in developed countries. tool, to observe non-invasively were found in the literature, in the average breathing relationship grew with GA. In collected under standard clinical
Thanks to epidemiological, the interaction between the ranging from cross-spectral frequency. With respect to the AS a balanced relationship was conditions with artifacts and noise.
animal and pathophysiological sympathetic and parasympathetic analysis to nonlinear methods. cardiorespiratory synchronization, present in Late Preterm and Moreover, these techniques were
studies, possible risk factors and systems and their capability to Nonetheless, a limitation of all our findings showed a significantly it moved toward a dominant capable of characterizing systems
mechanisms leading to this death respond properly to internal and these techniques was that they did higher value both in percentage of relationship from breathing to HR interactions. This supports our
are more understood, nonetheless external challenges. HRV is also not measure the directionality of time spent in phase-coupling and in Full Term. The hypothesis that aim to utilize nonlinear advanced
number of deaths has reached influenced by respiration: the the relationships. Transfer Entropy in length of the coupled epochs in directionality could depend on parameters to obtain reliable
a plateau in the last decade and neuronal control of breathing and (TE) was developed to address QS, both at the newborn and one- respiratory frequency was tested physiological and clinical indices.
no reliable quantitative tool to HR are closely linked, functionally precisely this issue. Its focus is month stage. When looking at the and we found a bimodal influence: This approach could lead to a
assess risk exists. The purpose of as well as anatomically. Thus, on tracking the information flow parameters behavior as a function concurrently with breathing quantitative autonomic profile to
this PhD thesis is to employ novel breathing and HR are the between two systems. Another of age, the ratio of the dominant frequency <0.6 Hz, an increased assess vulnerability in populations
signal processing methodologies output of a complex network of approach is that of describing synchronization shifted from a polarization toward values of at risk for SIDS. This could grant
to accurately estimate the effect controlling mechanisms, which the cardiorespiratory coupling majority of 3 beats in 1 breath in directionality from breathing to the possibility of the definition of
of several risk factors on the constantly adapt to the ever- as a dynamic synchronization newborns, to 4 beats in 1 breath in HR occurred. Nonetheless, the an “elastic” triple risk model, where
autonomic and cardiorespiratory changing need of the organism. process, meaning an interaction one-month infants. This is relevant mean breathing frequency did the contributions of the various
control in newborn infants. The Many efforts have been spent between two subsystems which since the peak of SIDS incidence is not change significantly in the GA risk factors could be weighted and
original contribution centers to describe the systems behind can be modeled as two weakly at 2-4 month of age. window analyzed (35-40 weeks); updated based on infants physical
on the use of noninvasive these signals, with time and self-sustained chaotic oscillators, Effect of position during sleep in thus, the significant change in and environmental conditions,
methodologies to analyze frequency domain approaches. quantifying measures like Phase premature infants. Results in the directionality with GA at birth hopefully improving predictability
physiological signals routinely Nonetheless, the complex Locking and directionality. newborn group showed clear could not be explained solely and generating novel monitoring
acquired in hospital/home origin of these signals makes These techniques were employed differences both in the long and by breathing frequency. Our solutions.
settings. Moreover, the approach traditional linear signal processing to quantify how SIDS risk factors short-term variability parameters, explanation for this phenomenon
to signal analysis incorporates approaches unsuitable or partially may alter cardiorespiratory with supine position having higher is that the threshold for the low
complex system generation and capable of characterizing the interaction and autonomic activity. variability but lower complexity. pass filter effect is still adapting
interaction, with a systemic view of systems generating the data. We investigated the influence At 2 months of age, sleep position between 35-40 weeks GA.
the cardiorespiratory physiology. One of the objectives of this of sleep state, sleep position, influenced mostly short-term Effect of prenatal alcohol/smoking
Different modes of interactions thesis was to implement complex prematurity and the combined variability, generally associated exposure on tilt response in
were explored, from amplitude to signal processing methods to effect of prenatal smoking and with parasympathetic regulation. newborns: The expected behavior
phase modulation. characterize newborn HRV and alcohol exposure on baseline The fact that at 2 months was displayed only by the
A complex interplay of autonomic cardiorespiratory activity. For this physiology and a physiological measures of vagal activity were unexposed group, with decreases
regulation, spontaneous reason, we selected entropies and challenge, i.e. the head up tilt. found to be diminished in prone of all the parasympathetic
contractility of the heart, phase rectified signal averaging These conditions were chosen to position suggests a suppressive parameters selected after tilt,
 140

Patient-specific modelling of stented developed methodology and was performed on one case evaluation of Absorb BVS stent

PhD Yearbook | 2019

the implications to intervention and the results were processed (Abbott Vascular, Abbott Park,
coronary bifurcations: a validated, OCT-based cardiology. This Chapter describes to quantify the effects on the IL, USA) at coronary bifurcations.
the imaging modalities that distribution of wall shear stress It included the acquisition of
reconstruction methodology for CFD analyses are mostly employed during along the geometry. Moreover, intravascular OCT images at
the interventions, focusing the chapter describes the baseline, after 9 and 18 months
on angiography and OCT. The collaborative work that was from the intervention. Patient-
Susanna Migliori
fluid dynamic mathematical performed with partners from the specific CFD simulations were
Advisors: Prof. Gabriele Angelo Dubini, Prof. Francesco Migliavacca formulations implemented for University of Sheffield as part of carried out and the blood flow 141
simulating the blood flow are the European Marie-Skłodowska was characterised in terms

bioENGINEERING
The clinical challenge of was achieved by means of a residence time showed similarities described, alongside the quantities Curie Innovative Training Network, of wall shear stress. The OCT
percutaneous coronary computer-aided design software. with the 2D map of the distance that are commonly considered Virtual Physiological Human- images at 9-months follow-up
interventions (PCI) is highly The 3D reconstruction of the between lumen contours at as descriptors for hemodynamic Cardiovascular Simulation were elaborated to evaluate the
dependent on the recognition stented phantom was assessed follow-up and baseline. Such conditions. Finally, the state-of-the- and Experimentation (VPH- neointima thickening which was
of the coronary anatomy that with the geometry that was findings were in agreement with art in patient-specific modelling is CaSE). A patient was found with presented in terms of restenosis
features each individual. The obtained from X-ray computed the literature, which supported briefly introduced. an occlusion at the left main area percentages. These last were
classic imaging modality used for micro tomography scan, used as the potential application in Chapter 3 presents the developed coronary artery bifurcation and compared with results from CFD
PCI is angiography, but advanced ground truth. Results reported cardiovascular intervention. OCT-based reconstruction underwent PCI procedure with simulations seeking to investigate
imaging techniques that are twist and distortions that were Objectives for future work can methodology and the validation the implantation of one stent. any relationship between blood
regularly performed during PCI, due to the catheter-based be a punctual comparison procedure. A typical geometry of The resultant hemodynamics was flow and vessel remodelling.
like optical coherence tomography imaging technique, these were between fluid dynamics superficial a human coronary segment with analysed with a CFD simulation.
(OCT), may provide detailed reduced with the centreline of quantities and neointima bifurcations was resembled as a Chapter 5 includes the elaboration
knowledge of the pre-intervention one side branch that was chosen thickening, enabling for a rigid phantom. A metallic stent of pre-operative OCT images for
vessel anatomy as well as the as reference for the error angle quantitative study. was deployed in the phantom, the simulation of highly disrupted
post-procedural assessment estimation. The 3D reconstruction Chapter 2 introduces the clinical then it was imaged with OCT blood flow in a coronary artery
of the specific stent-to-vessel was successfully used to perform motivation of the fulfilled and µCT. This latter provided a with spontaneous dissection. This
interactions. Computational fluid CFD analyses, demonstrating a research work by providing to reference geometry for validation last resulted in the generation of
dynamics (CFD) is an emerging great potential for patient-specific the reader an overview of the purposes. A proof-of-concept CFD an empty chamber into the vessel
investigation field in the setting investigations. coronary tree anatomy, with simulation was performed using wall. The developed reconstruction
of optimization of PCI results. The developed methodology atherosclerotic pathology genesis the achieved reconstruction. methodology was employed to
In this study, an OCT-based was employed to perform CFD and the main consequences. Chapter 4 reports the application achieve the geometry of a right
reconstruction methodology of simulations of blood flow across As the work here reported of the validated OCT-based coronary artery segment with
patient-specific coronary artery patient-specific coronary arteries has an application to coronary methodology to in-vivo medical bifurcations where an ulcerated
models, which include the actual with bifurcations after stent bifurcations, a brief insight of images that were acquired at the plaque was found. Patient-specific
geometry of the implanted stent, deployment. The developed the classification accepted by the Institute of Cardiology, Catholic CFD simulation revealed heavy
was developed for the execution framework was employed to study European Bifurcation Club for University of the Sacred Heart flow recirculation in the empty
of CFD simulations. the post-operative hemodynamics the different bifurcation lesions of Rome (Italy). Three patients’ plaque and at the interface with
The method was developed of sixteen patients. Among those a and treatment strategies are angiograms and OCT images the normal lumen.
and validated by means of a subset of twelve cases underwent described. Additional information were processed to achieve high Chapter 6 presents the application
rigid phantom that resembled OCT acquisitions after 9-months about single stent coronary fidelity 3D geometries of the of the validated OCT-based
a segment of the left anterior from the intervention. Such interventions is provided, as these stented coronary artery segments reconstruction method for
descending coronary artery images were used to compute are the resembled scenarios. with bifurcations. Patient-specific patients treated with a polymeric
where a stent was deployed. the percentage of restenosis The second part of the Chapter CFD simulations were carried bioresorbable scaffold at the
The methodology comprised area at the scaffolded segment, moves the reader closer to the out and results were processed Sussex Cardiac Centre, Brighton
the processing of OCT images which was then compared with topic of the dissertation. The for the characterisation of the and Sussex University Hospitals,
to retrieve the lumen contour the distribution of superficial information reported seeks to disrupted hemodynamics after Brighton, UK. The elaborated data
and the stent, then the three- hemodynamics quantities. The present the challenges of this stent implantation. An uncertainty were acquired during a clinical
dimensional (3D) reconstruction superficial distribution of relative research field, the novelty of the study on CFD boundary conditions trial whose objectives were the
 142

Supervised tissue classification in optical M2 by exploiting for the first the median penetration error images, overcoming challenges

PhD Yearbook | 2019

time in the literature intra- was 17 µm, which was 2 orders typical of intra-operative tissue
images: towards new applications of surgical operative in vivo multispectral-
imaging data acquisition
of magnitude smaller than
median vessel diameter.
classification to support surgeons
during interventional-medicine
data science and analysis. Multispectral
data were acquired during The PhD work lies in the wider
procedures.

laparoscopic procedures on research field of SDS, which aims

Sara Moccia - Advisor: Prof. Elena De Momi
7 pigs. Six abdominal organs at providing the surgeons with
Co-Advisor: Leonardo Mattos, Ph.D (abdominal wall, liver, spleen, decision support and context 143
Intra-operative tissue classification reliable tissue classification in classification to retrieve gallbladder, diaphragm and awareness during interventional

bioENGINEERING
plays a fundamental role in intraoperative optical images, reliable classification results intestine) were classified with medicine procedures. The
different clinical fields. The as to offer decision support and and improve classification multi-class SVM. The effect methodologies presented in
automatic image-based tissue provide context awareness during performance. M2 exploited of using multispectral data the PhD thesis demonstrated
classification is a valuable interventional-medicine processes. textural information and was an increase in accuracy the feasibility of using SDS
solution to provide decision The main contributions (Figure 1) supervised and semi- of 11% for the task of organ ML-based algorithms for intra-
support and context awareness of the PhD thesis are: supervised approaches classification and an increase of operative tissue classification
intra-operatively. The surgical coupled with confidence 23% for the task of automatic in several anatomical districts.
data science (SDS) community 1) A new method (M1) to estimation for pathological image tagging with respect to The methodological progress
is focusing more and more automatic learning- tissue classification. When standard RGB imaging. When made in this work highlights
on machine learning (ML) to based informative frame tested on 33 narrow-band exploiting the confidence the potential of SDS ML-based
perform tissue classification in the selection, which exploited laryngoscopic videos, which measure proposed in M2, algorithms in extracting useful
operating room (OR). However, a new set of features to refer to 33 different patients the result was a boost in information implicitly contained
several technical challenges are retrieve informative frames affected by early-stage classification accuracy of 38% in intra-operative optical
still present, hampering the clinical to be processed by tissue laryngeal cancer, a median (RGB) and 20% (MI).
translation of the developed classification algorithms. M1 classification recall of 93% was
methodology in the clinical exploited intensity, keypoint achieved. The method was also 4) Integration of the SDS
practice. Indeed, robust and and image spatial-content tested on the hepatic district, ML-based methodologies
reliable tissue classification is not features to classify, with multi- achieving a recall of 82% in within a handheld robotic
trivial due to image noise, varying class support vector machines classifying 40 RGB images tool to perform tissue
illumination level, different camera (SVM), endoscopic frames acquired in the OR of healthy avoidance in simulated
pose with respect to the tissues, as informative, blurred, with and pathological liver donors’ robotic-assisted surgical
and intra- and inter-patient tissue saliva or specular reflections, or grafts. The classification procedures (M4). With M4,
variability. There are several underexposed. When tested on recall was increased to 98% ML-based tissue segmentation
aspects that can be tackled to a balanced set of 720 images (laryngeal tissue) and to 86% was integrated within a
potentially overcome these issues, from 18 different narrow- (hepatic tissue) by estimating simulated robot-assisted
including hardware design (to band laryngoscopic videos, the confidence of the SVM surgical scenario. A forbidden-
allow high-quality optical imaging), a classification recall of 91% classification and excluding region virtual-fixture control
identification of images to be was achieved for informative low-confidence results. was exploited, as to prevent
processed (to avoid the processing frames, significantly unwanted robot-tissue
of uninformative images), and overcoming the state of the art 3) A new protocol (M3) interaction. In particular,
estimation of classification approaches (median recall = for endoscopic-image phantom vascular structures
confidence (to improve system 57%, maximum recall = 81%). analysis, which exploited were built and segmented
performance by excluding multispectral imaging (MI) using a deep–learning
uncertain results). 2) A new method (M2) for for data acquisition during approach. When including the
On this background, the overall reliable tissue classification endoscopic procedures with control, the error on the robotic Fig. 2 - Workflow of the PhD thesis. With the aim to support surgeons during
goal of the PhD work was to in endoscopic images, the goal of performing robust tip position when it entered interventional-medicine procedures, machine-learning methodologies were
develop a framework for ML-based which introduced a tissue classification and the forbidden zone (24% of developed to detect and classify anatomical tissues in intraoperative optical
images. Several anatomical districts were investigated for testing the proposed
SDS algorithms for robust and measure of confidence on image tagging. M3 expanded the time) was small. Indeed, methodologies.
 144

Novel approaches for context-awareness using a procedural knowledge The deep learning models were learn procedure-specific workflow

PhD Yearbook | 2019

(in the form of ontology), 3D used to recognize the step, which rules and analyze the contextual
and workflow analysis in surgery image processing and semantic
queries, which were used to
in progress and the consecutive
step. After the contextual
information and could a strong
technique for context-awareness
obtain contextual information recognition on the surgical step in surgery.
on the workflow. The framework is achieved, the information was
was able to retrieve contextual forwarded to ontology to find The overall results presented by
Nakawala Hirenkumar Chandrakant
information about current surgical other low-level surgical entities. this thesis demonstrates that
Advisors: Prof. Elena De Momi and Prof. Giancarlo Ferrigno activity along with the information We also developed the video knowledge-driven context aware 145
Computer-assisted surgery are adapt to the training context still have not achieved the desired on the need or presence of a annotation dataset on Robot- system, comprising knowledge

bioENGINEERING
increasingly becoming a part and surgical workflow. There are results for context recognition surgical instrument. To show the Assisted Partial Nephrectomy representation, computer vision,
of the operating rooms (OR). In several aspects that can be tackled and automatic workflow analysis applicability, as a paradigmatic (RAPN), which was used in this machine learning and logic
future, current OR are expected to potentially overcome these in surgery. On this background, scenario, Thoracentesis procedure work. The system was able to programming techniques, has
to include various technologies issues, including robust software the overall goal of this thesis was chosen. Further to that, based recognize 10 RAPN steps with potential to be a critical aspect of
such as context-awareness and architectures (to allow easy is to develop a knowledge- on the framework components, the prevalence-weighted macro- solving the problem of context-
automated surgical workflow integration of sensor data and driven context-aware system an intelligent training system for average (PWMA) recall of 0.83, awareness and automated surgical
analysis with robust sensors, surgical information), recognition for reliable recognition and Thoracentesis was developed, PWMA precision of 0.74, PWMA workflow analysis. The progress
robot-assisted surgical systems, of contextual information (to contextual information, aiding where formal knowledge about F1 score of 0.76, and the accuracy made in this thesis shows that
image-guided surgery, virtual guide and train surgeons on assistance to surgical training and the procedure workflow and of 74.29% on 9 videos of RAPN. combining these technologies help
and augmented reality systems, surgical workflows and removing providing decision support. The entities for object recognition is We found that the combined use overcoming potential barriers to
and better data analysis and unwanted information), and contributions of this PhD work are: specified in the form of production of deep learning and knowledge context-awareness in surgery. As
visualization systems. With the robust surgical workflow analysis rules, which used to recognise representation techniques is a a future perspective, this work
inclusion of new technologies (to improve system performance (1) A novel modular knowledge- surgical context, i.e. surgical promising approach for the multi- is a small step in the broader
e.g. robot-assisted surgeries by building and analyzing surgical driven context-aware system instruments and to automatically level recognition of RAPN surgical framework of context-aware
and change in the conventional workflow automatically for framework was developed, which interpret surgical workflow. workflow. surgical systems, autonomous and
surgical approaches, OR are context-awareness). Context- exploits the procedural knowledge Surgical training with the context- semiautonomous robotic systems
becoming highly involved with aware systems are expected to and computer vision-based aware system showed similar (3) This thesis also exploits a to support the surgeon during
numerous sensors, which produce analyze the intraoperative data methods to recognise surgical results as mentor-based training. new data mining approach using surgical activities.
multi-modal information. Despite and understand the surgical instruments and contextual With this framework, knowledge- inductive logic programming
the discernible advancement situation automatically, e.g. information. A knowledge- based context recognition and (ILP) was used to analyze the
of OR technologies, surgeons’ recognition of surgical steps. driven context-aware system situation interpretation have been surgical video annotation data to
decision-making capability has Despite being well researched, we framework was implemented achieved in the pre-operative and construct a surgical process model
not considerably influenced. intra-operative training scenarios and to analyze the contextual
Besides, surgeon’s cognitive in the laboratory-based setup. information. We used ILP to
load is increased analyzing such automatically develop production
extra surgical representational (2) Further to that, in this thesis, rules, in the first order logic,
information, which outpaced a new pipeline was developed based on the intraoperative video
human analysis capabilities and which integrates deep learning data. These rules were either
leads to preventable medical with knowledge representation constructed as to represent the
errors. For the advanced surgeries, techniques, called “Deep- surgical process model or to
surgical simulators are being used Onto” network as shown in understand the surgical context
for surgical training and replacing Fig. 1, to recognize surgical by analyzing semantic relations
the conventional training methods workflow. “Deep-Onto” network between the surgical entities.
such as lecturing, apprenticeship takes advantage of the formal Data mining was done on
and video demonstrations. knowledge and semantic relations Thoracentesis and RAPN video
However, current training regime encoded inside the ontology annotations datasets that were
and simulation-based training to recognize surgical steps and developed during this thesis
Fig. 1 - “Deep-Onto” network consists of deep learning models for current and
systems do not automatically next step recognition and knowledge model to identify other surgical contexts other low-level surgical activity. work. We found that ILP could
 146

Investigating vascular involvement in

PhD Yearbook | 2019

Multiple Sclerosis by Magnetic Resonance
Imaging
Laura Pelizzari
Advisors: Giuseppe Baselli, Maria Marcella Laganà, Francesca Baglio 147
vertebral artery - VA and internal correlation was found for HI (min In addition, since the regions

bioENGINEERING
Background and Aims reproducibility of the derived 26 HI were recruited to test if jugular veins - IJV) (on the whole RSN r = -0.433, p = 0.031, observed where hypoperfusion was found
Evidence is mounting that vascular CSA measures was assessed in a hypoperfusion in MS could be vessel: ICC > 0.9, p< 0.001 for CCA- within frontoparietal RSN; min did not coincide with GM atrophy
factors play an important role group of 36 healthy individuals related to GM volume loss. ICAs, ICC >0 .6, p< 0.001 for VAs, VT r = -0.422, p = 0.027, observed areas, hypoperfusion may not be
in the development of multiple (HI). In addition, scan-rescan Finally, 31 MS patients and 25 HI and ICC >0.7, p< 0.001 for IJVs). within left posterior cerebral artery merely a secondary phenomenon
sclerosis (MS). Some studies reproducibility was assessed by were scanned with ASL MRI in MS-vs-HI CSA comparison showed VT; WM r = -0.488, p = 0.025) but to GM volume reduction.
have reported neck vessel scanning a group of 9 HI twice, normocapnic and hypercapnic no significant  CSA differences not in MS patients. No significant Therefore, brain perfusion might
morphological and hemodinamical 5 days apart. Once the CSA conditions (Figure 1), and CVR between the two groups at correlation was observed between be used as a further marker of
alterations in MS patients. reproducibility was confirmed, alterations were tested within GM baseline and at follow-up. CVR and lesion load, EDSS, and tissue damage, in addition to GM
Furthermore, cerebral blood the method was applied in a parcels (resting state networks Nevertheless, in MS patients, disease duration. volume.
flow (CBF) and cerebrovascular longitudinal study including 69 MS and vascular territories) and white significant  CSA decrease was Finally, the loss of correlation
reactivity (CVR) impairment has patients and 22 HI, acquired twice, matter (WM). Bivariate correlation observed for the ICAs (C4:  p = Discussion and Conclusions between CVR and age in the MS
been observed. Nevertheless, five years apart. CSA differences between CVR, age and MS clinical 0.048; C7:  p = 0.005; WV:  p = The first study proposed a semi- group, compared to the significant
the exact role that vascular were tested at baseline (BL), at parameters was also computed. 0.012), for VAs (C3:  p = 0.028; C4:  automatic method to measure correlation observed in HI,
component plays in MS is still not follow-up (FU) and over the five Results p = 0.028; C7:  p = 0.028; WV:  p  = neck vessel CSA along the whole suggests that other factors related
completely understood. Therefore, years. The association between The semi-automatic method CSA p.012), and for IJVs (C3:  p = 0.014; vessel course. The method proved to the disease may influenced CVR
the aim of this doctoral work was CSA alterations, MS phenotypes evaluation showed excellent inter- C4:  p = 0.008; C5:  p = 0.010; C6:  to produce reproducible CSA in MS.
to investigate vascular involvement and cardiovascular co-morbidity and intra-rater repeatability at all p = 0.010; C7:  p = 0.008; WV:  p = measures, and the feasibility
in MS with magnetic resonance was tested. cervical levels and on the whole 0.002). Furthermore, patients with of CSA longitudinal studies was
imaging (MRI), from different In a separate study, CBF was vessel course (ICC>0.90, median MS without cardiovascular disease confirmed.
points of view (i.e. neck vessel investigated with arterial spin DSC>0.9), and good degree of showed significantly greater IJV No significant CSA differences
morphology, brain perfusion, CVR), labeling (ASL) MRI, together scan-rescan reproducibility for CSA change than MS patients between MS and HI were identified
in order to provide new insights with gray matter (GM) atrophy. all the considered neck vessels presenting with cardiovascular at the baseline. However,
about this issue. Specifically, 26 MS patients and (internal carotid artery - ICA; disease (C3: p= 0.018; C4: p=0.018; significant  CSA decrease over
C5: p=0.010; C6: p=0.015; C7: time of all major  neck  vessels
Methods p=0.018; WV: p=0.003). was observed for MS group, and
Firstly, a semi-automatic method Significant hypoperfusion was not for HI. Although Type 2 errors
to measure the cross sectional observed in MS patients within may have occurred preventing
area (CSA) of major neck vessels the anterior cingulate and showing any CSA difference over
on their whole course was paracingulate gyri, supplementary time in HC, the significant CSA
introduced for time of flight (TOF) motor cortex, precentral changes observed in MS patients
magnetic resonance angiography and superior frontal gyrus may be associated to MS-related
(MRA) data. A resampling (p ≤0.05). factors. In addition, a greater
FWE
procedure was implemented to No GM atrophy was found in these CSA reduction was observed for
normalize CSA measured on all regions. IJVs in patients without CVD with
slices, in order to allow group No significant CVR differences respect to MS patients with CVD,
comparisons at homologous levels were observed in MS group with suggesting that the changes in
and on the whole vessel. respect to HI within GM parcels IJV CSA are influenced by MS
Fig.1 - Experimental setting for ASL acquisition at normocapnia (left panel) and
Then, the intra- and inter-rater at hypercapnia (right panel). and WM. Significant CVR-age independently of CVD.
 148

The role of hypoxia-inducible transcription peculiar switch between M3 and activity of HIF-1A. In addition, due

PhD Yearbook | 2019

M4 (C). Moreover, we observed to the presence of genes involved of stemness properties, we
factor HIF-1A in acute myeloid leukemia that the same genes showing a
peculiar dysregulation pattern
in invasiveness processes and the
EMT master regulator ZEB2, this
found three miRNAs coherently
down-regulated. All of them are
across different AML subtypes subtype exhibited a motility and implicated in tumor suppressive
were also to distinguish malignant migratory behaviour. pathways in different tumor
from normal promyelocytes, Another EMT master regulator, types. Surprisingly, invasion,
Stefano Percio - Advisor: Linda Pattini
thus ruling out dependence on a ZEB1, was observed in the subset cell migration, chemotaxis, and
myeloid developmental stage. of genes highly distinctive for M0, transendothelial migration 149
With the diffusion and rapid key genes to be exploited as topological analysis by selecting, Interestingly, extending the the subtype correspondent to recurred in the subtypes highly

bioENGINEERING
development of high-throughput candidates to be tested in clinical only transcripts differentially analysis of the signature to the the undifferentiated leukemia. typified by the HIF-signalling.
technologies new data mining trials. expressed in APL respect to other other AMLs, general evidence Considering the involvement of Moreover, we observed that
strategies, based on biological In this study, we unearthed a AMLs (A). Resulting sub-network suggested a heterogeneous HIF-1A this gene in a mutually inhibition epigenetic modulators such as
knowledge and information prominent role for the HIF-1A showed two distinct communities involvement in the different network motif with some miRNAs non-coding RNA and chromatin
engineering, have been generated, signalling, letting to hypothesize with a different trend, each one subtypes. Employing a class well characterized in solid modifier played a crucial role in
laying the foundations for the the existence of HIF-dependent/ containing three HIF-1A direct prediction approach, M5 and tumors, we were prompted to EMT-like behaviour promotion and
interdisciplinary field of Systems driven leukemia subgroups. This target genes (B). M0 resulted more discriminated search for possible candidates leukemia development.
Biology. Specifically, in this study, gene is a master regulator of Specifically, this pattern evoked than other AMLs. For M5, which also in this context. Due to the Finally, findings from this study
we applied this holist approach cellular adaptation to a hypoxic typical characters of epithelial represents the acute monocityc overexpression of ZEB1 in this were also confirmed by the HIF-1A
to investigate the role of the microenvironment, but it also to mesenchymal transition leukemia, we found a prevalence subtype, of which co-expressed inhibition in vitro, resulting in
hypoxia-inducible transcription prompts a wide range of pathways (EMT), responsible for initiating of overexpressed genes, meaning genes were involved not only in impaired bone marrow homing
factor 1 alpha (HIF-1A) in acute like invasion, angiogenesis, metastatic processes through a highly incisive transcriptional EMT, but also in maintenance and leukemia progression in vivo.
myeloid leukemia. Generally, and tumor development. endeavouring the invasive In addition, the hypothesis that, in
leukemias are a composite class Previous studies have revealed phenotype of cancer cells. In A B
leukemic context, EMT-characters
of hematological malignancies an interaction between HIF-1A fact, the down-regulated genes are not a general phenomenon
HIF-1A direct targets
characterized by a block of the and a chimeric protein, PML- are significantly associated to ITGB2
of morphological transformation
differentiation at different stages RARA, characterizing a specific cell adhesion term, while the HMOX1 within the myeloid lineage, but
of hematopoietic lineage, following leukemic subtype. This subtype is three up-regulated targets LRP1
rather segregate with specific
by an abnormal proliferation of identified as M3, and derived from are involved in motility and leukemia subtypes targeting
MMP2

KRT18

cancer clones in bone marrow and the promyelocytic stage of the extracellular matrix degradation. IGFBP2 master regulators triggering EMT,
peripheral blood. Acute myeloid hematopoiesis, therefore called Since genes of the sub-network and could represent a promising
C
leukemia (AML) is a tumor of acute promyelocytic leukemia are mainly down-regulated, strategy to further increase the
the myeloid lineage with a rapid (APL). we wondered if microRNAs efficacy of AML treatment.
growth, and can be classified To this purpose, we started (miRNAs), which have a role in Overall, our data suggest that,
based on the maturity of the cell collecting a list of bona fide gene silencing, could generate in the era of precision medicine,
from which leukemia develops, HIF-1A direct targets, and we this inhibition. Surprisingly, the M0 M1 M2 M3 M4 M5 selected patients could benefit

going progressively from the applied reverse engineering to miR-181 family had the widest from combination approaches
most undifferentiated leukemia reconstruct an AML transcriptional number of potential targets, and with HIF or EMT inhibitors to
Fig. 1 -HIF-1A-dependent sub-network is specifically dysregulated in APL.
(M0) to the most differentiate network, selecting the genes it was recently discovered to A) Transcriptional network of gene interactions correspondent to the reduce leukemia dissemination.
forms such as M5 or M7. Although of the signature as seed. We promote EMT pathway in ovarian statistically significant pairwise dependencies of AML bone marrow expression
only a few effective therapeutic employed mutual information tumorigenesis. data. Each circle represents a transcript. Down-regulated transcripts are in blue
while up-regulated transcripts are in red. B) The sub-network was obtained by
options are available for this operator to estimate the pairwise Another feature of the sub- extracting from the overall AML network only transcripts that are differentially
disease, and relapse after initial correlation between transcripts network is a clear segregation of expressed (FDR q-value < 0.05) in the comparison between APL (M3) and other
treatment frequently occurs, the since it is able to detect also the the APL samples as emerged by AML subtypes. Down-regulated transcripts are in blue while up-regulated
transcripts are in red. Six HIF-1A direct target genes (highlighted in green) are
identification of novel targets non-linear dependencies and it hierarchical cluster. Specifically, present: MMP2, KRT18, and IGFBP2 in the up-regulated community, and ITGB2,
is mandatory. The molecular is reparametrization invariant. a progression in the number of HMOX1, and LRP1 in the vast community mostly down-regulated. C) The
profiling of different leukemia In a further step, we integrated differentially expressed genes APL-specific sub-network observed across different AML subtypes (from M0 to
M5). Down-regulated transcripts are in blue, up-regulated transcripts are in red,
subtypes could unravel novel gene expression analysis to seems to take place, with a non-differentially expressed transcripts are empty circles.
 150

Single cell fluid dynamics for the study of

PhD Yearbook | 2019

the liver microcirculation: computational
modelling and in vitro validation
Monica Piergiovanni - Advisor: prof. Gabriele Dubini
151
The development of µm/s - and pressure - 187Pa- were represented as a fluid droplet, infinitesimal element in a flowing Finally, the VoF model was the feasibility of using the VoF

bioENGINEERING
computational tools to support in good agreement with literature with specific physical properties. Newtonian fluid. From this, it was integrated with the sinusoidal model for application in the liver
biological research is now a central data. The model was also used to This model was used to investigate possible to estimate the forces network model, by reconstructing microfluidics. By means of this
topic in the field of biomedical evaluate the effect of an adhered the effect of the cell presence acting on the cell membrane the computational domain model it was also possible to
engineering. This is particularly leucocyte on the fluid dynamics on the fluid dynamics of the due to the simple flowing in a from the in vivo images and estimate the shear stresses and
crucial in those applications where of one of the branches. Wall whole microchannel, by varying capillary network (111÷203 pN for modelling a single cell flowing forces acting both on the cell and
experimental methods are not shear stress on the sinusoidal/ several parameters, specific of the RBC and 797÷849 nN for the in one of the branches. This on the endothelium, with values
used and/or do not provide a cellular wall are greatly influenced, the liver microcirculation. Mainly, leukocyte), an information that preliminary simulation showed ranging from 11.8 to 19.5 pN.
complete view of the process. reaching a disturbed condition the ratio between the cell and cannot be inferred by any existing
This is the case when studying with peaks of 5 Pa of the shear the plasma viscosity played a experimental methods.
the immune response to some stress for the highest occlusion crucial role on the deformation This VoF model was validated
pathologies of the liver, where rate tested (Figure 1). This of the cell. Less viscous cells (Red with an experimental campaign
the biochemical mechanisms approach was able to accurately Blood Cell) tended to deform on cell deformed under a shear
studied in vivo on animal models reproduce the local fluid dynamics when flowing in capillaries twice flow in microfluidic devices,
need to be supported also by at several stages of the leukocyte as big as them (Figure 2), while designed fabricated for this
a fluid dynamic investigation at adhesion. more viscous cells (leukocyte) specific application. By capturing
the level of microcirculation. The A Volume of Fluid (VoF) model, rotated and shifted towards the images of cells with a high speed
present dissertation is aimed at specific for the study of two- capillary wall, until they reached camera, the deformation of HL60
the development of computational phase flows, was then built to an equilibrium position. The VoF and Jurkat cells was estimated
fluid dynamics (CFD) models to be account for the flowing of a cell model provided all fluid dynamic at varying flow rates - 25 ÷
applied to liver capillaries - called in micro channels. With this parameters that, combined in 125 µl/min - and compared to
sinusoids, organised in the liver model, a single cell - leukocyte a post processing algorithm, computational simulations (Figure Fig. 2 - Results of VoF model for RBC model pRe 6:36 104;Ca 3 102; 1:5;D{H
functional unit, the lobule - and or Red Blood Cell (RBC) - was gave the state of stress on an 3). At increasing flow rate, the 0:5q. Deformation and equilibrium position at four time step - z = 31, 41, 62, 83
µm - and comparison with literature (Tomaiuolo et al., 2016)
at the study of cell motion inside cell is progressively deformed
them. by the fluid from a circular to an
The first model presented, elliptical shape up to a ratio = 1.35
reproduced a portion of the while maintaining a constant area
2
sinusoidal network, by means (about 70÷80 µm ).Even if the
of an adequate reconstruction cell populations showed a great
protocol applied on images variability in terms of deformability
captured in vivo on a mouse due the biological difference
liver. To set the pressure in the among single cells, a statistically
eleven outlet sinusoids, a lumped significant difference could be
parameter model was built, found at varying flow rates. The
representing the sinusoid potion computational model was able to
until the central vein. Resistances represent the deformation of a
were calculated with a Poiseuille Fig. 1 - 3D method: effect of leukocyte adhesion on WSS and streamlines, single cell in an extensional flow,
law with measures from the contributing with different occlusion percentage (from left to right: no with minor underestimation due Fig. 3 - Fost processing flow (images refer to HL60 at a flow rate of 30 ul/min).
occlusion, 42%; 70% and 86%). Estimated pressure drops created by the Top panel: automatic background subtraction and empty images removal.
images stack. Velocities - 90 ÷ 360 adhered cell and outflow from the occluded branch are also reported. to the 2D approximation. Bottom panel: definition of a ROI, image binarization and ellipse fitting.
 152

Modeling of microvasculature in uremic

PhD Yearbook | 2019

patients

Luca Possenti
Advisors: Maria Laura Costantino, Paolo Zunino 153
Microvascular alterations involved: (i) lumped parameter accounting for its geometry before being applied to address Prof. Roger Kamm and thanks the 3D-1D model, to investigate

bioENGINEERING
have recently been reported model of the arterial circulation; and solved by finite element uremic microvasculature, to the collaboration with the the actual rise of the trans-mural
in patients affected by chronic (ii) multiscale 3D-1D model of method. It was developed leveraging on available data from Cell and Tissue Engineering Lab pressure between the vasculature
kidney disease treated by the microvasculature; (iii) in vitro under the supervision of Prof. literature. Tests have assessed at IRCCS Galeazzi Orthopaedic and the gel surrounding it.
hemodialysis therapy. How these model of the microvasculature. Paolo Zunino, MOX laboratory, the contributions of the different Institute (Milan, Italy) - Dr. Matteo Finally the models’ improvements
microvascular alterations are The lumped parameter model Department of Mathematics, relevant factors and how test- Moretti. The Roberto Rocca and their interactions have
related to the pathology is still an was based on previous works Politecnico di Milano. It exploits a driven results actually match with Doctoral Fellowships (Spring 2018) been discussed, along with their
open and complex question. Such describing the arterial circulation framework of partial differential in vivo literature-driven data. supported the collaboration with limitations. This work has paved
alterations are likely to be related including peripheral vascular equations on domains with Moreover, a sensitivity analysis MIT. the way for a modeling support
to the non-physiological flow rates districts and vascular regulations. different dimensions (3D for was conducted to appreciate the This in vitro model consists in the to the current research activities
of fluids and solutes removed A single peripheral district was the interstitium and 1D for the effects induced by the alterations generation of microvasculature on microvascular alterations in
from blood in the artificial filter. analyzed to detail its description vasculature). Based on previous related to the pathology. Thanks on a microfluidic chip, achieving uremic patients.
The artificial treatment increases with particular reference to the works on advanced mathematical to this analysis, the central role a 3D microvascular network
the instantaneous mass- and fluid balance. Tests have been methods and their development, of the capillary wall hydraulic which can be perfused and
fluid-flow rates which have to be conducted by considering some the proposed model accounts conductivity was highlighted, pressurized. It was proposed to
washed out from the interstitium uremic parameters alterations for a non-linear contribution particularly motivating the in analyze alterations of the capillary
into the blood compartment, thus and highlighting the need for of the lymphatic system, the vitro analysis, which is aimed at membrane induced by the
over-stressing the microvascular a comprehensive modeling rheological effect of red blood describing its alterations. presence of uremic toxins. To this
wall membrane. This membrane approach. cells and their heterogeneous The in vitro model was developed aim, a trans-mural pressure was
alteration affects the fluid balance, The second computational distribution along the vascular at the Mechanobiology Lab, applied to study microvascular
the distribution of solutes, and approach consists in a multiscale network. The model was tested Massachusetts Institute of wall properties. In addition,
the delivery of nutrients to the model of the microvasculature, on a number of different cases Technology (Cambridge, MA, the experimental setup was
tissues and may contribute to an USA) under the supervision of computationally analyzed, by using
abnormal vascular development
and morphology. In addition,
the presence of uremic toxins
(currently more than one
hundred have been identified)
induces further alteration on the
microvascular membrane wall.
However, very few studies have
addressed how these toxins affect
the microvasculature.
To address this complex scenario,
a wide modeling approach was
designed. It is composed of three Fig. 2 - The in vitro model: microvasculature on a chip. (a) Geometry of the
different models, which can share Fig. 1 - 3D-1D model of the microvasculature. (a) Reduction to 1D for a generic chip used in this work. (b) Picture of the experimental setup on the confocal
information and results to better curved vessel. (b) Example of an artificial generated computational network. (c) microscope. (c) In vitro microvasculature on a chip perfused by dextran
One of the results obtained in this work. A portion of the interstitial domain has solution. Green: GFP positive endothelial cells. Red: Dextran solution. White
describe the complex phenomena not been displayed in order to show the microvascular network. bar: 200 µm.
 154

Development and validation of an eye

PhD Yearbook | 2019

tracking system for proton radiotherapy
treatment of ocular melanomas
Roberta Visone
Advisors: Prof. Marco Rasponi and Prof. Alberto Redaelli 155
Heart diseases still represent a in vitro cardiac cellular models. bidirectional perfusion on patch This platform was used to study be presented. The model was screening. These platforms were

bioENGINEERING
leading cause of death worldwide A biomimetic macroscale maturation were assessed. the effect of different electrical developed to better represent in pivotal in the establishment of
due to the limited capacity bioreactor will be presented, Subsequently, microfluidic stimulation patterns, alone or vitro the interplay occurring in vivo functional cardiac microtissues,
of mature cardiomyocytes to featuring an innovative geometry bioreactors will be presented, combined to mechanical stimuli, between different cell types (i.e., and eventually exploited to get
proliferate and regenerate necrotic of the cell culture chamber, each designed to address specific on neonatal rat cardiomyocyte fibroblasts and endothelial cells). new biological insights related to
myocardium. The ability to model designed to provide cardiac limitations of current Organs-on- microtissue functionality and By exploiting human endothelial cellular responses when subjected
human cardiac physiology in vitro patches with a bidirectional Chip devices. maturation. cells and cardiac fibroblasts, to combinations of physiologically
would represent a breakthrough interstitial perfusion combined An innovative biomimetic Furthermore, a new technology functional and perfusable relevant stimuli.
in the investigation of key with a precisely controlled microfluidic platform was allowing for the first time the vasculature networks were
biological aspects involved in electrical stimulation. The peculiar developed, able to integrate generation of multiple 3D cardiac developed and characterized.
heart development and disease design of the chamber allowed for for the first time biochemical, microtissues within microfluidic The collected preliminary data on
progression, possibly paving the the first time to directly evaluate electrical and mechanical platforms in an easy and vessel growth and maturation will
way to new strategies for drug individual cardiac patch during conditioning of 3D cardiac uniform fashion, was developed. serve as a basis to further studies
discovery and providing new culture, assessing its maturation, microtissues. An electrical system Microtissues were generated on myocardial vasculogenesis.
hints in the field of regenerative either by optical inspection or was embedded in a microfluidic from iPSC-CM and exploited in In the present PhD thesis both
medicine for the development of through direct tests of contraction platform designed to provide developmental biology studies technical and biological novelties
new cardiac therapies. (Figure 1). Of note, these controlled mechanical stimulation and drug screening assays in a were achieved. Technological
Bioengineering approaches are measurements could be carried (i.e., uniaxial strain) to 3D cardiac mid- to high-throughput fashion. advancements comprised: i) the
strongly needed in the relatively out without interrupting the microtissues. Stainless steel Microfluidic platforms were improvement of the biomimetic
new field of human induced culture operations. The bioreactor electrodes were used both to thus developed and validated as stimulation strategies in both
pluripotent stem cells (iPSC). In was validated with culture provide a tightly controlled electric alternative tools to standard 2D macro and micro bioreactors (i.e.
particular, new technological tools neonatal rat cardiomyocytes field, to stimulate microtissues cell culture systems, allowing to coupling different physical cues
are expected to provide significant seeded on collagen scaffolds; in culture, and to perform better reproduce physiological cell of native cardiac milieu) and ii)
environmental conditioning the effects of a biomimetic pacing tests, assessing their responses. a new technique enabling for
for iPSC manipulation and to electrical stimulation coupled with beating synchronization level. Finally, a cardiac-specific efficient generation of multiple
enhance currently limited- in vitro microvasculature model will tissues for high-throughput drug
maturation of cardiomyocytes
derived from them (iPSC-CMs).
The present PhD Thesis aimed
to develop novel technological
solutions to design advanced
cell culture platforms, both at
the macro (i.e., Bioreactors)
and micro scale (i.e. Organs-on-
Chip). By tailoring native cardiac-
Fig. 2 - a) Developed microfluidic platform for the electro-mechanical
specific cues, the ultimate goal stimulation of 3D microtissues. b) Initial cell population characterized from
was the generation and testing immunofluorescence images to be composed of cardiomyocytes (70.3%62.4%)
of physiologically relevant and Fig. 1 - a) The bioreactor allows the release of a single culture chamber b) to and stromal cells (29.7%62.4%). c) Immunofluorescent staining (cardiac
perform the live imaging of labeled cells of the cardiac construct by means of a Troponin I in green; Connexin-43 in purple) of the electrically stimulated cardiac
functional three-dimensional (3D) fluorescence microscope. microtissues (low voltage (LV) and high voltage (HV). Scale 10µm.