79 research outputs found
Human tissue-on-a-chip development for Type 2 Diabetes studies
No full textType 2 Diabetes (T2D) results from an insulin resistance condition that affects mainly adipose and skeletal muscle tissues by altering their glucose uptake and leading to major clinical complications. Moreover, it is known that these tissues interact each other with a negative cross-talk that worsen the pathology. Due to its clinical complexity, T2D would require patient-specific treatment. In this context it will be extremely helpful to have in vitro human tissue-based assays, able to provide biological responses representative of the in vivo patient-specific pathology.
The aim of this PhD thesis is the development and integration of human tissue models in a microfluidic technology, for in vitro T2D drug testing on patient-derived tissues. More specifically, to develop a new tool with the capability to dissect, reproduce and study tissue interactions in multi-organs derived pathologies, that allows to carry out biological investigations on human insulin-resistant tissue models which are exposed to fast physiological and pathological stimuli.
Integration “on-a-chip” of skeletal muscle and adipose tissues and their applications are presented in this thesis. These tissues were chosen for their involvement in T2D.
A myoblasts model integrated in a microfluidic technology and coupled with glucose FRET-based nanosensing is described. Through microfluidic technology it was possible to monitor extracellular glucose concentration with high temporal resolution with minimum disruption of cell culture condition, while glucose FRET nanosensing permitted to measure cytosolic glucose concentration in live cells. With the assistance of mathematical modeling and data analyses it was possible to derive from experimental data the kinetic parameters involved in glucose handling (glucose diffusion through plasma membrane and intracellular glucose phosphorylation).
On its side, adipose tissue organ cultures was integrated in an automated microfluidic chip which is able to provide temporally controlled stimulations (e.g. insulin or drugs), allowing significant flexibility of the experiments. This tissue model showed high viability and metabolic activity, high flow rate sensitivity in high resolution glucose uptake measurements and robustness.
Finally, it is presented the integration of human skeletal muscle in a microfluidic device. For the development of this in vitro tissue, micropattern techniques were coupled and applied in order to have a better control on cell topology. It has been studied the effect of different-width of pattern lanes on myoblasts proliferation and differentiation. Wider lanes negatively affect cell proliferation, whereas they have a positive effect on myoblasts differentiation. Human myoblasts were successfully integrated and differentiated in the microfluidic chip with a 300-μm lanes pattern. Myotubes showed a high sarcomeric organization, outlining the obtainment of a tissue model very similar to the in vivo muscle. Moreover, it has been demonstrated that insulin pathway activation is conserved in physiological conditions, and that it can be deeply investigate through conventional molecular biology techniques.
The skeletal muscle model on-a-chip was finally studied for the insulin resistance onset after treatment with adipose tissue conditioned medium.
These results show a good potential in future pharmaceutical and clinical experimentation. In fact they give proofs that a new tool able to dissect and reproduce tissue interactions in multi-organs derived diseases, can be generated. With its support, it would be possible to overcome current limitation in therapy design, by reproducing in vitro models of the disease, even in a patient-specific way, in order to perform individual therapeutic development.Il Diabete di tipo 2 insorge da una condizione di insulino-resistenza che colpisce soprattutto il tessuto muscolare e il tessuto adiposo, alterando il loro uptake di glucosio e portando a complicanze cliniche più gravi. È altresì noto che questi tessuti interagiscono tra loro con una comunicazione distruttiva che peggiora la patologia. A causa della sua complessità clinica, il Diabete di tipo 2 richiede spesso trattamenti paziente-specifici.
In questo scenario, nasce la necessità di sviluppare un un modello in vitro umano capace di fornire risposte biologiche rilevanti e che sia rappresentativo della patologia in vivo specifica del paziente.
L’obiettivo di questa tesi di dottorato è lo sviluppo e l’integrazione di modelli di tessuto umano derivati da pazienti affetti da Diabete di tipo 2 in una tecnologia microfluidica per svolgere screening di farmaci in vitro. Tale modello, permetterà di dissezionare, riprodurre e studiare le interazioni dei tessuti in patologie che coinvolgono più organi. In questo specifico caso, permetterà di investigare l’insulino-resistenza dei tessuti umani sottoposti a stimoli veloci, sia fisiologici che fisio-patologici.
Considerato il loro ruolo centrale nell’insorgenza del Diabete, in questa tesi è presentata l’integrazione in una tecnologia “Lab-on-a-chip” di tessuto muscolare e tessuto adiposo.
Un modello di mioblasti è stato utilizzato per lo studio delle dinamiche cellulari del glucosio. La tecnologia microfluidica in cui è stato integrato ha permesso di monitorare la concentrazione extracellulare di glucosio con un’alta risoluzione temporale, mantenendo integra nel contempo la coltura cellulare. Inoltre l’uso di un nanosensore intracellulare FRET specifico per il glucosio ha permesso di misurare la concentrazione citosolica del metabolita in mioblasti vivi. Avvalendosi di modellazione matematica e analitica dei dati sperimentali è stato possibile calcolare i parametri cinetici propri del glucosio in una cellula: nello specifico sono state calcolate le cinetiche del trasporto attraverso la membrana citosolica e della fosforilazione intracellulare.
Il tessuto adiposo è stato invece integrato come organo-coltura in un chip microfluidico automatizzato, il quale è in grado di fornire stimoli di insulina o farmaci controllati nel tempo, rendendo flessibile la possibilità di sperimentazione. Il tessuto adiposo da parte sua ha mostrato elevate vitalità e attività metabolica, un’alta sensibilità alla portata del medium durante le misure di uptake di glucosio e soprattutto una elevata ripetibilità sperimentale.
Infine, viene presentata l’integrazione in dispositivi microfluidici di tessuto muscolare umano accoppiando inoltre tecniche di micropattern per ottenere un migliore controllo della topologia cellulare in vitro. Innanzitutto è stato studiato l’effetto dovuto all’utilizzo di pattern a righe di diverse ampiezze sulla proliferazione e differenziamento dei mioblasti in vitro: maggiore è l’ampiezza delle righe del pattern, minore è la proliferazione e maggiore è il differenziamento. Il miglior pattern (composto da righe di 300 μm), è stato riprodotto all’interno di chip microfluidico. Mioblasti umani coltivati in questo sistema sono stati in grado di differenziare in miotubi caratterizzati da una elevata organizzazione sarcomerica. Inoltre è stato verificato il mantenimento dell’attivazione del pathway dell’insulina, risultato evidenziato adattando convenzionali tecniche di biologia molecolare ai piccoli volumi cellulari coinvolti. Il tessuto muscolare integrato in microfluidica è stato infine studiato sotto lo stimolo di medium condizionato da tessuto adiposo, mostrando l’insorgenza di insulino-resistenza.
Questi risultati mostrano un elevato potenziale nel futuro delle sperimentazioni cliniche e farmaceutiche grazie alla possibilità di poter riprodurre in vitro le interazioni multi-organo di patologie complesse. Con il supporto di tali strumenti sarà possibile superare le attuali limitazioni presenti nello sviluppo di nuove terapie. Sarà infatti possibile riprodurre in vitro la patologia, anche in una maniera paziente-specifica, permettendo quindi l’attuazione di terapie personalizzate
“Bioartificial” scaffolds for the development of in vitro models of human cardiac fibrotic tissue
No full textBackground Myocardial infarction causes the loss of billions of cardiomyocytes and the remodelling of local extracellular matrix (ECM), leading to the progressive formation of a stiff fibrotic tissue mainly populated by cardiac fibroblasts. In vitro models of human pathological cardiac tissue able to closely reproduce post-infarct microenvironment could greatly improve preclinical experimentation on human heart, providing predictive tools to study new therapies. In this work, the structure and composition of bi- and three-dimensional scaffolds were tailored to obtain models of human pathological cardiac tissue with different thicknesses and severity degrees. Methods Polycaprolactone (PCL) was processed by electrospinning (Linari Engineering) and fused-deposition modelling (Rokit Invivo) to obtain 2D and 3D scaffolds, respectively. Gelatin (G) was grafted on scaffold surface through a mussel-inspired approach based on two steps: (i) 3,4-Dihydroxy-D,L-phenylalanine (DOPA) polymerisation on PCL surface; (ii) incubation in G solution. After each functionalization step, physicochemical, morphological and mechanical characterizations were performed. Cardiac fibroblasts isolated from human ventricle (HCFs, PromoCell) were cultured on the scaffolds at a density of 7x104 cells/cm2, and their adhesion, proliferation and protein expression were analysed. Results SEM analysis showed that 2D electrospun membranes consisted of a nanofiber network free of defects, while a reproducible interconnected porous structure was obtained for 3D scaffolds. QCM-D, ATR-FTIR and XPS analyses confirmed successful surface modification after each step, while the amount of grafted G was quantified by a colorimetric assay. Mechanical and thermal properties of scaffolds did not vary after functionalization. HCFs cultured on G grafted scaffolds showed better attachment and proliferation compared to non-functionalized scaffolds. The expression of fibroblast markers (α-SMA, DDR2) and secretion of typical cardiac ECM proteins (Fibronectin, Laminin, Tenascin and Collagen IV) were confirmed by immunofluorescence and western blot analysis. Conclusions 2D and 3D bioartificial scaffolds supported long-term HCF culture, and their composition and structure affected HCF markers and protein deposition. In the future, new therapies will be tested in vitro using such constructs. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme grant agreement No 772168
PEGDA-Gelatin/PEDOT:PSS hydrogels as electroconductive and 3D-printable scaffolds for cardiac tissue engineering
No full textINTRODUCTION
Hydrogels are hydrophilic polymeric networks, able to mimic the microenvironment of human tissues and therefore they are widely studied in tissue engineering (TE). Electroactive tissues, such as cardiac, neural and muscle, strictly depends on electrochemical signaling between cells. Therefore, TE scaffolds interacting with those tissues should be designed with electroconductive properties [1]. Electroconductive hydrogels (ECHs), are a class of smart biomaterials that merge the electrical properties of intrinsically conductive materials with hydrogel networks. In recent studies, the in vivo application of conductive hydrogels demonstrated their ability to re-synchronize heart contraction, after myocardial infarction [2]. Nevertheless, a hydrogel-based scaffold with highly tunable electrical and mechanical properties, showing also bioactivity, biocompatibility and biodegradability, is still missing [1]. Furthermore, the heart tissue has an highly hierarchical and anisotropic microstructure [3]. In cardiac TE, scaffolds able to support alignment of contractile cells, are demanded. Bioprinting methods are promising as they can print oriented constructs. Furthermore, the application of bioprinting to photo-crosslinkable hydrogels may allow high spatiotemporal control of scaffold structure [3]. The aims of this work, were: (i) the development of photo-curable ECHs based on polyethylene glycol diacrylate (PEGDA), gelatin and poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) PEDOT:PSS, with tunable electrical, mechanical and bioactive properties for cardiac tissue engineering application; (ii) to investigate the suitability of PEGDA-Gelatin/PEDOT:PSS hydrogels as inks for prospective biofabrication of engineered cardiac tissues.
MATERIALS AND METHODS
Following previous studies by the authors, photo-cured PEGDA-gelatin hydrogels were optimized. Herein, Riboflavin was used as a biocompatible photoinitiator and different PEGDA/gelatin hydrogels were tested. PEDOT:PSS was added to hydrogels to impart electrical conductivity. Photopolymerization was analyzed by photorheology. Mechanical compression properties were studied, while electrical properties were evaluated by sheet resistance and dielectric spectroscopy. In vitro degradation properties of hydrogels were also evaluated.
As a proof of concept for cardiac tissue engineering use, in vitro biocompatibility and adhesion tests with human cardiac fibroblasts (HCFs) were performed on hydrogels. Finally, printability of hydrogels was also preliminarily assessed.
RESULTS AND DISCUSSION
Hydrogel gelation time, final cross-linking density, microstructure, swelling and degradation properties were finely modulated by PEGDA/gelatin ratio. By its increase, hydrogels with increasing stiffness were obtained, with elastic moduli close to that of healthy native cardiac tissue. The addition of PEDOT:PSS into the hydrogels reduced gelation time and increased surface and bulk electrical properties. As a bioactive component, gelatin was successfully integrated into the hydrogel network. Hydrogels were also cytocompatible and promoted the adhesion of HCFs up to 5 days. Finally, PEGDA-Gelatin/PEDOT:PSS hydrogels were micro-extruded into grid-shaped scaffolds.
CONCLUSIONS
Electroconductive photo-curable PEGDA-gelatin/PEDOT:PSS hydrogels were developed as promising for future bioprinting of cardiac tissues.
REFERENCES
1. Rogers, Z. J., Zeevi, M. P., Koppes, R. and Bencherif, S. A., Bioelectricity, 2 (3): 279-292, 2020.
2. Zhang, C. et al., Biomaterials, 231: 2020
3. Zenobi-wong, M., Lee, M. and Rizzo R., Chem. Rev., 120: 10950-11027, 2020
ACKNOWLEDGEMENTS
This project is supported from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (BIORECAR GA N° 772168)
Plasma-activated hydrogels for in vitro fibrotic cardiac tissue modelling
No full textIschemic heart disease is caused by cardiomyocyte death under hypoxic conditions, followed by the formation of a dysfunctional fibrotic scar. Reactive oxygen species (ROS) play an important role in this process. Physical atmospheric pressure plasmas (APP), generate unique mixtures of ROS and started to be considered for medical therapy. Due to the many potentials of this blooming new field ahead, there is a need to exploit the main concepts derived from plasma research in chemistry and biology as a mechanistic link between the ROS production from APP and their medical benefit (1). Indeed, different types and doses of in vivo-like ROS can be generated by APP treatment of physiological solutions, and their effects on different cell types have been studied, including ROS-induced cell apoptosis (2). On the other hand, the exploitation of APP treatment to model in vitro diseased tissues has not been reported up to now. The aim of this work was to develop an in vitro cardiac fibrotic tissue model for preclinical studies of drugs. An APP was used to generate ROS at a proper amount to induce oxidative stresses into human adult cardiac fibroblasts (AHCFs) without affecting their viability. Cells were exposed to plasma-activated medium for 24 h and their viability was evaluated after 1, 3 and 7 days. AHCF activation into myofibroblasts was also analyzed versus control conditions. Then, ROS-loaded hydrogels were prepared with proper concentration and crosslinking degree to match the stiffness of healthy and fibrotic cardiac tissues. ROS stability into the starting polymer solutions and their release kinetics from freshly-prepared hydrogels were studied. ROS-loaded hydrogels were used for AHCF culture. In the future, an in vitro model of the fibrotic cardiac tissue will be prepared using the selected ROS-loaded hydrogel and its structural and functional properties will be analyzed
3D IN VITRO MODELS OF EARLY AND LATE HUMAN CARDIAC FIBROSIS BY TUNING 3D ARCHITECTURE OF BIOARTIFICIAL SCAFFOLDS
No full text
Engineering of injectable multifunctional polyurethane-based hydrogels for the advanced treatment of hard-to-close wounds
No full textElectroconductive photo-curable PEGDA-Gelatin/PEDOT:PSS hydrogels for prospective cardiac tissue engineering application
No full textIntroduction
Hydrogels are hydrophilic cross-linked polymeric materials that have been widely studied in tissue engineering (TE) to mimic human tissues [1]. The functionality of electroactive tissues, such as cardiac, neural and muscle, strictly depend on electrochemical signaling between cells. Therefore, TE scaffolds interacting with those tissues should be designed with electroconductive properties [2]. Recently, electroconductive hydrogels (ECHs), combining intrinsically conductive materials with hydrogels networks, have demonstrated to be able to promote the formation of electroactive engineered tissues both in vitro and in vivo. Poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), a conductive polymer, presents good biocompatibility and dispersibility in aqueous solution. Hence, it has already been involved in the development of ECHs for engineering cardiac or neural tissue [3][4]. Nevertheless, an hydrogel-based scaffold with highly tunable electrical and mechanical properties, showing also bioactivity, biocompatibility and biodegradability, is still missing [2]. The aim of this work, was the development of photo-curable ECHs based on polyethylene glycol diacrylate (PEGDA), gelatin and PEDOT:PSS, with tunable electrical, mechanical and bioactive properties, for cardiac tissue engineering application.
Methodology
In previous studies by the authors, UV-cured hydrogels based on PEGDA and gelatin were obtained [5]. Riboflavin was selected as a biocompatible photoinitiator and three different initial PEGDA/gelatin weight ratios were tested. PEDOT:PSS was incorporated, to impart electrical conductivity to the final system. The photopolymerization process was analyzed through photorheology. Physico-chemical properties of hydrogels were investigated. Mechanical characterization was carried out through compression tests while electrical properties were evaluated by means of sheet resistance and dielectric spectroscopy measurements. In vitro degradation properties of hydrogels were also evaluated. Finally, as a proof of concept for cardiac tissue engineering application, in vitro biocompatibility and adhesion tests with human cardiac fibroblasts (HCFs) were performed on the developed hydrogels.
Results
The gelation time of hydrogels as well as their final cross-linking density, microstructure, swelling and degradation properties were finely modulated by varying the ratio between PEGDA and gelatin. Accordingly, by increasing PEGDA/gelatin ratio, hydrogels with increasing stiffness were obtained, with elastic moduli similar to those reported for healthy native cardiac tissue. The addition of PEDOT:PSS within the hydrogels reduced their gelation time while increasing both their surface and bulk electrical properties. As a fundamental bioactive component, gelatin was successfully bonded to the final hydrogel network structure. Additionally, hydrogels were cytocompatible and promoted the adhesion of HCFs.
Conclusions
Electroconductive photo-curable PEGDA-gelatin/PEDOT:PSS hydrogels developed in this study are promising candidates for cardiac tissue engineering applications, deserving future investigations.
1. Hoffman, A. S. et al., Adv. Drug Deliv. Rev. 64, 18–23 (2012).
2. Rogers, Z. J. et al., Bioelectricity 2, 279–292 (2020).
3. Roshanbifar, K. et al., Adv. Funct. Mater. 28 (2018).
4. Heo, D.N. et al., Mater. Sci. Eng. C 99, 582–590 (2019).
5. Cosola, A. et al., Polymers 11, 1-9 (2019).
BIORECAR project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme grant agreement No 77216
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