1. Aims Engineered tissues are developed by seeding cells onto biocompatible scaffolds and culturing them over time. The aim of this project is to understand how varying microstructures of synthetic scaffolds affect cell behavior in static and dynamic environments. The overarching goal of this project can be explained by three different specific aims. The first specific aim is to characterize the mechanical properties of silicone membranes and to quantify how mechanical stimuli imposed at the scaffold scale propagate to cells. The project includes the study of two different patterns of membranes. 3D printed silicone membranes with micron-resolution will be employed in order to obtain highly controllable scaffold micro-architectures. The same characterization will be done with Poly (4-hydroxybutyric acid) (P4HB) scaffolds. Characterization will consist on microstructure characterization though Scanning Electron Microscopy (SEM), mechanical properties will be measured by uniaxial stretch tests. I will use Finite Element simulations to estimate and visualize the deformation of scaffolds under different mechanical loads. The second specific aim is to seed the 3D-printed membranes and the P4HB scaffolds with vascular smooth muscle cells and cultured them up to 2 weeks under static and dynamic conditions: the expectation is to achieve appreciable ECM depositions. High-resolution fluorescent microscopy/live cell imaging will be employed for computational model construction, and FRET force sensors in cell-ECM adhesions will be used for model validation. The third specific aim regards the optimization of the silicone scaffold micro-architecture and mechanical stimulation to achieve engineered tissues of targeted density and stiffness. I will apply the bio-chemo-mechanical data of the in silico and in vitro systems in order to develop and calibrate a multiscale “systems”-like model of ECM growth and remodeling (E&R) with scaffold micro-architecture topology and mechanical stimulation as inputs and ECM density and stiffness as outputs. The expectation is to be able to predict, design, and target the density and stiffness of engineered tissue in order to modulate and enhance the synthetic behavior of cells seeded on synthetic scaffolds. 2. State of the art ‘Tissue Engineering (TE) is the application of principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationship in normal and pathological mammalian tissue and the development of biological substitutes to restore, maintain or improve tissue function.’ This was the first definition of TE given at a National Sciences Foundation workshop in 1988. [1] However, the fields of tissue engineering, biomaterials, and regenerative medicine have grown synergistically over the last decade at an exponential rate. The fundamental components of a regenerative therapy are cells, biomaterial scaffolds that support cell adhesion and growth, and signals that induce cell responses such as proliferation and differentiation. The first use of biomaterials is to be attributed to the Ancient Egyptians, Greeks and Romans. They were the first populations that used biomaterials like vegetable fibers or animal sinew for making sutures as well as wood for prosthetic limbs. The National Institutes of Health (NIH) describes a biomaterial as ‘’any substance (other than a drug) or combination of substances synthetic or natural in origin, which can be used for any period of time, as a whole or part of a system which treats, augments, or replaces tissue, organ, or function of the body.’’ [5] According to the great strides that tissue engineering and regenerative medicine have made in recent years, the definition has to include ‘’any material used in a medical device intended to interact with biological systems’’. This integration broadened the field of biomaterials to include all the structures and combinations of devices that interact with the body. The main classes of biomaterials are: a) Metals: Metals are inorganic materials and have mechanical, thermal, and electrical properties thanks to their atomic arrangements and bonding characteristics. These materials have conductivity and mechanical strength that are ideal for several medical applications, such as prosthesis, dental implants and fixation devices. b) Polymers: Polymers are materials composed of large molecular chains of monomers linked together forming a backbone. They are very versatile in their composition and properties and are used in surgical tools, device coatings, vascular grafts, injectable biomaterials and catheters. They can be distinguished in synthetic and natural polymers. c) Ceramics: Ceramics are inorganic, orthopedic materials. Their compressive strength and biological inertness are attractive features, but they are limited by their brittleness and poor mechanical properties. Low electrical conductivity and low thermal conductivity are other advantages of ceramics for use as biomaterials. Ceramics can be classified as completely re-absorbable, bioreactive and biointert. d) Composites: The combinations of two or more distinct biomaterials on a macroscopic scale allows the creation of composite materials. In this way, it is possible to combine the desirable physical and mechanical properties of each of them. [10], [9] There are two different phases: discontinuous phase and continuous phase (the matrix). Composites are typically composed of continuum phases where there is at least one discontinuous phase immortalized or imbedded. As the forces are transferred at the interface of the materials, this is crucial to the overall behavior of composites. Along with the interfacial interaction, the shape, orientation, and volume fraction of the inclusive moieties have to be included in the considerations. Particulate and fibrous composites are two different types of composite biomaterials. The choice of the biomaterial to use depends on which characteristics we need. The main features that we usually look for in a biomedical field are the following. [1] a) Biocompatibility: The most important feature for the scaffold that is sought in tissue engineering is biocompatibility. In tissue engineering, especially if we are talking about scaffolds, cells must be able to function normally on the surface. They can adhere onto the surface and eventually migrate through the scaffold and begin to proliferate and lay down new matrix only if the biocompatibility is respected b) Mechanical Properties: The production of scaffolds with adequate mechanical properties is one of the biggest challenges for engineers. Ideally, the mechanical properties should be as similar as possible to the characteristics of the anatomical site into which it is to be implanted. In addition, it is important that the sample is strong enough to withstand surgical handling during implantation. This aspect introduces some challenges for cardiovascular and orthopaedic applications in particular. c) Scaffold Architecture: Regarding the scaffold architecture, it is of crucial importance to guarantee a right cell penetration and adequate diffusion of nutrients into the scaffold. In order to ensure that, an interconnected pore structure and high porosity are two fundamental features of the structure. Furthermore, it is equally important to properly eliminate waste products. So, a porous interconnected structure helps to allow diffusion of waste products out of the scaffold, and the products of scaffold degradation should be able to exit the body without interference with other organs and surrounding tissues. The failure of vascularization and elimination of waste from the center of tissue engineered constructs causes core degradation. In this project, silicone and Poly (4-hydroxybutyric acid) (P4HB) were studied as potential biomaterials where cells could be able to adhere and proliferate in order to reproduce vascular smooth muscle tissue. Silicone In 1901, Frederic Stanley Kipping, an English chemist, used for the first time the word ‘Silicone’. Silicones are compounds that contain silicon, carbon, hydrogen, oxygen, and perhaps other kinds of atoms as well, and have very different physical and chemical properties. Silicones are inorganic polymers made up of repeating units of siloxane. This is a chain of alternating silicon atoms and oxygen atoms, that formed the backbone chain (...-Si-O-Si-O-Si-O-...). There are organic groups linked to the silicon atoms. Silicone is stable both chemically and mechanically: this means it does not react and not change its chemical structure when in contact with biological tissue for examples, and neither if stressed by forces. It is biocompatible: so, it does not have an immune-reaction with the other tissues around itself. It is not biodegradable: indeed, silicone cannot be decomposed by bacteria or other natural organisms. For this specific project the biodegradability is not a requisite for the biomaterial. Moreover, silicone is 3D printable: it can be used as an ink of the 3D printer. So, all the production process is easier and faster. This aspect is very relevant, since it is possible to strongly control the micro-structure of the scaffolds, comparing it with other types of materials used in biomedical applications, such as electrospun poly-.caprolactone (PCL), or nonwoven Poly (4-hydroxybutyric acid), in which their structure is random and not-controllable. Poly (4-hydroxybutyric acid) Poly (4-hydroxyutyric acid) (P4HB) is a homopolymer composed of 4-hydroxybutyrate (4HB) and belongs to a diverse class of materials called polyhydroxyalkanoates (PHAs), produced naturally by microorganisms. The P4HB material was supplied by Tepha. The TephaFLEX® polymer is a thermoplastic linear polyester and is produced by means of a recombinant fermentation process. After this process, the material is isolated and, then, purified in order to achieve a high purity of the material. Starting from this pure material, it is possible to convert it into a large range of medical devices, especially in sutures, films, and textile products. According to [12], P4HB is currently the only PHA to be used clinically in a medical device cleared by the FDA or CE marked for use in Europe. This is possible because, once implanted, P4HB will degrade in the body primarily by hydrolysis to produce 4HB. A variety of tissues, such as liver, lung, heart, kidney, muscle and brown fat is composed of 4HB monomer. Indeed, the body is able to metabolize quickly this monomer. 4HB is eliminated from the body primarily by metabolism via the Krebs Cycle and secondarily by beta-oxidation ultimately to carbon dioxide and water. [13] The P4HB biomaterial is excellent biocompatibility and passed tests for cytotoxicity irritation and sensitization, systemic toxicity, genotoxicity, subchronic and chronic implantation, and hemolysis. 3. Materials and Methods a) 3D-Bioplotter for silicone scaffolds To obtain highly controllable scaffold micro-architectures, I used 3D-bioplotter in collaboration with the Micro/nano Transport and Printed devices Laboratory led by Prof. Hong Zhao. Silicone has been chosen due to its properties that make it printable. In Figure 1, we can see the 3D-bioplotter and its process of printing membrane. According to [14], the 3D-Bioplotter System can transform 3D CAD models in physical 3D scaffold with a designed and defined outer form and an open inner structure. This is possible thanks to its versatile rapid prototyping tool. The ink of the 3D-bioplotter is made by 6 gr of Silicone and 5 drops of catalyst. This quantity is enough for three rectangular samples of dimensions 40X0.9X20 mm with 12 layers. The silicone is bought from MOMENTIVE. In this project, I studied the interaction of cells with four different micro-architectures of the membranes: the fibers are oriented in 0-90 degree and 45-45 degree. Both these orientations were tested aligned and staggered. They are shown in the following Figure 2. b) P4HB scaffolds The scaffolds were obtained from a piece of nonwoven Tepha biomaterial and had a rectangle-shape of 20mm of width and 40mm of length, as showed in the Figure. 3 c) Cells The aim of this project regards the reproduction of a tissue-like structure into the silicone and P4HB membranes. In this project, I studied the behavior of two different kind of cells: rat vascular smooth muscle cells (rVSMCs) acquired from ATCC (American Type Culture Collection Manassas, VA,USA) and the Primary Cells extracted directly from media-layer of the rat aorta. In the adult animal, the main function of the VSMCs is to adjust the blood vessel diameters after physiological stimuli. In fact, they are blood vessel components characterized by specific contractile proteins, calcium handling proteins, ion channels, and cell surface receptors that regulate the contraction of the cells. [15] According to [16], a good performance of the vasculature is strictly dependent to smooth muscle cells. By contraction and relaxation, they alter the luminal diameter, which enables blood vessels to maintain an appropriate blood pressure. However, vascular SMCs also perform other functions, which become progressively more important. For the seeding of both the scaffolds, I performed four different protocols in order to compare the results and established which one guarantees the best result. d) Mechanical Characterization All mechanical testing is performed in custom built uniaxial stretcher. One side is fixed with a sensor load cell model LSB210, and on the other side is a NA0830 actuator (Zaber, Vancouver, British Columbia, Canada). Four black markers are placed on the sample at the vertex of a square and 3 hooks are pierced at each end. A nylon wire is passed through the hooks and pulleys to mount the sample on the stretcher. The samples for pre mechanical testing are preconditioned 30 cycles, with a 1mm amplitude, and 10% strain for silicone scaffolds and 5% for P4HB scaffolds, and then stretched at a rate of 1 mm per minute to a strain of 15% and 10%, for silicone and P4HB scaffolds respectively. From the data collected from these mechanical tests, I obtained the stiffness of each sample by graphing the Force in Newton and the strain Exx expressed in percentage. The stiffness of a material is the extent to which an object resists deformation in response to an applied force. The complementary concept is flexibility or pliability: the more flexible an object is, the less stiff it is. e) SEM analysis The micro-architecture of the silicone and P4HB samples were evaluated with the Scanning Electon Microscope (SEM) analysis. In particular for silicone scaffolds, I checked if the fiber diameter, the distance center-to-center of the fibers and the dimensions of the holes of the membranes respected the features designed through solidworks blueprints. For the P4HB, I used SEM analysis to explore in a deeper way the nonwoven micro-architecture of the material. f) Stretchers Static and dynamic stretchers were used to better understand the mechanical properties of the virgin materials and explore the behavior of cells seeded on the scaffolds. g) Cells Characterization Cells were characterized through western blot and immunofluorescence. The first is a technique that involves the separation of proteins based on their molecular weight by gel electrophoresis, their transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semi-quantitative identification of specific proteins and their molecular weight from a complex mixture. [26] For western blot, I used -SMA, Vimentin, N-cadherin and collagen I as primary antibodies. Then, I performed immunofluorescence as technique to characterize plated cells and detect the cells seeded on the scaffolds. In this case, I stained the samples with the following antibodies: -SMA, Vimentin, N-cadherin. h) Finite Element Simulations Based on SEM data, I built an idealized geometric model with perfect cylinders intersecting each other using SOLIDWORKS software. The model consisted on 20x6 cylinder arrangement on each orientation forming a 6x6x1.5 mm structure. The geometric model was imposed to ANSYS Mechanical APDL. A Finite Element mesh was generated with tetrahedral quadratic elements with element size dependent on curvature and proximity to fiber intersection. For simulation purposes I assumed Silicone as a hyperelastic material 4. Results, discussion and conclusions Mechanical characterization showed that mechanical properties of silicone scaffolds are slightly affected by the structure orientation. SEM images showed that P4HB scaffold was less porous, and more fibers packed than silicone scaffolds. It also showed to be way stiffer than silicone scaffolds. Nevertheless, it was also less compliant and was permanently damaged at strains close to 15% whereas strains up to 40% were applied on silicone scaffolds with no signs of permanent damage. As showed in the previous chapter, the simulations predicted 0-90 arrays to be stiffer than 45-45 configurations. The rationale is that in 0-90 configurations the only mode of deformation is the actual stretching of the fibers. On the other hand, on 45-45 degree configuration fibers will not only stretch but also rotate, thus, as the scaffolds is pulled the diamond-shape holes become slender. This results in lower load requirements to achieve the same displacement. From experimental results we found that silicone scaffolds with 0-90 configuration were slightly stiffer than 45-45 arrays. Suggesting that the different geometric configurations can affectively affect the mechanical behaviour. Nevertheless, difference among configuration resulted lower in experiments than what was predicted by simulations. This difference could be a consequence of: • Occurrence of defects during the manufacture process such as: non effective fibre attachment, voids, discontinuities, impurities, etc. • Damaging of the scaffolds during manipulation and mounting. • Difference in real and simulation conditions, i.e. pulling is performed by three hooks in the experimental setup, whereas simulations were done assuming uniform pulling of all the fiber ends. According to the seeding of the scaffolds, the third strategy had the good results, even if it is only a first step, because it will be important improve also the growth of the cells as a second step. However, these issues were not found in the P4HB material, that resulted to be a good option.
1. Scopo I tessuti ingegnerizzati si sono sviluppati grazie alla semina delle cellule in scaffolds biocompatibili e alla loro coltura nel tempo. Lo scopo di questo progetto è quello di capire come le variazioni della micro-architettura di scaffolds sintetici possano influenzare il comportamento delle cellule sia in condizioni di coltura statiche che dinamiche. L’obiettivo generale del progetto può essere schematizzato in tre scopi specifici. Il primo scopo specifico è di caratterizzare le proprietà meccaniche delle membrane di silicone e quantificare come gli stimoli meccanici applicati allo scaffold possano propagarsi alle cellule. Il progetto include lo studio di due differenti modelli di membrane. Le membrane saranno ottenute attraverso la stampa 3D con una risoluzione dell’ordine del micron, in modo da ottenere uno scaffold con una micro-architettura altamente controllabile. Lo stesso tipo di caratterizzazione verrà svolta anche con scaffolds di Poli (acido-4-idrossibutirrico) (P4HB). La caratterizzazione consisterà sia nel caratterizzare la micro-architettura attraverso il microscopio a scansione elettrica (SEM), che nel valutare le proprietà meccaniche misurate attraverso prove di trazione uniassiali. Simulazioni ad Elementi finiti saranno utilizzate per stimare e visualizzare la deformazione degli scaffolds applicando carichi meccanici differenti. Il secondo scopo specifico è di seminare le membrane stampate 3D e gli scaffolds di P4HB con cellule muscolari lisce vascolari e coltivare questi fino a due settimane applicando condizioni statiche o dinamiche: l’aspettativa è quella di ottenere depositi di ECM in quantità apprezzabile. La microscopia fluorescente ad alta risoluzione / imaging di cellule vive sarà utilizzata per la costruzione di modelli computazionali e i sensori di forza FRET nelle aderenze cellula-ECM saranno utilizzati per la validazione del modello. Il terzo scopo specifico riguarda l’ottimizzazione della micro-architettura degli scaffolds in silicone e la stimolazione meccanica in modo da ottenere tessuti ingegnerizzati di rigidezza e densità stabilita. Applicherò i dati biochimico-meccanici dei sistemi in silico e in vitro al fine di sviluppare e calibrare un modello multiscala simile a "sistemi" di crescita e rimodellamento dell'ECM (E&R) con topologia di micro-architettura dello scaffold e stimolazione meccanica come input, e densità e rigidità dell'ECM come output. L'aspettativa è di essere in grado di prevedere, progettare e indirizzare la densità e la rigidità del tessuto ingegnerizzato al fine di modulare e migliorare il comportamento sintetico delle cellule seminate su scaffold sintetici. 2. Stato dell’arte .L’ingegneria tissutale (TE) è l’applicazione di principi e metodi di ingegneria e le scienze della vita verso la comprensione fondamentale della relazione struttura-funzione nel tessuto sano e patologico dei mammiferi e lo sviluppo di sostituti biologici per ripristinare, mantenere o migliorare la funzione dei tessuti.’ Questa è stata la prima definizione di TE data al National Sciences Foundation workshop nel 1988. [1] Tuttavia, i campi dell’ingegneria tissutale, dei biomateriali e della medicina rigenerativa sono cresciuti in modo sinergico e con velocità esponenziale nell’ultima decade. I componenti fondamentali per la terapia rigenerativa sono cellule, scaffolds prodotti con biomateriali che supportano l’adesione e la crescita delle cellule, e i segnali che inducono risposte cellulari come proliferazione e differenziazione. Il primo uso dei biomateriali è da attribuire agli Antichi Egizi, ai Greci e ai Romani. Loro furono le prime popolazioni ad usare i biomateriali come fibre vegetali o siero animale per produrre suture, o legno per le protesi di arto. Gli Istituti Nazionali di Sanità (NIH) descrivono un biomateriale come ‘ogni sostanza (oltre che un farmaco) o combinazione di sostanze di origine sintetica o naturale, che può essere usata per qualsiasi periodo di tempo, nell’ intero o parte di un sistema che tratta, aumenta o sostituisce il tessuto, l'organo o la funzione del corpo. [5] Secondo i grandi passi avanti compiuti negli ultimi anni dall'ingegneria dei tessuti e dalla medicina rigenerativa, la definizione deve includere "qualsiasi materiale utilizzato in un dispositivo medico destinato a interagire con i sistemi biologici". Questa integrazione ha ampliato il campo dei biomateriali per includere tutte le strutture e combinazioni di dispositivi che interagiscono con il corpo. Le principali classi di biomateriali sono: a) Metalli: i metalli sono materiali inorganici e hanno proprietà meccaniche, termiche ed elettriche grazie alle loro disposizioni atomiche e alle caratteristiche di legame. Questi materiali hanno conduttività e resistenza meccanica ideali per diverse applicazioni mediche, quali protesi, impianti dentali e dispositivi di fissaggio. b) Polimeri: i polimeri sono materiali composti da grandi catene molecolari di monomeri collegati tra loro formando una sorta di ‘spina dorsale’. Sono molto versatili nella loro composizione e proprietà e sono utilizzati in strumenti chirurgici, rivestimenti di dispositivi, innesti vascolari, biomateriali iniettabili e cateteri. Si possono distinguere in polimeri sintetici e naturali. c) Ceramici: i ceramici sono materiali inorganici, usati principalmente nell’ortopedia. La loro resistenza a compressione e inerzia biologica sono caratteristiche interessanti, ma sono limitati dalla loro fragilità e dalle scarse proprietà meccaniche. Bassa conducibilità elettrica e bassa conducibilità termica sono altri vantaggi dei ceramici per quanto riguarda il loro uso come biomateriali. I ceramici possono essere classificati come completamente riassorbibile, bioattivi e biointerti. d) Compositi: la combinazione di due o più biomateriali distinti su scala macroscopica consentono la creazione di materiali compositi. In questo modo, è possibile combinare le proprietà fisiche e meccaniche desiderabili di ciascuno di essi. [10], [9] Esistono due fasi diverse: fase discontinua e fase continua (la matrice). I compositi sono tipicamente composti da fasi continue in cui vi è almeno una fase discontinua immortalata o incorporata. Poiché le forze vengono trasferite all'interfaccia dei materiali, questo è un aspetto cruciale per il comportamento generale dei compositi. Insieme all'interazione interfacciale, la forma, l'orientamento e la frazione di volume delle frazioni inclusive devono essere incluse nelle considerazioni. I compositi particolati e fibrosi sono due diversi tipi di biomateriali compositi. La scelta del biomateriale da usare dipende da quali caratteristiche abbiamo bisogno. Le caratteristiche principali che, di solito, vengono richieste nel campo biomedico sono descritte di seguito. [1] a) Biocompatibilità: la caratteristica più importante per lo scaffold che si cerca nell'ingegneria dei tessuti è la biocompatibilità. Nell'ingegneria dei tessuti, specialmente se si parla di scaffolds, le cellule devono essere in grado di svolgere il loro normalmente ciclo vitale sulla superficie. Possono aderire alla superficie ed eventualmente migrare attraverso la struttura dello scaffold e iniziare a proliferare e creare nuova matrice solo se la biocompatibilità è rispettata. b) Proprietà Meccaniche: la produzione di scaffolds con adeguate proprietà meccaniche è una delle maggiori sfide per gli ingegneri. Idealmente, le proprietà meccaniche dovrebbero essere il più simili possibile alle caratteristiche del sito anatomico in cui deve essere impiantato. Inoltre, è importante che il campione sia abbastanza forte da resistere alla manipolazione chirurgica durante l'impianto. Questo aspetto introduce alcune sfide per le applicazioni cardiovascolari e ortopediche in particolare. c) Architettura dello scaffold: per quanto riguarda l'architettura dello scaffold, è di fondamentale importanza garantire una corretta penetrazione cellulare e un'adeguata diffusione delle sostanze nutritive nell'impalcatura. Per garantire questo, due caratteristiche sono fondamentali nella struttura: pori interconnessi ed elevata porosità. È altrettanto importante, inoltre, eliminare correttamente i prodotti di scarto. Quindi, una struttura porosa interconnessa aiuta a consentire la diffusione dei prodotti di scarto dallo scaffold e i prodotti di degradazione dello scaffold dovrebbero essere in grado di uscire dal corpo senza interferenze con altri organi e tessuti circostanti. Il fallimento della vascolarizzazione e l'eliminazione dei rifiuti dal centro dei costrutti di ingegneria tissutale provoca il degrado del nucleo. In questo progetto, Silicone e P4HB sono stati studiati come potenziali biomateriali in cui le cellule possano aderire e proliferare allo scopo di riprodurre tessuto muscolare liscio vascolare. Silicone Nel 1901, Frederic Stanley Kipping, un chimico inglese, usò per la prima volta la parola "silicone". I siliconi sono composti che contengono silicio, carbonio, idrogeno, ossigeno e anche altri tipi di atomi e hanno proprietà fisiche e chimiche molto diverse. I siliconi sono polimeri inorganici costituiti da unità ripetitive di silossano. Questa è una catena di atomi di silicio e atomi di ossigeno alternati, che forma la catena (...- Si-O-Si-O-Si-O -...). Esistono gruppi organici collegati agli atomi di silicio. Il silicone è stabile sia chimicamente che meccanicamente: ciò significa che non reagisce e non cambia la sua struttura chimica quando viene a contatto con il tessuto biologico per esempio, e nemmeno se sollecitato da forze. È biocompatibile: quindi non determina una reazione immunitaria con gli altri tessuti attorno a sé. Non è biodegradabile: infatti, il silicone non può essere decomposto da batteri o altri organismi naturali. Per questo specifico progetto la biodegradabilità non è un requisito per il biomateriale. Inoltre, il silicone è stampabile in 3D: può essere utilizzato come inchiostro della stampante 3D. Quindi, tutto il processo di produzione è più semplice e veloce. Questo aspetto è molto rilevante, poiché è possibile controllare fortemente la microstruttura delle impalcature, confrontandola con altri tipi di materiali utilizzati in applicazioni biomediche, come il poli--caprolattone (PCL) o Poli (4 -acido idrossibutirrico), in cui la loro struttura è casuale e non controllabile. Poli (4-acido idrossibutirrico) Il poli (acido 4-idrossiutirrico) (P4HB) è un omopolimero composto da 4-idrossibutirrato (4HB) e appartiene a una diversa classe di materiali chiamati poliidrossialcanoati (PHA), prodotti naturalmente da microrganismi. Il materiale P4HB è stato fornito da Tepha. Il polimero TephaFLEX® è un poliestere lineare termoplastico ed è prodotto mediante un processo di fermentazione ricombinante. Dopo questo processo, il materiale viene isolato e, quindi, purificato per ottenere un'elevata purezza del materiale. A partire da questo materiale puro, è possibile convertirlo in una vasta gamma di dispositivi medici, in particolare in suture, pellicole e prodotti tessili. Secondo [12], il P4HB è attualmente l'unico PHA utilizzato clinicamente in un dispositivo medico autorizzato dalla FDA o marcato CE per l'utilizzo in Europa. Ciò è possibile perché, una volta impiantato, il P4HB si degrada nel corpo principalmente per idrolisi per produrre 4HB. Una varietà di tessuti, come fegato, polmone, cuore, reni, muscoli e grasso bruno, è composta da monomero 4HB. In effetti, il corpo è in grado di metabolizzare rapidamente questo monomero. Esso viene eliminato dall'organismo principalmente attraverso il metabolismo con il ciclo di Krebs e in secondo luogo dalla beta-ossidazione e, in ultima analisi, con l'anidride carbonica e l'acqua. [13] Il biomateriale P4HB ha un'eccellente biocompatibilità e ha superato i test per l'irritazione e la sensibilizzazione della citotossicità, la tossicità sistemica, la genotossicità, l'impianto subcronico e cronico e l'emolisi. 3. Materiali e Metodi a) 3D-Bioplotter per scaffolds di silicone Per ottenere scaffolds con micro-architettura altamente controllabile, ho usato il bioplotter 3D in collaborazione con ‘Micro/nano Transport and Printed devices Laboratory’ della Professoressa Hong Zhao. Il silicone è stato scelto per le sue proprietà che lo rendono stampabile. Nella Figura 4, possiamo vedere il bioplotter 3D e il suo processo di stampa di una membrana. Secondo [14] , il 3D-Bioplotter può trasformare modelli CAD 3D in scaffold 3D fisici con una forma esterna progettata e definita e una struttura interna aperta. Ciò è possibile grazie al suo versatile strumento di prototipazione rapida. L'inchiostro del bioplotter 3D è composto da 6 gr di silicone e 5 gocce di catalizzatore. Questa quantità è sufficiente per tre campioni rettangolari di dimensioni 40X0.9X20 mm con 12 strati. Il silicone viene acquistato da MOMENTIVE. Per questo progetto, Ho studiato l’interazione delle cellule con 4 differenti micro-architetture delle membrane: le fibre sono orientate 0-90 gradi e 45-45 gradi. Entrambi questi due modelli sono stati studiati con le fibre allineate e non allineate. Sono mostrati nella Figura 5 di seguito. b) Scaffolds in P4HB Gli scaffolds in P4HB sono stati ottenuti a partire da un pezzo di tessuto nonwoven fornito dalla Tepha, ed hanno una forma rettangolare di larghezza 20mm e lunghezza 40mm, come mostrato nella Figura 6 seguente. c) Cellule Lo scopo del progetto riguarda la riproduzione di una struttura simile al tessuto negli scaffolds di silicone e P4HB. In questo progetto ho studiato il comportamento di due differenti tipi di cellule: le cellule di tessuto muscolare liscio vascolare del ratto (rVSMCs), acquistate dall’ATCC (American Type Culture Collection Manassas, VA,USA), e le cellule primarie estratte direttamente dalla media dell’aorta di ratto. Nell’animale adulto la principale funzione delle VSMCs è quella di variare il diametro dei vasi sanguigni dopo stimoli fisiologici. Infatti, sono componenti dei vasi sanguigni caratterizzati da specifiche proteine contrattili, proteine di gestione del calcio, canali ionici e recettori della superficie cellulare che regolano la contrazione delle cellule. [15] Secondo [16] , una buona prestazione del sistema vascolare dipende strettamente dalle cellule muscolari lisce. Per la contrazione e il rilassamento, alterano il diametro luminale, che consente ai vasi sanguigni di mantenere una pressione sanguigna adeguata. Tuttavia, le SMC vascolari svolgono anche altre funzioni, che diventano progressivamente più importanti. Per la semina di entrambi gli scaffold, ho eseguito quattro protocolli diversi al fine di confrontare i risultati e stabilire quale garantisce il miglior risultato. d) Caratterizzazione Meccanica Tutti i test meccanici vengono eseguiti attraverso uno stretcher uniassiale costruito su misura. Un lato è fissato con una cella di carico del sensore modello LSB210 e dall'altro c’è un attuatore NA0830 (Zaber, Vancouver, British Columbia, Canada). Quattro marker neri sono posizionati sul campione in modo da formare un quadrato e 3 ganci sono inseriti su ciascuna estremità. Un filo di nylon viene fatto passare attraverso i ganci e le pulegge per montare il campione sullo stretcher. I campioni per i test pre-meccanici sono precondizionati con 30 cicli, di un'ampiezza 1 mm e una deformazione del 10% per gli scaffolds in silicone e il 5% per gli scaffolds in P4HB, successivamente allungati a una velocità di 1 mm al minuto a una deformazione del 15% e del 10% , rispettivamente per ponteggi in silicone e P4HB. Dai dati raccolti da questi test meccanici, ho ottenuto la rigidità di ciascun campione rappresentando graficamente la Forza di Newton e la deformazione Exx espressa in percentuale. La rigidità di un materiale è la misura in cui un oggetto resiste alla deformazione in risposta a una forza applicata. Il concetto complementare è flessibilità o flessibilità: più un oggetto è flessibile, meno è rigido. e) Analisi SEM La micro-architettura degli scaffolds in silicone e in P4H è stata valutata attraverso il microscopio a scansione elettrica (SEM). In particolare per gli scaffolds in silicone, ho controllato che il diametro delle fibre, la distanza tra i centri di due fibre adiacenti e le dimensioni dei pori dello scaffold rispettassero le caratteristiche progettate con Solidworks. Per gli scaffolds in P4HB, invece, attraverso l’SEM ho esplorato in modo più profondo la micro-architettura nonwoven del materiale. f) Stretchers Stretchers static e dinamici sono stati utilizzati per approfondire la conoscenza delle proprietà meccaniche dei materiali vergini e per esplorare il comportamento delle cellule seminate sugli scaffolds. g) Caratterizzazione delle cellule Le cellule sono state caratterizzate mediante Western Blot e immunofluorescenza. La prima è una tecnica che prevede la separazione delle proteine in base al loro peso molecolare mediante elettroforesi su gel, il loro trasferimento su una membrana e l'immunodeficienza selettiva di un antigene immobilizzato. Questo è un metodo importante e di routine per l'analisi delle proteine che dipende dalla specificità dell'interazione anticorpo-antigene ed è utile per l'identificazione qualitativa o semi quantitativa di proteine specifiche e il loro peso molecolare da una miscela complessa. [26] Per il Western Blot ho usato -SMA, Vimentina, N-caderina e collagene I come anticorpi primari. Invece, ho eseguito l'immunofluorescenza come tecnica per caratterizzare le cellule piastrate e per rilevare le cellule seminate sugli scaffold. In questo caso, ho colorato i campioni con i seguenti anticorpi: -SMA, Vimentina e N-caderina. h) Simulazioni ad elementi finiti Sulla base dei dati SEM, ho creato un modello geometrico ideale con cilindri perfetti che si intersecano tra loro utilizzando il software SOLIDWORKS. Il modello consisteva in una disposizione del cilindro 20x6 su ciascun orientamento formando una struttura 6x6x1,5 mm. Il modello geometrico è stato imposto ad ANSYS Mechanical APDL. È stata generata una mesh di elementi finiti con elementi quadratici tetraedrici con dimensioni degli elementi dipendenti dalla curvatura e dalla vicinanza all'intersezione delle fibre. Ai fini della simulazione ho assunto il silicone come materiale iperelastico. 4. Risultati, discussione e conclusioni La caratterizzazione meccanica ha mostrato che le proprietà meccaniche degli scaffolds in silicone sono leggermente influenzate dall'orientamento della struttura. Le immagini SEM hanno mostrato che lo scaffold in P4HB è meno poroso e fibre più compatte rispetto agli scaffolds in silicone. Ha anche dimostrato di essere molto più rigido degli scaffolds in silicone. Tuttavia, è risultato essere anche meno compliante ed è stato danneggiato in modo permanente a deformazioni vicine al 15%, mentre le deformazioni fino al 40% sono state applicate su scaffolds in silicone senza segni di danni permanenti. Come mostrato nel capitolo precedente, le simulazioni hanno previsto che gli scaffolds 0-90 fossero più rigidi delle configurazioni 45-45. La logica è che nelle configurazioni 0-90 l'unica modalità di deformazione è l'effettivo allungamento delle fibre. D'altra parte, su una configurazione a 45-45 gradi le fibre non solo si allungano ma ruotano, quindi, man mano che gli scaffolds vengono tirati, i fori a forma di diamante diventano sottili. Ciò comporta requisiti di carico inferiori per ottenere lo stesso spostamento. Dai risultati sperimentali abbiamo scoperto che gli scaffolds in silicone con configurazione 0-90 sono leggermente più rigidi rispetto agli scaffolds 45-45. Questo suggerisce che le diverse configurazioni geometriche possono influenzare in modo efficace il comportamento meccanico. Tuttavia, la differenza tra le configurazioni è risultata inferiore negli esperimenti rispetto a quanto previsto dalle simulazioni. Questa differenza potrebbe essere una conseguenza di: • Presenza di difetti durante il processo di fabbricazione come: un non efficace attaccamento della fibra, vuoti, discontinuità, impurità, ecc. • Danneggiamento degli scaffolds durante la manipolazione e il montaggio. • Differenza nelle condizioni reali e di simulazione, vale a dire che la trazione viene eseguita da tre ganci nella configurazione sperimentale, mentre le simulazioni sono state fatte ipotizzando una trazione uniforme di tutte le estremità della fibra. Secondo la semina delle impalcature, la terza strategia ha dato buoni risultati, anche se è solo un primo passo, perché sarà importante migliorare anche la crescita delle cellule come secondo passo. Tuttavia, questi problemi non sono stati trovati nel materiale P4HB, risultando essere una buona opzione.
Mechanical stimulation of engineered tissues in bioprinted silicone scaffolds and its synergy with scaffold micro-architecture
POTERE, FEDERICA
2018/2019
Abstract
1. Aims Engineered tissues are developed by seeding cells onto biocompatible scaffolds and culturing them over time. The aim of this project is to understand how varying microstructures of synthetic scaffolds affect cell behavior in static and dynamic environments. The overarching goal of this project can be explained by three different specific aims. The first specific aim is to characterize the mechanical properties of silicone membranes and to quantify how mechanical stimuli imposed at the scaffold scale propagate to cells. The project includes the study of two different patterns of membranes. 3D printed silicone membranes with micron-resolution will be employed in order to obtain highly controllable scaffold micro-architectures. The same characterization will be done with Poly (4-hydroxybutyric acid) (P4HB) scaffolds. Characterization will consist on microstructure characterization though Scanning Electron Microscopy (SEM), mechanical properties will be measured by uniaxial stretch tests. I will use Finite Element simulations to estimate and visualize the deformation of scaffolds under different mechanical loads. The second specific aim is to seed the 3D-printed membranes and the P4HB scaffolds with vascular smooth muscle cells and cultured them up to 2 weeks under static and dynamic conditions: the expectation is to achieve appreciable ECM depositions. High-resolution fluorescent microscopy/live cell imaging will be employed for computational model construction, and FRET force sensors in cell-ECM adhesions will be used for model validation. The third specific aim regards the optimization of the silicone scaffold micro-architecture and mechanical stimulation to achieve engineered tissues of targeted density and stiffness. I will apply the bio-chemo-mechanical data of the in silico and in vitro systems in order to develop and calibrate a multiscale “systems”-like model of ECM growth and remodeling (E&R) with scaffold micro-architecture topology and mechanical stimulation as inputs and ECM density and stiffness as outputs. The expectation is to be able to predict, design, and target the density and stiffness of engineered tissue in order to modulate and enhance the synthetic behavior of cells seeded on synthetic scaffolds. 2. State of the art ‘Tissue Engineering (TE) is the application of principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationship in normal and pathological mammalian tissue and the development of biological substitutes to restore, maintain or improve tissue function.’ This was the first definition of TE given at a National Sciences Foundation workshop in 1988. [1] However, the fields of tissue engineering, biomaterials, and regenerative medicine have grown synergistically over the last decade at an exponential rate. The fundamental components of a regenerative therapy are cells, biomaterial scaffolds that support cell adhesion and growth, and signals that induce cell responses such as proliferation and differentiation. The first use of biomaterials is to be attributed to the Ancient Egyptians, Greeks and Romans. They were the first populations that used biomaterials like vegetable fibers or animal sinew for making sutures as well as wood for prosthetic limbs. The National Institutes of Health (NIH) describes a biomaterial as ‘’any substance (other than a drug) or combination of substances synthetic or natural in origin, which can be used for any period of time, as a whole or part of a system which treats, augments, or replaces tissue, organ, or function of the body.’’ [5] According to the great strides that tissue engineering and regenerative medicine have made in recent years, the definition has to include ‘’any material used in a medical device intended to interact with biological systems’’. This integration broadened the field of biomaterials to include all the structures and combinations of devices that interact with the body. The main classes of biomaterials are: a) Metals: Metals are inorganic materials and have mechanical, thermal, and electrical properties thanks to their atomic arrangements and bonding characteristics. These materials have conductivity and mechanical strength that are ideal for several medical applications, such as prosthesis, dental implants and fixation devices. b) Polymers: Polymers are materials composed of large molecular chains of monomers linked together forming a backbone. They are very versatile in their composition and properties and are used in surgical tools, device coatings, vascular grafts, injectable biomaterials and catheters. They can be distinguished in synthetic and natural polymers. c) Ceramics: Ceramics are inorganic, orthopedic materials. Their compressive strength and biological inertness are attractive features, but they are limited by their brittleness and poor mechanical properties. Low electrical conductivity and low thermal conductivity are other advantages of ceramics for use as biomaterials. Ceramics can be classified as completely re-absorbable, bioreactive and biointert. d) Composites: The combinations of two or more distinct biomaterials on a macroscopic scale allows the creation of composite materials. In this way, it is possible to combine the desirable physical and mechanical properties of each of them. [10], [9] There are two different phases: discontinuous phase and continuous phase (the matrix). Composites are typically composed of continuum phases where there is at least one discontinuous phase immortalized or imbedded. As the forces are transferred at the interface of the materials, this is crucial to the overall behavior of composites. Along with the interfacial interaction, the shape, orientation, and volume fraction of the inclusive moieties have to be included in the considerations. Particulate and fibrous composites are two different types of composite biomaterials. The choice of the biomaterial to use depends on which characteristics we need. The main features that we usually look for in a biomedical field are the following. [1] a) Biocompatibility: The most important feature for the scaffold that is sought in tissue engineering is biocompatibility. In tissue engineering, especially if we are talking about scaffolds, cells must be able to function normally on the surface. They can adhere onto the surface and eventually migrate through the scaffold and begin to proliferate and lay down new matrix only if the biocompatibility is respected b) Mechanical Properties: The production of scaffolds with adequate mechanical properties is one of the biggest challenges for engineers. Ideally, the mechanical properties should be as similar as possible to the characteristics of the anatomical site into which it is to be implanted. In addition, it is important that the sample is strong enough to withstand surgical handling during implantation. This aspect introduces some challenges for cardiovascular and orthopaedic applications in particular. c) Scaffold Architecture: Regarding the scaffold architecture, it is of crucial importance to guarantee a right cell penetration and adequate diffusion of nutrients into the scaffold. In order to ensure that, an interconnected pore structure and high porosity are two fundamental features of the structure. Furthermore, it is equally important to properly eliminate waste products. So, a porous interconnected structure helps to allow diffusion of waste products out of the scaffold, and the products of scaffold degradation should be able to exit the body without interference with other organs and surrounding tissues. The failure of vascularization and elimination of waste from the center of tissue engineered constructs causes core degradation. In this project, silicone and Poly (4-hydroxybutyric acid) (P4HB) were studied as potential biomaterials where cells could be able to adhere and proliferate in order to reproduce vascular smooth muscle tissue. Silicone In 1901, Frederic Stanley Kipping, an English chemist, used for the first time the word ‘Silicone’. Silicones are compounds that contain silicon, carbon, hydrogen, oxygen, and perhaps other kinds of atoms as well, and have very different physical and chemical properties. Silicones are inorganic polymers made up of repeating units of siloxane. This is a chain of alternating silicon atoms and oxygen atoms, that formed the backbone chain (...-Si-O-Si-O-Si-O-...). There are organic groups linked to the silicon atoms. Silicone is stable both chemically and mechanically: this means it does not react and not change its chemical structure when in contact with biological tissue for examples, and neither if stressed by forces. It is biocompatible: so, it does not have an immune-reaction with the other tissues around itself. It is not biodegradable: indeed, silicone cannot be decomposed by bacteria or other natural organisms. For this specific project the biodegradability is not a requisite for the biomaterial. Moreover, silicone is 3D printable: it can be used as an ink of the 3D printer. So, all the production process is easier and faster. This aspect is very relevant, since it is possible to strongly control the micro-structure of the scaffolds, comparing it with other types of materials used in biomedical applications, such as electrospun poly-.caprolactone (PCL), or nonwoven Poly (4-hydroxybutyric acid), in which their structure is random and not-controllable. Poly (4-hydroxybutyric acid) Poly (4-hydroxyutyric acid) (P4HB) is a homopolymer composed of 4-hydroxybutyrate (4HB) and belongs to a diverse class of materials called polyhydroxyalkanoates (PHAs), produced naturally by microorganisms. The P4HB material was supplied by Tepha. The TephaFLEX® polymer is a thermoplastic linear polyester and is produced by means of a recombinant fermentation process. After this process, the material is isolated and, then, purified in order to achieve a high purity of the material. Starting from this pure material, it is possible to convert it into a large range of medical devices, especially in sutures, films, and textile products. According to [12], P4HB is currently the only PHA to be used clinically in a medical device cleared by the FDA or CE marked for use in Europe. This is possible because, once implanted, P4HB will degrade in the body primarily by hydrolysis to produce 4HB. A variety of tissues, such as liver, lung, heart, kidney, muscle and brown fat is composed of 4HB monomer. Indeed, the body is able to metabolize quickly this monomer. 4HB is eliminated from the body primarily by metabolism via the Krebs Cycle and secondarily by beta-oxidation ultimately to carbon dioxide and water. [13] The P4HB biomaterial is excellent biocompatibility and passed tests for cytotoxicity irritation and sensitization, systemic toxicity, genotoxicity, subchronic and chronic implantation, and hemolysis. 3. Materials and Methods a) 3D-Bioplotter for silicone scaffolds To obtain highly controllable scaffold micro-architectures, I used 3D-bioplotter in collaboration with the Micro/nano Transport and Printed devices Laboratory led by Prof. Hong Zhao. Silicone has been chosen due to its properties that make it printable. In Figure 1, we can see the 3D-bioplotter and its process of printing membrane. According to [14], the 3D-Bioplotter System can transform 3D CAD models in physical 3D scaffold with a designed and defined outer form and an open inner structure. This is possible thanks to its versatile rapid prototyping tool. The ink of the 3D-bioplotter is made by 6 gr of Silicone and 5 drops of catalyst. This quantity is enough for three rectangular samples of dimensions 40X0.9X20 mm with 12 layers. The silicone is bought from MOMENTIVE. In this project, I studied the interaction of cells with four different micro-architectures of the membranes: the fibers are oriented in 0-90 degree and 45-45 degree. Both these orientations were tested aligned and staggered. They are shown in the following Figure 2. b) P4HB scaffolds The scaffolds were obtained from a piece of nonwoven Tepha biomaterial and had a rectangle-shape of 20mm of width and 40mm of length, as showed in the Figure. 3 c) Cells The aim of this project regards the reproduction of a tissue-like structure into the silicone and P4HB membranes. In this project, I studied the behavior of two different kind of cells: rat vascular smooth muscle cells (rVSMCs) acquired from ATCC (American Type Culture Collection Manassas, VA,USA) and the Primary Cells extracted directly from media-layer of the rat aorta. In the adult animal, the main function of the VSMCs is to adjust the blood vessel diameters after physiological stimuli. In fact, they are blood vessel components characterized by specific contractile proteins, calcium handling proteins, ion channels, and cell surface receptors that regulate the contraction of the cells. [15] According to [16], a good performance of the vasculature is strictly dependent to smooth muscle cells. By contraction and relaxation, they alter the luminal diameter, which enables blood vessels to maintain an appropriate blood pressure. However, vascular SMCs also perform other functions, which become progressively more important. For the seeding of both the scaffolds, I performed four different protocols in order to compare the results and established which one guarantees the best result. d) Mechanical Characterization All mechanical testing is performed in custom built uniaxial stretcher. One side is fixed with a sensor load cell model LSB210, and on the other side is a NA0830 actuator (Zaber, Vancouver, British Columbia, Canada). Four black markers are placed on the sample at the vertex of a square and 3 hooks are pierced at each end. A nylon wire is passed through the hooks and pulleys to mount the sample on the stretcher. The samples for pre mechanical testing are preconditioned 30 cycles, with a 1mm amplitude, and 10% strain for silicone scaffolds and 5% for P4HB scaffolds, and then stretched at a rate of 1 mm per minute to a strain of 15% and 10%, for silicone and P4HB scaffolds respectively. From the data collected from these mechanical tests, I obtained the stiffness of each sample by graphing the Force in Newton and the strain Exx expressed in percentage. The stiffness of a material is the extent to which an object resists deformation in response to an applied force. The complementary concept is flexibility or pliability: the more flexible an object is, the less stiff it is. e) SEM analysis The micro-architecture of the silicone and P4HB samples were evaluated with the Scanning Electon Microscope (SEM) analysis. In particular for silicone scaffolds, I checked if the fiber diameter, the distance center-to-center of the fibers and the dimensions of the holes of the membranes respected the features designed through solidworks blueprints. For the P4HB, I used SEM analysis to explore in a deeper way the nonwoven micro-architecture of the material. f) Stretchers Static and dynamic stretchers were used to better understand the mechanical properties of the virgin materials and explore the behavior of cells seeded on the scaffolds. g) Cells Characterization Cells were characterized through western blot and immunofluorescence. The first is a technique that involves the separation of proteins based on their molecular weight by gel electrophoresis, their transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semi-quantitative identification of specific proteins and their molecular weight from a complex mixture. [26] For western blot, I used -SMA, Vimentin, N-cadherin and collagen I as primary antibodies. Then, I performed immunofluorescence as technique to characterize plated cells and detect the cells seeded on the scaffolds. In this case, I stained the samples with the following antibodies: -SMA, Vimentin, N-cadherin. h) Finite Element Simulations Based on SEM data, I built an idealized geometric model with perfect cylinders intersecting each other using SOLIDWORKS software. The model consisted on 20x6 cylinder arrangement on each orientation forming a 6x6x1.5 mm structure. The geometric model was imposed to ANSYS Mechanical APDL. A Finite Element mesh was generated with tetrahedral quadratic elements with element size dependent on curvature and proximity to fiber intersection. For simulation purposes I assumed Silicone as a hyperelastic material 4. Results, discussion and conclusions Mechanical characterization showed that mechanical properties of silicone scaffolds are slightly affected by the structure orientation. SEM images showed that P4HB scaffold was less porous, and more fibers packed than silicone scaffolds. It also showed to be way stiffer than silicone scaffolds. Nevertheless, it was also less compliant and was permanently damaged at strains close to 15% whereas strains up to 40% were applied on silicone scaffolds with no signs of permanent damage. As showed in the previous chapter, the simulations predicted 0-90 arrays to be stiffer than 45-45 configurations. The rationale is that in 0-90 configurations the only mode of deformation is the actual stretching of the fibers. On the other hand, on 45-45 degree configuration fibers will not only stretch but also rotate, thus, as the scaffolds is pulled the diamond-shape holes become slender. This results in lower load requirements to achieve the same displacement. From experimental results we found that silicone scaffolds with 0-90 configuration were slightly stiffer than 45-45 arrays. Suggesting that the different geometric configurations can affectively affect the mechanical behaviour. Nevertheless, difference among configuration resulted lower in experiments than what was predicted by simulations. This difference could be a consequence of: • Occurrence of defects during the manufacture process such as: non effective fibre attachment, voids, discontinuities, impurities, etc. • Damaging of the scaffolds during manipulation and mounting. • Difference in real and simulation conditions, i.e. pulling is performed by three hooks in the experimental setup, whereas simulations were done assuming uniform pulling of all the fiber ends. According to the seeding of the scaffolds, the third strategy had the good results, even if it is only a first step, because it will be important improve also the growth of the cells as a second step. However, these issues were not found in the P4HB material, that resulted to be a good option.File | Dimensione | Formato | |
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https://hdl.handle.net/10589/151085