Loss of vascular function associated with cardiovascular disease, such as arthrosclerosis, represents the leading medical epidemic in the United States and typically requires surgical intervention through synthetic or autologous vascular grafts. To overcome the limitations associated with adult cell sources, which are often restricted by supply or compromised by disease, mesenchymal stem cells (MSCs) have emerged as potential candidates for vascular tissue engineering. While evidence suggests the roles of several factors influencing MSC differentiation into vascular phenotypes, including matrix rigidity, geometry and chemistry, the phenomena associated with these events are still largely unknown. Further, the development of mature vascular phenotypes, such as vascular smooth muscle cells (vSMCs), with functional behavior remains elusive to the research community. This thesis proposed to engineer and direct specific and mature vascular differentiation from MSCs by way of highly tailored matrices mimicking the vascular niche environment. Taking inspiration from natural organization, we contend that a biomimetic design approach to tissue scaffolds that display features of the natural cellular microenvironment whilst mimicking the bulk tissue properties may elicit highly specific differentiation of MSCs to vascular phenotypes. To validate our hypothesis, we employed a systemic approach incorporating physical and chemical microenvironmental cues, i.e. stiffness, biological ligands and chemical factors, with the aim to augment vascular phenotype expression, functionality, and final incorporation into a tailored biomaterial scaffolds. First, we present a novel technique for the preparation of silk hydrogels directly from high pressure CO2 environments without the need for crosslinking agents or additional additives such as surfactants or co-solvents. Through this novel method, we demonstrate the utility of CO2 as a volatile electrolyte, capable of sufficiently influencing the sol-gel transition of silk proteins, resulting in the formation of stable hydrogels with properties suitable for biomedical applications. Second, we hypothesized that suitable soluble factor regimen and matrix rigidity can instruct MSC differentiation towards more mature, functional vSMCs. To address this, we investigated cellular differentiation on tunable SF hydrogels prepared using a solvent-free CO2 processing method. The focus of this portion of the thesis is on exploiting the combined use of substrate stiffness and growth factor (TGF- β1) on SF matrices, with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells (hMSCs). Our data reveal that hMSC differentiation into mature SMCs can be achieved within modest culture periods (72 h) by combining appropriate SF hydrogel stiffness (33 kPa) with growth factor (TGF-β1). These findings advance our understanding of how complex multicomponent biomaterials, whereby mimicking the intricacy of natural tissue environments, can play a significant role in developing optimal stem cell differentiation protocols. Third, we postulated that the presentation of ECM proteins on 3D matrices with tunable stiffness will augment the differentiation of MSCs to vascular lineages. To address this, we established a high-throughput ECM platform based on soft, fibrous PEG hydrogels meanwhile highly-tunable in stiffness and 3-dimensional geometry. Using this technique, we identified several microenvironments supporting MSC adhesion, spreading and differentiation toward early vascular lineages. This portion of the thesis supports the hypothesis that a complex milieu exists coupling protein functional behavior with substrate rigidity and that this phenomenon may potentially be exploited through proper application of high-throughput screening methodologies. In the final work of this thesis, we explored the integration of ECM-derived small engineered peptides with 3D soft matrices to refine the differentiation of MSCs to vascular phenotypes, and further successfully recapitulate the complex vascular niche necessary for specific and efficient MSC differentiation into vascular lineages. In line with this, we report the development of a microarray platform based on electrospun nanofibrous hydrogels of photoclickable thiol-ene poly(ethylene glycol) (PEG) hydrogels. Here, we demonstrate the ability to control primary cell adhesion to soft, fibrous hydrogels functionalized with RGD peptide. However, future work will be focused on designing combinatorial peptide studies, whereby, the integration of several biological ligands of interest with tunable physical properties can instruct stem cell differentiation in a highly specific manner. This thesis has provided fundamental insights into the effects of physiological stimuli on vascular differentiation of MSC in terms of the specificity and maturity of the final differentiated cells. Better understanding of such mechanisms will prove paramount in the sequential stages of MSC differentiation to mature vascular cells. Additionally, the findings of this thesis will help to better define the process of regenerating functional healthy vascular tissue from MSCs. Altogether, a combinatorial approach investigating the effects of matrix elasticity, biological ligands and growth factors on MSC differentiation in a 3D nanofiber culture will be critical towards understanding and recapitulating MSC differentiation in the in vivo vascular environment.

Cell-based therapies have a relatively long tradition in modern medicine. Since the 70s surgeons tried to treat malignant and non-malignant disease with direct injection of bone marrow cells. Other cell-based therapies have been proposed after these initial achievements, but it was only in the late eighties that a new concept of therapy, based on cells, has been organically developed. In that years, R. Langer, J. and C. Vacanti proposed the combined use of cells and materials (i.e., scaffolds) to repair tissues and organs, so overcoming the several problems associated with the use of transplants. They coined the term “tissue engineering” as “an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ”. The practical application of these concepts started at the Howard Green & Associates with the researches on cultured sheets of autologous epidermis transplanted to patients suffering from different types of skin lesions1. Other remarkable examples followed this initial attempt. Autologous osteoblast cells, taken from the periosteum and seeded into coral scaffolds, have been used to reconstruct the traumatically lost thumb of a patient. Occluded pulmonary arteries, replaced with a polycaprolactone-polyglycolic acid copolymers scaffold, seeded with own patient peripheral blood vessels cells, gave positive results. Similarly, isolated vascular smooth muscles and endothelial cells were used to reconstruct arteries. Another example is the attempted substitution of surgical bladder augmentation in favour of tissue engineered bladders made by collagen in which urothelial and smooth muscle cells have been seeded. The therapeutic approaches on which tissue engineering has been initially based can be divided in two major techniques: i) the use of scaffold embedded with cells that adhered and proliferated in it and ii) direct seeding of isolated cells in the injured part to promote regeneration. In more recent time, however, an new approach has been developed by a Japanese research group coordinated by prof. Okano. This has been named by him “cell sheet engineering”. The technique is based on the possibility to harvest an undamaged sheet of cells that can be directly transplanted to the injured organ and promote its recovery. Cell sheet engineering possess some advantages over the other techniques as will be clear from the next chapter. Nevertheless, it needs to be improved and, in particular, further studies are necessary to better comprehend the mechanisms by which the cell layer is harvested. This process is based on the behaviour of a “smart polymer” called poly(N-isopropylacrylamide) (PNIPAM) that is capable to trigger cells adhesion simply varying temperature. At 37 °C, cells can adhere and proliferate on substrates grafted with this peculiar polymer, but, once temperature is decreased, it modifies its structure causing cell detachment. If the cells are confluent, then a cell sheet can be harvested and, consequently, used for tissue engineering applications. The focus of the present work has been the study and characterization of smart substrates employed for cell sheet engineering. A general overview on tissue engineering and “cell sheet engineering” applications are summarized in the background (Chapter 1). The state of the art on the different substrates employed and the behaviour of smart polymer are introduced. The general introduction is concluded with the basic concepts on the synthesis route adopted (Chapter 2). The experimental section is divided in two distinct parts: 1) the first part (Chapter 3) is focused on PNIPAM. A deeper description of the characteristics and the applications for this polymer are presented in a brief introduction. Then, the synthesis and general characterization of the polymer are discussed. The smart properties of tethered PNIPAM are tested by in vitro cell cultures and cell sheets, harvested from the obtained samples, characterized. The behaviour of the outermost region of the PNIPAM-coating are deeply investigated by means of Wilhelmy plate technique. A possible model for the evolution of the observed phenomena is given. In the end, an analysis related to the influence of PNIPAM thickness is presented. In particular, the correlation between the polymer chins length and the smart behaviour is investigated by cell culture test and dynamic contact angle. 2) The second part of the of the work (Chapter 4) is dedicated to a different approach to obtain a cell sheet. In the initial section of the chapter, a possible electroactive substrate is examined as an alternative to PNIPAM. The unexpected results, however, led to a different strategy that is presented. Despite limited to a specific cell line, this method allowed for a simple cell sheet harvesting that is described. A possible application is proposed and the characterization of the substrates used for this approach are exposed. Finally, the biological response and the cell sheets obtained by this method are studied.

Recent experimental results have suggested important direct implications of viscoelasticity of human cells and cell cytoskeleton dynamics on some relevant collective and at single-cell behaviors such as migration, adhesion, and morphogenesis. Other experimental studies have been performed on individual cancer and healthy cells of different types, demonstrating that the former were about 70% softer than the latter. In this thesis with the aim of characterizing — and gaining insights into — the frequency response of single-cell systems to mechanical stimuli (typically LITUS), a generalized viscoelastic paradigm which combines classical and spring-pot based (fractional derivative) models is presented. Than the modelling has been enriched considering the non-linear effect of the prestress, induced in protein filaments during cell adhesion and in the cell membrane (with a simple multiscale scheme that incorporates finite elasticity and a 3-D circus tent-like model), on the overall cell stiffness and finally determining its influence on the in-frequency response of the cell. The theoretical results have shown that the differences in stiffness — at least in principle — allow us to mechanically discriminate between tumor and normal cells: the critical frequencies associated with oscillation magnitude peaks (from tens to hundreds of kilohertz) could be helpfully utilized for targeting or ad hoc altering the functions of cancer cells. An experimental validation of the theoretical results is an ongoing work and the preparation of the experimental setup is also presented. In this thesis some first models have been presented to replicate in-vivo collective behavior of cells. Coherent angular rotation of epithelial cells has been reproduced by a cell-centered based mechanical model in which units are polarized, motile, and interact with the neighboring cells via harmonic forces. Starting from this model a continuum non-linear viscoelastic model incorporating the dynamics of liquid crystals has been studied and some preliminary numerical simulations have been performed.

A computer aided manufacturing approach to encapsulate viable mammalian cells in hydrogels and use these capsules as the building blocks for scaffolds. A novel 3D capable contactless bioprinter is presented that encapsulates cells in a alginate hydrogel through an electro hydro dynamic process and deposit these capsules on a specifically engineered substrate manufacturing scaffold without the need for further postprocessing.

Stroke and traumatic brain injury are among the leading causes of death in the world. Until now, there are no effective treatments available. Current pharmaceutical treatments have limited benefits to repair the damaged tissue. Brain tissue engineering is a promising strategy to help brain regeneration after the damage induced by stroke or traumatic brain injury. In this thesis, our work focused on designing and evaluating appropriate silk fibroin-based hydrogels combined with stem cells therapy for brain tissue regeneration. The work initially started from looking for appropriate silk fibroin-based hydrogel substrates which can support the viability and neural differentiation of pluripotent cells. Mouse embryonic stem cells (mESC) were used as a model. Different processing procedures of silk fibroin-based hydrogel substrates were prepared by chemical genipin crosslinking and physical sonication crosslinking. The viability and neural differentiation of pluripotent cells on these hydrogel substrates were evaluated, using tissue culture plates (TCP) as control. Different crosslinking processes were found to modulate the neural differentiation of pluripotent cells. Chemical genipin crosslinked hydrogel substrates could inhibit the neural differentiation of mESC compared to control TCP, while the physical sonication crosslinked hydrogel substrates could support the neural differentiation as TCP. According to the results obtained in the first stage, the physically sonication-crosslinked 3D silk fibroin hydrogel was produced to encapsulate human neural stem cells (hNSC). In order to improve the hNSC attachment and neuronal differentiation, the isoleucine-lysine-valine-alanine-valine (IKVAV) peptide derived from laminin was covalently conjugated to the silk fibroin. The viability and neural differentiation of hNSC were evaluated in the unmodified and IKVAV peptide modified silk fibroin hydrogels. We found that the IKVAV peptide modified silk fibroin hydrogel could increase the viability, proliferation and neuronal differentiation of hNSC. Furthermore, the angiogenesis potential of sonication-induced 3D silk fibroin unmodified and modified with IKVAV and a scramble peptide VVIAK (as control) were evaluated in a human outgrowth endothelial cells (OEC) mono-culture system and a co-culture system in which OEC were cultured with human bone marrow mesenchymal stem cells (BM-MSC). Both the silk fibroin unmodified and modified with IKVAV peptide could not induce angiogenesis in the mono-culture system under the VEGF condition. However, in the co-culture system, we found that unmodified, IKVAV-modified and VVIAK-modified silk fibroin hydrogels all could support angiogenesis. Furthermore, there were no significant differences among unmodified, IKVAV modified and VVIAK modified silk fibroin hydrogels influencing on angiogenesis structure and gene expression related to angiogenesis. The thesis will introduce the detailed work in three different chapters (from chapter 3 to chapter 5) respectively.

Organ failure is one the biggest problems, doctors face every day. Many patients are not able to get a transplant, but even those who recieved it, may undergo painful process of organ rejection and be on the transplant waiting list again. Organ transplants shortage is severe problem in current medicine that has many ethical and medical issues. To solve this problem, the new direction in regenerative medicine was formed, organ prinitng. The main goal of organ printing is fabrication of organ replacements that would mimic the original ones in terms of complexity and functionality. By direct fabrication and maturation of organs in vitro, the problem of organ shortage can be solved, moreover, based on the advances in cell therapy, these organs can be printed with patients own cells, which will eliminte the problem of transplant rejection. Organ printing is multistep and complex process, composed of three main steps: tissue design, or theoretical modelling of replacement composition; tissue fabrication, or direct cell encapsulation and controlled assembly of building units; at last, tissue maturation to reach desirable functionality of the replacement. In the past decade, there was developed a variety of methods for the second step of organ printing, cell encapsulation, which is practicaly the main procedure for tissue fabrication. However, all these methods of cell encapsulation are complex and they might affect cells viability and functionality, which will result in changed tissue function. Thus, starting from the detailed analysis of the tissue fabrication process (encapsulation and assembly methods) the list of possible cell behavior affectors was composed. Based on this list, we designed a multistep protocol for coherent evaluation of cells behavior parameters, in terms of viability, functionality and activity during the tissue fabrication and its maturation steps. Three main materials were used for this study, two naturally (alginate and modified gelatin) and one synthetically (polyethilene glycol) derived polymers. The encapsulation step was performed with two different methods based on chemical or photo crosslinking of the material. Cell parameteres were evaluated on the molecular level for variety of parameters, including viability, activity, proliferation, stress markers expression, at last ability to adapt artificial environment to the cell functional niche with extracellular matrix markers expression, and proteoglycans. The innovation of the presented study consists in the developing a unique protocol for detailed cell functionality evaluation during the organ printing procedures. In fact, based on the conducted study, it was proved the safety of the encapsulation methods. Moreover, based on the cell parameters post-encapsulation, there was suggested the optimal time for tissue maturation for application of the fabricated structures in organ printing, but also in other fileds, like developmental and pathological biology, or drug screening. Eventully, a novel way of simple blocks assembly into 3D complex structures was developed and proved to be safe for cell parameters. At last, for the future research in organ printing, a detailed study over a cell behavior and functionality has to be performed for every fabrication method, what will improve the organ production process drastically.

The objective of this dissertation is to develop and test a predictive model for the passive kinematics of human joints based on the energy minimization principle. To pursue this goal, the tibio-talar joint is chosen as a reference joint, for the reduced number of bones involved and its simplicity, if compared with other sinovial joints such as the knee or the wrist. Starting from the knowledge of the articular surface shapes, the spatial trajectory of passive motion is obtained as the envelop of joint configurations that maximize the surfaces congruence. An increase in joint congruence corresponds to an improved capability of distributing an applied load, allowing the joint to attain a better strength with less material. Thus, joint congruence maximization is a simple geometric way to capture the idea of joint energy minimization. The results obtained are validated against in vitro measured trajectories. Preliminary comparison provide strong support for the predictions of the theoretical model.

The main bulk of this Thesis is focused on the response of cell membranes due to chemical and mechanical stimuli. Henceforth, it is mainly devoted to deduce how the key aspect of the cell response activated by chemical signaling can be predicted by a simplified energetics, making use of both theoretical models and numerical simulations. The a ention is focused on cell membranes embedding G protein-coupled receptors (GPRCs). By analyzing the behavior of cell mem- branes, one can isolate three main contributions in order to model their respon- se: (1) diffusion of receptors and transporters embedded in the lipid membrane; (2) conformational changes of the receptors; (3) membrane elasticity. Moreover, the interplay between TM confomational changes and lateral pressure of the lipid membrane against such TMs is introduced. The chemical potential of the receptor-ligand compound, deduced as the variational derivative of such energy, is compared with the one calculated by accounting for the work done by the lateral pressure. The result yields a relationship between the conformational field, the mechanical field (interpreted as either the thickness change or the areal change) and the distribution of the compounds receptor-ligand. The analysis of such resulting constitutive equation among those three quantities shows that, essentially, the reason why ligand-GPRCs compounds prefer to live on lipid ra is a necessity involving the interplay between the work performed by the lateral pressure and the need of TMs to change their conformation during ligand binding. Henceforth, mechanobiology gives a justification to the experimental findings of Kobilka and Lei ovitz, Chemistry Nobel Prizes 2012.