Giorgia Imparato

The role of microscaffold properties in controlling the collagen assembly in 3D dermis equivalent using modular tissue engineering.

The realization of thick and viable tissues equivalents in vitro is one of the mayor challenges in tissue engineering, in particular for their potential use in tissue-on-chip technology. In the present study we succeeded in creating 3D viable dermis equivalent tissue, via a bottom-up method, and proved that the final properties, in terms of collagen assembly and organization of the 3D tissue, are tunable and controllable by micro-scaffold properties and degradation rate. Gelatin porous microscaffolds with controlled stiffness and degradation rate were realized by changing the crosslinking density through different concentrations of glyceraldehyde. Results showed that by modulating the crosslinking density of the gelatin microscaffolds it is possible to guide the process of collagen deposition and assembly within the extracellular space and match the processes of scaffold degradation, cell traction and tissue maturation to obtain firmer collagen network able to withstand the effect of contraction.

Complementary therapeutic effects of dual delivery of insulin-like growth factor-1 and vascular endothelial growth factor by gelatin microspheres in experimental heart failure

Strategies to prevent adverse left ventricular (LV) remodelling after myocardial infarction have included several traditional approaches and novel cell‐based or gene therapies. Delivery of growth factors in post‐infarction heart failure has emerged as a valuable alternative strategy. Our aim was to investigate the effects of sequential release of vascular endothelial growth factor (VEGF) and insulin‐like growth factor‐1 (IGF‐1) from biodegradable gelatin microspheres in experimental heart failure.

Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors.

Tissue-engineered constructs can be fabricated by the assembly of smaller building blocks in order to mimic much of the native biology that is often made from repeating functional units. Our aim was to realize a three-dimensional (3-D) tissue-like construct in vitro by inducing the assembly of functional micrometric tissue precursors (microTPs). MicroTPs were obtained by dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. During the dynamic seeding, cells adhered, proliferated and synthesized a thin layer of extracellular matrix (ECM) in and around the macroporous beads, generating the microTPs. The analysis showed that the ECM produced was rich in type I collagen. The cells and ECM layer around the microTPs allowed their biological sintering via cell-cell and cell-matrix interaction after only a few days of dynamic seeding. The assembling ability of microTPs was exploited by placing them in a maturation chamber. After 1 week of culture disc-shaped constructs (1cm in diameter, 1mm in thickness) of completely assembled microTPs were collected. The biohybrid obtained presented both a homogeneous and compact aspect. Moreover, histological and immunohistochemical analyses revealed an abundant ECM, rich in type I collagen, interconnecting the microTPs. The results obtained in this survey pave the way to realizing a 3-D dermal tissue equivalent by means of a bottom-up tissue engineering approach.

Biophysical properties of dermal building-blocks affect extra cellular matrix assembly in 3D endogenous macrotissue

The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.

An Engineered Breast Cancer Model on a Chip to Replicate ECM-Activation In Vitro during Tumor Progression.

In this work, a new model of breast cancer is proposed featuring both epithelial and stromal tissues arranged on a microfluidic chip. The main task of the work is the in vitro replication of the stromal activation during tumor epithelial invasion. The activation of tumor stroma and its morphological/compositional changes play a key role in tumor progression. Despite emerging evidences, to date the activation of tumor stroma in vitro has not been achieved yet. The tumor-on-chip proposed in this work is built in order to replicate the features of its native counterpart: multicellularity (tumor epithelial cell and stromal cell); 3D engineered stroma compartment composed of cell-assembled extracellular matrix (ECM); reliable 3D tumor architecture. During tumor epithelial invasion the stroma displayed an activation process at both cellular and ECM level. Similarly of what repeated in vivo, ECM remodeling is found in terms of hyaluronic acid and fibronectin overexpression in the stroma compartment. Furthermore, the cell-assembled ECM featuring the stromal tissue, allowed on-line monitoring of collagen remodeling during stroma activation process via real time multiphoton microscopy. Also, trafficking of macromolecules within the stromal compartment has been monitored in real time.

Bioengineered tumoral microtissues recapitulate desmoplastic reaction of pancreatic cancer

Many of the existing three-dimensional (3D) cancer models in vitro fail to represent the entire complex tumor microenvironment composed of cells and extra cellular matrix (ECM) and do not allow a reliable study of the tumoral features and progression. In this paper we reported a strategy to produce 3D in vitro microtissues of pancreatic ductal adenocarcinoma (PDAC) for studying the desmoplastic reaction activated by the stroma-cancer crosstalk. Human PDAC microtissues were obtained by co-culturing pancreatic cancer cells (PT45) and normal or cancer-associated fibroblasts within biodegradable microcarriers in a spinner flask bioreactor. Morphological and histological analyses highlighted that the presence of fibroblasts resulted in the deposition of a stromal matrix rich in collagen leading to the formation of tumor microtissues composed of a heterotypic cell population embedded in their own ECM. We analyzed the modulation of expression of ECM genes and proteins and found that when fibroblasts were co-cultured with PT45, they acquired a myofibroblast phenotype and expressed the desmoplastic reaction markers. This PDAC microtissue, closely recapitulating key PDAC microenvironment characteristics, provides a valuable tool to elucidate the complex stroma-cancer interrelationship and could be used in a future perspective as a testing platform for anticancer drugs in tissue-on-chip technology. STATEMENT OF SIGNIFICANCE Tumor microenvironment is extremely complex and its organization is due to the interaction between different kind of cells and the extracellular matrix. Tissue engineering could give the answer to the increasing need of 3D culture model that better recapitulate the tumor features at cellular and extracellular level. We aimed in this work at developing a microtissue tumor model by mean of seeding together cancer cells and fibroblasts on gelatin microsphere in order to monitor the crosstalk between the two cell populations and the endogenous extracellular matrix deposition. Results are of particular interest because of the need of heterotypic cancer model that can replicate the complexity of the tumor microenvironment and could be used as drug screening platform.

In vitro three-dimensional models in cancer research: a review

Abstract Three-dimensional (3D) cell cultures have recently garnered great attention because they promote levels of cells differentiation and tissue organisation not possible in conventional two-dimensional (2D) culture systems. Cancer development is a complex process regulated by interactions between epithelial cells, activated stromal cells, and soluble and insoluble components of the extracellular matrix (ECM). As a consequence, in the field of cancer biology a 3D tumour model that accurately recreates the in vivo tumour phenotype would be a valuable tool for studying tumour biology and would allow better pre-clinical evaluation of anticancer drug candidates. Here, we review the 3D tumour models currently available and the more advanced techniques from the tissue-engineering field used to create a more clinically accurate ex vivo tumour model. Moreover, we highlight the drastic differences in drug responses between 3D and 2D models and give a glance to the emerging multi-organ microdevices that can mimic in vivo tissue–tissue interactions.

A novel engineered dermis for in vitro photodamage research.

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three‐dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non‐human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast‐populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo‐aged dermis could be detected in the fibroblast‐populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright

Fabrication of 3D tissue equivalent: an in vitro platform for understanding collagen evolution in healthy and diseased models

In this study we realized a three-dimensional human dermis equivalent (3D-HDE) and, by exploiting multi-photon microscopy (MPM) we validated its use as an in vitro model to study collagen network re-arrangement under simulated solar exposure. The realization of 3D-HDE has been pursed by means of a bottom-up tissue engineering strategy that comprises firstly the fabrication of micron sized tissue building blocks and then their assembly in a 3D tissue construct. The building blocks injected in a maturation chamber, and cultured under optimized culture condition, were able to fuse due to the establishment of cell-cell and cell-extra cellular matrix (ECM) interactions that induced a biological sintering process resulting in 3D-HDE production. The final 3D tissue was made-up by fibroblasts embedded in their own ECM rich in endogenous collagen type I, resembling the composition and the architecture of native human dermis. Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) imaging have been exploited to assess modification in collagen assembly before and after UV irradiation. Textural features and SHG to TPFE ratio of the endogenous ECM within 3D-HDE have been shown to vary after UVA irradiation, proving the hypothesis that the 3DHDE realized can be used as biological platform in vitro to study ECM modifications induced by photo-damage.

Engineered cardiac micromodules for the in vitro fabrication of 3D endogenous macro-tissues

The in vitro fabrication of an endogenous cardiac muscle would have a high impact for both in vitro studies concerning cardiac tissue physiology and pathology, as well as in vivo application to potentially repair infarcted myocardium. To reach this aim, we engineered a new class of cardiac tissue precursor (CTP), specifically conceived in order to promote the synthesis and the assembly of a cardiac extracellular matrix (ECM). The CTPs were obtained by culturing a mixed cardiac cell population, composed of myocyte and non-myocyte cells, into porous gelatin microspheres in a dynamic bioreactor. By engineering the culture conditions, the CTP developed both beating properties and an endogenous immature cardiac ECM. By following a bottom-up approach, a macrotissue was fabricated by molding and packing the engineered tissue precursor in a maturation chamber. During the macrotissue formation, the tissue precursors acted as cardiac tissue depots by promoting the formation of an endogenous and interconnected cardiac network embedding the cells and the microbeads. The myocytes cell fraction pulled on ECM network and induced its compaction against the internal posts represented by the initial porous microbeads. This reciprocal interplay induced ECM consolidation without the use of external biophysical stimuli by leading to the formation of a beating and endogenous macrotissue. We have thus engineered a new class of cardiac micromodules and show its potential for the fabrication of endogenous cardiac tissue models useful for in vitro studies that involve the cardiac tissue remodeling.