Francesco Urciuolo

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.

Osteogenic differentiation and mineralization in fibre-reinforced tubular scaffolds: theoretical study and experimental evidences

The development of composite scaffolds with well-organized architecture and multi-scale properties (i.e. porosity, degradation) represents a valid approach for achieving a tissue-engineered construct capable of reproducing the medium- and long-term in vitro behaviour of hierarchically complex tissues such as spongy bone. To date, the implementation of scaffold design strategies able to summarize optimal scaffold architecture as well as intrinsic mechanical, chemical and fluid transport properties still remains a challenging issue. In this study, poly ɛ-caprolactone/polylactid acid (PCL/PLA) tubular devices (fibres of PLA in a PCL matrix) obtained by phase inversion/salt leaching and filament winding techniques were proposed as cell instructive scaffold for bone osteogenesis. Continuous fibres embedded in the polymeric matrix drastically improved the mechanical response as confirmed by compression elastic moduli, which vary from 0.214 ± 0.065 to 1.174 ± 0.143 MPa depending on the relative fibre/matrix and polymer/solvent ratios. Moreover, computational fluid dynamic simulations demonstrated the ability of composite structure to transfer hydrodynamic forces during in vitro culture, thus indicating the optimal flow rate conditions that, case by case, enables specific cellular events—i.e. osteoblast differentiation from human mesenchymal stem cells (hMSCs), mineralization, etc. Hence, we demonstrate that the hMSC differentiation preferentially occurs in the case of higher perfusion rates—over 0.05 ml min–1—as confirmed by the expression of alkaline phosphate and osteocalcin markers. In particular, the highest osteopontin values and a massive mineral phase precipitation of bone-like phases detected in the case of intermediate flow rates (i.e. 0.05 ml min–1) allows us to identify the best condition to stimulate the bone extracellular matrix in-growth, in agreement with the hydrodynamic model prediction. All these results concur to prove the succesful use of tubular composite as temporary device for long bone treatment.

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

Hemoglobin-Conjugated Gelatin Microsphere as a Smart Oxygen Releasing Biomaterial.

In this study, a novel micrometric biomaterial acting as a cyclic oxygen releasing system is designed. Human hemoglobin (Hb) is conjugated to the surface of gelatin microspheres (GM) to produce gelatin hemoglobin oxygen depot (G-HbOD). G-HbOD is obtained by means of two different conjugation strategies. The degree of conjugation of GM surfaces in terms of free amino groups by using HPLC is first evaluated. By following the strategy A (G-HbOD_A), Hb is conjugated to GM by means of the formation of a polyurethane linker. In the strategy B (G-HbOD_B) the conjugation occurs via amide bound formation. Physical and morphological differences between G-HbOD_A and G-HbOD_B are investigated by means of Fourier Transform Infrared Spectroscopy (FTIR), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Differences in oxygen uptake/release kinetics are found depending on the conjugation strategy and it is proved that G-HbOD works under repeated cycles in microfluidic chip. Moreover, G-HbOD is also able to work as oxygen depot in the early stages of 3D cell cultures.