Costantino Casale

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.

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.

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.

Recapitulating spatiotemporal tumor heterogeneity in vitro through engineered breast cancer microtissues

Tumor and microenvironmental heterogeneity hinders the study of breast cancer biology and the assessment of therapeutic strategies, being associated with high variability and drug resistance. In this context, it is mandatory to develop three-dimensional breast tumor models able to reproduce this heterogeneity and the dynamic interaction occurring between tumor cells and microenvironment. Here we show a new breast cancer microtissue model (T-µTP) uniquely able to present intra-tumor morphological heterogeneity in a dynamic and responsive endogenous matrix. T-µTP consists of adenocarcinoma cells, endothelial cells and stromal fibroblasts. These three kinds of cells are totally embedded into an endogenous matrix which is rich in collagen and hyaluronic acid and it is directly produced by human fibroblasts. In this highly physiologically relevant environment, tumor cells evolve in different cluster morphologies recapitulating tumor spatiotemporal heterogeneity. Moreover they activate the desmoplastic and vascular reaction with affected collagen content, assembly and organization and the presence of aberrant capillary-like structures (CLS). Thus, T-µTP allows to outline main crucial events involved in breast cancer progression into a single model overcoming the limit of artificial extra cellular matrix surrogates. We strongly believe that T-µTP is a suitable model for the study of breast cancer and for drug screening assays following key parameters of clinical interest. STATEMENT OF SIGNIFICANCE Tumor and microenvironmental heterogeneity makes very hurdle to find a way to study and treat breast cancer. Here we develop an innovative 3D tumor microtissue model recapitulating in vitro tumor heterogeneity. Tumor microtissues are characterized by the activation of the stromal and vascular reaction too. We underline the importance to mimic different microenvironmental tumor features in the same time and in a single tissue in order to obtain a model of spatiotemporal tumor genesis and progression, suitable for the study of tumor treatment and resistance.

Engineering a human skin equivalent to study dermis remodelling and epidermis senescence in vitro after UVA exposure

Utra Violet type A (UVA) exposure strongly affects the ageing of human skin by modifying both epidermis and dermis and their cross talk as well. The possibility to get a deep understanding in vitro of such crucial mechanism would have a huge impact in the development of antiageing compounds. Here, we present a full thickness model of human skin equivalent formed by a millimeter‐sized dermis completely composed of fibroblasts embedded in their own extracellular matrix. We show that such endogenous nature of the dermis compartment allows the replication of the complexity of the mutual interactions occurring between cellular and extracellular components of the skin under UVA exposure: (a) oxidative stress formation in the whole tissue (dermis and epidermis); (b) senescence of germinative layer of epidermal tissue in terms of p63, ki67, and activated caspase‐3 regulation; (c) modification of the collagenous network architecture in the dermis compartment. By using this human skin model, it is possible to study a widely shared assumptions not yet proved in vitro such the effect of UVA on the self‐renewal capability of skin stem cells.

Spatiotemporal Evolution of the Wound Repairing Process in a 3D Human Dermis Equivalent

Several skin equivalent models have been developed to investigate in vitro the re-epithelialization process occurring during wound healing. Although these models recapitulate closure dynamics of epithelial cells, they fail to capture how a wounded connective tissue rebuilds its 3D architecture until the evolution in a scar. Here, the in vitro tissue repair dynamics of a connective tissue is replicated by using a 3D human dermis equivalent (3D-HDE) model composed of fibroblasts embedded in their own extracellular matrix (ECM). After inducing a physical damage, 3D-HDE undergoes a series of cellular and extracellular events quite similar to those occurring in the native dermis. In particular, fibroblasts differentiation toward myofibroblasts phenotype and neosynthesis of hyaluronic acid, fibronectin, and collagen during the repair process are assessed. Moreover, tissue reorganization after physical damage is investigated by measuring the diameter of bundles and the orientation of fibers of the newly formed ECM network. Finally, the ultimate formation of a scar-like tissue as physiological consequence of the repair and closure process is demonstrated. Taking together, the results highlight that the presence of cell-assembled and responsive stromal components enables quantitative and qualitative in vitro evaluation of the processes involved in scarring during wound healing.