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Despite advances in the development of in vitro tissue models such as reconstructed human skin, the questions in dermatological research, which can be addressed with these models, are limited. This is mainly due to the lack of key extra-cellular components, which makes dermal compartment an incomplete approach to human ECM in vivo. Today there is a growing awareness of the fact that the ECM has a functional importance as a dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes. We established a human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In our model dermal ECM presents laminin, fibronectin, hyaluronic acid, elastin and collagen arranged and organized as in the human counterpart. As proof of the physiological relevance of such tissue environment we demonstrate that -by using adult human skin cells-it is possible to generate follicle-like structures in vitro resembling what occurs in vivo in the fetal skin. Immunotypization evidences an inward-oriented differentiation of the follicular-like structures through immunopositivity for epithelial stem cell markers such as p63 and K19. Moreover we succeeded in innervating this human skin by inducing rat dorsal root ganglion neurons axon ingrowth and demonstrate the effective functionality of the nerve network. Neurofilaments network infiltrates the entire native dermis ECM until reaching the epidermis, as demonstrated by immunopositivity to neurofilament-M and second harmonic generation imaging. Calcium imaging demonstrates that electrical current travel in the neuronal network proving the its sensing functionality. Our results demonstrate the fundamental role of ECM in morphogenesis, corroborating the awareness of its importance in building up functional in vitro skin model for fundamental research applications.

During the development of de novo synthesized cartilage tissue engineered constructs, transport and biophysical properties are expected to change in time and space. Monitoring and control of the evolution of these parameters are of crucial importance to process biohybrid constructs in vitro. The aim of this work was to measure fluid and macromolecular transport and evolution of mechanical properties of tissue-engineered cartilage constructs as a function of culture time and extracellular matrix (ECM) production. It was found, in agreement with other literature reports, that mechanical and fluid transport properties of the constructs correlated well with time of culture and glycosaminoglycan (GAG) content. Further, diffusion coefficients of 2 probes, dextran (500 kDa) and bovine serum albumin (BSA), correlated well with GAG production. Diffusion coefficients (D) were measured with high spatial and temporal resolution by fluorescent recovery after photobleaching (FRAP). Diffusivity steadily decreases with time while it does not vary through the thickness of the specimen. On the basis of these results, an empirical relationship between diffusion coefficient and GAG content was proposed for the 2 probes analyzed. The results of this study provide useful information to optimize and control the tissue culture process in vitro.

An intestine-on-chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine-on-chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well-oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.

Here, we propose an immune-responsive human Microbiota-Intestine axis on-chip as a platform able to reproduce the architecture and vertical topography of the microbiota with a complex extracellular microenvironment consisting of a responsive extra cellular matrix (ECM) and a plethora of immune-modulatory mediators released from different cell populations such as epithelial, stromal, blood and microbial species in homeostatic and inflamed conditions. Firstly, we developed a three-dimensional human intestine model (3D-hI), represented by an instructive and histologically competent ECM and a well-differentiated epithelium with mucus-covered microvilli. Then, we replicated the microenvironmental anaerobic condition of human intestinal lumen by fabricating a custom-made microbiota chamber (MC) on the apical side of the Microbiota-human Intestine on chip (MihI-oC), establishing the physiological oxygen gradient occurring along the thickness of human small intestine from the serosal to the luminal side. The complexity of the intestinal extracellular microenvironment was improved by integrating cells populations that are directly involved in the inflammatory response such as peripheral blood mononuclear cells (PBMCs) and two species of the intestinal commensal microbiota (Lactobacillus rhamnosus and Bifidobacterium longum). We found that lipopolysaccharide (LPS)-induced inflammation elicits microbiota's geographical change and induce Bifidobacterium longum iper-proliferation, highlighting a role of such probiotic in anti-inflammatory process. Moreover, we proved, for the first time, the indirect role of the microbiota on stromal reshaping in immune-responsive MihI-oC in terms of collagen fibers orientation and ECM remodeling, and demonstrated the role of microbiota in alleviating gastrointestinal, immunological and infectious diseases by analyzing the release of key immune-mediators after inflammatory stimulus (reactive oxygen species (ROS), pro- and anti-inflammatory cytokines).

The intestine is a highly heterogeneous hollow organ with biological, mechanical and chemical differences between lumen and wall. A functional human intestine model able to recreate the in vivo dynamic nature as well as the native tissue morphology is demanded for disease research and ​drug discovery. Here, we present a system, which combines an engineered three-dimensional (3D) tubular-shaped intestine model (3D In-tube) with a custom-made microbioreactor to impart the key aspects of the in vivo microenvironment of the human intestine, mimicking the rhythmic peristaltic movement. We adapted a previously established bottom-up tissue engineering approach, to produce the 3D tubular-shaped lamina propria and designed a glass microbioreactor to induce the air–liquid interface ​condition and peristaltic-like motion. Our results demonstrate the production of a villi-like protrusion and a correct spatial differentiation of the intestinal epithelial cells in enterocyte-like as well as mucus-producing-like cells on the lumen side of the 3D In-tube. This dynamic platform offers a proof-of-concept model of the human intestine. Keywords: 3D engineered tubular-shaped intestine model, Air--liquid interface, Peristaltic-like motion, Extracellular matrix, Microbioreactor

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

Therapeutic approaches based on nanomedicine have garnered great attention in cancer research. In vitro biological models that better mimic in vivo conditions are crucial tools to more accurately predict their therapeutic efficacy in vivo. In this work, a new 3D breast cancer microtissue has been developed to recapitulate the complexity of the tumor microenvironment and to test its efficacy as screening platform for drug delivery systems. The proposed 3D cancer model presents human breast adenocarcinoma cells and cancer-associated fibroblasts embedded in their own ECM, thus showing several features of an in vivo tumor, such as overexpression of metallo-proteinases (MMPs). After demonstrating at molecular and protein level the MMP2 overexpression in such tumor microtissues, we used them to test a recently validated formulation of endogenous MMP2-responsive nanoparticles (NP). The presence of the MMP2-sensitive linker allows doxorubicin release from NP only upon specific enzymatic cleavage of the peptide. The same NP without the MMP-sensitive linker and healthy breast microtissues were also produced to demonstrate NP specificity and selectivity. Cell viability after NP treatment confirmed that controlled drug delivery is achieved only in 3D tumor microtissues suggesting that the validation of therapeutic strategies in such 3D tumor model could predict human response. Statement of Significance A major issue of modern cancer research is the development of accurate and predictive experimental models of human tumors consistent with tumor microenvironment and applicable as screening platforms for novel therapeutic strategies. In this work, we developed and validated a new 3D microtissue model of human breast tumor as a testing platform of anti-cancer drug delivery systems. To this aim, biodegradable nanoparticles responsive to physiological changes specifically occurring in tumor microenvironment were used. Our findings clearly demonstrate that the breast tumor microtissue well recapitulates in vivo physiological features of tumor tissue and elicits a specific response to microenvironmentally-responsive nanoparticles compared to healthy tissue. We believe this study is of particular interest for cancer research and paves the way to exploit tumor microtissues for several testing purposes.

Despite advances in the development of in vitro tissue models such as reconstructed human skin, the questions in dermatological research, which can be addressed with these models, are limited. This is mainly due to the lack of key extra-cellular components, which makes dermal compartment an incomplete approach to human ECM in vivo. Today there is a growing awareness of the fact that the ECM has a functional importance as a dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes. We established a human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In our model dermal ECM presents laminin, fibronectin, hyaluronic acid, elastin and collagen arranged and organized as in the human counterpart. As proof of the physiological relevance of such tissue environment we demonstrate that -by using adult human skin cells-it is possible to generate follicle-like structures in vitro resembling what occurs in vivo in the fetal skin. Immunotypization evidences an inward-oriented differentiation of the follicular-like structures through immunopositivity for epithelial stem cell markers such as p63 and K19. Moreover we succeeded in innervating this human skin by inducing rat dorsal root ganglion neurons axon ingrowth and demonstrate the effective functionality of the nerve network. Neurofilaments network infiltrates the entire native dermis ECM until reaching the epidermis, as demonstrated by immunopositivity to neurofilament-M and second harmonic generation imaging. Calcium imaging demonstrates that electrical current travel in the neuronal network proving the its sensing functionality. Our results demonstrate the fundamental role of ECM in morphogenesis, corroborating the awareness of its importance in building up functional in vitro skin model for fundamental research applications.

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.

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time-consuming and expensive. In this work, we propose a cost-effective, easy-to-produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

Solute transport through soft gels and tissues is intimately coupled to mechanical stress and deformation of the macromolecular network. The aim of this study was to investigate the effect of periodic mechanical stimuli upon solute transport through agarose gels at different concentrations. For this purpose it was experimentally evaluated the materials parameters that govern the coupling between elasto-dynamic and solute transport: hydraulic conductivity (K), elastic modulus (HA), and macromolecular diffusivity (Dg) along with their strain dependence behavior. Mechanical activated solute transport simulation was carried out in order to elucidate the role of amplitude and frequency of soliciting mechanical stimuli on mass kinetics release. Results show that mechanical loading affects the release of macromolecules from a gel in a frequency and strain dependent manner. These findings pave the way for novel strategies for the design and engineering of smart drug delivery devices with transport mechanisms triggered by mechanical stimuli. © 2008 American Institute of Chemical Engineers AIChE J, 2008

Objective: To identify possible sonographic prenatal parameters and postnatal parameters in order to obtain more bankable cord blood units (CBUs) containing a high number of primitive progenitor cells, allowing CBUs to be used as a source of haematopoietic progenitors for clinical transplantation. Study design: Prospective study undertaken in the Department of Gynaecology, Obstetrics and Reproductive Science, Second University of Naples, Italy. In total, 219 unrelated CBU donors were enrolled. Ultrasound parameters (biparietal diameter, head circumference, abdominal circumference, femur length, estimation of fetal weight, umbilical artery pulsatility index), collected at hospital admission, together with birth weight and placental weight, were correlated with bankable CBU parameters (CBU volume, total nucleated cell count, CD34+ cell count). Results: Femur length and abdominal circumference correlated positively with bankable CBUs. Receiver operating curve analysis showed that these parameters can identify bankable CBUs. Conclusions: This is the first prospective study to show the relationship between ultrasonographic fetal parameters at term and the possibility of obtaining high-quality CBUs. As such, cord blood banking could be improved worldwide by performing low-cost ultrasonographic scans. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. In this chapter, we examine the promise and shortcomings of “top-down” and “bottom-up” approaches for creating engineered biological tissues. In top-down approaches, the cells are expected to populate the scaffold and create the appropriate extracellular matrix and microarchitecture often with the aid of a bioreactor that furnish the set of stimuli required for an optimal cellular viability. Specifically, we survey the role of cell material interaction on oxygen metabolism in three-dimensional (3D) in vitro cultures as well as the time and space evolution of the transport and biophysical properties during the development of de novo synthesized tissue-engineered constructs. We show how to monitor and control the evolution of these parameters that is of crucial importance to process biohybrid constructs in vitro as well as to elaborate reliable mathematical model to forecast tissue growth under specific culture conditions. Furthermore, novel strategies such as bottom-up approaches to build tissue constructs in vitro are examined. In this fashion, tissue building blocks with specific microarchitectural features are used as modular units to engineer biological tissues from the bottom up. In particular, the attention will be focused on the use of cell seeded microbeads as functional building blocks to realize 3D complex tissue. Finally, a challenge will be the potential integration of bottom-up techniques with more traditional top-down approaches to create more complex tissues than are currently achievable using either technique alone by optimizing the advantages of each technique.

Bottom-up approach is an appealing strategy to build complex three-dimensional (3D) viable tissues in vitro starting from microtissue precursors (μTP). In this work we biofabricated a thick dermal-like tissue by sequentially combining two steps: a μTPs production and assembly followed by tissue maturation in a purpose-built bioreactor. The μTPs were produced by first seeding bovine primary fibroblasts on gelatine microparticles and then cultivating them in stirring conditions until a thick layer of ∼80μm of de novo synthesized extracellular matrix uniformly covered the microparticle surface. The μTPs were then loaded into a cylindrical chamber (2mm in depth and 35mm in diameter) and let to maturate and assemble into a 3D viable biohybrid tissue under specific fluid flow conditions. Several combinations of perfusion and/or tangential fluid flow were applied and their effect on the tissue formation and maturation was assessed. Results show that structural composition and mechanical features of the final 3D bioengineered tissue are strongly affected by the hydrodynamic environment and demonstrate that by optimizing culture conditions a 3D viable tissue with properties similar to that of native derma could be produced. © 2011 Mary Ann Liebert, Inc.