Multistage Nanocarrier Based on an Oil Core-Graphene Oxide Shell.

Potent synthetic drugs, as well as biomolecules extracted from plants, have been investigated for their selectivity toward cancer cells. The main limitation in cancer treatment is the ability to bring such molecules within each single cancer cell, which requires accumulation in the peritumoral region followed by homogeneous spreading within the entire tissue. In the last decades, nanotechnology has emerged as a powerful tool due to its ability to protect the drug during blood circulation and allow enhanced accumulation around the leaky regions of the tumor vasculature. However, the ideal size for accumulation of around 100 nm is too large for effective penetration into the dense collagen matrix. Therefore, we propose a multistage system based on graphene oxide nanosheet-based quantum dots (GOQDs) with dimensions that are 12 nm, functionalized with hyaluronic acid (GOQDs-HA), and deposited using the layer-by-layer technique onto an oil-in-water nanoemulsion (O/W NE) template that is around 100 nm in size, previously stabilized by a biodegradable polymer, chitosan. The choice of a biodegradable core for the nanocarrier is to degrade once inside the tumor, thus promoting the release of smaller compounds, GOQDs-HA, carrying the adsorbed anticancer compound, which in this work is represented by curcumin as a model bioactive anticancer molecule. Additionally, modification with HA aims to promote active targeting of stromal and cancer cells. Cell uptake experiments and preliminary penetration experiments in three-dimensional microtissues were performed to assess the proposed multistage nanocarrier.

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

A functional 3D full-thickness model for comprehending the interaction between airway epithelium and connective tissue in cystic fibrosis.

Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodeling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodeling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to fully replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodeling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed gland-like structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

Bioengineered Wound Healing Skin Models: The Role of Immune Response and Endogenous ECM to Fully Replicate the Dynamic of Scar Tissue Formation In Vitro.

The healing of deep skin wounds is a complex phenomenon evolving according with a fine spatiotemporal regulation of different biological events (hemostasis, inflammation, proliferation, remodeling). Due to the spontaneous evolution of damaged human dermis toward a fibrotic scar, the treatment of deep wounds still represents a clinical concern. Bioengineered full-thickness skin models may play a crucial role in this direction by providing a deep understanding of the process that leads to the formation of fibrotic scars. This will allow (i) to identify new drugs and targets/biomarkers, (ii) to test new therapeutic approaches, and (iii) to develop more accurate in silico models, with the final aim to guide the closure process toward a scar-free closure and, in a more general sense, (iv) to understand the mechanisms involved in the intrinsic and extrinsic aging of the skin. In this work, the complex dynamic of events underlaying the closure of deep skin wound is presented and the engineered models that aim at replicating such complex phenomenon are reviewed. Despite the complexity of the cellular and extracellular events occurring during the skin wound healing the gold standard assay used to replicate such a process is still represented by planar in vitro models that have been largely used to identify the key factors regulating the involved cellular processes. However, the lack of the main constituents of the extracellular matrix (ECM) makes these over-simplistic 2D models unable to predict the complexity of the closure process. Three-dimensional bioengineered models, which aim at recreating the closure dynamics of the human dermis by using exogenous biomaterials, have been developed to fill such a gap. Although interesting mechanistic effects have been figured out, the effect of the inflammatory response on the ECM remodelling is not replicated yet. We discuss how more faithful wound healing models can be obtained by creating immunocompetent 3D dermis models featuring an endogenous ECM.

Geometrical confinement controls cell, ECM and vascular network alignment during the morphogenesis of 3D bioengineered human connective tissues.

Engineered tissues featuring aligned ECM possess superior regenerative capabilities for the healing of damaged aligned tissues. The morphofunctional integration in the host's injury site improves if the aligned ECM elicits the unidirectional growth of vascular network. In this work we used a bottom-up tissue engineering strategy to produce endogenous and highly aligned human connective tissues with the final aim to trigger the unidirectional growth of capillary-like structures. Engineered microtissues, previously developed by our group, were casted in molds featured by different aspect ratio (AR) to obtain final centimeter-sized macrotissues differently shaped. By varying the AR from 1 to 50 we were able to vary the final shape of the macrotissues, from square to wire. We demonstrated that by increasing the AR of the maturation space hosting the microtissues, it was possible to control the alignment of the neo-synthesized ECM. The geometrical confinement conditions at AR = 50, indeed, promoted the unidirectional growth and assembly of the collagen network. The wire-shaped tissues were characterized by parallel arrangement of the collagen fiber bundles, higher persistence length and speed of migrating cells and superior mechanical properties than the square-shaped macrotissues. Interestingly, the aligned collagen fibers elicited the unidirectional growth of capillary-like structures. STATEMENT OF SIGNIFICANCE: Alignment of preexisting extracellular matrices by using mechanical cues modulating cell traction, has been widely described. Here, we show a new method to align de novo synthesized extracellular matrix components in bioengineered connective tissues obtained by means of a bottom-up tissue engineering approach. Building blocks are cast in maturation chambers, having different aspect ratios, in which the in vitro morphogenesis process takes place. High aspect ratio chambers (corresponding to wire-shaped tissues) triggered spontaneous alignment of collagenous network affecting cell polarization, migration and tensile properties of the tissue as well. Aligned ECM provided a contact guidance for the formation of highly polarized capillary-like network suggesting an in vivo possible application to trigger fast angiogenesis and perfusion in damaged aligned tissues.

Intrinsic Abnormalities of Cystic Fibrosis Airway Connective Tissue Revealed by an In Vitro 3D Stromal Model.

Cystic fibrosis is characterized by lung dysfunction involving mucus hypersecretion, bacterial infections, and inflammatory response. Inflammation triggers pro-fibrotic signals that compromise lung structure and function. At present, several in vitro cystic fibrosis models have been developed to study epithelial dysfunction but none of these focuses on stromal alterations. Here we show a new cystic fibrosis 3D stromal lung model made up of primary fibroblasts embedded in their own extracellular matrix and investigate its morphological and transcriptomic features. Cystic fibrosis fibroblasts showed a high proliferation rate and produced an abundant and chaotic matrix with increased protein content and elastic modulus. More interesting, they had enhanced pro-fibrotic markers and genes involved in epithelial function and inflammatory response. In conclusion, our study reveals that cystic fibrosis fibroblasts maintain in vitro an activated pro-fibrotic state. This abnormality may play in vivo a role in the modulation of epithelial and inflammatory cell behavior and lung remodeling. We argue that the proposed bioengineered model may provide new insights on epithelial/stromal/inflammatory cells crosstalk in cystic fibrosis, paving the way for novel therapeutic strategies.

Bioengineered Skin Substitutes: the Role of Extracellular Matrix and Vascularization in the Healing of Deep Wounds.

The formation of severe scars still represents the result of the closure process of extended and deep skin wounds. To address this issue, different bioengineered skin substitutes have been developed but a general consensus regarding their effectiveness has not been achieved yet. It will be shown that bioengineered skin substitutes, although representing a valid alternative to autografting, induce skin cells in repairing the wound rather than guiding a regeneration process. Repaired skin differs from regenerated skin, showing high contracture, loss of sensitivity, impaired pigmentation and absence of cutaneous adnexa (i.e., hair follicles and sweat glands). This leads to significant mobility and aesthetic concerns, making the development of more effective bioengineered skin models a current need. The objective of this review is to determine the limitations of either commercially available or investigational bioengineered skin substitutes and how advanced skin tissue engineering strategies can be improved in order to completely restore skin functions after severe wounds.

Pre-vascularized dermis model for fast and functional anastomosis with host vasculature.

Skin engineering for clinical applications has gained numerous advances, however, most of the available dermis substitutes are exogenous matrices acting for a limited time. Indeed, after implantation these matrices need to be colonized by host cells such as fibroblast and endothelial cells which respectively produce their own extracellular matrix and set a vascular network within the construct. These steps are essential to guarantee implant efficacy, but they may require a long time depending on tissue dimension and lesion severity. Here we show the pre-vascularization process of a dermis equivalent featured by an endogenous matrix produced by human dermal fibroblasts. In this environment, endothelial cells were able to develop mature capillary-like-structures (CLS) as demonstrated by both the inner lumen and the positivity for alpha-SMA, laminin and collagen. The pre-vascularized dermis model (PVD) so obtained had a human matrix populated by fibroblasts as well as a complex capillary network making the construct ready to be implanted. These features make the graft very easy to handle during the surgery. In vivo results showed that 7 days after implantation CLS effectively anastomosed with host vessels. Therefore we argue that the proposed PVD may represent a new class of dermis substitute of strong clinical interest.

Non-invasive Production of Multi-Compartmental Biodegradable Polymer Microneedles for Controlled Intradermal Drug Release of Labile Molecules.

Transdermal drug delivery represents an appealing alternative to conventional drug administration systems. In fact, due to their high patient compliance, the development of dissolvable and biodegradable polymer microneedles has recently attracted great attention. Although stamp-based procedures guarantee high tip resolution and reproducibility, they have long processing times, low levels of system engineering, are a source of possible contaminants, and thermo-sensitive drugs cannot be used in conjunction with them. In this work, a novel stamp-based microneedle fabrication method is proposed. It provides a rapid room-temperature production of multi-compartmental biodegradable polymeric microneedles for controlled intradermal drug release. Solvent casting was carried out for only a few minutes and produced a short dissolvable tip made of polyvinylpyrrolidone (PVP). The rest of the stamp was then filled with degradable poly(lactide-co-glycolide) (PLGA) microparticles (µPs) quickly compacted with a vapor-assisted plasticization. The outcome was an array of microneedles with tunable release. The ability of the resulting microneedles to indent was assessed using pig cadaver skin. Controlled intradermal delivery was demonstrated by loading both the tip and the body of the microneedles with model therapeutics; POXA1b laccase from Pleurotus ostreatus is a commercial enzyme used for the whitening of skin spots. The action and indentation of the enzyme-loaded microneedle action were assessed in an in vitro skin model and this highlighted their ability to control the kinetic release of the encapsulated compound.

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.

Controlling the orientation of a cell-synthesized extracellular matrix by using engineered gelatin-based building blocks.

In tissue engineering there is growing interest in fabricating highly engineered platforms designed to instruct cells towards the synthesis of tissues that reproduce their natural counterpart. In this context, a fundamental factor to take into account is the control over the final tissue orientation, especially for what concerns the replication of load-bearing tissues whose functions are strictly related to their microstructural organization. Starting from this point, in this work we have engineered a gelatin-based hydrogel in order to be patterned by 2-photon polymerization (2PP) lithography for the fabrication of instructive free standing building blocks designed to produce anisotropic collagen-based µtissues. Biological results clearly highlighted the strong relationship between µtissue orientation and such topographies, which resulted in a crucial element in the production of highly anisotropic µtissues.

In vitro activation of the neuro-transduction mechanism in sensitive organotypic human skin model.

Recent advances in tissue engineering have encouraged researchers to endeavor the production of fully functional three-dimensional (3D) thick human tissues in vitro. Here, we report the fabrication of a fully innervated human skin tissue in vitro that recapitulates and replicates skin sensory function. Previous attempts to innervate in vitro 3D skin models did not demonstrate an effective functionality of the nerve network. In our approach, we initially engineer functional human skin tissue based on fibroblast-generated dermis and differentiated epidermis; then, we promote rat dorsal root ganglion (DRG) neurons axon ingrowth in the de-novo developed tissue. Neurofilaments network infiltrates the entire native dermis extracellular matrix (ECM), as demonstrated by immunofluorescence and second harmonic generation (SHG) imaging. To prove sensing functionality of the tissue, we use topical applications of capsaicin, an agonist of transient receptor protein-vanilloid 1 (TRPV1) channel, and quantify calcium currents resulting from variations of Ca++ concentration in DRG neurons innervating our model. Calcium currents generation demonstrates functional cross-talking between dermis and epidermis compartments. Moreover, through a computational fluid dynamic (CFD) analysis, we set fluid dynamic conditions for a non-planar skin equivalent growth, as proof of potential application in creating skin grafts tailored on-demand for in vivo wound shape.

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.

Endogenous human skin equivalent promotes in vitro morphogenesis of follicle-like structures.

Three-dimensional constructs formed by cells embedded in an exogenous scaffold could not represent a faithful in vitro replica of native and functional tissues. In this work we produced an endogenous human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In such an organotypic context we demonstrated 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 detected the presence of versican within the intricate network of the dermal compartment, suggesting its role as an inductive factor for the morphogenesis of appendage-like structures. These results support the importance of the repository and regulatory role of the endogenous extra cellular matrix in guiding tissue morphogenesis. The microenvironment provided by the endogenous human skin equivalent preserves p63 and K19 positive cells and could finally be involved in: (i) triggering the arrangement of the keratinocytes in follicle-like structures; (ii) promoting the convolute profile of the derma-epidermal junction and (iii) improving epidermis barrier function. We argue that the nature of dermal compartment plays a role in directing epithelial cell fate and function in vitro.

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.