3D COMPLEX ENDOGENOUS TISSUE EQUIVALENT IN VITRO: PROCESSING AND BIOTECHNOLOGICAL APPLICATION

In this PhD thesis a novel bottom-up approach, recently published, has been improved and used to produce thick functional tissues. First of all the chapter 1 deals with a critical analysis of the literature which highlights how the morphological, mechanical e functional properties of tissue in vivo are strictly regulated by extra cellular matrix and cell cross talking has been. Then in chapter 2 the realization of 3D dermis equivalent in vitro tissue completely made up of endogenous extracellular matrix by assembling functional microtissues precursor is reported. In particular it is highlighted how the microscaffold degradation rate affects the assembly and maturation of de novo synthesized ECM in a 3D dermis equivalent in vitro by means of morphological analysis that highlights the composition and of ECM’s main components. In the chapter 3 the processing of a millimetric thick 3D dermis equivalent model is described underlining the capability of the process to overcome the classical diffusion limits of traditional top-down tissue engineering. A comparative survey between 3D dermis realized and native human dermis has been reported by means of immunofluorescence analyses. Moreover multiphoton microscopy, a non invasive microscopic techniques that guarantee high resolution capability, has been used to assess the capability of our model of responding to external stimuli such as the UVA, stating that: the 3D dermis model is able to recapitulate the intricate pathway that in vivo plays a fundamental role in phenomena in which the structural proteins of ECM are involved. In chapter 4 is reported the bio-fabrication of a skin full thickness in vitro model by testing different co-coltures condition in order to adjust the keratinocytes differentiation properties that are strongly affected by the growth factor present in the medium and by the interaction with an endogenous ECM such as the dermis equivalent tissue realized. Since the human skin model realized, is made up of endogenous ECM it could be used in vitro both as screening tool and as model for the study of healthy and disease skin, and in vivo as skin substitute in clinical application. At last in chapter 5 an industrial application of the tissue engineering strategy developed has been explored. Dermis equivalent of animal origin having different shape such as fiber, disk, sheet and “doll shoes” has been produced demonstrating the versatility of the bottom-up that guarantee control of the final tissues shape . Moreover, the realized tissues equivalent have been subjected to chrome tanning to demonstrate its possible application as an useful alternative to animal skin in the textile fields

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

Anti-Inflammatory activity of a polyphenolic extract from Arabidopsis thaliana in in vitro and in vivo models of Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the primary form of dementia in the elderly. One of the main features of AD is the increase in amyloid-beta (Aβ) peptide production and aggregation, leading to oxidative stress, neuroinflammation and neurodegeneration. Polyphenols are well known for their antioxidant, anti-inflammatory and neuroprotective effects and have been proposed as possible therapeutic agents against AD. Here, we investigated the effects of a polyphenolic extract of Arabidopsis thaliana (a plant belonging to the Brassicaceae family) on inflammatory response induced by Aβ. BV2 murine microglia cells treated with both Aβ25⁻35 peptide and extract showed a lower pro-inflammatory (IL-6, IL-1β, TNF-α) and a higher anti-inflammatory (IL-4, IL-10, IL-13) cytokine production compared to cells treated with Aβ only. The activation of the Nrf2-antioxidant response element signaling pathway in treated cells resulted in the upregulation of heme oxygenase-1 mRNA and in an increase of NAD(P)H:quinone oxidoreductase 1 activity. To establish whether the extract is also effective against Aβ-induced neurotoxicity in vivo, we evaluated its effect on the impaired climbing ability of AD Drosophila flies expressing human Aβ1⁻42. Arabidopsis extract significantly restored the locomotor activity of these flies, thus confirming its neuroprotective effects also in vivo. These results point to a protective effect of the Arabidopsis extract in AD, and prompt its use as a model in studying the impact of complex mixtures derived from plant-based food on neurodegenerative diseases

Nasal delivery devices: A comparative study on cadaver model

Nasal nebulization is a more effective method of delivering topical medication than nasal spray. The purpose of this study was to assess the deposition patterns of nebulization in delivering topical agents to the nasal cavities in the human cadaveric model using a color-based method. We have compared these following nasal devices: single-dose vial irrigation, syringe-irrigation, common nasal spray, Spray-sol, MAD nasal, and Rinowash nasal douche. Endoscopic images were recorded at six anatomical regions prior to and following each nasal device application and four reviewers evaluated the amount of surface area staining. At the nasal vestibule, the blue dye distribution achieved with Spray-sol was more extensive than nasal sprays. At inferior turbinate and nasal cavity floor, single dose vial, syringe, MAD nasal, Spray-sol, and Rinowash demonstrated a greater extent of dye distribution than nasal spray. At the middle turbinate, the average score of both Spray-sol and MAD nasal was significantly higher than other nasal investigated devices. At the nasopharynx, Spray-sol nebulization covers a surface significantly greater than other devices. Compared to traditional sprays, Spray-sol and MAD nasal provided a more effective method of delivering topical agents to the deeper and higher portions of the nasal cavities

Shared polygenic risk and causal inferences in amyotrophic lateral sclerosis

Objective To identify shared polygenic risk and causal associations in amyotrophic lateral sclerosis (ALS). Methods Linkage disequilibrium score regression and Mendelian randomization were applied in a large-scale, data-driven manner to explore genetic correlations and causal relationships between >700 phenotypic traits and ALS. Exposures consisted of publicly available genome-wide association studies (GWASes) summary statistics from MR Base and LD-hub. The outcome data came from the recently published ALS GWAS involving 20,806 cases and 59,804 controls. Multivariate analyses, genetic risk profiling, and Bayesian colocalization analyses were also performed. Results We have shown, by linkage disequilibrium score regression, that ALS shares polygenic risk genetic factors with a number of traits and conditions, including positive correlations with smoking status and moderate levels of physical activity, and negative correlations with higher cognitive performance, higher educational attainment, and light levels of physical activity. Using Mendelian randomization, we found evidence that hyperlipidemia is a causal risk factor for ALS and localized putative functional signals within loci of interest. Interpretation Here, we have developed a public resource () which we hope will become a valuable tool for the ALS community, and that will be expanded and updated as new data become available. Shared polygenic risk exists between ALS and educational attainment, physical activity, smoking, and tenseness/restlessness. We also found evidence that elevated low-desnity lipoprotein cholesterol is a causal risk factor for ALS. Future randomized controlled trials should be considered as a proof of causality. Ann Neurol 2019;85:470-481Peer reviewe

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

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

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 vitr

Realization a complex skin equivalent tissue

The aim of this work is the realization of a three-dimensional human skin equivalent tissue (3D-HSE) completely made up by endogenous extra cellular matrix (ECM). The realization of 3D-HSE has been pursued by means of a bottom-up tissue engineering strategy that firstly comprises the fabrication of functional micro-metric tissue precursors (mTPs) through dynamic seeding of fibroblasts on biodegradable porous microbeads with a controlled and tunable degradation rate. Then mTPs have been assembled in a 3D dermis tissue and the epidermal cells are lastly seeded on the top of dermal equivalent. In the development of epithelial stratum is important to maintein the culture submerged to allow complete epidermal cells coverage, melanocytes organization along epidermal basal membrane and early keratinocytes stratification, and later raising the developing culture to the air-liquid interface thus promoting epidermal differentiation. Epidermal differentiation markers have been investigated by means of the immunofluorescence analysis while collagen was assesses by multiphoton microscopy analysis. The functional endogenous dermis has a crucial role in the morphogenesis of a pluristratified epidermis. Transmission electron microscopy well show the fine structure of the dermal-epidermal junction confirming that epidermal layer is well anchored to the dermis. Moreover dermis-epidermis cross-talking is guaranteed as proved by the presence of basal membrane and this structure has a characteristic rete ridge morphology with typical epidermis appendages going deep through the underlying dermis resembling bulge-like structure. In view of its complexity, this skin model can be specifically used for tackling various problems in the chemical/pharmaceutical and cosmetics industry. Since an endogenous ECM is present it is possible to study not only the cellular response but also the ECM response in terms of change in assembly as well as composition to a drug