Casale, Costantino (2013) 3D COMPLEX ENDOGENOUS TISSUE EQUIVALENT IN VITRO: PROCESSING AND BIOTECHNOLOGICAL APPLICATION. [Tesi di dottorato]

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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.

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