Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. Synthetic polymers gather a series of advantages because they can be chemically modified obtaining hydrogels with desired biochemical, mechanical and degradation properties. Among the synthetic class of polymer, poly(amidoamine)s (PAAs) based hydrogels, containing the 2,2-bisacrylamidoacetic acid-agmatine monomeric unit, are known to have promising biological properties because they can enhance cellular adhesion by interacting with the arginine-glycine-aspartate (RGD)-binding motif of integrins. In this thesis, we first exploited the potential of RGD-mimetic PAA based hydrogels for the establishment of three dimensional (3D) cell culture systems with tunable mechanical and degradation properties. We presented two different approaches for the fabrication of PAA based hydrogels. In the first approach, we copolymerized the PAA oligomers (OPAA) with poly(ethylene glycol) (PEG) to fabricate hydrogels with improved biological, mechanical and optical transparency properties. In the second approach, a synthetic polymeric precursor for cell encapsulation was realized with the aim to combine, by grafting, the adhesive properties of PAAs with the good mechanical integrity of poly(2-hydrox-yethyl)methacrylate (PHEMA) based hydrogels. We demonstrated that with our approaches it is possible to tune the mechanical properties of our hydrogels keeping the adhesive properties typical of pure PAA and providing a tunable platform for 3D cell culture. Once evaluated the PAA potential to support 3D cell culture, we adopted a biologically and mechanically driven design to fabricate OPAAs macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. OPAAs were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. Furthermore, OPAAF supported cell adhesion, proliferation and adipogenesis in vitro together with adipose tissue and vessels infiltration in vivo. ECM is known to be the ideal scaffold for tissue engineering application. In the detail, decellularized adipose tissue proved to have an adipoinuctive effect on adipose stromal cells. Therefore we decided to further implement the biological properties of the OPAAF by decoration with a devitalized adipose tissue matrix. We first optimized a protocol for the 3D adipogenic differentiation of human primary cells in vitro using a perfusion bioreactor system. The OPAAF efficiently supported human adipose stromal cells (hASCs) differentiation into mature adipocytes. We then decorated the OPAAF with engineered devitalized adipose tissue matrix deposited by hASCs. Two different protocols have been adopted for the decoration, in order to investigate the role of a differentiated and stromal matrix in adipogenic induction. With our approach we efficiently generated hybrid scaffolds combining the positive features of both synthetic and natural biomaterials. The obtained hybrid constructs showed to have an adipoinductive effect on hASCs in absence of any growth factor in vitro and promoted adipogenesis in vivo. Overall, these results proved that our approach can provide an alternative strategy for adipose tissue reconstruction based on the use of patients cells for the generation of custom made hybrid scaffolds. The engineered adipose tissue could also serve as a 3D model of fat tissue at different stages of differentiation with potential use in drug testing.

AN ALTERNATIVE STRATEGY FOR ADIPOSE TISSUE RECONSTRUCTION / E. Rossi ; internal advisor: P. Milani; external advisor: I. Martin ; supervisor: C. Lenardi. - : . DIPARTIMENTO DI FISICA, 2017 Mar 02. ((28. ciclo, Anno Accademico 2016. [10.13130/e-rossi_phd2017-03-02].

AN ALTERNATIVE STRATEGY FOR ADIPOSE TISSUE RECONSTRUCTION

E. Rossi
2017-03-02

Abstract

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. Synthetic polymers gather a series of advantages because they can be chemically modified obtaining hydrogels with desired biochemical, mechanical and degradation properties. Among the synthetic class of polymer, poly(amidoamine)s (PAAs) based hydrogels, containing the 2,2-bisacrylamidoacetic acid-agmatine monomeric unit, are known to have promising biological properties because they can enhance cellular adhesion by interacting with the arginine-glycine-aspartate (RGD)-binding motif of integrins. In this thesis, we first exploited the potential of RGD-mimetic PAA based hydrogels for the establishment of three dimensional (3D) cell culture systems with tunable mechanical and degradation properties. We presented two different approaches for the fabrication of PAA based hydrogels. In the first approach, we copolymerized the PAA oligomers (OPAA) with poly(ethylene glycol) (PEG) to fabricate hydrogels with improved biological, mechanical and optical transparency properties. In the second approach, a synthetic polymeric precursor for cell encapsulation was realized with the aim to combine, by grafting, the adhesive properties of PAAs with the good mechanical integrity of poly(2-hydrox-yethyl)methacrylate (PHEMA) based hydrogels. We demonstrated that with our approaches it is possible to tune the mechanical properties of our hydrogels keeping the adhesive properties typical of pure PAA and providing a tunable platform for 3D cell culture. Once evaluated the PAA potential to support 3D cell culture, we adopted a biologically and mechanically driven design to fabricate OPAAs macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. OPAAs were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. Furthermore, OPAAF supported cell adhesion, proliferation and adipogenesis in vitro together with adipose tissue and vessels infiltration in vivo. ECM is known to be the ideal scaffold for tissue engineering application. In the detail, decellularized adipose tissue proved to have an adipoinuctive effect on adipose stromal cells. Therefore we decided to further implement the biological properties of the OPAAF by decoration with a devitalized adipose tissue matrix. We first optimized a protocol for the 3D adipogenic differentiation of human primary cells in vitro using a perfusion bioreactor system. The OPAAF efficiently supported human adipose stromal cells (hASCs) differentiation into mature adipocytes. We then decorated the OPAAF with engineered devitalized adipose tissue matrix deposited by hASCs. Two different protocols have been adopted for the decoration, in order to investigate the role of a differentiated and stromal matrix in adipogenic induction. With our approach we efficiently generated hybrid scaffolds combining the positive features of both synthetic and natural biomaterials. The obtained hybrid constructs showed to have an adipoinductive effect on hASCs in absence of any growth factor in vitro and promoted adipogenesis in vivo. Overall, these results proved that our approach can provide an alternative strategy for adipose tissue reconstruction based on the use of patients cells for the generation of custom made hybrid scaffolds. The engineered adipose tissue could also serve as a 3D model of fat tissue at different stages of differentiation with potential use in drug testing.
LENARDI, CRISTINA
RGD mimetic ; Poly(amidoamine) ; 3D porous network ; mechanical properties ; extracellular Matrix ; decoration ; adipose tissue regeneration
Settore FIS/03 - Fisica della Materia
Centro Interdisciplinare Materiali ed Interfacce Nanostrutturati - CIMAINA
AN ALTERNATIVE STRATEGY FOR ADIPOSE TISSUE RECONSTRUCTION / E. Rossi ; internal advisor: P. Milani; external advisor: I. Martin ; supervisor: C. Lenardi. - : . DIPARTIMENTO DI FISICA, 2017 Mar 02. ((28. ciclo, Anno Accademico 2016. [10.13130/e-rossi_phd2017-03-02].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/466585
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