Despite several progresses in terms of early diagnosis and prevention, coronary heart disease and heart attack are still the most common causes of death in Western countries. Cardiac Regenerative Medicine appears to be a promising alternative to pharmacologic treatment or organ transplantation although cellular therapies based on progenitor cell injection are still problematic. Myocardial Tissue Engineering (MTE) is a Regenerative Medicine approach, able to integrate cell therapy with the use of polymeric substrates (myocardial scaffolds or heart patches) that can reduce cell loss and potentially prevent remodeling and fibrotic processes. In in vivo MTE strategies, scaffolds should be capable to recruit cardiac progenitor and differentiated cells which are present in the adult heart and promote their proliferation/differentiation. Consequently, these substrates have to meet strict requirements in terms of biological, mechanical, surface, biodegradability properties. In particular, they should mimic the natural Extracellular Matrix (ECM), achieving a chemical, morphological and mechanical biomimicry. In this thesis work, biomimetic polymeric constructs are proposed as heart patches for myocardial functions restoration and cardiac tissue regeneration after a myocardial infarction. These constructs were prepared as dense (films) and porous scaffolds from synthetic biodegradable polyurethanes (PURs), that were selected because of their chemical versatility and elastomeric mechanical behavior. In detail, poly(ester urethanes) and poly(ether ester urethanes) were synthesized starting from poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) as macrodiols, 1,4-diisocyanatobutane (BDI) as diisocyanate, L-Lysine Ethyl Ester and Alanine-Alanine-Lysine (AAK) as chain extenders. PCL was selected to confer biodegradability to the final PUR, while PEG was added in low amounts to tune wettability, mechanical and biological properties of films and scaffolds. BDI was selected since it is an aliphatic diisocyanate and its biodegradation products are non toxic. L-Lysine Ethyl Ester and Alanine-Alanine-Lysine were selected as chain extenders for their biocompatible degradation products and because biodegradability properties can be tuned thanks to the introduction of AAK peptide in the polymer chain, since the Alanine-Alanine sequence is a target for the elastase enzyme. Spectroscopic and chromatographic analysis demonstrated the successful synthesis of the designed PURs. Films, obtained by hot pressing, were thermally and mechanically characterized. They were all characterized by an elastomeric behaviour with elastic moduli ranging from 7 to 14 MPa. Contact Angle measurements revealed slightly hydrophobic film surfaces with contact angle values in the range 78-94°. Based on mechanical testing results, two formulations (KBC1250 and KBC1250-E1500-20) were processed into scaffolds by Thermally Induced Phase Separation (TIPS) with the application of a thermal gradient, that allowed the formation of stretched and unidirectional pores. These microstructures, that were studied trough Scanning Electron Microscopy (SEM) micrographs, mimicked the striated muscle tissue. Tensile tests revealed lower mechanical properties for scaffolds with respect to films (elastic moduli of about 2 MPa, maximum stress in the range 0.3-0.6 MPa and maximum strain in the range 120-160%). Nevertheless, both porous substrates have suitable elastomeric behaviours for contractile tissues regeneration, with elastic moduli closer to that of myocardial tissue (20 kPa-0.5 MPa) for porous constructs. Viability tests on cardiomyocytes revealed the best cell response for dense film and porous scaffold obtained from the polyurethane KBC1250, with an increasing viability for the porous substrate, which is ascribable to its microstructure features. Hydrolytic and enzymatic degradation tests showed a faster weight loss for the scaffolds in the presence of the enzyme (lipase), probably because the enzymatic degradation mechanism takes place on surface and porous constructs exhibit a larger exposed area. Moreover, elastase degradation tests demonstrated that additional degradation through biological processes can be achieved for these polymers by the simple introduction of specifically designed peptide sequences in the PUR backbone. Based on biological and mechanical characterization, dense and porous KBC1250 constructs were selected to be surface functionalized by the covalent attachment of Arginine-Glycine-Aspartic Acid (RGD) peptides, in order to promote cell adhesion and proliferation and obtain a “chemical biomimicry”. These peptide is the active sequence of laminin and fibronectin proteins, that are responsible for the adhesion of cells to the ECM. This chemical modification was performed on films homogeneously or through a silicone mask, in order to create a linear RGD micropattern. Analogous laminin and fibronectin patterns revealed promising from literature data in promoting mesenchymal stem cell differentiation and cardiomyocyte spatial organization. Spectroscopic analysis, increase in surface wettability and colorimetric assays demonstrated the successful surface modification of PUR films and scaffolds. Functionalized films were characterized by an optimal hydrophylicity (65°) and peptide density (3.2 and 1.7 nmol/mm2) for cell adhesion promotion. Peptide quantification and cell viability tests demonstrated indirectly the successful use of the siloxane mask in allowing the RGD attachment on the uncovered areas. Cell viability tests and SEM micrographs showed the positive effect of film modification on cardiomyocyte viability and adhesion. Although the same successful peptide attachment was obtained on scaffold surfaces, the biological response on this type of substrate was just slightly higher after the surface modification. This result can be explained considering that the porous surfaces, although an increase in wettability after the functionalization, are still far from the optimal contact angle value promoting cell attachment or that some not covalently bound peptides inside the scaffold microstructure can induce cell detachment and apoptosis. KBC1250 substrates were also tested with human cardiac Mesenchymal Stem Cells (MSCs) and Cardiosphere Derived Cells (CDCs), which belong to the progenitor cell reservoir present in the adult myocardium. MSCs were detected and extracted for the first time in a human heart by Dr Rachel Oldershaw and Dr Annette Meeson of Newcastle University, while CDCs were extracted from human biopsies through the creation of Cardiospheres (cell clusters containing cardiac stem cells, differentiating progenitors and spontaneously differentiated cardiomyocytes). Subsequent culture of these CDCs and MSCs on KBC1250 films and scaffolds revealed promising results for the application of the porous constructs in MTE. Viability cell test showed that scaffold promoted CDC and MSC growth. Although a decrease in cell health after 14 culture days was observed for MSCs, these preliminary tests on human cardiac progenitor cells showed that KBC1250 porous scaffolds are suitable substrates for both CDC and MSC proliferation.

Materials and Nanostructured Coatings for Soft Tissue Regeneration / Silvestri, Antonella. - STAMPA. - (2013). [10.6092/polito/porto/2507432]

Materials and Nanostructured Coatings for Soft Tissue Regeneration

SILVESTRI, ANTONELLA
2013

Abstract

Despite several progresses in terms of early diagnosis and prevention, coronary heart disease and heart attack are still the most common causes of death in Western countries. Cardiac Regenerative Medicine appears to be a promising alternative to pharmacologic treatment or organ transplantation although cellular therapies based on progenitor cell injection are still problematic. Myocardial Tissue Engineering (MTE) is a Regenerative Medicine approach, able to integrate cell therapy with the use of polymeric substrates (myocardial scaffolds or heart patches) that can reduce cell loss and potentially prevent remodeling and fibrotic processes. In in vivo MTE strategies, scaffolds should be capable to recruit cardiac progenitor and differentiated cells which are present in the adult heart and promote their proliferation/differentiation. Consequently, these substrates have to meet strict requirements in terms of biological, mechanical, surface, biodegradability properties. In particular, they should mimic the natural Extracellular Matrix (ECM), achieving a chemical, morphological and mechanical biomimicry. In this thesis work, biomimetic polymeric constructs are proposed as heart patches for myocardial functions restoration and cardiac tissue regeneration after a myocardial infarction. These constructs were prepared as dense (films) and porous scaffolds from synthetic biodegradable polyurethanes (PURs), that were selected because of their chemical versatility and elastomeric mechanical behavior. In detail, poly(ester urethanes) and poly(ether ester urethanes) were synthesized starting from poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) as macrodiols, 1,4-diisocyanatobutane (BDI) as diisocyanate, L-Lysine Ethyl Ester and Alanine-Alanine-Lysine (AAK) as chain extenders. PCL was selected to confer biodegradability to the final PUR, while PEG was added in low amounts to tune wettability, mechanical and biological properties of films and scaffolds. BDI was selected since it is an aliphatic diisocyanate and its biodegradation products are non toxic. L-Lysine Ethyl Ester and Alanine-Alanine-Lysine were selected as chain extenders for their biocompatible degradation products and because biodegradability properties can be tuned thanks to the introduction of AAK peptide in the polymer chain, since the Alanine-Alanine sequence is a target for the elastase enzyme. Spectroscopic and chromatographic analysis demonstrated the successful synthesis of the designed PURs. Films, obtained by hot pressing, were thermally and mechanically characterized. They were all characterized by an elastomeric behaviour with elastic moduli ranging from 7 to 14 MPa. Contact Angle measurements revealed slightly hydrophobic film surfaces with contact angle values in the range 78-94°. Based on mechanical testing results, two formulations (KBC1250 and KBC1250-E1500-20) were processed into scaffolds by Thermally Induced Phase Separation (TIPS) with the application of a thermal gradient, that allowed the formation of stretched and unidirectional pores. These microstructures, that were studied trough Scanning Electron Microscopy (SEM) micrographs, mimicked the striated muscle tissue. Tensile tests revealed lower mechanical properties for scaffolds with respect to films (elastic moduli of about 2 MPa, maximum stress in the range 0.3-0.6 MPa and maximum strain in the range 120-160%). Nevertheless, both porous substrates have suitable elastomeric behaviours for contractile tissues regeneration, with elastic moduli closer to that of myocardial tissue (20 kPa-0.5 MPa) for porous constructs. Viability tests on cardiomyocytes revealed the best cell response for dense film and porous scaffold obtained from the polyurethane KBC1250, with an increasing viability for the porous substrate, which is ascribable to its microstructure features. Hydrolytic and enzymatic degradation tests showed a faster weight loss for the scaffolds in the presence of the enzyme (lipase), probably because the enzymatic degradation mechanism takes place on surface and porous constructs exhibit a larger exposed area. Moreover, elastase degradation tests demonstrated that additional degradation through biological processes can be achieved for these polymers by the simple introduction of specifically designed peptide sequences in the PUR backbone. Based on biological and mechanical characterization, dense and porous KBC1250 constructs were selected to be surface functionalized by the covalent attachment of Arginine-Glycine-Aspartic Acid (RGD) peptides, in order to promote cell adhesion and proliferation and obtain a “chemical biomimicry”. These peptide is the active sequence of laminin and fibronectin proteins, that are responsible for the adhesion of cells to the ECM. This chemical modification was performed on films homogeneously or through a silicone mask, in order to create a linear RGD micropattern. Analogous laminin and fibronectin patterns revealed promising from literature data in promoting mesenchymal stem cell differentiation and cardiomyocyte spatial organization. Spectroscopic analysis, increase in surface wettability and colorimetric assays demonstrated the successful surface modification of PUR films and scaffolds. Functionalized films were characterized by an optimal hydrophylicity (65°) and peptide density (3.2 and 1.7 nmol/mm2) for cell adhesion promotion. Peptide quantification and cell viability tests demonstrated indirectly the successful use of the siloxane mask in allowing the RGD attachment on the uncovered areas. Cell viability tests and SEM micrographs showed the positive effect of film modification on cardiomyocyte viability and adhesion. Although the same successful peptide attachment was obtained on scaffold surfaces, the biological response on this type of substrate was just slightly higher after the surface modification. This result can be explained considering that the porous surfaces, although an increase in wettability after the functionalization, are still far from the optimal contact angle value promoting cell attachment or that some not covalently bound peptides inside the scaffold microstructure can induce cell detachment and apoptosis. KBC1250 substrates were also tested with human cardiac Mesenchymal Stem Cells (MSCs) and Cardiosphere Derived Cells (CDCs), which belong to the progenitor cell reservoir present in the adult myocardium. MSCs were detected and extracted for the first time in a human heart by Dr Rachel Oldershaw and Dr Annette Meeson of Newcastle University, while CDCs were extracted from human biopsies through the creation of Cardiospheres (cell clusters containing cardiac stem cells, differentiating progenitors and spontaneously differentiated cardiomyocytes). Subsequent culture of these CDCs and MSCs on KBC1250 films and scaffolds revealed promising results for the application of the porous constructs in MTE. Viability cell test showed that scaffold promoted CDC and MSC growth. Although a decrease in cell health after 14 culture days was observed for MSCs, these preliminary tests on human cardiac progenitor cells showed that KBC1250 porous scaffolds are suitable substrates for both CDC and MSC proliferation.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11583/2507432
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