Ischemic heart diseases are the leading cause of death worldwide: approximately 3.8 million men and 3.4 million women die each year from coronary heart diseases (World Health Organization data). Among them, acute myocardial infarction (MI), usually known as a heart attack, accounts for about 23% of deaths each year. The term MI refers to the acute coronary syndrome due to the obstruction of one or more branches of the coronary arteries that blocks blood flow and determines necrosis of the myocardium. The process of cardiac myocyte death and regeneration is part of the normal heart homeostasis; however, in pathological conditions the balance between renewal and death is altered, the proliferative capacity remains only in the non-infarcted area and in the border zone (interface between the infarcted and healthy tissue), the process of myocyte death begins to predominate, hypertrophy becomes evident and the number of ventricular myocytes starts to decrease. For this reason, a MI causes a permanent damage: the infarcted area turns into a fibrous scar and the left ventricle undergoes a detrimental remodeling process, characterized by dilatation, hypertrophy and worsening of cardiac function. MI treatment is usually based on drug administration, surgery or minimally invasive therapies, that, unfortunately, are unable to completely restore the functionality of the damaged myocardium; for this reason, at least one third of patients who survive undergoes heart failure, i.e. heart inability to provide sufficient pump action to maintain blood flow to meet the needs of the body. At present, cardiac transplantation remains the gold standard of cardiac replacement therapy in patients showing heart failure symptoms. However, the supply of donor hearts is limited and anti-rejection immunosuppressive therapies need to be administered. Alternative forms of cardiac replacement therapy are being investigated (ventricle assist devices and total artificial heart) to bridge the time to heart transplantation or to permanently replace the heart when organ transplantation is impossible because of age and other comorbid conditions. In this context, the need to develop therapies able to stimulate cardiac tissue repair, sustain a functional improvement of the adjacent muscle areas and limit ventricular remodeling has emerged as a challenging target in the last years. This thesis work faces the need of new alternative therapies to treat infarcted patients through the design of bioengineered constructs (scaffolds), both cellularized and not, to be implanted on the damaged area, with the final aim of stimulating cardiac tissue repair. Such an application requires compliance with strict requirements: biocompatibility, biomimetics, promotion of cell adhesion, proliferation and eventually differentiation, degradation post-implantation, adequate mechanical properties, interconnected porosity, moderate hydrophilicity. The first experimental step faced in this work was the selection of the scaffold forming material. To this aim, attention was directed to the large family of polyurethanes (PURs), that, thanks to their high chemical versatility, allow a fine tuning of the physico-chemical, biological and mechanical properties of the resulting polymer. A series of poly(ester urethane)s based on poly(ε-caprolactone) diol (PCL, Mn=2000 g/mol), 1,4-diisocyanatobutane (BDI) and different chain extenders was synthesized and thoroughly characterized, with the final aim of selecting the most promising polymer for the intended application. For instance, the chain extenders were an amino acid derivative diamine (L-Lysine ethyl ester), a cyclic diol (1,4-cyclohexane dimethanol -CDM-), an amino acid derivative diol (N-Boc-serinol) and a custom made diamine, containing an enzymatically degradable peptide (Ala-Ala sequence). PCL was selected to confer biodegradability to the final PUR, while BDI is an aliphatic diisocyanate, whose degradation product is expected to be putrescine, a non-toxic diamine, essential for cell growth and differentiation. Spectroscopic and chromatographic analysis demonstrated the successful synthesis of the designed PURs. Thermal characterization highlighted that all the synthesized PURs can be suitable substrates for biomedical applications. Contact angle measurements revealed slightly hydrophobic surfaces with contact angle values in the range 76-90°. Moreover, a correlation between surface domain morphologies and thermal properties was highlighted and a relationship between biological response and surface morphology was observed. PURs that possessed the highest crystallinity (PURs synthesized by using N-Boc serinol or the custom made peptide as chain extender) showed high order spherulitic morphologies; on the opposite, the PUR with the lowest crystallinity (PUR synthesized by using L-Lysine ethyl ester as chain extender) showed a clear phase separation, but no spherulites and lamellar structures were detected. In addition, a better C2C12 myoblast adhesion and proliferation were observed for polymers having surface with less ordered structure (PURs synthesized by using L-Lysine ethyl ester or CDM as chain extender). In the series of polyurethanes studied, the most promising polymer for the repair of soft contractile tissues was that synthesized by using L-Lysine ethyl ester as chain extender (acronym K-BC2000), since it better matched the elastomeric behavior of muscle tissues (Young Modulus 8.5 ± 0.5 MPa, stress at break 9.8 ± 1.7 MPa, strain at break 682.7 ± 41.3 %) and, in the meantime, cell tests performed with myoblasts C2C12 on this substrate showed high viability, adequate cell adhesion, spreading and proliferation. Moreover, durability tests (strain 15 %, frequency 1 Hz, 432000 cycles) performed on compression molded K-BC2000 films revealed the capability of the selected PUR to withstand cyclic stresses and the absence of macroscopic damages or fracture. Hydrolytic degradation of K-BC2000 dense films was negligible within 8 weeks, while they completely degraded enzymatically in 6 weeks. Once the scaffold forming material was selected, it was processed in the form of porous matrices by Thermally Induced Phase Separation (TIPS) using dimethyl sulfoxide (DMSO) as solvent. With the final aim of developing non-cellularized cardiac patches, two scaffolds differing in the concentration of the starting PUR solution in DMSO (5 or 10 %w/v) were fabricated by applying a thermal cooling gradient during the quenching phase, to produce scaffolds with oriented pores, that mimic muscle tissue anisotropy. Scaffolds were obtained with open and interconnected pores, having sizes ranging from tens μm to more than 200 μm and porosity of 80-90%. As desired, the pores were elongated in the direction of the applied cooling gradient. The scaffold fabricated using a 10 %w/v concentrated PUR solution (acronym K-BC2000 10% w/v) demonstrated potential for future application in cardiac tissue engineering/regenerative medicine from a mechanical (Young Modulus 1.6 ± 0.6 MPa, stress at break 0.2 ± 0.0 MPa, strain at break 30.0 ± 1.7 % in dry state), thermal and biological point of view. Biological tests with human bone marrow-derived mesenchymal stem cells (hBMMSCs), indeed, showed its ability to support cell adhesion and growth. As a consequence of scaffold morphology, cells cultured on it adopted a stretched spindle-shape morphology and aligned in parallel to each other. Furthermore, very preliminary investigations on the differentiation potential of hBMMSCs seeded on it revealed loss of stemness (C-kit down-regulation) and upregulation of a myogenic marker (Mef-2C), after 8 days of static cell culture. However, further work must be conducted to better understand cell differentiation pathways. In addition, the scaffold K-BC2000 10 %w/v turned out easy to sew up and showed good handiness and a satisfactory resistance to suture, that made it potentially suturable on heart infarcted region. This scaffold was further mechanically characterized at both the macro- and nano-scale by tensile tests and Indentation-Type Atomic Force Miscoscopy (IT-AFM), respectively. Both tests were conducted in wet conditions to mimic in vivo environment. Moreover, the construct was morphologically characterized at the nanoscale through Atomic Force Miscoscopy (AFM). The interest in scaffold nanoscale properties arose from the growing evidence that both nanoscale features and stiffness influence cell behavior in terms of cytoskeletal organization, adhesion and differentiation. The study of scaffold nano-morphology showed, for the first time, that thermally induced phase separation under application of a thermal cooling gradient can be exploited to successfully produce oriented scaffolds not only at the microscale (aligned fibers), as assessed by Scanning Electron Microscopy (SEM), but also at the nanoscale (aligned fibrils, due to polymer chain orientation in the direction of the applied cooling gradient). As a consequence, cells interacting with the developed substrates sense the presence of an anisotropic structure at two different scales: at a larger scale, cells sense pore orientation that should favor their alignment in a preferred direction, thus mimicking striated muscle fiber organization; at the nanoscale, cells sense polymer chain orientation that can positively influence the cytoskeletal organization and morphology of the single cell. At the nanoscale, the scaffold K-BC2000 10 %w/v exhibited Young Modulus values of hundreds kPa, mimicking the stiffness of native cardiac cells, and Young Modulus values of few MPa that could favor cell adhesion and electromechanical coupling. At the macro-scale, the scaffold showed a Young Modulus of about 300 kPa in wet conditions, in accordance with the stiffness required for its application in cardiac tissue engineering/regenerative medicine. Finally, thermally induced phase separation under application of a thermal cooling gradient was exploited to fabricate matrices able to withstand up to 10 days of dynamic cell culture in a stress-controlled bioreactor, with the final aim of developing cellularized constructs for application in cardiac TERM. Three different scaffolds were produced by changing the quenching parameters (-20 °C for 5 h, -20 °C overnight, -80 °C for 3 h) adopted to induce phase separation in a 12 %w/v K-BC2000 solution in DMSO. The application of a thermal cooling gradient during the quenching phase resulted in scaffolds with oriented and stretched pores in a preferred direction, i.e. the direction of the applied cooling gradient, only when the quenching phase was conducted at -80 °C for 3 h. The lack of stretched and elongated pores in both the scaffolds quenched at -20 °C is probably correlated to the concurrent effects of both the freezing conditions and the concentration of the starting solution. However, from a structural point of view, i.e. porosity (about 80%), pore size and pore distribution (pore size ranges between tens and hundreds μm), all the scaffolds turned out suitable for application in cardiac TERM. The scaffold fabricated by quenching the 12 %w/v PUR solution at -20 °C for 5 h was selected for further studies since its mechanical properties (Young Modulus 2.5 ± 0.3 MPa, stress at break 0.4 ± 0.0 MPa, strain at break 127.5 ± 4.8 % in dry state, Young Modulus 1.1 ± 0.1 MPa, stress at break 0.3 ± 0.0 MPa, strain at break 171.3 ± 5.0 % in wet state) matched at best the requirements for application in cardiac TERM and it turned out suitable to withstand 10 days of mechanical stimulation (0.4 ± 0.2 N, 1 Hz) without rupture or damages. Adipose tissue-derived stromal cells (ADSCs) were seeded on it and subjected to dynamic cell culture in a stress-controlled bioreactor in the presence of a serum free medium enriched with a cardiogenic cocktail developed and patented by the Swiss Stem Cell Foundation (Lugano, Switzerland). Differently from bioreactors typically described in literature, that apply an uniaxial sinusoidal strain (usually in the range 5-15% to mimic cardiac muscle deformation during each cardiac cycle) to the grasped patches, the dynamic cell culture system used in this work applied an uniaxial sinusoidal stress to the cellularized patch, thus making it possible to mechanically stimulate cells with a mechanical stress approximately equivalent to the diastolic stress of the native heart. In addition, in view of the potential clinical application of the developed cellularized patch, both the cell culture medium and the cardiogenic cocktail did not contain any animal derivatives or chemical agents that may negatively affect cell genomic stability. Cells seeded on the porous substrates adopted a stretched spindle-shape morphology and aligned in the direction of the applied mechanical stimulus. The combination of a 3D support and mechanical stimulation, in the presence of serum free medium enriched with the cardiogenic cocktail, led to an at least 2 fold increase in mRNA expression of several cardiac markers (Nkx2.5, Mef-2C, HAND2 and MHC), compared to cardiogenic induction conducted in 2D (cell culture plates). These results suggest, for the first time, that even a weak mechanical stimulus (the applied cyclic stress (0.4 ± 0.1 N, 1 Hz) resulted in a cyclic strain of about 1%) can have significant effects on stem cell commitment towards a cardiac phenotype. However, further studies are necessary to confirm these promising results and assess the influence of the amplitude of the applied stress on cardiac marker expression. Moreover, strategies to promote cell migration need to be implemented, since cells mainly localized on scaffold surface and penetrated within approximately 200-300 μm.

Bioengineered Patches in the Regeneration of Infarcted Myocardial Tissue / Boffito, Monica. - (2014).

Bioengineered Patches in the Regeneration of Infarcted Myocardial Tissue.

BOFFITO, MONICA
2014

Abstract

Ischemic heart diseases are the leading cause of death worldwide: approximately 3.8 million men and 3.4 million women die each year from coronary heart diseases (World Health Organization data). Among them, acute myocardial infarction (MI), usually known as a heart attack, accounts for about 23% of deaths each year. The term MI refers to the acute coronary syndrome due to the obstruction of one or more branches of the coronary arteries that blocks blood flow and determines necrosis of the myocardium. The process of cardiac myocyte death and regeneration is part of the normal heart homeostasis; however, in pathological conditions the balance between renewal and death is altered, the proliferative capacity remains only in the non-infarcted area and in the border zone (interface between the infarcted and healthy tissue), the process of myocyte death begins to predominate, hypertrophy becomes evident and the number of ventricular myocytes starts to decrease. For this reason, a MI causes a permanent damage: the infarcted area turns into a fibrous scar and the left ventricle undergoes a detrimental remodeling process, characterized by dilatation, hypertrophy and worsening of cardiac function. MI treatment is usually based on drug administration, surgery or minimally invasive therapies, that, unfortunately, are unable to completely restore the functionality of the damaged myocardium; for this reason, at least one third of patients who survive undergoes heart failure, i.e. heart inability to provide sufficient pump action to maintain blood flow to meet the needs of the body. At present, cardiac transplantation remains the gold standard of cardiac replacement therapy in patients showing heart failure symptoms. However, the supply of donor hearts is limited and anti-rejection immunosuppressive therapies need to be administered. Alternative forms of cardiac replacement therapy are being investigated (ventricle assist devices and total artificial heart) to bridge the time to heart transplantation or to permanently replace the heart when organ transplantation is impossible because of age and other comorbid conditions. In this context, the need to develop therapies able to stimulate cardiac tissue repair, sustain a functional improvement of the adjacent muscle areas and limit ventricular remodeling has emerged as a challenging target in the last years. This thesis work faces the need of new alternative therapies to treat infarcted patients through the design of bioengineered constructs (scaffolds), both cellularized and not, to be implanted on the damaged area, with the final aim of stimulating cardiac tissue repair. Such an application requires compliance with strict requirements: biocompatibility, biomimetics, promotion of cell adhesion, proliferation and eventually differentiation, degradation post-implantation, adequate mechanical properties, interconnected porosity, moderate hydrophilicity. The first experimental step faced in this work was the selection of the scaffold forming material. To this aim, attention was directed to the large family of polyurethanes (PURs), that, thanks to their high chemical versatility, allow a fine tuning of the physico-chemical, biological and mechanical properties of the resulting polymer. A series of poly(ester urethane)s based on poly(ε-caprolactone) diol (PCL, Mn=2000 g/mol), 1,4-diisocyanatobutane (BDI) and different chain extenders was synthesized and thoroughly characterized, with the final aim of selecting the most promising polymer for the intended application. For instance, the chain extenders were an amino acid derivative diamine (L-Lysine ethyl ester), a cyclic diol (1,4-cyclohexane dimethanol -CDM-), an amino acid derivative diol (N-Boc-serinol) and a custom made diamine, containing an enzymatically degradable peptide (Ala-Ala sequence). PCL was selected to confer biodegradability to the final PUR, while BDI is an aliphatic diisocyanate, whose degradation product is expected to be putrescine, a non-toxic diamine, essential for cell growth and differentiation. Spectroscopic and chromatographic analysis demonstrated the successful synthesis of the designed PURs. Thermal characterization highlighted that all the synthesized PURs can be suitable substrates for biomedical applications. Contact angle measurements revealed slightly hydrophobic surfaces with contact angle values in the range 76-90°. Moreover, a correlation between surface domain morphologies and thermal properties was highlighted and a relationship between biological response and surface morphology was observed. PURs that possessed the highest crystallinity (PURs synthesized by using N-Boc serinol or the custom made peptide as chain extender) showed high order spherulitic morphologies; on the opposite, the PUR with the lowest crystallinity (PUR synthesized by using L-Lysine ethyl ester as chain extender) showed a clear phase separation, but no spherulites and lamellar structures were detected. In addition, a better C2C12 myoblast adhesion and proliferation were observed for polymers having surface with less ordered structure (PURs synthesized by using L-Lysine ethyl ester or CDM as chain extender). In the series of polyurethanes studied, the most promising polymer for the repair of soft contractile tissues was that synthesized by using L-Lysine ethyl ester as chain extender (acronym K-BC2000), since it better matched the elastomeric behavior of muscle tissues (Young Modulus 8.5 ± 0.5 MPa, stress at break 9.8 ± 1.7 MPa, strain at break 682.7 ± 41.3 %) and, in the meantime, cell tests performed with myoblasts C2C12 on this substrate showed high viability, adequate cell adhesion, spreading and proliferation. Moreover, durability tests (strain 15 %, frequency 1 Hz, 432000 cycles) performed on compression molded K-BC2000 films revealed the capability of the selected PUR to withstand cyclic stresses and the absence of macroscopic damages or fracture. Hydrolytic degradation of K-BC2000 dense films was negligible within 8 weeks, while they completely degraded enzymatically in 6 weeks. Once the scaffold forming material was selected, it was processed in the form of porous matrices by Thermally Induced Phase Separation (TIPS) using dimethyl sulfoxide (DMSO) as solvent. With the final aim of developing non-cellularized cardiac patches, two scaffolds differing in the concentration of the starting PUR solution in DMSO (5 or 10 %w/v) were fabricated by applying a thermal cooling gradient during the quenching phase, to produce scaffolds with oriented pores, that mimic muscle tissue anisotropy. Scaffolds were obtained with open and interconnected pores, having sizes ranging from tens μm to more than 200 μm and porosity of 80-90%. As desired, the pores were elongated in the direction of the applied cooling gradient. The scaffold fabricated using a 10 %w/v concentrated PUR solution (acronym K-BC2000 10% w/v) demonstrated potential for future application in cardiac tissue engineering/regenerative medicine from a mechanical (Young Modulus 1.6 ± 0.6 MPa, stress at break 0.2 ± 0.0 MPa, strain at break 30.0 ± 1.7 % in dry state), thermal and biological point of view. Biological tests with human bone marrow-derived mesenchymal stem cells (hBMMSCs), indeed, showed its ability to support cell adhesion and growth. As a consequence of scaffold morphology, cells cultured on it adopted a stretched spindle-shape morphology and aligned in parallel to each other. Furthermore, very preliminary investigations on the differentiation potential of hBMMSCs seeded on it revealed loss of stemness (C-kit down-regulation) and upregulation of a myogenic marker (Mef-2C), after 8 days of static cell culture. However, further work must be conducted to better understand cell differentiation pathways. In addition, the scaffold K-BC2000 10 %w/v turned out easy to sew up and showed good handiness and a satisfactory resistance to suture, that made it potentially suturable on heart infarcted region. This scaffold was further mechanically characterized at both the macro- and nano-scale by tensile tests and Indentation-Type Atomic Force Miscoscopy (IT-AFM), respectively. Both tests were conducted in wet conditions to mimic in vivo environment. Moreover, the construct was morphologically characterized at the nanoscale through Atomic Force Miscoscopy (AFM). The interest in scaffold nanoscale properties arose from the growing evidence that both nanoscale features and stiffness influence cell behavior in terms of cytoskeletal organization, adhesion and differentiation. The study of scaffold nano-morphology showed, for the first time, that thermally induced phase separation under application of a thermal cooling gradient can be exploited to successfully produce oriented scaffolds not only at the microscale (aligned fibers), as assessed by Scanning Electron Microscopy (SEM), but also at the nanoscale (aligned fibrils, due to polymer chain orientation in the direction of the applied cooling gradient). As a consequence, cells interacting with the developed substrates sense the presence of an anisotropic structure at two different scales: at a larger scale, cells sense pore orientation that should favor their alignment in a preferred direction, thus mimicking striated muscle fiber organization; at the nanoscale, cells sense polymer chain orientation that can positively influence the cytoskeletal organization and morphology of the single cell. At the nanoscale, the scaffold K-BC2000 10 %w/v exhibited Young Modulus values of hundreds kPa, mimicking the stiffness of native cardiac cells, and Young Modulus values of few MPa that could favor cell adhesion and electromechanical coupling. At the macro-scale, the scaffold showed a Young Modulus of about 300 kPa in wet conditions, in accordance with the stiffness required for its application in cardiac tissue engineering/regenerative medicine. Finally, thermally induced phase separation under application of a thermal cooling gradient was exploited to fabricate matrices able to withstand up to 10 days of dynamic cell culture in a stress-controlled bioreactor, with the final aim of developing cellularized constructs for application in cardiac TERM. Three different scaffolds were produced by changing the quenching parameters (-20 °C for 5 h, -20 °C overnight, -80 °C for 3 h) adopted to induce phase separation in a 12 %w/v K-BC2000 solution in DMSO. The application of a thermal cooling gradient during the quenching phase resulted in scaffolds with oriented and stretched pores in a preferred direction, i.e. the direction of the applied cooling gradient, only when the quenching phase was conducted at -80 °C for 3 h. The lack of stretched and elongated pores in both the scaffolds quenched at -20 °C is probably correlated to the concurrent effects of both the freezing conditions and the concentration of the starting solution. However, from a structural point of view, i.e. porosity (about 80%), pore size and pore distribution (pore size ranges between tens and hundreds μm), all the scaffolds turned out suitable for application in cardiac TERM. The scaffold fabricated by quenching the 12 %w/v PUR solution at -20 °C for 5 h was selected for further studies since its mechanical properties (Young Modulus 2.5 ± 0.3 MPa, stress at break 0.4 ± 0.0 MPa, strain at break 127.5 ± 4.8 % in dry state, Young Modulus 1.1 ± 0.1 MPa, stress at break 0.3 ± 0.0 MPa, strain at break 171.3 ± 5.0 % in wet state) matched at best the requirements for application in cardiac TERM and it turned out suitable to withstand 10 days of mechanical stimulation (0.4 ± 0.2 N, 1 Hz) without rupture or damages. Adipose tissue-derived stromal cells (ADSCs) were seeded on it and subjected to dynamic cell culture in a stress-controlled bioreactor in the presence of a serum free medium enriched with a cardiogenic cocktail developed and patented by the Swiss Stem Cell Foundation (Lugano, Switzerland). Differently from bioreactors typically described in literature, that apply an uniaxial sinusoidal strain (usually in the range 5-15% to mimic cardiac muscle deformation during each cardiac cycle) to the grasped patches, the dynamic cell culture system used in this work applied an uniaxial sinusoidal stress to the cellularized patch, thus making it possible to mechanically stimulate cells with a mechanical stress approximately equivalent to the diastolic stress of the native heart. In addition, in view of the potential clinical application of the developed cellularized patch, both the cell culture medium and the cardiogenic cocktail did not contain any animal derivatives or chemical agents that may negatively affect cell genomic stability. Cells seeded on the porous substrates adopted a stretched spindle-shape morphology and aligned in the direction of the applied mechanical stimulus. The combination of a 3D support and mechanical stimulation, in the presence of serum free medium enriched with the cardiogenic cocktail, led to an at least 2 fold increase in mRNA expression of several cardiac markers (Nkx2.5, Mef-2C, HAND2 and MHC), compared to cardiogenic induction conducted in 2D (cell culture plates). These results suggest, for the first time, that even a weak mechanical stimulus (the applied cyclic stress (0.4 ± 0.1 N, 1 Hz) resulted in a cyclic strain of about 1%) can have significant effects on stem cell commitment towards a cardiac phenotype. However, further studies are necessary to confirm these promising results and assess the influence of the amplitude of the applied stress on cardiac marker expression. Moreover, strategies to promote cell migration need to be implemented, since cells mainly localized on scaffold surface and penetrated within approximately 200-300 μm.
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