Ανάπτυξη εμφυτευμάτων για χρήση στο μυοκάρδιο της δεξιάς κοιλίας μετά από πνευμονική υπέρταση

Abstract

Pulmonary hypertension is defined as an increase in pulmonary arterial pressure at values higher than the normal ones. This increase has as a direct consequence of causing right ventricular pressure overload, which initially leads to hypertrophy and ultimately to right heart failure or even death. Indeed, pulmonary arterial hypertension (PAH) is a lethal disease with high rates of mortality and morbidity. Also, if not diagnosed and treated promptly, then average survival is only 2.8 years. Nonetheless, prognosis and diagnosis is still determined by the hypertrophy and by the right ventricular dilation and dysfunction. There are three main therapeutic options for the treatment of pulmonary hypertension, pharmacological treatments, mechanical support of the right ventricle (using special devices), and the use of biomaterials and implants. Regarding the latter option, a thorough literature review shows that there is no scientific documentation reporting on implants developed for use in right ventricular myocardium after pulmonary hypertension. The presentstudy aims at covering this gap by proposing a Road Map for the development of biomaterials for implants that can be used to treat (prevent, treat) pulmonary hypertension. Thus, this investigation comprises two distinct stages. The first one concerns the selection, qualification and optimization of a biomaterial that can be a candidate for being used for this purpose. The second one highlights the development (in terms of biology, surgery practice and performance, microstructure, chemistry, and mechanics) of the interface and the interactions between the implant and the outer surface of the myocardium, where the membrane is placed, as a major issue for predicting and understanding the performance of a biomaterial in the treatment of the disease, following the appropriate medicalprotocol proposed by the surgeon as far as its application in the heart is concerned. In the light of the specific application in the present case and the level of the research undertaken in this study, the most suitable geometry of the biomaterial was selected to be that of a membrane. Then, eight qualification criteria were defined, which must satisfy a biomaterial in order to be selected as candidate for use in the treatment of the disease. These are (1) the ability to create a membrane, (2) the good quality of the membrane, (3) the flexibility in order to be able to wrap around the epicardial tissue, (4) the tuning ability to regulate the mechanical properties in order to approach those of the cardiac tissue, (5) the tuning ability to regulate the thickness, (6) neutral pH, (7) in vitro and in vivo biocompatibility, and (8) biodegradability within a specific period of time. A series of hydrogels were produced, starting with PEG (poly (ethylene glycol)) and its derivatives ofPEGSDA (PEG sebacate diacrylate) and OPF (oligo poly (ethylene glycol) fumarate), followed by hydrogels of natural polysaccharides based on alginic acid and chitosan. The hydrogels based on PEG and alginic acid did not meet the qualification criteria and were, therefore, rejected from further consideration and experimentation. However, chitosan hydrogels showed better behavior. Therefore, various preparation methods were tested in order to produce a membrane that meets the qualification criteria. The parameters tested were the molecular weight (medium and low molecular weight chitosan was used), the neutralization solution (NaOH, KOH, β-GP), and theneutralization procedure (immersion or drop wise). Any combination of these three factors affects the mechanism of hydrogel formation and therefore the quality of the final product. The membrane with the optimal properties was prepared by the gelation process, using NaOH solution as a neutralizing agent without solvent evaporation. The properties of this membrane, which met the selection criteria, were determined experimentally. The determination of the degree of deacetylation, of the structural features of the membrane, by using X-ray diffraction and infrared (FT-IR) spectroscopy, of the microstructure and the texture (porosity) of the membrane, of the thermal properties, the sorption ability in PBS and water at 37 ° C, and the mechanical properties, was experimentally carried out. The mechanical properties were measured by tensile strength experiments in dry and wet environment as well as after the membrane immersion in blood plasma and PBS at 37° C for estimating the degradation of mechanical properties in a physiological environment over time, and by dynamic mechanical analysis (DMA) measurements in a dry and wet environment, to determine the storage modulus (E ') and of the damping factor (tanδ). The above properties were measured in order to evaluate whether this membrane mimics the properties ofthe heart tissue. The results showed that the physicochemical properties and the microstructure allow the membrane to be regarded as a candidate for use in the particular application, while its mechanical properties are in good agreement with the values of the myocardial tissue. Then, the interface developed between the selected membrane and cells or cardiac tissue was studied. Invitro biological tests were performed in fibroblast cell culture (NIH3T3). The results showed evidence of viability and growth of fibroblasts in the chitosan membrane, suggesting the biocompatibility of the membrane. Then, in vivo tests, by implanting the membrane in Wistar rats, were conducted. The results showed that a surgeon can easily and reliably handle the membrane, the membrane was positioned accurately around the heart and remained firmly in place within the period of implantation (30 days), without causing any problems or death in animals. There was also evidence of degradation. After the animals euthanasia, the foreign body response recorded the bestpossible biological response to the implanted material, where a significant reduction in inflammatory and a prominent increase of newly formed vessels were recorded. Then, the chemistry of the interface between the membrane and the myocardial tissue after implantation of 2 and 7 days was studied. The heart tissue wrapped around with membrane was obtained from the animals and theinterface was separated. The detached surfaces were examined by infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The experimental findings were interpreted with the aid of results calculated theoretically by using the density functional theory (DFT) according to the B3LYP / 6-31G method. Both experimental and theoretical results suggested a strong interaction between membrane and tissue, attributed to ionic and hydrogen bonds. Finally, computational modeling was performed by developing a realistic simulation model of the human heart, in order to evaluate the effect of the membrane prepared on wall stress, deformation, and displacement inthe case of pulmonary hypertension. Before solving the problem, the three-dimensional geometry of the right ventricle was first constructed, followed by creating fluid and solid mesh and finally by defining the boundary conditions and the properties of the fluid and solid ventricular wall. Various thicknesses of membranes (1, 2, 3 mm) and Young modulus (0.3 - 0.7 MPa) were tested. The work ends with a discussion, the conclusions, and proposals for future research.