טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGvirtz Markish Maskit
SubjectRemodeling Porcine Extra Cellular Matrix in a Perfused
Bioreactor with Mechanical and Electrical
Stimulation
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Marcelle Machluf


Abstract

Scaffolds for cardiac tissue regeneration serve as a potential therapeutic approach for progressive heart failure following myocardial infarction (MI), and aim to improve MI recovery by replacing the scar tissue with a cardiac patch, thus providing mechanical support and avoiding hypertrophy. Nonetheless, seeding the cardiac patches offers additional advantage for heart regeneration by utilizing cells ability to produce inherent ECM and conduct electrical current.

We have developed a 15mm thickness scaffold, produced from decellularized porcine heart. The acellular scaffold allows perfusion of nutrients through its existing blood vessels to the entire tissue, without immunogenic response.

In the current research, we hypothesized that providing the recellularized patch with synchronized electrical-mechanical stimulation will promote expression of cardiac markers, improve cell viability, and increase cell penetration.

We have, therefore, designed and built a new modular; user-defined, synchronized electrical-mechanical stimulation apparatus, and constructed it into a newly developed perfusion bioreactor. The electrical signal was designed as a user-defined signal, and electrical pulses were given both in rectangular form as is practiced by other groups, as well as action potential shaped signal, which has not been done before, to mimic the cardiac surrounding for BmMSCs with no beating capabilities.

The cultivation studies included mechanical stimulation, electrical stimulation or their synchronized combination, thus aiming to mimic cardiac unique mechanical electrical coupling. During cultivation, we studied pH, glucose and cell viability. Following one months’ cultivation, the scaffolds were removed from the bioreactor and cell dispersion and differentiation were studied. Additional studies, in which MSCs were cocultured with neonatal rat cardiomyocytes, were performed to assess the influence of stimulations with cardiomyocytes on MSCs culture and differentiation. In addition, we explored the influence of action potential like stimulation on MSCs.

Our results demonstrated the significant effect that the different stimulations or combinations had on cell viability, with best results attributed to the mechanical stimulation, secondly, the electrical stimulation and synchronized dual-stimulation presented better viability results than perfusion and the control non-stimulated tissue. PH and glucose maintained steady state condition thus supporting the stability of cell growth.  H&E and Masson's trichrome staining revealed greater cell density at the mechanical stimulation cultivation. The electrical stimulation study implies to promote differentiation more than cell proliferation. Co-culture studies have an early suggestion of promoting differentiation toward cardiac lineage. Moreover, mechanical electrical stimulation with action potential shaped electrical stimulation maintains same viability results as synchronized mechanical electrical stimulation with rectangular stimuli.

In conclusion, we have demonstrated the effect of electrical, mechanical and synchronized stimulations to improve cell viability and proliferation, along with the importance of synchronized mechanical electrical stimulations to encourage differentiation from mesenchymal expression. Furthermore, co-cultured mesenchymal cells with cardiomyocytes presented initial indication for early cardiac markers expression, finally synchronized mechanical electrical - action potential shaped stimulations, presented the ability to maintain cell viability despite of the additional overload stimulation. Overall, we established the importance of mimicking physiological stimulation conditions and support its necessity to advance recellularization process for replacing MI necrotic tissue and improving the life quality and expectancy of MI patients.