טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLevy-Mishali Meital
SubjectDifferentiation of Skeletal Muscle Cells on PLLA/PLGA
Composite Scaffolds
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Shulamit Levenberg
Full Thesis textFull thesis text - English Version


Abstract

Cells feel and respond to the stiffness of their substrate. In order to advance tissue engineering research, scaffold stiffness must be optimized for a given application and cell type. The goal of this study was to investigate the effect of scaffold stiffness on seeded myoblast cells. Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization and/or viability accordingly. Here, myoblasts were cultured on composite 3 Dimensional (3D) Poly lactic acid (PLLA)/Poly lactic co glycolic acid (PLGA) scaffolds of varied elasticity. 5 various scaffolds were created with gradient of PLLA and PLGA concentrations using salt leaching technique and characterized by scanning electron microscopy (SEM), stress-strain curves and degradation experiment. The shrinking effect of the scaffolds caused by cell contraction forces, as well as cell organization, myotube formation, viability, and protein/gene expression were also examined. The results of the research suggest that scaffold stiffness has effect on cell response. As PLLA concentration in the scaffold increased, it presented firmer pore structure, higher young's modulus and slower degradation rate. Alternatively, high PLGA concentration induced more amorphous pore structure, lower young's modulus and higher degradation rate. The PLLA polymer provided stiffness for scaffold which supported myotube formation (EPLLA ~ 15.2 kPa) while PLGA scaffold (EPLGA ~ 0.3 kPa) failed to support cell viability and fusion into myotubes. Yet, as indicated by 2D culturing experiment, myoblasts fusion into myotubes and expression of muscle markers was not affected by the polymer composition. Introduction of collagen microsponges into the PLLA/PLGA scaffolds moderately effected the cells, but the PLGA-collagen scaffold showed significantly improved mechanical properties (EPLGA-Collagen ~ 7.3 kPa) and cell organization. To confirm the understanding that scaffold stiffness was the reason for cell response, elimination of scaffold shrinking by affixing apparatus was applied and comparable cell organization with the non-shrinking scaffolds was obtained. Overall, the results indicate that compliant scaffold as the PLGA is insufficient for myoblast differentiation into myotubes. On the other hand, excessively firm scaffold could not lead to parallel myotube organization. Hence, optimal stiffness of scaffold for mayoblasts can be achieved by PLLA /PLGA blend.