Investigation of poly(3-hydroxybutyrate) (P3HB) - poly-β-alanine (PBA) fibrous tissue scaffolds in the effectiveness of skeletal muscle regeneration

The aim of the thesis is to obtain an in vitro functional skeletal muscle using tissue engineering approaches for repairing damage in skeletal muscle tissue. To accomplish this aim, the research utilizes fibrous matrices of poly-β-alanine (PBA) doped poly(3-hydroxybutyrate) (P3HB) which can mimic natural muscle tissue and exhibit piezoelectric characteristics. The effectiveness of the matrices in myogenic differentiation was assessed in static culture conditions with the C2C12 mouse myoblast cell line and dynamic culture conditions using a hybrid bioreactor that provides mechanical, electrical, and electromechanical stimulations. In the first part of the thesis, random and aligned P3HB/PBA nanofiber matrices were produced using the electrospinning method. The matrices were characterized for their chemical, thermal, morphological, mechanical, crystallographic, and piezoelectric properties. In contrast to the literature, both random and aligned P3HB-based matrices exhibited an increase in the d33 piezoelectric constant, measured as 5 pC/N and 5.3 pC/N, respectively. In static culture studies, cells on aligned matrices formed regular myotubular structures and showed dense microvilli. Viability analyses, morphological examinations, myogenic gene expression and marker analyses determined that aligned matrices were myogenically compatible and well supported myogenic differentiation. In the second part, the effect of mechanical and electrical stimulations on the mechanical and morphological properties of the matrices was investigated before cell culture studies. Subsequently, dynamic culture studies were conducted using a hybrid bioreactor providing mechanical, electrical, and electromechanical stimulations. Notably, mechanical stimulation with 5% strain, 0.5 Hz frequency, and a 20 min stretching/2 h resting (M-D1 group) and electrical stimulation with a 2 ms pulsing period, 0.4 V voltage, and a 20 min pulsing/2 h resting (E-D2 group) yielded the most successful results in terms of myogenic differentiation and maturation. Electromechanical stimulation studies (EM-D) were carried out by combining M-D1 and E-D2 stimulation parameters to investigate the synergistic effects on myogenic differentiation and maturation. The EM-D group showed a 105% increase in tensile strength. The gene expressions related to myogenic maturation were analyzed in the EM-D group and it was determined that the expression of the myosin heavy chain (MHC) was ≈ 23 times higher compared to the M-D1 group and ≈ 13 times higher compared to the E-D2 group. Similarly, the expression of neurofilament (NF-H) showed a ≈ 95-fold increase compared to the M-D1 group and ≈ 11 times higher compared to the E-D2 group. The expression of acetylcholine receptor-α (AChR-α) was found to increase ≈ 13 times compared to the M-D1 group and ≈ 1.5 times compared to the E-D2 group. These findings indicate that myogenic maturation is highly supported in the EM-D group. The thesis demonstrates that aligned P3HB/PBA nanofiber matrices successfully support myogenic differentiation through their morphological, mechanical, and piezoelectric properties. Additionally, it has been shown that when these matrices are simultaneously supported with controlled electromechanical stimulations, an in vitro functional skeletal muscle tissue can be obtained. For the future clinical use of this engineered tissue product, the matrices should be interacted with satellite cells, and in vivo animal studies should be conducted.