Aloe Vera (Av) Katkılı Poli (Bütılen Adipat-Ko-Tereftalat) (Pbat) Doku İskelesi ile Fotobiyomodüle Kalp Yaması Geliştirilmesi
Özet
This thesis study has been financially supported by Hacettepe University Research Fund
(Project No: FDK-2020-18721).
The damage to the muscle that loses its ability to contract after myocardial infarction (MI) is not repaired. ‘Cardiac tissue engineering’ studies offer a new perspective on treating diseases that lead to heart failure due to the rest of the heart trying to fulfill the contraction function of the muscles in that area. In this study, it was aimed to produce Aloe vera (AV)-doped poly (butylene adipate-co-terephthalate) (PBAT) nanofibers that can be used as a cardiac patch after MI and to support in-vitro cardiomyogenic differentiation of the H9C2 rat cardiomyoblast cell line-cardiac microvascular endothelial cell (KMEH) co-culture cultured in these matrices under dynamic conditions in the presence and absence of infrared radiation.
In the first part, it was proposed to evaluate basal membrane-supported random (R-PBAT) and aligned (A-PBAT) nanofibrous matrices as a cardiac patch. Using Response Surface Methodology (RSM), electrospinning parameters were optimized to produce nanofibers with the desired average diameter and alignment values at a flow rate of 0.1 mL/hr of 10% (w/v) polymer solution in an electric field of 15 kV at 20 cm between the needle tip and collector. The average diameters were found for R-PBAT and A-PBAT nanofibers as 555126 nm and 417137 nm (91% alignment), respectively. In this section, it was determined that the A-PBAT structure improved H9C2 adhesion, spreading, and proliferation behavior. Therefore, it was decided to use aligned nanofibrous matrices for further studies.
In the second part, H9C2 and CMEC cells were characterized, and their specific growth rates and doubling times were found as 0.017 hr-1 – 41 hours and 0.0454 hr-1 – 22 hours, respectively. A cytotoxicity test was applied to determine the doping amount of AV in PBAT nanofibrous matrices, and it was decided that the doping concentration of AV as 10 mg/mL. Cell culture studies were performed to determine the effects of AV and PBM on cellular responses. Optimized PBM conditions were application from 20 cm distance for 3 minutes every other day.
In the last part of the thesis study, AV was doped into the A-PBAT nanofibrous matrix structure during the electrospinning process, and AV-PBAT nanofibers with an average diameter of 430±116 nm, 85% aligned, were produced. The doping process decreased the tensile strength of the matrices from 8.5±0.9 MPa to 5.9±0.4 MPa, the elastic modulus from 29.2±4.5 MPa to 18.4±0.8 MPa, and the elongation at break values from 48.0±10.55% to 25.7±2.72%. ATR-FTIR, Raman, EDX and EDX mapping, elemental analysis, and XPS methods were used to prove the chemical existence of AV in nanofibers. After the preliminary tests, it was decided to operate the electromechanical stimulation bioreactor by applying a 0.4 V electrical current with a 2 Hz stretching – 10 Hz resting pattern, 5% strain, and a 2 msec pulse period to samples regularly for 7 days under aseptic conditions. RT-qPCR analysis results revealed that PBM application, especially under dynamic conditions, caused a high increase in the expression levels of significant genes such as Cacnal1c, Cacnal1s, FGF-2, Slc29a1, and VEGF-A.
In conclusion, the thesis study revealed that AV-PBAT nanofibrous matrices support cardiomyogenic differentiation and vascularization when stimulated every other day with polychromatic light and infrared energy provided by a plasma arc light source (PAC), emitting light in the wavelength range of 600-1200 nm with an energy density of 3.5 J/cm2min from a distance of 20 cm and delivering a daily dose of 10.4 J/cm2 of infrared radiation in an electromechanical stimulation bioreactor.
