Stimulation of MSCs Osteogenic Differentiation on a Micro/ Nanofibrous Scaffold Using Mechanical Stimulation in the Perfusion Bioreactor

Abstract

In tissue engineering, the 3D constructs seeded with cells tested in vitro in static conditions suffer from poor cellular penetration and non-homogenous matrix deposition, due to the insufficient flow of nutrients, gases and signal molecules through the scaffold volume. However, a dynamic culture of cells can improve these drawbacks and simulate physiological conditions, such as flow in the canalicular system of bones. The purpose of this comparative study was to evaluate the different flow rates of 1 and 3 mL/min of perfusion bioreactor on human mesenchymal stromal cells (hMSCs), seeded in an osteogenic media on 3D porous constructs. After three weeks of dynamic cultivation, the metabolic activity and proliferation of the cells was evaluated, as well as the alkaline phosphatase (ALP) activity and the expression of bone related genes, RunX2, collagen type I and osteocalcin. The dynamic culture conditions did not influence the metabolic activity of hMSCs. The highest cell proliferation was detected on the scaffold cultured at a flow rate of 3 mL/min. The dynamic culture did not prove the penetration of cells into the scaffold's volume. The ALP activity, and PCR analysis of osteogenic markers RunX2 and collagen type I were highest on the scaffolds cultured at 1 mL/min, compared to the static culture and even to the dynamic culture at 3 mL/min. The study shows that hMSCs behave differently in different flow rates. While the higher flow rate (3 mL/min, shear stress 479 mPa) improved cell proliferation, the slower flow rate of 1 mL/min (shear stress 160 mPa) supported the ALP activity and expression of osteogenic marker genes. Interestingly, our expectation that dynamic culture improves cell penetration into the scaffold was not proved.