Proliferation studies and extracellular matrix deposition in dynamic, 3-dimensional fibroblast culture

Abstract: Tissue engineering is a growing multidisciplinary field that works towards restoring, maintaining, and/or enhancing tissue and organ functions. One of the challenges is developing biodegradable and biocompatible scaffolds (matrices) that promote cell maturation and organization in vivo. There is a number of studies that demonstrate an increased matrix synthesis in vitro from cell cultures grown under mechanical stimulation. This project investigated the effect of dynamic stimulation on murinae fibroblasts, cultured on a non-biodegradable, 3-dimensional scaffold, polyethyleneterephtalate (PET). Cell proliferation was evaluated with an MTT assay (Thiazolyl Blue Tetrasolium Bromide), and cell-extracellular matrix (ECM) distribution was studied with scanning electron microscopy (SEM), Scanning Laser Confocal microscopy, and histology. A bioreactor and silicon tube with PET knitting implemented a dynamic cell culture. A physiological pressure on the cells was simulated by expansion and relaxation of the silicon tube with the help of a medium and a peristaltic pump. Cell cultures in dynamic conditions showed a considerably higher proliferation and growth rate than cells grown in static conditions. Dynamically cultured fibroblasts increased their proliferation nearly 10 times within 1 week compared with static cultures. The static cultures reached their highest cell count at three weeks, which was half the dynamic proliferation's highest value. The results showed that cells were tightly bound to the PET scaffold, and the ECM proteins had initiated a matrix bridging over the fibre distances in the PET scaffold. Immunostaining confirmed the production of ECM by verifying the presence of collagen type I.