Three-Dimensionally Enhanced Extra Cellular Matrix Formation for Bone Regeneration

Objectives: Spatiotemporal organization of rich extra cellular matrix (ECM) with embedded cell population is essential for success of bone regeneration. We studied the effect of cyclic strain, creating fluid flow by means of mechanotransduction, in ECM production and proliferation into bone-like biogenic microenvironment scaffold (BMS) using MC3T3 cells.
Methods: Hydroxyapatite based BMS, mimicking trabecular architecture with a mean pore size of 320 um, were prepared. Each 10mm height x 10mm diameter cylindrical BMS was placed into the 6-well plates (n=3) and 2.5 x 10⁵ MC3T3 cells were seeded. After 10 minutes, BMS was inverted to minimize the gravity effect of sinking cells. 10 ml of α-MEM medium was added and incubated in 37°C for 24h. Then, each BMS was horizontally rotated under 0 rpm, 20 rpm, or 40 rpm for 1 minute every 24h. The efficacy of ECM organization and cell population was evaluated using H&E staining at time points of 1, 2, and 3 weeks.
Results: Cyclic strain throughout BMS structure induced effective three-dimensional (3-D) ECM formation, which was not significantly evident in static environment (0 rpm). With rotational fluid movement by means of physical change, fabrication of ECM was accelerated and 3-D ECM structures were demonstrated. Static environment showed the highest and even distribution of cell population, whereas 20 rpm and 40 rpm had less. However, stronger actin stretching and ECM crosslinking was evidenced under cyclic strain, indicating active 3-D ECM formation to fill micropores. Comparing to other groups, most organized, rich 3-D ECM structure was observed under 20 rpm at 2 weeks time point.
Conclusions: Cyclic mechanical strain affects the 3-D ECM formation to fill empty space. ECM provides a base for further cell ingress and habitation into space. Thus, enhanced complex 3-D ECM with BMS was successfully achieved for the application of bone regeneration.