Collagen cross-linking in osteoblastic cells promoted by randomized electrical stimulation

Abstract

Collagen cross-linking enhances the mechanical properties of bone by improving toughness of the bone matrix. Randomized electrical stimulation contributes positively to the behavior of osteoblastic cell in vitro by promoting osteogenic differentiation and mineral deposition. However, the effects of randomized electrical stimulation on collagen crosslink formation, which are associated with bone toughness, are yet to be elucidated. In this study, we identify the effects of randomized electrical stimulation on collagen cross-linking of osteoblast cells in vitro. MC3T3-E1 osteoblastic cells are stimulated with two electric stimulation (ES) patterns: a periodic pulse train (PrPT) with a constant pulse rate of 500 Hz, and a random pulse train (RdPT) composed of randomized pulse rates up to 500 Hz for 3 min/day for the first 3 days. After the stimulation, lysyl oxidase (LOX) and type I collagen (COL) gene expression, LOX activity, and collagen insolubility are investigated. In addition, Fourier transform infrared spectroscopy (FTIR) is performed to evaluate the degree of collagen maturity, i.e., collagen cross-linking. RdPT ES does not affect LOX and COL mRNA expression levels after a single stimulation of 3 min. However, on day 14 of RdPT ES, the LOX activity is significantly increased compared with the control. The ratio of insoluble collagen in the RdPT-stimulated sample is significantly higher than that of the control on day 21. FTIR exhibits significantly higher collagen maturity (1652/1680 cm⁻¹ area ratio) in the RdPT-stimulated sample than in the control and PrPT groups on day 21. In conclusion, randomized electrical stimulation promotes bone collagen cross-linking in osteoblastic cells through the activation of LOX.

Graphical Abstract