In vivo intervertebral disc remodeling: Kinetics of mRNA expression in response to a single loading event

Abstract

Kinetics of mRNA expression following a single loading event was measured using an in vivo rat tail model. Animals were instrumented and loaded in compression for 1.5 h at 1 MPa and 1 Hz. Real-time RT-PCR was used to measure mRNA levels 0, 8, 24 and 72 h after mechanical stimulation for genes associated with matrix proteins (aggrecan, collagen-I, collagen-II), proteases (MMP-2, MMP-3, MMP-13, ADAMTS-4), and their inhibitors (TIMP-1, TIMP-3) in anulus fibrosus and nucleus pulposus regions. Baseline mRNA levels were of greatest abundance for matrix proteins and lowest for proteases. The mRNA levels reached maximum levels 24 h following mechanical stimulation for the majority of genes evaluated, but some had maximum levels 8 and 72 h following loading. The mRNA levels returned to baseline levels for all genes in the nucleus 72 h following loading, but the majority of genes in the anulus remained upregulated. Results support a coordinated strategy of relative mRNA expression that varied over time beginning with inhibition of tissue breakdown, followed by synthesis of aggrecan and matrix degrading enzymes, and eventually collagen metabolism days following loading. Consequently, optimal time for tissue harvest for mRNA measurements depends on genes of interest. Results suggest attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur, and that changes in TIMP and MMP levels may have greater potency in affecting structural protein abundance than direct changes in the structural protein messages. Results have important implications for disc remodeling and tissue engineering.