Abstract
Introduction Bone morphogenetic protein-7 (BMP-7) is known to stimulate both cellular proliferation and extracellular matrix synthesis in the intervertebral disc but its protective role in apoptosis is unknown. The aim of this study was to determine whether BMP-7 protect cultured intervertebral disc cells following stimulation of apoptosis.
Methods Nucleus pulposus tissues were obtained from consent individuals under surgical procedures and digested with collagenase prior to culturing. Cellular apoptosis was achieved by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide (H2O2) incubation. BMP-7 (Stryker) was used at 100ng/ml, 5 hours prior to the addition of apoptotic stimulation. Cellular apoptosis was detected by TUNEL assay, caspase-3 activity and caspase-3 protein expression. Cellular proliferation and viability was assayed by H3-thymidine incorporation and MTS assay respectively. Collagen II and aggrecan protein levels were measured using western blots and immunostaining. Proteoglycan synthesis was determined by (35)S-sulfate incorporation method. Nitric oxide and alkaline phosphatase activity were measured.
Results Both extrinsic and intrinsic apoptotic pathways were induced by TNF-β or hydrogen peroxide with increased proteolytic activity of caspase-3 as well as cellular shrinkage and nuclear condensation. Addition of BMP-7 prior to stimulation of apoptosis resulted in complete block of the apoptotic effects of both inducers as well as the cellular nitric oxide induced by TNF-β and BMP-7 increases cellular viability, proliferation and extracellular matrix production in an apoptotic environment with no osteoblastic activity induction of discal cells.
Discussion BMP-7 prevents apoptosis of cultured human disc cells induced by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide. Induction of apoptosis led to down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3, however addition of BMP-7 alone prevented the effects observed. One possible mechanism of the anti-apoptotic effects of BMP-7 was shown by its retardation of the elevated levels of TNF-β induced nitric oxide.
The abstracts were prepared by Assoc Prof Bruce McPhee. Correspondence should be addressed to him at the Division of Orthopaedics, The University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Herston, Brisbane, 4029, Australia.
