Abstract
Introduction: The concept of cell senescence has been described as the mechanism responsible for the ageing of tissues, that is a finite ability to replicate and produce new tissue. The senescent cell population is separate and distinct from the cells which are undergoing programmed cell death (apoptosis), and those which are necrosing acutely. Cells reaching the senescent state have an increase in β-galactosidase activity, which is detectable using an established technique for soft tissues including fibroblasts and epithelial tissues. Senescence has not previously been investigated in bone. We have investigated this and hypothesise that new bone formed by distraction osteogenesis will have fewer senescent cells than the adult cortical “old” bone.
Methods: Eight New Zealand white rabbits underwent application of a M100 Orthofix external fixator to the tibia and creation of a mid-diaphyseal osteotomy, using a hand saw. After a seven day latency period, distraction was commenced (0.5mm twice daily) to twenty percent lengthening. After 3 weeks consolidation, the tibae were harvested for histological analysis.Senescent Staining:The sections were stained using a technique described by Faragher, using an X-gal based stain. Sections were incubated for 16 hours at 37 degrees centigrade before counter staining with DAPI. Sections underwent histological analysis and total cell and senescent cell counts performed.
Results: Surprisingly, large numbers of cells within the bone regenerate stained for cell senescence. A mixture of multinucleate and mononucleate cells were present. The location and appearance of the multinucleate cells prompted the use of TRAP staining. This provided support for these cells being osteoclasts.
Discussion: Previously, a high percentage of apoptotic cells and a high rate of cell division has been reported in bone regenerate. The surprisingly high numbers of cells within the newly formed bone staining positively for senescence suggest that there may also be a high senescent cell population. Alternatively, the positive TRAP staining may indicate that the stain is less specific than reported and may be staining osteoclasts and mature macrophages within the bone regenerate.
Correspondence should be addressed to Mr Carlos Wigderowitz, Honorary Secretary BORS, University Dept of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School, Dundee DD1 9SY.
