RELATIVE QUANTITATIVE RT-PCR ANALYSIS OF CYTOKINE EXPRESSION BY NON-UNITING FRACTURES – SUPPRESSION OF INTERLEUKIN-1 AND TNF-ALPHA EXPRESSION IN FRACTURE NON-UNIONS

Abstract

Background: Non-union of fractures is a common problem faced by orthopaedic surgeons. Although the basic processes of fracture healing have been better elucidated in recent years, in terms of their cellular and molecular biology, the pathogenesis of fracture non-union remains poorly understood.

Aims: To examine the pattern of cytokine expression in established non-unions, in particular the inflammatory cytokines interleukin 1 and tumour necrosis factor alpha.

Materials and Methods: Tissue was taken from 7 non united fractures at the time of a surgical procedure aimed at effecting union. Part of the tissue was snap-frozen in liquid nitrogen, and a portion of the sample was processed for routine histology. Normal bone tissue was taken from the femoral shaft at the time of arthoplasty, to provide normal control tissue. Total RNA was extracted from the frozen tissue by means of a mortar and pestle and a modified phenol-chloroform extraction protocol. Cytokine expression patterns were examined using the Cytokine Gene Expression plate I (PE Biosystems) and analysed using the Sequence Detection Software and Microsoft Excel.

Results: A consistent pattern of cytokine expression was seen in all non-union tissue samples. There was marked suppression of interleukin 1 beta, interleukin 8, interleukin 10 and TNF-alpha when compared to resting bone. This environment is thus one where the stimulus for bone resorption is suppressed, with consequent loss of stimulation of bone formation (theory of “bone coupling”), directly and also possibly through interaction with prostaglandin production. In addition, collagen production is stimulated preferentially. These findings argue against the traditional definitions of fracture non-union, and suggest a possible adjunctive role for the administration of interleukins in the treatment of non-united fractures.