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IL-6 IS THE PRO-INFLAMMATORY CYTOKINE RESPONDING TO TENSION STRESS IN DISTRACTION OSTEOGENESIS. Cho, T.-J.(A-KRF); Choi, I.H.;Chung, C.Y. ;Kim, E.-H.(A-KRF) Seoul National University, Seoul, Korea Introduction Distraction osteogenesis, which is a process of strain-induced new bone formation, plays a powerful role in limb reconstruction by either limb lengthening or bone transport. Many growth factors such as BMP’s, TGF beta’s, vasculoendothelial growth factor, insulin-like growth factors have been reported to express and to regulate new bone formation during this process, and enhancement of bone formation by those growth factors are under development. The pro-inflammatory cytokines have been shown not only to coordinate the hematopoietic and immune systems, but also to contribute to bone repair by regulating osteoclastogenesis and the early recruitment and differentiation of osteoblastic lineage cells ( 1). In order to investigate the role of pro-inflammatory cytokines during distraction osteogenesis, we quantified the mRNA expression of those cytokines over 3 weeks period, and compared with non-distraction control animals. The spatial expression pattern of IL-6, which showed a significant temporal expression pattern, was also investigated by immunohistochemistry. Materials and Methods The left tibiae of 16 week-old Sprague-Dawley rats weighing 350 - 400gm were fixed with a pair of mini-mono fixators which enabled gradual lengthening, and osteotomized at its diaphysis. The fragments were distracted by the rate of 0.5mm/day in two steps from day 7 to day 14. Non-distraction control group had the same operative procedure only without any distraction procedure. Total RNA was extracted from the regenerating tissues at the postoperative 1 day, 3 day, 5 day, 7 day, 9 day, 14 day, 21 day, and before operation. The mRNA expression for IL-1alpha, IL-1beta, IL-6, TNFalpha, TNFbeta, and two housekeeping genes (L32 and GAPDH) by RNase protection assay using RiboQuant(Pharmingen, San Diego, CA). Intensity of the protected bands on electrophoresis gel were quantified, and standardized by those of housekeeping genes. Tibial segments including the distraction gap were harvested after perfusion with 4% paraformaldehyde solution. Following overnight fixation, the tissues were decalcified with 10% EDTA buffer. Immunohistochemical study was performed for IL-6 using ABC method (LSAB-2 rat system, DAKO, Carpinteria, CA) with monoclonal antibody for IL-6 (Santa Cruz Biotechnology, Santa Cruz, CA). Results Among the cytokines tested, IL-1beta and IL-6 produced detectable signals by RNase protection assay, while IL-1alpha, TNFalpha and TNFbeta did not produce any detectable signals throughout the experiment period. The mRNA expressions of IL-1beta and IL-6 were upregulated at postoperative 1 day by 2.0 and 3.5 folds, respectively (Fig. 1). The mRNA expression of IL-1beta returned to non-operated state from postoperative 3 days throughout the experiment period. The upregulation IL-6 mRNA also subsided on postoperative 3, 5, 7 days after 1 day peak, however, it reactivated on postoperative 9 days, which could not be observed in the non-distraction control group. Immunohistochemistry for IL-6 revealed its expression in the hypertrophic chondrocyte, young osteoblast, as well as primitive mesenchymal cells at the distraction gap of postoperative 9 days specimen. Discussion This study revealed the temporal expression pattern of proinflammatory cytokines during the distraction osteogenesis. Comparing with previous report on the fracture model in which intramedullary nail and blunt trauma were used(1), induction of TNFalpha mRNA was quite negligible in this external fixator – open osteotomy model. This may be due to relatively mild degree of tissue injury and rigid fixation. We intended to detect which molecule was up-regulated in response to the distraction stess, and IL-6 was found to be the straininduced cytokine among the tested ones. IL-6 was suggested to mediate osteoblast recruitment (2). IL-6 immunoreactivity localized at the primitive mesenchymal cells of the distraction gap also suggests its role in osteoblast recruitment by distraction strain. Although its reactivation by distraction procedure did not sustain throughout the distraction phase, IL-6 can be considered as a unique cytokine acting as an important signal in the early phase of distraction in response to tension stress during distraction osteogenesis. Fig. 1 Result of RNase protection assay. IL-6 showed reactivation at day 9 in D.O. D.O.: distraction osteogenesis group, Non-D.O.: non-distraction control group. References 1. Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC, Einhorn TA. J Bone Miner Res, 2001, 16:1004-1014. 2. Karadag A, Scutt AM, Croucher PI. J Bone Miner Res 2000, 15:1935-1943 49th Annual Meeting of the Orthopaedic Research Society Poster #0391