Koelbl O, Knaus P, Pohl F, Flentje M, Sebald W
Department of Radiation Therapy, University of Würzburg, Germany.
Strahlenther Onkol. 2001 Aug;177(8):432-6. doi: 10.1007/pl00002425.
Heterotopic ossification (HO) is a common complication following total hip replacement. Clinical studies showed the effectiveness of irradiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to find a suitable cell system, which can reproduce in-vitro data resulting from clinical in-vivo studies. The establishment of such a cell model allows detailed analyses of the mechanism of radiotherapy.
The chicken limb bud test was used as an in-vitro model. The cells acquired by the limb bud test were irradiated with different doses (0 Gy, 3 Gy, 7 Gy, 10 Gy, 20 Gy). Irradiation was set either 1 hour before, or 1 or 3 days after BMP-2 incubation. The synthesis of proteoglycans (PGS) upon treatment with bone morphogenetic protein (BMP)-2 was measured in cells incubated with BMP-2 for 4 days followed by 35SO4(2-) labeling for 6 hours. Labeled proteoglycans were precipitated using Alcian blue and measured in a raytest radio-TLC analyzer. The incubation with BMP-2 was defined to correlate the in-vivo stimulus meaning the operation.
The proteoglycan synthesis was significantly reduced by irradiation 1 hour before or 1 day after BMP-2 incubation, if the dosage was at least 7 Gy. Higher doses than 7 Gy did not lead to lower proteoglycan levels. There was only a trend for a reduction of proteoglycan synthesis by 3 Gy irradiation, but no significant difference compared to the non-irradiated control. An irradiation 3 days after BMP-2 incubation had no effect on proteoglycan.
A dose and time dependent effect of radiation on BMP-2-induced proteoglycan synthesis was observed. Therefore the results of clinical in-vivo studies were reproduced exactly by the limb bud test. We established an in-vitro cell model to analyze the mechanism of the prevention of heterotopic ossification by radiotherapy on cellular or sub-cellular level.
异位骨化（HO）是全髋关节置换术后的常见并发症。临床研究表明放射治疗对预防异位骨化有效。放射治疗减少异位骨化的机制尚不清楚。本研究的目的是找到一种合适的细胞系统，该系统能够重现临床体内研究产生的体外数据。建立这样的细胞模型可以详细分析放射治疗的机制。
采用鸡胚肢体芽试验作为体外模型。通过肢体芽试验获得的细胞用不同剂量（0 Gy、3 Gy、7 Gy、10 Gy、20 Gy）进行照射。照射设置在骨形态发生蛋白（BMP）-2孵育前1小时，或BMP-2孵育后1天或3天。在用BMP-2处理4天，随后用35SO4(2-)标记6小时的细胞中，测量骨形态发生蛋白（BMP）-2处理后蛋白聚糖（PGS）的合成。使用阿尔新蓝沉淀标记的蛋白聚糖，并在raytest放射性薄层色谱分析仪中进行测量。将与BMP-2的孵育定义为关联体内刺激，即手术。
如果剂量至少为7 Gy，在BMP-2孵育前1小时或孵育后1天进行照射可显著降低蛋白聚糖的合成。高于7 Gy的剂量并未导致蛋白聚糖水平更低。3 Gy照射仅有降低蛋白聚糖合成的趋势，但与未照射对照组相比无显著差异。BMP-2孵育后3天进行照射对蛋白聚糖无影响。
观察到辐射对BMP-2诱导的蛋白聚糖合成具有剂量和时间依赖性效应。因此，肢体芽试验准确重现了临床体内研究的结果。我们建立了一个体外细胞模型，以在细胞或亚细胞水平分析放射治疗预防异位骨化的机制。
