Double and quadruple tetracycline labeling of bone: impact of the label itself.
Double and quadruple tetracycline labeling of bone: impact of the label itself.
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骨的双重和四环素标记：标记本身的影响。
DOI：
10.1002/jbmr.1818
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Dempster,David
中科院分区：
文献类型：
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作者：
Lindsay,Robert;Zhou,Hua;Cosman,Felicia;Nieves,Jeri;Dempster,David
骨的定量组织形态测量是一种公认的技术，在临床患者评价和临床研究和药物开发中具有特定应用，可以评估药物的安全性及其作用机制。最近，两个出版物审查了现代组织形态计量学的问题，并更新了ASBMR组织形态计量学命名委员会关于命名、符号和单位的建议。（1，2）在此，我们提请注意最初由Parfitt及其同事（3）描述的一个问题，该问题对从骨形成的动态评估中可能得出的结论具有影响。在大多数组织形态学应用中，骨用结合到骨矿化表面的四环素标记。这已经成为一种标准方法，可以计算所有四个骨室（松质骨、皮质内骨、皮质内骨和骨膜骨）的骨形成率。在大多数临床情况下，给予相同形式的四环素（通常为盐酸四环素、土霉素或去甲基金霉素）的两个单独标签，每个标签与另一个标签间隔12或14天，并在第二个标签完成后约1周进行活检。对于一些应用，不同的标记可用于两个标记中的第二个;两个标记在UV光下的颜色差异允许将单个标记明确地分配给第一或第二标记周期。几年前，我们描述了一种四重标记技术（4），该技术允许在单次活检中获得短期纵向数据，前两个四环素标记以通常的格式给出，但与第二组标记间隔数周。为了在UV光下区分第一组和第二组标记，我们对第一组两种标记使用相同的四环素，对第二组使用不同的四环素。使用该技术，以去甲基金霉素作为第一对标记，盐酸四环素作为第二对标记，我们证明了特立哌酮在细胞水平上对骨形成的早期影响。使用该序列，我们发现在未接受骨质疏松症药物治疗的受试者的松质骨中，第二组标记几乎总是短于第一组，因此被标记覆盖的表面的比例，矿化表面（MS/BS），计算为单标记表面的两倍+ 50%，使用第二组标记物时，差异较小（2.94 ± 1.13%去甲基金霉素vs 2.14 ± 0.83%盐酸四环素），差异未达到统计学显著性（n= 9）。在皮质内连接处也观察到类似的趋势。考虑到许多松质骨包的新月形解剖结构，我们可能已经预料到，如果有的话，第二组标签应该更长，即使在未经治疗的对照受试者中也是如此。特立哌酮的作用非常明显，MS/BS显著增加，（由于四环素的不同形式，超过了标记长度的任何预期差异的数倍）。我们现在在一个单独的实验中报告，其中盐酸四环素作为初始标记组，去甲基金霉素作为最终标记组，在未经治疗的受试者的松质骨中，第二标记现在比第一组长（2.82 ± 0.76盐酸四环素对3.76 ± 0.65去甲基金霉素; p< 0.05，n= 14）。这有力地支持了Parfitt及其同事（3）的原始数据，该数据表明，无论标签的给药顺序如何，去甲基金霉素标签总是长于土霉素标签。我们相信我们的数据证实了建议（3）。
Quantitative histomorphometry of bone is a well-recognized technique with selected applications in clinical patient evaluation and in clinical research and drug development that allows assessment of the safety of drugs and their mechanism of action. Recently, two publications have examined issues in modern histomorphometry and have updated the ASBMR Histomorphometry Nomenclature Committee’s recommendations on nomenclature, symbols, and units.(1, 2) Here we draw attention to an issue originally described by Parfitt and colleagues (3) which has implications for the conclusions that might be drawn from dynamic assessment of bone formation. In most histomorphometry applications, the bone is labeled with a tetracycline that binds to the mineralizing surface of bone. This has become a standard method that allows calculation of the rate of bone formation in all four bone compartments (cancellous, endocortical, intracortical, and periosteal bone). In most clinical situations, two separate labels of the same form of tetracycline (usually tetracycline HCl, oxytetracycline, or demethylchlortetracycline) are administered, each separated from the other by a period of 12 or to 14 days, and the biopsy is performed about 1 week after completion of the second label. For some applications, a different label may be used for the second of the two labels; the difference in the color of the two labels under UV light allows unambiguous assignment of single labels to the first or second labeling period. Several years ago we described a quadruple-labeling technique (4) that allows short-term longitudinal data in a single biopsy with the first two tetracycline labels given in the usual format, but separated by several weeks from the second set of labels. In order to distinguish the first and second sets of labels under UV light, we used the same tetracycline for the first set of two labels and a different one for the second set. Using that technique, with demethylchlortetracycline as the first pair of labels and tetracycline HCl for the second set, we demonstrated the early effects of teriparatide on bone formation at the cellular level. With that sequence, we found that in cancellous bone of subjects on no osteoporosis medication, the second set of labels was almost always shorter than the first set, and consequently the proportion of the surface covered by label, the mineralized surface (MS/BS), calculated as double+ 50% of the singlelabeled surface, was less with the second set of labels (2.94+ 1.13% demethylchlortetracycline versus 2.14+ 0.83% tetracycline HCl), a difference that did not achieve statistical significance (n= 9). A similar trend was seen at the endocortical junction. Given the crescent-shaped anatomy of many cancellous bone packets, we might have expected that, if anything, the second set of labels should be longer, even in untreated control subjects. The effects of teriparatide were clearly evident, with a marked increase in MS/BS (that exceeded by several-fold any expected differences in label length due to the different forms of tetracycline).We now report in a separate experiment, in which tetracycline HCl was given as the initial set of labels and demethylchlortetracycline as the final set, in cancellous bone of untreated subjects, that the second labels are now longer than the first set (2.82+ 0.76 tetracycline HCl versus 3.76+ 0.65 demethylchlortetracycline; p< 0.05, n= 14). This strongly supports the original data from Parfitt and colleagues (3) which suggested that the demethylchlortetracycline label was always longer than the oxytetracycline label, irrespective of the order in which the labels were administered. We believe that our data confirms the recommendation (3 …