Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis

Pulsed Electromagnetic Frequencies (PEMF) have shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment we present a three-dimensional ring culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group . Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control) and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.脉冲电磁场（Pulsed Electromagnetic Frequencies, PEMF）已被证实可有效促进骨修复，但该干预手段的最优参数及其分子机制仍未明确。为探究脉冲电磁场干预时机的影响，本研究构建了三维环体成骨培养模型，该模型可高效新生胶原与矿化基质，并在62天的培养周期内多个阶段对脉冲电磁场刺激产生响应。研究人员将出生后2天的小鼠颅盖骨成骨前体细胞，通过纤维蛋白原聚合作用包被于特氟龙环内外，并以悬浮方式培养，避免与组织培养塑料表面接触。环体构建物分别接受以下四种脉冲电磁场干预方案：全程每日暴露4小时（Daily组）、仅第1-10天暴露、仅第11-27天暴露、仅第28-63天暴露，其余时间不施加脉冲电磁场，并设置无脉冲电磁场干预的对照组。本研究采用的脉冲电磁场参数为HF Physio型号，频率40.85 kHz，爆发周期67 ms。成骨过程通过持续存在的茜素红S（Alizarin Red S）荧光检测进行动态监测，并通过微型计算机断层扫描（micro-CT）定期验证。当脉冲电磁场全程干预或在第1-10天、第11-27天干预时，可诱导矿化速率出现早期发作且具有统计学意义的瞬时提升（约4倍）；若仅在第28-63天施加干预，茜素红S检测显示的刺激效应未达到统计学显著性，但在培养第63天时微型计算机断层扫描检测结果显示该效应具有统计学意义。此外，各脉冲电磁场干预组的微型计算机断层扫描密度分布均向更高密度区间偏移。通过热重分析（thermogravimetric analysis）测定，环体培养生成的组织矿质与基质比值为2.0（为颅盖骨对照组的80%）；X射线衍射（X-ray diffraction）检测显示，沉积的晶体结构中50%为羟基磷灰石（hydroxyapatite）（为颅盖骨与股骨对照组的63%），且该比例不受脉冲电磁场干预的影响。上述结果与扫描电子显微镜（scanning electron microscopy）的背散射、二次电子及元素分析结果一致。综上，在明确且强成骨的培养环境中，不同时机施加的脉冲电磁场可进一步促进成骨作用，具备增强骨修复的潜力。