Institute of Materials Research, Division of Metallic Biomaterials, Helmholtz-Zentrum Geesthacht (HZG), 21502 Geesthacht, Germany; Department of Plastic Surgery and Hand Surgery, Klinikum Rechts der Isar, Technical University Munich (TUM), D-81675 Munich, Germany.
Institute of Materials Research, Division of Metallic Biomaterials, Helmholtz-Zentrum Geesthacht (HZG), 21502 Geesthacht, Germany.
Acta Biomater. 2015 Nov;27:294-304. doi: 10.1016/j.actbio.2015.08.042. Epub 2015 Aug 28.
Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment.
An attractive human osteoblasts and osteoclasts cocultivation regime was developed as an in vitro cytocompatibility model for magnesium implants. Parameters in terms of cellular proliferation and differentiation behaviors were investigated and we conclude that high concentration of magnesium extract could lead to a promotion in osteoblastogenesis but an inhibition in osteoclastogenesis. It could contribute to the repeated observations of enhanced bone growth adjacent to degradable magnesium alloys. More interestingly, it demonstrates that compared to monoculture, osteoclasts in cocultures with osteoblasts exhibited higher tolerance to the culture environment with high magnesium extract. It might attribute to the neutralization process of the alkaline medium by acid generated by increased amount of osteoblasts in the condition with high concentration of Mg extract. The submitted work could be of significant importance to other researchers working in the related field(s), thus appealing to the readership of Acta Biomaterialia.
成骨细胞和破骨细胞的共培养是理解镁（Mg）基植入物在降解过程中如何影响骨代谢和重塑的一个研究课题。首先，用人端粒酶逆转录酶（hTERT）转导的间充质干细胞（SCP-1）在成骨补充剂的作用下分化为成骨细胞，然后在没有添加破骨细胞生成促进因子的情况下进一步与外周血单核细胞（PBMC）共培养。同时，将培养物暴露于不同浓度的 Mg 提取物稀释液（0、30×、10×、5×、3×、2×和 1×）中。表型特征表明，尽管 2×稀释的 Mg 提取物对破骨细胞单培养具有极强的毒性，但与成骨细胞共培养的单核细胞对更高浓度的 Mg 提取物表现出更强的耐受性。1×稀释的 Mg 提取物培养物中大量成骨细胞的密集生长表明，高浓度的 Mg 提取物促进了成骨细胞的增殖/分化行为。细胞内碱性磷酸酶（ALP）和抗酒石酸酸性磷酸酶（TRAP）活性以及核因子 kappa-B 配体（RANKL）、巨噬细胞集落刺激因子（M-CSF）和破骨细胞相关受体（OSCAR）的蛋白和基因表达的结果表明，高浓度的 Mg 提取物显著增强了成骨细胞的形成，而减少了破骨细胞的形成。结论是，虽然已经证明了成骨诱导性的增加，但也应该考虑到镁基植入物周围潜在的破骨细胞生成减少的影响。应优先进行含有成骨细胞和破骨细胞的共培养，以作为镁基植入物的体外细胞相容性评估模型，因为它们更接近体内环境。
