Introduction

Currently, various kinds of materials are used for the treatment of bone defects. In general, these materials have a problem of formativeness. The three -dimensional (3D) printing technique has been introduced to fabricate artificial bone with arbitrary shapes, but poor bone replacement is still problematic.

Our group has created a β⁻tricalcium phosphate (β⁻TCP) scaffold by applying 3D printing technology. This scaffold has an arbitrary shape and an internal structure suitable for cell loading, growth, and colonization. The scaffold was coated with a recombinant collagen peptide (RCP) to promote bone replacement.

As indicated by several studies, cells loaded to scaffolds promote bone regeneration, especially when they are induced osteoblastic differentiation before transplantation. In this study, culture duration for bone marrow cells was optimized before being loaded to this new scaffold material.

Method

Bone marrow cells isolated from C57BL/6J mice were subjected to osteogenic culture for 4, 7, and 14 days. The differentiation status of the cells was examined by alkaline phosphatase staining, alizarin red staining, and real-time RT-PCR for differentiation markers. In addition, the flow of changes in the abundance of endothelial cells and monocytes was analyzed by flow cytometry according to the culture period of bone marrow cells.

Next, cells at days 4, 7, and 14 of culture were placed on a β-TCP/RCP scaffold and implanted subcutaneously into the back of C57BL/6J mice. Grafts were harvested and evaluated histologically 8 weeks later. Finally, Cells cultured for 7 days were also transplanted subperiosteally in the skull of the mouse with scaffolds.

Result

Alkaline phosphatase staining was most prominent at 7 days, and alizarin red staining was positive at 14 days. Real-time RT-PCR revealed that Runx2 and Alp peaked at 7 days, while expression of Col1a1 and Bglap was highest at 14 days. Flow cytometry indicated that endothelial cells increased from day 0 to day 7, while monocytes increased continuously from day 0 to day 14. When transplanted into mice, the scaffold with cells cultured for 7 days exhibited the most prominent osteogenesis. The scaffold, which was transplanted subperiosteally in the skull, retained its shape and was replaced with regenerated bone over a large area of the field of view.

Conclusion

Osteoblasts before full maturation are most efficient for bone regeneration, and the pre-culture period suitable for cells to be loaded onto a β-TCP/RCP hybrid scaffold is approximately 7 days.

This β-TCP/RCP hybrid scaffolds will also be useful for bone augmentation.

中文翻译：

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使用β-磷酸三钙/重组胶原肽混合支架移植前优化骨髓细胞的培养时间。

介绍

目前，有多种材料用于治疗骨缺损。一般来说，这些材料存在成形性问题。三维（3D）打印技术已被引入来制造任意形状的人造骨，但骨替代效果不佳仍然是一个问题。

我们课题组利用3D打印技术制作了β⁻磷酸三钙（β⁻TCP）支架。该支架具有任意形状和适合细胞装载、生长和定植的内部结构。该支架涂有重组胶原肽（RCP）以促进骨替代。

多项研究表明，装载到支架上的细胞可促进骨再生，特别是当它们在移植前诱导成骨细胞分化时。在这项研究中，在加载到这种新支架材料之前，优化了骨髓细胞的培养时间。

方法

从 C57BL/6J 小鼠分离的骨髓细胞进行成骨培养 4、7 和 14 天。通过碱性磷酸酶染色、茜素红染色和分化标记物的实时RT-PCR检查细胞的分化状态。另外，根据骨髓细胞的培养期间，通过流式细胞术分析内皮细胞和单核细胞的丰度变化的流程。

接下来，将培养第 4、7 和 14 天的细胞置于 β-TCP/RCP 支架上，并皮下植入 C57BL/6J 小鼠背部。8周后收获移植物并进行组织学评估。最后，将培养7天的细胞也用支架移植到小鼠颅骨骨膜下。

结果

碱性磷酸酶染色在第 7 天时最为明显，茜素红染色在第 14 天时呈阳性。实时RT-PCR显示Runx2和Alp的表达在7天时达到峰值，而Col1a1和Bglap的表达在14天时达到最高。流式细胞术显示内皮细胞从第0天到第7天增加，而单核细胞从第0天到第14天持续增加。当移植到小鼠体内时，培养7天的细胞支架表现出最显着的成骨作用。该支架被移植到颅骨骨膜下，保留了其形状，并在大范围的视野范围内被再生骨所取代。

结论

完全成熟之前的成骨细胞对骨再生最有效，适合将细胞装载到β-TCP/RCP混合支架上的预培养期约为7天。

这种 β-TCP/RCP 混合支架也可用于骨增强。