Purpose: Healing of osteoporotic defects is challenging and requires innovative approaches to elicit molecular mechanisms promoting osteoblasts-osteoclasts coupling and bone homeostasis.
Methods: Cytocompatibility and biocompatibility of previously characterised nanocomposites, i.e Ca₅(PO₄)₃OH/Fe₃O₄ (later called nHAp/IO) functionalised with microRNAs (nHAp/[email protected]/124) was tested. In vitro studies were performed using a direct co-culture system of MC3T3-E1 pre-osteoblast and 4B12 pre-osteoclasts. The analysis included determination of nanocomposite influence on cultures morphology (confocal imaging), viability and metabolic activity (Alamar Blue assay). Pro-osteogenic signals were identified at mRNA, miRNA and protein level with RT-qPCR, Western blotting and immunocytochemistry. Biocompatibility of biomaterials was tested using bilateral cranial defect performed on a senescence-accelerated mouse model, ie SAM/P6 and Balb/c. The effect of biomaterial on the process of bone healing was monitored using microcomputed tomography.
Results: The nanocomposites promoted survival and metabolism of bone cells, as well as enhanced functional differentiation of pre-osteoblasts MC3T3-E1 in co-cultures with pre-osteoclasts. Differentiation of MC3T3-E1 driven by nHAp/[email protected]/124 nanocomposite was manifested by improved extracellular matrix differentiation and up-regulation of pro-osteogenic transcripts, ie late osteogenesis markers. The nanocomposite triggered bone healing in a cranial defect model in SAM/P6 mice and was replaced by functional bone in Balb/c mice.
Conclusion: This study demonstrates that the novel nanocomposite nHAp/IO can serve as a platform for therapeutic miRNA delivery. Obtained nanocomposite elicit pro-osteogenic signals, decreasing osteoclasts differentiation, simultaneously improving osteoblasts metabolism and their transition toward pre-osteocytes and bone mineralisation. The proposed scaffold can be an effective interface for in situ regeneration of osteoporotic bone, especially in elderly patients.

Keywords: senile osteoporosis, osteoblasts, osteoclasts, nanohydroxyapatite, iron oxide nanoparticles, small non-coding RNA


中文翻译：

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新型纳米羟基磷灰石 (nHAp) 基支架掺杂氧化铁纳米颗粒 (IO)，用小非编码 RNA (miR-21/124) 功能化可调节矮小相关转录因子 2 和骨桥蛋白的表达，促进双侧骨质疏松骨的再生衰老加速小鼠模型 (SAM/P6) 中的颅骨缺陷。第2部分

目的：骨质疏松缺损的愈合具有挑战性，需要创新的方法来引发促进成骨细胞-破骨细胞偶联和骨稳态的分子机制。
方法：先前表征的纳米复合材料的细胞相容性和生物相容性，即 Ca ₅ (PO ₄ ) ₃ OH/Fe ₃ O ₄（后来称为 nHAp/IO）用 microRNAs（nHAp/[电子邮件保护]/124）功能化进行了测试。使用 MC3T3-E1 前成骨细胞和 4B12 前破骨细胞的直接共培养系统进行体外研究。分析包括确定纳米复合材料对培养物形态（共聚焦成像）、活力和代谢活性（Alamar Blue 测定）的影响。通过 RT-qPCR、Western 印迹和免疫细胞化学在 mRNA、miRNA 和蛋白质水平上鉴定出促成骨信号。使用衰老加速小鼠模型（即 SAM/P6 和 Balb/c）进行的双侧颅骨缺损测试了生物材料的生物相容性。使用微型计算机断层扫描监测生物材料对骨愈合过程的影响。
结果：纳米复合材料促进骨细胞的存活和代谢，以及增强前成骨细胞 MC3T3-E1 在与前破骨细胞共培养中的功能分化。由 nHAp/[email protected]/124 纳米复合材料驱动的 MC3T3-E1 分化表现为改善的细胞外基质分化和促成骨转录物（即晚期成骨标志物）的上调。纳米复合材料在 SAM/P6 小鼠的颅骨缺损模型中引发骨愈合，并在 Balb/c 小鼠中被功能性骨取代。
结论：该研究表明，新型纳米复合材料 nHAp/IO 可以作为治疗性 miRNA 传递的平台。获得的纳米复合材料引发促成骨信号，减少破骨细胞分化，同时改善成骨细胞代谢及其向前骨细胞和骨矿化的转变。所提出的支架可以成为骨质疏松骨原位再生的有效界面，特别是在老年患者中。

关键词：老年性骨质疏松症，成骨细胞，破骨细胞，纳米羟基磷灰石，氧化铁纳米颗粒，非编码小RNA