A pilot study: Nano-hydroxyapatite-PEG/PLA containing low dose rhBMP2 stimulates proliferation and osteogenic differentiation of human bone marrow derived mesenchymal stem cells

IF 3.4 3区医学 Q1 ORTHOPEDICS JOR Spine Pub Date : 2023-06-01 DOI:10.1002/jsp2.1258

Eda Çiftci Dede, Merve Gizer, Feza Korkusuz, Zeynep Bal, Hiroyuki Ishiguro, Hideki Yoshikawa, Takashi Kaito, Petek Korkusuz

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Abstract

Background

Bone morphogenetic protein 2 (BMP2) can enhance posterolateral spinal fusion (PLSF). The minimum effective dose that may stimulate mesenchymal stem cells however remains unknown. Nano-hydroxyapatite (nHAp) polyethylene glycol (PEG)/polylactic acid (PLA) was combined with recombinant human BMP2 (rhBMP2). We in vitro evaluated proliferation, differentiation, and osteogenic genes of human bone marrow mesenchymal stem cells with 0.5, 1.0, and 3.0 μg/mL rhBMP2 doses in this study.

Methods

In vitro experimental study was designed to proliferation by a real-time quantitative cell analysis system and the osteogenic differentiation by alkaline phosphatase (ALP) activity and osteogenic marker (Runx2, OPN, and OCN) gene expressions of human derived bone marrow mesenchymal stem cells (hBMMSCs). nHAp was produced by wet chemical process and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, and energy-dispersive x-ray spectroscopy. PEG/PLA polymer was produced at a 51:49 molar ratio. 0.5, 1.0, and 3.0 μg/mL rhBMP2 and nHAp was combined with the polymers. hBMMSCs were characterized by multipotency assays and surface markers were assessed by flow cytometer. The hBMMSC-rhBMP2 containing nHAp-PEG/PLA composite interaction was evaluated by transmission electron microscopy. Proliferative effect was evaluated by real-time proliferation analysis, and osteogenic capacity was evaluated by ALP activity assay and qPCR.

Results

hBMMSC proliferation in the 0.5 μg/mL rhBMP2 + nHAp-PEG/PLA and the 1.0 μg/mL rhBMP2 + nHAp-PEG/PLA groups were higher compared to control. 1.0 μg/mL rhBMP2 + nHAp-PEG/PLA and 3.0 μg/mL rhBMP2 + nHAp-PEG/PLA containing composites induced ALP activity on days 3 and 10. 0.5 μg/mL rhBMP2 + nHAp-PEG/PLA application stimulated Runx2 and OPN gene expressions.

Conclusion

rhBMP2 + nHAp-PEG/PLA composites stimulate hBMMSC proliferation and differentiation. The nHAp-PEG/PLA composite with low dose of rhBMP2 may enhance bone formation in future clinical PLSF applications.

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一项初步研究：含有低剂量rhBMP2的纳米羟基磷灰石PEG/PLA刺激人骨髓间充质干细胞的增殖和成骨分化。

背景：骨形态发生蛋白2（BMP2）可增强后外侧脊柱融合（PLSF）。然而，刺激间充质干细胞的最小有效剂量仍然未知。将纳米羟基磷灰石（nHAp）聚乙二醇（PEG）/聚乳酸（PLA）与重组人BMP2（rhBMP2）复合。我们在体外评估了0.5、1.0和3.0的人骨髓间充质干细胞的增殖、分化和成骨基因 μg/mL rhBMP2剂量。方法：通过实时定量细胞分析系统设计体外实验研究人骨髓间充质干细胞（hBMMSCs）的增殖和碱性磷酸酶（ALP）活性和成骨标志物（Runx2、OPN和OCN）基因表达的成骨分化。采用湿法化学法制备了nHAp，并用傅立叶变换红外分光光度计、扫描电子显微镜和能量色散x射线光谱对其进行了表征。PEG/PLA聚合物以51:49的摩尔比制备。0.5、1.0和3.0 μg/mL rhBMP2和nHAp与聚合物结合。hBMMSCs通过多能性测定进行表征，表面标记物通过流式细胞仪进行评估。通过透射电子显微镜评价了含有nHAp-PEG/PLA的hBMMSC-rhBMP2复合物的相互作用。通过实时增殖分析评估增殖效果，并通过ALP活性测定和qPCR评估成骨能力。结果：hBMMSC在0.5 μg/mL rhBMP2 + nHAp-PEG/PLA和1.0 μg/mL rhBMP2 + nHAp-PEG/PLA组与对照组相比更高。1 μg/mL rhBMP2 + nHAp-PEG/PLA和3.0 μg/mL rhBMP2 + 含nHAp-PEG/PLA的复合物在第3天和第10天诱导ALP活性。0.5 μg/mL rhBMP2 + nHAp-PEG/PLA应用刺激Runx2和OPN基因表达。结论：rhBMP2 + nHAp-PEG/PLA复合物刺激hBMMSC增殖和分化。具有低剂量rhBMP2的nHAp-PEG/PLA复合物可能在未来的临床PLSF应用中增强骨形成。

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