Experimental groups.

Aim
To evaluate in vitro the effect of laser photobiomodulation (PBM) combined or not with 30-nm hydroxyapatite nanoparticles (HANp), on the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) by morphometric analysis using artificial intelligence programs (TensorFlow and ArcGIS).
Methods
UC-MSCs were isolated and cultured until 80% confluence was reached. The cells were then plated according to the following experimental groups: G1 –control (DMEM), G2 –BMP-2, G3 –BMP-7, G4 –PBM (660 nm, 10 mW, 2.5 J/cm2, spot size of 0.08 cm2), G5 –HANp, G6 –HANp + PBM, G7 –BMP-2 + PBM, and G8 –BMP-7 + PBM. The MTT assay was used to analyze cell viability at 24, 48 and 72 h. Osteogenic differentiation was assessed by Alizarin Red staining after 7, 14 and 21 days. For morphometric analysis, areas of osteogenic differentiation (pixel2) were delimited by machine learning using the TensorFlow and ArcGIS 10.8 programs.
Results
The results of the MTT assay showed high rates of cell viability and proliferation in all groups when compared to control. Morphometric analysis revealed a greater area of osteogenic differentiation in G5 (HANp = 142709,33±36573,39) and G6 (HANp + PBM = 125452,00±24226,95) at all time points evaluated.
Conclusion
It is suggested that HANp, whether combined with PBM or not, may be a promising alternative to enhance the cellular viability and osteogenic differentiation of hUC-MSCs.

研究目的：本研究旨在通过人工智能程序（TensorFlow与ArcGIS）开展形态计量分析，体外评估激光光生物调节作用（Laser Photobiomodulation, PBM）联合或不联合30纳米羟基磷灰石纳米颗粒（30-nm hydroxyapatite nanoparticles, HANp）对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）骨向分化的影响。
研究方法：本研究分离培养人脐带间充质干细胞（hUC-MSCs）至细胞汇合度达80%，随后按以下实验组接种细胞：G1为对照组（DMEM培养基）、G2为骨形态发生蛋白2（Bone Morphogenetic Protein 2, BMP-2）组、G3为骨形态发生蛋白7（Bone Morphogenetic Protein 7, BMP-7）组、G4为PBM组（波长660 nm，功率10 mW，能量密度2.5 J/cm²，光斑面积0.08 cm²）、G5为HANp组、G6为HANp联合PBM组、G7为BMP-2联合PBM组、G8为BMP-7联合PBM组。分别于24 h、48 h、72 h采用MTT检测分析细胞活力；于诱导7、14、21 d后采用茜素红染色评估细胞骨向分化水平。形态计量分析阶段，通过机器学习结合TensorFlow与ArcGIS 10.8程序划定骨向分化区域（单位：像素²）。
研究结果：MTT检测结果显示，与对照组相比，所有实验组的细胞活力与增殖率均处于较高水平。形态计量分析结果表明，在所有检测时间点内，G5组（HANp组：142709.33±36573.39）与G6组（HANp联合PBM组：125452.00±24226.95）的骨向分化区域面积更大。
研究结论：本研究提示，无论是否联合PBM，HANp均有望成为提升人脐带间充质干细胞（hUC-MSCs）细胞活力与骨向分化能力的潜在可行方案。