Study on Osteogenic Ability of Dental Pulp Stem Cells at Different Concentrations
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摘要:
目的 探究不同浓度的牙髓干细胞（dental pulp stem cell，DPSC）在定量Bio-Oss骨粉中最佳的成骨比例。 方法 收集牙髓组织、组织块贴壁法分离培养原代细胞，流式细胞术鉴定DPSC表面标志物，诱导成骨及成脂鉴定DPSC多向分化潜能；GFP慢病毒转染DPSC，嘌呤霉素筛选GFP-DPSC；设置1×105、2×105、4×105、8×1054组细胞数接种于0.05 g骨粉并成骨培养，对照组不加骨粉，第3天和第7天进行碱性磷酸酶活性测定；培养第7天在扫描电镜下观察细胞在骨粉内的形态变化。 结果 原代分离培养成功的DPSC符合间充质干细胞来源，相关表面标记物高表达，可向成脂和成骨分化；GFP成功转染DPSC，第3天和第7天进行碱性磷酸酶活性检测，实验组均高于对照组，差异具有统计学意义（P < 0.05）；扫描电镜发现DPSC在骨粉表面爬行，部分细胞伸出伪足，4×10 5和8×105 2组细胞较为密集。 结论 4×105～8×105个DPSC与0.05 g Bio-Oss骨粉复合培养成骨效果较好，可以缩短成骨周期，细胞若接种过多，成骨效果反而抑制，造成干细胞的浪费。 Abstract:Objective To explore the optimal osteogenic ratio of Dental pulp stem cells （DPSC） with different concentrations in quantitative Bio-Oss bone powder. Methods The primary cells were isolated and cultured by adherent method. The surface markers of DPSC were identified by flow cytometry, and the multidirectional differentiation potential of DPSC was identified by osteogenesis and adipogenesis. DPSC was transfected with GFP lentivirus, and GFP-DPSC was screened with puromycin. The cells in groups 1×105, 2×105, 4×105 and 8×105 were inoculated with 0.05 g bone powder and cultured for osteogenesis, while the control group did not add bone powder. Alkaline phosphatase activity was measured on day 3 and day 7. On the 7th day of culture, the morphological changes of the cells in bone powder were observed under scanning electron microscope. Results The DPSC was derived from mesenchymal stem cells with high expression of related surface markers and could differentiate into adipogenic and osteogenic cells. GFP was successfully transfected into DPSC, and alkaline phosphatase activity was detected on the 3rd and 7th day, and the experimental group was higher than the control group, and the difference was statistically significant （P < 0.05）. Scanning electron microscopy showed that DPSC crawled on the surface of bone powder, and some cells extended pseudopodia, and the cells of 4×10 5 and 8×105 groups were more dense. Conclusions The combined culture of 4×105-8×105 DPSC and 0.05 g Bio-Oss bone powder has a good osteogenic effect, which can shorten the osteogenic cycle. If the cells are inoculated too much, the osteogenic effect is inhibited, resulting in the waste of stem cells. -
图 1 DPSC的培养及多向分化能力鉴定
A：DPSC原代细胞（×50）；B：扩大培养第三代细胞（×50）；C：DPSC成骨对照组组染色所见（×100）；D：DPSC成骨诱导组染色所见（×100）；E：DPSC成脂对照组染色所见（×200）；F：DPSC成脂诱导组染色所见（×200）。
Figure 1. Culture of DPSC and appraisal of multi -directional differentiation ability
图 3 GFP转染DPSC不同时间荧光表达（×100）
A：36 h GFP转染DPSC的荧光表达；B：72 h GFP转染DPSC的荧光表达；C：96 h GFP转染DPSC的荧光表达。
Figure 3. Fluorescence expression of GFP transfected DPSC at different time
图 4 GFP-DPSC在Bio-Oss骨粉中的生长（×100）
A：第3天时GFP-DPSC在Bio-Oss骨粉中的状态；B：第14天时GFP-DPSC在Bio-Oss骨粉中的状态。
Figure 4. Growth of GFP-DPSC in Bio-Oss bone powder
图 7 1×105组同一视野下SEM图像
A：100 μm ；B：50 μm。
Figure 7. SEM images of 1×105 groups in the same field of view
图 8 2×105组同一视野下SEM图像
A：100 μm ；B：50 μm。
Figure 8. SEM images of 2×105 groups in the same field of view
图 9 4×105组同一视野下SEM图像
A：100 μm ；B：50 μm。
Figure 9. SEM images of 4×105 groups in the same field of view
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