Otero L, Carrillo N, Calvo-Guirado J L, Villamil J, Delgado-Ruíz R A
Pontificia Universidad Javeriana, Carrera 7 No. 40-62, Bogotá, Colombia.
Stony Brook University School of Dental Medicine, New York, United States.
Br J Oral Maxillofac Surg. 2017 Sep;55(7):697-702. doi: 10.1016/j.bjoms.2017.05.005. Epub 2017 Jun 13.
The purpose of this study was to analyse the potential of platelet-rich plasma (PRP) culture media to induce osteogenic differentiation of periodontal ligament stem cells and dental pulp stem cells compared with four other methods of culture. Both types of cell were collected from 35 healthy patients and cultured in five different media (Dulbecco's modified eagle's medium (DMEM); DMEM and melatonin; DMEM and PRP; DMEM and ascorbic acid 200μmol; DMEM and l-ascorbate 2-phosphate 50μmol). Cells were characterised by flow cytometry. Alizarin Red stain, alkaline phosphatase stain, and the expression of collagen type 1 (Col-1), runt-related transcription factor (RUNX2), osteoprotegerin, and osteopontin (quantified by qRT-PCR) were used to detect the osteogenic profile in each culture. Flow cytometry showed that both types of stem cell were a homogeneous mixture of CD90(+), CD105(+), STRO-1(+), CD34 (-), and CD45 (-) cells. Dental pulp stem cells that were cultured with PRP showed the best osteogenic profile (RUNX2 p=0.0002; osteoprotegerin p=0.001). The group of these stem cells that showed the best osteogenic profile was also cultured with PRP (osteoprotegerin p=0.001). Medium five (with l-ascorbate 2-phosphate 50μmol added) showed an increase in all osteogenic markers for periodontal ligament stem cells after PRP, while the best culture conditions for osteogenic expression of dental pulp stem cells after PRP was in medium four (ascorbic acid 200μmol added). These results suggested that culture in PRP induces osteogenic differentiation of both types of stem cell, modulating molecular pathways to promote bony formation.
本研究的目的是分析富含血小板血浆（PRP）培养基与其他四种培养方法相比，诱导牙周膜干细胞和成牙本质干细胞成骨分化的潜力。从35名健康患者中收集这两种类型的细胞，并在五种不同的培养基中培养（杜氏改良 Eagle 培养基（DMEM）；DMEM 与褪黑素；DMEM 与 PRP；DMEM 与 200μmol 抗坏血酸；DMEM 与 50μmol 1-抗坏血酸-2-磷酸酯）。通过流式细胞术对细胞进行表征。采用茜素红染色、碱性磷酸酶染色以及Ⅰ型胶原（Col-1）、 runt 相关转录因子（RUNX2）、骨保护素和骨桥蛋白的表达（通过 qRT-PCR 定量）来检测每种培养物中的成骨情况。流式细胞术显示，这两种类型的干细胞均为 CD90(+)、CD105(+)、STRO-1(+)、CD34 (-)和 CD45 (-)细胞的均匀混合物。用 PRP 培养的牙髓干细胞显示出最佳的成骨情况（RUNX2 p = 0.0002；骨保护素 p = 0.001）。显示出最佳成骨情况的这些干细胞组也用 PRP 培养（骨保护素 p = 0.001）。培养基五（添加 50μmol 1-抗坏血酸-2-磷酸酯）显示，PRP 处理后牙周膜干细胞的所有成骨标志物均增加，而 PRP 处理后牙髓干细胞成骨表达的最佳培养条件是在培养基四（添加 200μmol 抗坏血酸）中。这些结果表明，在 PRP 中培养可诱导这两种类型干细胞的成骨分化，调节分子途径以促进骨形成。
