Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, Debrecen, Hungary.
Department of Applied Mathematics and Probability Theory, Faculty of Informatics, University of Debrecen, Debrecen, Hungary.
Bone. 2020 Mar;132:115214. doi: 10.1016/j.bone.2019.115214. Epub 2019 Dec 26.
Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily, it is known to be a factor involved in skeletal development and capable of inducing in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) isolated from extracted third molar teeth are an ideal resource for bone tissue engineering and regeneration applications, due to their convenient isolation, safe cryopreservation, and easy maintenance in cell cultures. The aims of this study were to deliver BMP-2 under control of the tetracycline-inducible (tet-on) promoter into dental pulp stem cells and to examine whether these BMP-2 expressing cell lines are capable of promoting osteogenic differentiation in vitro. BMP-2 gene was cloned into the lentiviral transfer plasmid pTet-IRES-EGFP and used to establish the DPSC-BMP-2 cell line. DPSC, DPSC-GFP (mock) and DPSC-BMP-2 cell lines were cultured in growth medium or osteogenic medium in the presence or absence of 100 ng/ml doxycycline. To assess differentiation, alkaline phosphatase activity, calcium accumulation and gene transcription levels of different genes involved in osteogenic differentiation (BMP-2, Runx2, alkaline phosphatase, and noggin) were measured. Doxycycline-induced BMP-2 expression induced the differentiation of DPSCs into the preosteoblastic stage but could not favor the further maturation into osteoblasts and osteocytes. We found that while Runx2 gene transcription was continuously upregulated in doxycycline-treated DPSC-BMP-2 cells, the alkaline phosphatase activity and the accumulation of minerals were reduced. As a result of the increased BMP-2 expression, the transcription level of the BMP antagonist noggin was also upregulated, and probably caused the observed effects regarding alkaline phosphatase (ALP) activity and mineral deposition. Our study shows that this system is effective in controlling transgene expression in DPSC cell line. Exploration of all known factors affecting osteogenic differentiation and their interactions is of major importance for the field of regenerative medicine. As the metabolic reaction to the upregulated transgene transcription appears to be cell line-specific, a wrongly selected target gene and/or regulation system could have adverse effects on differentiation.
骨形态发生蛋白 2（BMP-2）是转化生长因子-β超家族的成员，已知其是参与骨骼发育并能诱导间充质干细胞（MSCs）体外成骨分化的因子。从拔除的第三磨牙中分离的牙髓干细胞（DPSCs）是骨组织工程和再生应用的理想资源，因为它们易于分离、安全冷冻保存，并且在细胞培养中易于维持。本研究的目的是在四环素诱导（tet-on）启动子的控制下将 BMP-2 递送至牙髓干细胞，并研究这些表达 BMP-2 的细胞系是否能够在体外促进成骨分化。BMP-2 基因被克隆到慢病毒转移质粒 pTet-IRES-EGFP 中，并用于建立 DPSC-BMP-2 细胞系。在存在或不存在 100ng/ml 强力霉素的情况下，将 DPSC、DPSC-GFP（对照）和 DPSC-BMP-2 细胞系在生长培养基或成骨培养基中培养。为了评估分化，测量碱性磷酸酶活性、钙积累以及不同成骨分化基因（BMP-2、Runx2、碱性磷酸酶和 noggin）的转录水平。强力霉素诱导的 BMP-2 表达诱导 DPSCs 分化为成骨前体细胞，但不能促进其进一步成熟为成骨细胞和骨细胞。我们发现，虽然 Runx2 基因转录在强力霉素处理的 DPSC-BMP-2 细胞中持续上调，但碱性磷酸酶活性和矿物质积累减少。由于 BMP-2 表达增加，BMP 拮抗剂 noggin 的转录水平也上调，可能导致碱性磷酸酶（ALP）活性和矿物质沉积观察到的影响。我们的研究表明，该系统可有效控制 DPSC 细胞系中转基因的表达。探索所有已知影响成骨分化的因素及其相互作用对于再生医学领域非常重要。由于对上调的转基因转录的代谢反应似乎是细胞系特异性的，因此错误选择的靶基因和/或调节系统可能会对分化产生不利影响。
