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Effects of calcium concentration on nonviral gene delivery to bone marrow-derived stem cells.
Journal of Tissue Engineering and Regenerative Medicine ( IF 3.3 ) Pub Date : 2019-11-11 , DOI: 10.1002/term.2971
Timothy M Acri 1 , Noah Z Laird 1 , Sean M Geary 1 , Aliasger K Salem 1 , Kyungsup Shin 2
Affiliation  

BACKGROUND Calcium ions (Ca2+ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth. METHODS Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in media with Ca2+ concentrations ranging from 0 to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, messenger RNA (mRNA) for osteocalcin and CBFA1 was quantified using real time-polymerase chain reaction (PCR). Calcium deposition was qualitatively assessed after three and 14 days using Alizarin Red staining. RESULT Our results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP-2). CONCLUSION Ca2+ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of polyethyleneimine-plasmid bone morphogenetic protein 2 (PEI-pBMP-2) and 10 mM Ca2+ as compared with PEI-pBMP-2 alone.

中文翻译:

钙浓度对非病毒基因传递至骨髓干细胞的影响。

背景技术钙离子(Ca 2+)影响自然的骨愈合,并且钙经常用于骨组织工程支架和水泥中。支架还可整合基因递送系统,以进一步促进成骨细胞分化。因此,我们的目标是确定Ca2 +浓度是否影响骨髓基质细胞的转染,因为这些细胞在骨愈合中起着重要作用,并且可以渗入旨在促进骨生长的基因激活支架。方法将骨髓来源的间充质干细胞(BMSC)培养在Ca2 +浓度为0至20 mM的培养基中,并用聚乙烯亚胺-质粒DNA(PEI-pDNA)复合物转染。分别使用MTS分析和流式细胞仪确定细胞活力和转染效率。使用荧光显微镜法评估BMSC中PEI-pDNA复合物的定位。为了确定BMSC的分化,使用实时聚合酶链反应(PCR)对骨钙蛋白和CBFA1的信使RNA(mRNA)进行了定量。在三天和十四天后使用茜素红染色定性评估钙沉积。结果我们的结果表明,在细胞活力和转染效率方面,介于8和12 mM之间的Ca2 +水平对BEIs用PEI-pDNA复合物的转染产生积极影响。当用编码骨形态发生蛋白2(BMP-2)的质粒DNA转染细胞时,Ca2 +浓度为10 mM也会增加成骨基因骨钙蛋白的表达。结论与PEI-pDNA复合物结合使用时,浓度为10 mM的Ca2 +可以显着降低毒性并提高转染效率,
更新日期:2019-11-11
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