Biomaterials & Tissue Engineering Division, Dept. of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA.
Biomaterials. 2013 Oct;34(32):7862-72. doi: 10.1016/j.biomaterials.2013.07.029. Epub 2013 Jul 24.
Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising choice of patient-specific stem cells with superior capability of cell expansion. There has been no report on bone morphogenic protein 2 (BMP2) gene modification of iPSC-MSCs for bone tissue engineering. The objectives of this study were to: (1) genetically modify iPSC-MSCs for BMP2 delivery; and (2) to seed BMP2 gene-modified iPSC-MSCs on calcium phosphate cement (CPC) immobilized with RGD for bone tissue engineering. iPSC-MSCs were infected with green fluorescence protein (GFP-iPSC-MSCs), or BMP2 lentivirus (BMP2-iPSC-MSCs). High levels of GFP expression were detected and more than 68% of GFP-iPSC-MSCs were GFP positive. BMP2-iPSC-MSCs expressed higher BMP2 levels than iPSC-MSCs in quantitative RT-PCR and ELISA assays (p < 0.05). BMP2-iPSC-MSCs did not compromise growth kinetics and cell cycle stages compared to iPSC-MSCs. After 14 d in osteogenic medium, ALP activity of BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs (p < 0.05), indicating that BMP2 gene transduction of iPSC-MSCs enhanced osteogenic differentiation. BMP2-iPSC-MSCs were seeded on CPC scaffold biofunctionalized with RGD (RGD-CPC). BMP2-iPSC-MSCs attached well on RGD-CPC. At 14 d, COL1A1 expression of BMP2-iPSC-MSCs was 1.9 times that of iPSC-MSCs. OC expression of BMP2-iPSC-MSCs was 2.3 times that of iPSC-MSCs. Bone matrix mineralization by BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs at 21 d. In conclusion, iPSC-MSCs seeded on CPC were suitable for bone tissue engineering. BMP2 gene-modified iPSC-MSCs on RGD-CPC underwent osteogenic differentiation, and the overexpression of BMP2 in iPSC-MSCs enhanced differentiation and bone mineral production on RGD-CPC. BMP2-iPSC-MSC seeding on RGD-CPC scaffold is promising to enhance bone regeneration efficacy.
人诱导多能干细胞衍生的间充质干细胞（iPSC-MSCs）是一种有前途的患者特异性干细胞选择，具有卓越的细胞扩增能力。目前尚无关于骨形态发生蛋白 2（BMP2）基因修饰 iPSC-MSCs 用于骨组织工程的报道。本研究的目的是：（1）基因修饰 iPSC-MSCs 以递送 BMP2；（2）将 BMP2 基因修饰的 iPSC-MSCs 接种在 RGD 固定的磷酸钙水泥（CPC）上用于骨组织工程。iPSC-MSCs 被感染绿色荧光蛋白（GFP-iPSC-MSCs）或 BMP2 慢病毒（BMP2-iPSC-MSCs）。检测到高水平的 GFP 表达，超过 68%的 GFP-iPSC-MSCs 呈 GFP 阳性。定量 RT-PCR 和 ELISA 检测显示，BMP2-iPSC-MSCs 的 BMP2 表达水平高于 iPSC-MSCs（p<0.05）。与 iPSC-MSCs 相比，BMP2-iPSC-MSCs 的生长动力学和细胞周期阶段没有受到影响。在成骨培养基中培养 14 天后，BMP2-iPSC-MSCs 的碱性磷酸酶（ALP）活性是 iPSC-MSCs 的 1.8 倍（p<0.05），表明 BMP2 基因转导增强了 iPSC-MSCs 的成骨分化。BMP2-iPSC-MSCs 接种在 RGD 生物功能化的 CPC 支架上（RGD-CPC）。BMP2-iPSC-MSCs 很好地附着在 RGD-CPC 上。在 14 天时，BMP2-iPSC-MSCs 的 COL1A1 表达是 iPSC-MSCs 的 1.9 倍。BMP2-iPSC-MSCs 的 OC 表达是 iPSC-MSCs 的 2.3 倍。在 21 天时，BMP2-iPSC-MSCs 的骨基质矿化是 iPSC-MSCs 的 1.8 倍。总之，接种在 CPC 上的 iPSC-MSCs 适合用于骨组织工程。在 RGD-CPC 上进行 BMP2 基因修饰的 iPSC-MSCs 经历了成骨分化，并且 iPSC-MSCs 中 BMP2 的过表达增强了 RGD-CPC 上的分化和骨矿物质生成。在 RGD-CPC 支架上接种 BMP2-iPSC-MSCs 有望增强骨再生效果。
