Research ArticleBone Regeneration

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

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Science Translational Medicine  17 May 2017:
Vol. 9, Issue 390, eaal3128
DOI: 10.1126/scitranslmed.aal3128
  • Fig. 1. Reporter gene expression in mini-pig tibial fractures after ultrasound-mediated gene delivery.

    (A) Ex vivo fluorescence imaging of an exposed GFP-treated fracture 5 days after treatment. Fracture margins are represented by a dashed rectangle. (B) Flow cytometry analysis of the percentage of GFP+ cells isolated from fracture sites 5 days after treatment with or without ultrasound (*P = 0.049). (C) Mean fluorescence intensity per cell isolated from fractures 5 days after treatment with or without ultrasound (**P = 0.0013). (D) Luciferase enzyme activity in cells isolated from fracture sites 5 days after treatment with or without ultrasound (****P = 0.0001). Percentage of (E) CD29+ (****P = 0.0001), (F) CD90+ (**P = 0.0057), and (G) CD44+ cells (P = 0.0531) out of GFP+ cells isolated from fractures 5 days after treatment with or without ultrasound. Data are means ± SEM; P values were determined by two-tailed Student’s t tests (No US, n = 3; US, n = 4; RLU, relative light units).

  • Fig. 2. BMP-6 expression after ultrasound-mediated gene delivery to tibia fractures in mini-pigs.

    (A) Gene [relative quantification (RQ); *P = 0.0111, ****P = 0.0001] and (B) protein expression (**P = 0.0085, ****P = 0.0001) in tibial fracture sites 2, 5, and 10 days after ultrasound-mediated BMP-6 gene delivery. Data are means ± SEM; P values were determined by two-way analysis of variance (ANOVA) with multiple comparisons (n = 3 per experimental group).

  • Fig. 3. Ultrasound-mediated BMP-6 gene delivery to mini-pig tibial bone fractures.

    Quantitative analysis of bone formation in the tibial fractures, including (A) bone volume density (****P = 0.0001) and (B) apparent density (**P = 0.0029). Data are means ± SEM; P values were determined by one-way ANOVA with multiple comparisons. (C) Representative μCT slices of the fractures 8 weeks after surgery. Asterisks represent new bone formation within the fracture. Arrows point to cortical discontinuity indicating nonunion within the fracture. Autograft margins are represented by a dashed square. (D) Masson’s trichrome staining of tibial fractures 8 weeks after surgery at low magnification (upper subfigures) and high magnification of the yellow square (lower subfigures). Arrows point to the border between native bone and newly formed bone in the fracture. Scale bars, 1 mm. (Autograft, n = 7; BMP-6 + US, n = 8; BMP-6, n = 6; US only, n = 3; Scaffold only, n = 4; HA, hydroxyapatite.)

  • Fig. 4. Biomechanical properties of treated tibiae.

    Torsion testing was performed on tibiae harvested from treated mini-pigs. (A) Photograph of a tibia reaching its failure point during biomechanical testing. Analysis of load and rotation was performed to determine the (B) maximum torque (strength; **P = 0.0048), (C) torsional rigidity (stiffness; *P = 0.02), and (D) energy to maximum (toughness; **P = 0.0077) of treated bones. Data are means ± SEM; P values were determined by one-way ANOVA with multiple comparisons (N·m, Newton meter; n = 5 per experimental group).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/390/eaal3128/DC1

    Fig. S1. Critical-sized bone fracture model in Yucatán mini-pigs’ tibia.

    Fig. S2. Endogenous mesenchymal progenitor cell recruitment to mini-pig tibial fracture site.

    Fig. S3. Ultrasound treatment setup.

    Fig. S4. Biodistribution of BMP-6 expression after ultrasound-mediated gene delivery.

    Fig. S5. Comparison of bone formation between male and female mini-pigs.

    Table S1. Primary data.

    Data file S1. MATLAB code of biomechanical analysis.

  • Supplementary Material for:

    In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

    Maxim Bez, Dmitriy Sheyn, Wafa Tawackoli, Pablo Avalos, Galina Shapiro, Joseph C. Giaconi, Xiaoyu Da, Shiran Ben David, Jayne Gavrity, Hani A. Awad, Hyun W. Bae, Eric J. Ley, Thomas J. Kremen, Zulma Gazit, Katherine W. Ferrara, Gadi Pelled, Dan Gazit*

    *Corresponding author. Email: dan.gazit{at}csmc.edu

    Published 17 May 2017, Sci. Transl. Med. 9, eaal3128 (2017)
    DOI: 10.1126/scitranslmed.aal3128

    This PDF file includes:

    • Fig. S1. Critical-sized bone fracture model in Yucatán mini-pigs’ tibia.
    • Fig. S2. Endogenous mesenchymal progenitor cell recruitment to mini-pig tibial fracture site.
    • Fig. S3. Ultrasound treatment setup.
    • Fig. S4. Biodistribution of BMP-6 expression after ultrasound-mediated gene delivery.
    • Fig. S5. Comparison of bone formation between male and female mini-pigs.
    • Legend for table S1
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.
    • Data file S1 (.m format). MATLAB code of biomechanical analysis.