Acceleration of Fracture Healing by Overexpression of Basic Fibroblast Growth Factor in the Mesenchymal Stromal Cells

Abstract

In this study, we engineered mesenchymal stem cells (MSCs) to over-express basic fibroblast growth factor (bFGF) and evaluated its effects on fracture healing. Adipose-derived mouse MSCs were transduced to express bFGF and green fluorescence protein (ADSC^bFGF -GFP). Closed-femoral fractures were performed with osterix-mCherry reporter mice of both sexes. The mice received 3 × 10⁵ ADSCs transfected with control vector or bFGF via intramuscular injection within or around the fracture sites. Mice were euthanized at days 7, 14, and 35 to monitor MSC engraftment, osteogenic differentiation, callus formation, and bone strength. Compared to ADSC culture alone, ADSC^bFGF increased bFGF expression and higher levels of bFGF and vascular endothelial growth factor (VEGF) in the culture supernatant for up to 14 days. ADSC^bFGF treatment increased GFP-labeled MSCs at the fracture gaps and these cells were incorporated into the newly formed callus. quantitative reverse transcription polymerase chain reaction (qRT-PCR) from the callus revealed a 2- to 12-fold increase in the expression of genes associated with nervous system regeneration, angiogenesis, and matrix formation. Compared to the control, ADSC^bFGF treatment increased VEGF expression at the periosteal region of the callus, remodeling of collagen into mineralized callus and bone strength. In summary, MSC^bFGF accelerated fracture healing by increasing the production of growth factors that stimulated angiogenesis and differentiation of MSCs to osteoblasts that formed new bone and accelerated fracture repair. This novel treatment may reduce the time required for fracture healing. Stem Cells Translational Medicine 2017;6:1880-1893.

Keywords: Basic fibroblast growth factor; Bone strength; Callus; Mesenchymal stromal cells; Osteoblasts.

Figure 1

bFGF levels in MSCs and culture supernatant. Adipose‐derived mouse MSCs were transduced with bFGF or control vectors and grown to 60% confluence at P0. bFGF and VEGF levels were measured by enzyme‐linked immunosorbent assay (ELISA) at days 3, 7, 10, and 14 post‐bFGF transduction in both cell lysates and culture medium. Abbreviations: bFGF, basic fibroblast growth factor; MSCs, mesenchymal stromal cells; VEGF, vascular endothelial growth factor.

Figure 2

Engraftment of transplanted MSCs in fracture callus at days 14 and 21 post‐fracture and cell transplantations. Closed‐femoral fractures were performed in the right femurs of 2‐month‐old female osterix‐mcherry mice. These mice received 3 × 10⁵ ADSCs transfected with control vector or ADSC^bFGF via IM injection adjacent to fracture sites. (A): Mice were sacrificed at day 14 post‐fracture. They were injected with 100 μl of 20mg/mL D‐Luciferin Firefly 10 minutes prior to sacrifice. Frozen sections of callus were stained with anti‐luciferase antibody followed by Alexa‐Fluo 488‐conjugated secondary antibody. Scale bar 50 µm. Mice were sacrificed at day 21. Some transplanted cells (in green) were retained in the bone marrow space within the callus (white arrows) in ADSC (B) or ADSC^bFGF (C) treated groups. Scale bar 100 µm. Abbreviations: ADSC, Adipose‐derived mouse MSCs; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; Luc, luciferase.

Figure 3

Paracrine signals that contributed to callus formation. Mice were treated as described in Figure 2 and sacrificed at day 14. Photos were taken from central regions of the callus (small insert to the right). Femurs were fractured in osterix‐mcherry WT mice: (A): low magnification showing the fractured callus. Scale bar 100 µm. (B): Callus were stained with anti‐αSMA conjugated to FITC (green arrows). Scale bar 50 µm. (C): Fractures in osterix‐mcherry WT mice were stained with anti‐VEGF and Alexa‐Fluo 594‐conjugated secondary antibody. (D): Fractures in osterix‐mcherry WT mice were stained with anti‐PDGF‐BB and Alexa‐Fluo 488‐conjugated secondary antibody. Abbreviations: αSMA+, smooth muscle α–actin; ADSC, adipose‐derived mouse MSCs; bFGF, basic fibroblast growth factor; C, callus; PDGF, platelet‐derived growth factor PDGF; Ps, periosteal surface. Scale bar 50 µm.

Figure 4

Bone formation at day 21. Mice were treated as described in Figure 2 and sacrificed at day 21. Calcein (10 mg/kg) was injected s.c. in mice at 9 and 2 days before sacrifice. Endogenous osterix + cells are in red and green is calcein labeling, corresponding to mineral deposition. ADSC^bFGF increased the green‐labeled mineralized surface and bone formation rate in both the female (A) and male (B) mice. Scale bar 100 µm. *, Significant difference between indicated group by Wilcoxon ranked‐sum comparison test. Abbreviations: ADSC, adipose‐derived mouse MSCs; bFGF, basic fibroblast growth factor; DAPI, 4′,6‐diamidino‐2‐phenylindole; PBS, phosphate buffered saline.

Figure 5

Callus formation and bone strength at days 21 and 35. Mice were treated as described in Figure 2 and sacrificed at days 21 or 35 post‐fracture. Callus structure was first measured by microCT, then the femurs were subjected to three point‐bending tests for both female (A–D) and male (E–H) mice. Representative two‐dimensional images (A, E) or 3D thickness mappings (B, F) are presented for indicated groups. Red represents highly mineralized tissue, and green represents less mineralized tissue (B, F). * Significant difference between indicated group by Wilcoxon ranked‐sum comparison test. Abbreviations: ADSC, adipose‐derived mouse MSCs; bFGF, basic fibroblast growth factor; PBS, phosphate buffered saline.

Figure 6

Effects of ADSC^bFGF treatment on chondrogenesis and osteogenesis during fracture healing. Col2‐CreERT mice were crossed with tdTomato‐mCherry reporter mice so that Col2+ cells and their descendants expressed tdTomato. Cre was activated by IP injection of tamoxifen (3 mg/kg × 2 days) prior to femoral fracture. Mice were sacrificed at days 7 or 14. Scale bar 100 µm. Abbreviations: ADSC, adipose‐derived mouse MSCs; bFGF, basic fibroblast growth factor; PBS, phosphate buffered saline.