Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model

Abstract

Bone Tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments for large non-healing bone defects. BTE approaches mimic autologous bone grafts, by combining cells, scaffold, and growth factors, and have the added benefit of being able to manipulate these constituents to optimize healing. Electrical stimulation (ES) has long been used to successfully treat non-healing fractures and has recently been shown to stimulate bone cells to migrate, proliferate, align, differentiate, and adhere to bio compatible scaffolds, all cell behaviors that could improve BTE treatment outcomes. With the above in mind we performed in vitro experiments and demonstrated that exposing Mesenchymal Stem Cells (MSC) + scaffold to ES for 3 weeks resulted in significant increases in osteogenic differentiation. Then in in vivo experiments, for the first time, we demonstrated that exposing BTE treated rat femur large defects to ES for 8 weeks, caused improved healing, as indicated by increased bone formation, strength, vessel density, and osteogenic gene expression. Our results demonstrate that ES significantly increases osteogenic differentiation in vitro and that this effect is translated into improved healing in vivo. These findings support the use of ES to help BTE treatments achieve their full therapeutic potential.

Figure 1

Cell viability and Alkaline Phosphatase activity measurements, in vitro. (A) ß-TCP scaffold granules. (B) AT-MSCs were seeded on ß-TCP granules and stained with DAPI. Red arrows show cell nuclei and black arrows show the pores in the scaffold. (C) Effect of electrical stimulation on the viability of AT-MSCs seeded on ß-TCP scaffold, during 21 days in culture. Cell viability measured by MTT assay, shown as fold change relative to day 0. There was no significant difference in viability between cells exposed, and not (controls) exposed to electrical stimulation. (D) Effect of electrical stimulation on Alkaline Phosphatase activity of AT-MSCs during 21 days. Data are presented as mean ± SD (n = 3), (*p < 0.05).

Figure 2

Osteogenic marker gene expression, in vitro. Expression of (A) TGF-β1, (B) BMP2, (C) Osteopontin, and (D) Calmodulin were measured by qRT-PCR and compared between electrically stimulated and control groups. Relative expressions were normalized to RPLP1 and YWHAZ (housekeeping genes). In the experimental group expression of genes TGF-ß1 (day 7), BMP2 (days 3, 7, 14 and 21), Osteopontin (days 3, 7 and 14), and Calmodulin (day 21) was significantly increased in comparison to controls. Data are presented as mean ± SD (n = 3), (*p < 0.05).

Figure 3

Histological sections of femur defect. After one week post-surgery (A) Control, (B) Sham, and (C) Electrically stimulated groups were stained with Alcian Blue, Orange-G and Hematoxylin. Histological assessment at this time point showed no regeneration in control and sham groups. Arrow shows endochondral ossification center in electrically stimulated group, (scale bar = 1 mm). At eight weeks post-surgery (D) Control, (E) Sham, and (F) Electrically stimulated groups, were assessed with the same histological staining, (Scale bar = 500 μm). Bottom rows are higher magnification images from the location of interest; (d1) fibrous connective tissue within the defect in the control group, (d2) scaffold material covered with fibrous tissue, (e1) fibrous connective tissue within the defect in the sham group, (e2) scaffold material covered with mixed tissues, (f1) cartilaginous and bony tissues within the defect in the experimental group, and (f2) scaffold material covered with cartilaginous and bony tissues, (20×) (scale bar = 50 μm).

Figure 4

Bone healing score and tissue constituent percentage of the defect. At eight weeks post-surgery, (A) Healing scores were calculated using histological image analysis. Data are shown as mean ± SD (n = 5). Experimental group scored significantly higher than control and sham groups, (#p < 0.1; *p < 0.05; **p < 0.01) (B) Histomorphometric distribution of newly formed bone, fibrous, and cartilage tissues within the defect, at eight weeks post-surgery. Percentage measured from the whole defect area using ImageJ. Data are shown as mean ± SD (n = 5).

Figure 5

Vascularization in the defect area 8 weeks post-surgery. (A) Histological sections of electrically stimulated tissue stained with anti-alpha smooth muscle actin (α-SMA) antibodies (scale bar 1 mm). Higher magnification images of vessels in the fibrous tissue (a1) and in bone tissue (a2) (20×, scale bar 100 μm) (B) Vessel density (b1) was calculated for the entire defect area, (#p < 0.1). Vessel density calculated in the fibrous tissue and non-fibrous tissues in controls (b2), sham (b3) and experimental (b4) groups. Data are presented as mean ± SD (n = 5). Vessel density in fibrous tissue was significantly (**p < 0.01) higher than non-fibrous tissue in all groups. Vessel density in fibrous tissue was lower in the experimental than in the sham group (*p < 0.1).

Figure 6

Mechanical properties of newly formed bone in the defect. (A) Load displacement diagram of representative femur samples from sham (black), experimental (red) group and intact bone (blue). (B) Maximum load before fracture was measured at eight weeks after surgery. Maximum load was higher in experimental compared to sham group (p < 0.1). (C) Yield load, in the experimental group was higher than in the sham group (p = 0.058). (D) Stiffness was not different between sham and experimental groups (#p < 0.1).

Figure 7

Osteogenic marker gene expression, in vivo. At eight weeks post-surgery, osteogenic marker gene expression measured by means of qRT-PCR and normalized to RPLP1 and YWHAZ (housekeeping genes). (A) TGF-ß1, (B) BMP2, (C) RunX2, (D) ColIa2, (E) Osterix, (F) Osteopontin and (G) Calmodulin expression were significantly higher at eight weeks in the experimental in comparison to sham and control groups. Data are presented as mean ± SD (n = 5), (*p < 0.05).

Figure 8

In vivo rat model and electrical stimulation set-up. (A) Schematic shows the electrical stimulation device and the location of the anode and cathode in the defect. Device consists of a 1.2 V battery, 10 MΩ resistor, stainless steel cathode (shown in black) and platinum anode (shown in red). Cathode located within the defect, and the anode located in muscle tissue close to the defect area. (B) Dorsal view of rat with surgical incision and implanted device (arrow). (C) Surgical incision exposing right femur defect stabilized with stainless steel plate and screws, and electrodes (arrow) tunneled from the device to the defect area.