Effect of dietary calcium on spinal bone fusion in an ovariectomized rat model

Abstract

Objective: To evaluate the effect of calcium supplementation on spinal bone fusion in ovariectomized (OVX) rats.

Methods: Sixteen female Sprague Dawley rats underwent bilateral ovariectomy at 12 weeks of age to induce osteoporosis and were randomly assigned to two groups : control group (n=8) and calcium-supplemented group (OVX-Ca, n=8). Autologous spinal bone fusion surgery was performed on both groups 8 weeks later. After fusion surgery, the OVX-Ca group was supplemented with calcium in drinking water for 8 weeks. Blood was obtained 4 and 8 weeks after fusion surgery. Eight weeks after fusion surgery, the rats were euthanized and the L4-5 spine removed. Bone fusion status and fusion volume were evaluated by manual palpation and three-dimensional computed tomography.

Results: The mean fusion volume in the L4-5 spine was significantly greater in the OVX-Ca group (71.80±8.06 mm(3)) than in controls (35.34±8.24 mm(3)) (p<0.01). The level of osteocalcin, a bone formation marker, was higher in OVX-Ca rats than in controls 4 weeks (610.08±10.41 vs. 551.61±12.34 ng/mL) and 8 weeks (552.05±19.67 vs. 502.98±22.76 ng/mL) after fusion surgery (p<0.05). The level of C-terminal telopeptide fragment of type I collagen, a bone resorption marker, was significantly lower in OVX-Ca rats than in controls 4 weeks (77.07±12.57 vs. 101.75±7.20 ng/mL) and 8 weeks (69.58±2.45 vs. 77.15±4.10 ng/mL) after fusion surgery (p<0.05). A mechanical strength test showed that the L4-5 vertebrae in the OVX-Ca group withstood a 50% higher maximal load compared with the controls (p<0.01).

Conclusion: Dietary calcium given to OVX rats after lumbar fusion surgery improved fusion volume and mechanical strength in an ovariectomized rat model.

Keywords: Calcium; Osteoporosis; Ovariectomized rat; Spinal bone fusion.

Fig. 1

Experimental groups and schedule. Sixteen female Sprague Dawley rats underwent bilateral ovariectomy at 12 weeks of age to induce osteoporosis. Then, autologous spinal bone fusion surgery was performed on both groups (Control; n=8, OVX-Ca; n=8) 8 weeks later. Eight weeks after fusion surgery, the rats were euthanized and the L4-5 spine was removed. The serum concentrations of markers of bone metabolism were measured by ELISA at baseline, 8 weeks after ovariectomy, and 4 and 8 weeks after fusion surgery. The bone fusion status and fusion volume were evaluated by manual palpation and 3D-µCT scanning. The mechanical strength was also tested. OVX-Ca : calcium-supplemented group.

Fig. 2

Fusion volume. At 8 weeks after fusion surgery, the fusion masses comprised a mixture of dispersed grafted bone fragments and newly formed bone in the OVX-Ca group (p<0.01). ^*p<0.01 for OVX-Ca vs. control group. OVX-Ca : calcium-supplemented group.

Fig. 3

Representative µCT images of vertebrae from the two groups. OVX-Ca rats show larger fusion mass than controls eight weeks after surgery. ▸ : fusion mass, OVX-Ca : calcium-supplemented group.

Fig. 4

Serum osteocalcin concentration (A) as a marker of bone formation. The concentration of C-terminal telopeptide fragment of type I collagen (CTX-1) (B) was used as a marker of bone resorption. Values are represented as the mean±standard deviation. ^*p<0.05 for the OVX-Ca vs. control group. OVX-Ca : calcium-supplemented group.

Fig. 5

The loading force to the maximal load on the fusion mass in L4 and L5 vertebrae determined using a three-point bending test. The OVX-Ca group significantly increases the maximal load as compared with to the control group (p<0.01). ^*p<0.01 for OVX vs. control group comparison. OVX-Ca : calcium-supplemented group.