Effect of alendronate or 8-prenylnaringenin applied as a single therapy or in combination with vibration on muscle structure and bone healing in ovariectomized rats

Abstract

Bisphosphonate alendronate (ALN), phytoestrogen 8-prenylnaringenin (8-PN) and the whole body vibration exert a favorable effect on osteoporotic bone. However, the impact of these treatments and the combination of pharmacological therapies with biomechanical stimulation on muscle and bone has not yet been explored in detail. The effect of ALN and 8-PN and their combination with the vibration (Vib) on skeletal muscle and bone healing was investigated in ovariectomized (Ovx) rats. Three-month old rats were Ovx (n = 78), or left intact (Non-Ovx; n = 12). Five weeks after Ovx, all rats were treated according to the group assignment (n = 12/13): 1) Non-Ovx; 2) Ovx; 3) Ovx + Vib; 4) Ovx + ALN; 5) Ovx + ALN + Vib; 6): Ovx + 8-PN; 7) Ovx + 8-PN + Vib. Treatments with ALN (0.58 mg/kg BW, in food), 8-PN (1.77 mg/kg BW, daily s.c. injections) and/or with vertical vibration (0.5 mm, 35 Hz, 1 g, 15 min, 2×/day, 5×/week) were conducted for ten weeks. Nine weeks after Ovx, all rats underwent bilateral tibia osteotomy with plate osteosynthesis and were sacrificed six weeks later. Vibration increased fiber size and capillary density in muscle, enlarged callus area and width, and decreased callus density in tibia, and elevated alkaline phosphatase in serum. ALN and ALN + Vib enhanced capillarization and lactate dehydrogenase activity in muscle. In tibia, ALN slowed bone healing, ALN + Vib increased callus width and density, enhanced callus formation rate and expression of osteogenic genes. 8-PN and 8-PN + Vib decreased fiber size and increased capillary density in muscle; callus density and cortical width were reduced in tibia. Vibration worsened 8-PN effect on bone healing decreasing the callus width and area. Our data suggest that Vib, ALN, 8-PN, or 8-PN + Vib do not appear to aid bone healing. ALN + Vib improved bone healing; however application is questionable since single treatments impaired bone healing. Muscle responds to the anti-osteoporosis treatments and should be included in the evaluation of the drugs.

Keywords: Bisphosphonate alendronate; Bone healing; Muscle; Osteoporosis; Phytohormone 8-prenylnaringenin; Vibration.

Fig. 1

Schematic flowchart of the experiment. Twelve-week-old female rats were either ovariectomized (Ovx) or left intact (Non-Ovx). Five weeks after the rats were Ovx, the treatments with alendronate (ALN), 8-prenylnaringenin (8-PN), Vibration (Vib) or combined treatments ALN + Vib or 8-PN + Vib were started. Nine weeks after the rats were Ovx, all the rats underwent bilateral osteotomy of the tibia. After osteotomy, Vib treatments were interrupted for 5 days. Samples were analyzed 6 weeks after the osteotomy.

Fig. 2

(A) Body weight of the rats (g) either ovariectomized (Ovx) or left intact (Non-Ovx) at week 0 and treated with ALN, 8-PN, and/or vibration (Vib) during 10 weeks. Mean ± standard deviation (SD) values of at least eleven replications. (B) Food intake of the experimental animals (g/rat/day). (C) ALN doses in ALN treated groups (mg/kg BW/day). Mean ± SD values of three replications. Statistical analysis was done between the treatment groups at the respective week. Asterisk: means of Non-Ovx rats differ from those of the other groups at the respective week. (b–e) Ovx + 8-PN + Vib differs vs. Ovx (b), Ovx + Vib (c), Ovx + ALN (d), Ovx + ALN + Vib (e) (p < 0.05, Scheffé-test).

Fig. 3

Cross-sectional area of slow-twitch oxidative (SO) (A,D,G), fast-twitch oxidative (FO) (B,E), and fast-twitch glycolytic (FG) (C,F) muscle fibers in musculus soleus (MS), musculus gastrocnemius (MG), and musculus longissimus (ML), as well as the weight of MS (H) and MG (I). Mean ± standard deviation (SD) values of at least eleven replications. (a) Means differ vs. Non-Ovx, (b) vs. Ovx, (c) vs. Ovx + Vib, (d) vs. Ovx + ALN, (f) vs. Ovx + 8-PN, (g) vs. Ov + -PN + Vib (p < 0.05). Dunn-test: A,C,E. Scheffé-test: B,D,F–I.

Fig. 4

Analyses of micro-CT (A,B) and the microradiographs (C–I) of tibiae made at the osteotomy site divided into craniomedial (cran), caudal (caud) and endosteal (e) regions in Non-Ovx and Ovx rats treated with ALN, 8-PN and/or Vib during 10 weeks. Cl: Osseous callus tissue, Ct: Cortical bone, Wi: Width, Dn: Density, BV/TV: Bone volume fraction, Total BMD: Total bone mineral density. Means ± SD of at least eleven replications. (a) means differ vs. Non-Ovx, (b) vs. Ovx, (c) vs. Ovx + Vib, (d) vs. Ovx + ALN, (e) vs. Ovx + ALN + Vib, (f) vs. Ovx + 8-PN, (g) vs. Ovx + 8-PN + Vib (p < 0.05). Dunn-test: E–G. Scheffé-test: A–D,H,I.

Fig. 5

Callus area (μm²) measured in fluorescence-labeled sections of the tibia divided into craniomedial (A), caudal (B), and endosteal (C) regions in Non-Ovx and Ovx rats treated with ALN, 8-PN and/or Vib. (D) Total callus area (μm²). CG: calcein green stained callus area built within 0–22 days, AC: alizarin complexon stained area built within 23–32 days, TC: tetracycline stained area formed within 33–41 days after osteotomy. Means ± SD of at least eleven replications. Asterisk: means differs from all other groups, (a) vs. Non-Ovx, (b) vs. Ovx, (c) vs. Ovx + Vib, (d) vs. Ovx + ALN, (e) vs. Ovx + ALN + Vib, (f) vs. Ovx + 8-PN, (g) vs. Ovx + 8-PN + Vib (p < 0.05). Scheffé-test: CG endosteal, CG caudal, total caudal. Dunn-test: all other parameters.

Fig. 6

Results of the peripheral quantitative computed tomography (pQCT) measurements of the vertebrae (D–I) and abdominal CSA (A–C) in vivo at the beginning of the study, at 5 (A,D,G), 9 (B,E,H) and 15 (C,F,I) weeks. At the beginning of the study, CSA: 1361 ± 101 mm², total BMD: 506 ± 25 mg/cm³, SSI: 11 ± 2. Asterisk: means of Non-Ovx rats differed from those of the other groups, (a) vs. Non-Ovx, (b) vs. Ovx, (c) vs. Ovx + Vib, (d) vs. Ovx + ALN, (e) vs. Ovx + ALN + Vib (p < 0.05, Scheffé-test).