The beneficial effects of simultaneous supplementation of Lactobacillus reuteri and calcium fluoride nanoparticles on ovariectomy-induced osteoporosis

Abstract

Background: The development of new strategies to inhibit and/or treat osteoporosis as a chronic systemic disease is one of the most crucial topics. The present study aimed to investigate the simultaneous effects of calcium fluoride nanoparticles (CaF₂ NPs) and lactobacillus reuteri ATCC PTA 6475 (L. reuteri) against osteoporosis in an ovariectomized rat model (OVX).

Methods: In this study, 18 matured Wistar female rats were randomly assigned into 6 groups, including control, OVX, sham, OVX + L. reuteri, OVX + CaF₂ NPs, and OVX + L. reuteri + CaF₂ NPs. We used OVX rats to simulate post-menopausal osteoporosis, and the treatments were begun two weeks before OVX and continued for four weeks. All groups' blood samples were collected, and serum biomarkers (estrogen, calcium, vitamin D₃, and alkaline phosphatase (ALP)) were measured. The tibia and Femur lengths of all groups were measured. Histopathological slides of tibia, kidney, and liver tissues were analyzed using the Hematoxylin and Eosin staining method.

Results: Our results revealed that dietary supplementation of L. reuteri and CaF₂ NPs in low doses for 6 weeks did not show adverse effects in kidney and liver tissues. The tibial and femoral lengths of OVX rats as well as the population of osteoblasts and osteocytes and newly generated osteoid in the tibia remarkably increased in the combination therapy group. Moreover, there was a significant increase in serum estrogen levels and a significant decrease in serum calcium and alkaline phosphatase levels in combination treatment groups compared to the OVX groups not receiving the diet.

Conclusions: Our results suggest the favorable effects of the simultaneous supplementation of L. reuteri and CaF₂ NP to reduce post-menopausal bone loss.

Keywords: Bone biomarkers; Calcium fluoride nanoparticles; Lactobacillus reuteri; Osteoporosis; Ovariectomy.

Fig. 1

X-ray diffraction (XRD) spectrum of CaF₂ nanoparticles

Fig. 2

a) ICP-OES analysis. The amount of elements in CaF₂ nanoparticles (ppm) b) XRF analysis. Percentage of elements in synthesized nanoparticle c) Hydrodynamic diameter of CaF₂ nanoparticles measured by dynamic light scattering (DLS)

Fig. 3

Transmission electron microscopy (TEM) images of CaF₂ nanoparticles at different scale bars: a) 40 nm b) 60 nm c) 80 nm d) 100 nm e) 150 nm

Fig. 4

a) Average weight of all groups during different weeks of study b) tibial bone length c) femoral bone length. (** P < 0.01, *** P < 0.001, **** P < 0.0001 based on oneway ANOVA, followed by Tukey’s Multiple Comparison Test)

Fig. 5

The histological light microscopy images of H&E stained sections in tibia bone of rats

Fig. 6

The histological light microscopy images of H&E stained sections in liver of rats

Fig. 7

The histological light microscopy images of H&E stained sections in kidney of rats

Fig. 8

Evaluation of biochemical parameters in experimental groups. a) serum estrogen level. b) serum vitamin D3 level. (* P < 0.05, ** P < 0.01, *** P < 0.001 based on oneway ANOVA, followed by Tukey’s Multiple Comparison Test)

Fig. 9

Evaluation of biochemical parameters in experimental groups. a) serum calcium level. b) serum ALP level. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 based on oneway ANOVA, followed by Tukey’s Multiple Comparison Test)