Fluorine-contained hydroxyapatite suppresses bone resorption through inhibiting osteoclasts differentiation and function in vitro and in vivo

Abstract

Objectives: Fluorine, an organic trace element, has been shown to unfavourably effect osteoclasts function at a low dose. Use of hydroxyapatite (HA) has been effective in exploring its roles in promoting bone repair. In this study, we used HA modified with fluorine to investigate whether it could influence osteoclastic activity in vitro and ovariectomy-induced osteoclasts hyperfunction in vivo.

Materials and methods: Fluorohydroxyapatite (FHA) was obtained and characterized by scanning electron microscope (SEM). Osteoclasts proliferation and apoptosis treated with FHA were assessed by MTT and TUNEL assay. SEM, F-actin, TRAP activity and bone resorption experiment were performed to determine the influence of FHA on osteoclasts differentiation and function. Moreover, HA and FHA were implanted into ovariectomized osteoporotic and sham surgery rats. Histology and Micro-CT were examined for further verification.

Results: Fluorine released from FHA slowly and sustainably. FHA hampered osteoclasts proliferation, promoted osteoclasts apoptosis, suppressed osteoclasts differentiation and function. Experiments in vivo validated that FHA participation brought about an inhibitory effect on osteoclasts hyperfunction and less bone absorption.

Conclusion: The results indicated that FHA served as an efficient regulator to attenuate osteoclasts formation and function and was proposed as a candidature for bone tissue engineering applications.

Keywords: bone resorption; fluorohydroxyapatite; osteoclast; osteoporosis.

Figure 1

The sketch of fluorohydroxyapatite (FHA) structures (plane projection): Fluorine partially replaced the hydroxyl in hydroxyapatite (HA)

Figure 2

Characterization of FHA. A, presented the SEM image of FHA with porous morphology. B, EDS analysis illustrated the presence of fluorine element in FHA, including fluorine element. C, XRD patterns of FHA and HA. D, Fluorine released from FHA at a relatively slow speed and maintained a certain concentration per day, the gross concentration increased with the constant fluorine release

Figure 3

Fluorohydroxyapatite inhibited osteoclasts proliferation and promoted apoptosis. A, Representative images of TUNEL assay at 24 and 48 h, respectively, after osteoclasts were induced. B, SEM images of osteoclasts on HA and FHA, the white arrows indicated the osteoclasts. C, TUNEL staining results shown significantly more apoptotic cells in FHA group. D, Significantly less number osteoclasts were formed in FHA group. E, Induced osteoclasts were treated with fluorine‐contained FHA and fluorine‐free HA, respectively, and were then exposed to MTT assay. *: differences were considered to be statistically significant (P < 0.05)

Figure 4

FHA inhibited osteoclasts formation and function. A, Representative images of bone resorption experiment (white arrows, bone lacunae), F‐actin immunofluorescence staining (white arrows, F‐actin ring) and TRAP staining (black arrows, TRAP‐positive osteoclasts); B, Number of bone lacunae in FHA group was significantly lower than HA group; C, Bone lacunae area in FHA group was significantly lower than HA group; D, Fluorescence intensity of F‐actin staining was compared between FHA and HA group. Significant difference was observed. E.TRAP‐positive cells in FHA group had a statistical difference from HA group. *: differences were considered to be statistically significant (P < 0.05)

Figure 5

Fluorohydroxyapatite inhibited osteoclasts formation and function in vivo. A, Representative images of TRAP, H&E and Masson staining. (HB, host bone; NB, new bone) B, TRAP‐positive cells were significantly different between HA OVX and FHA OVX groups. C, New bone formation of H&E staining in HA OVX group was less than FHA OVX significantly. D, Collagen formed of Masson staining in FHA OVX group was significantly more than HA OVX group. *: differences were considered to be statistically significant (P < 0.05)

Figure 6

Fluorohydroxyapatite inhibited excessive bone resorption induced by osteoclasts hyperfunction. A, Bone tissue in the mandible bone defect was observed by Micro‐CT and three‐dimensional (3D) images were acquired and utilized for quantitative evaluation by the manufacturer's morphometric software (VGStudio MAX). Red boxes referred the interested areas. B,C, Trabecular thickness (Tb.Th, μm) and trabecular number (Tb.N, 1/mm) of HA, FHA, HA OVX and FHA OVX groups were calculated. *: differences were considered to be statistically significant (P < 0.05)