Estradiol and zinc-doped nano hydroxyapatite as therapeutic agents in the prevention of osteoporosis; oxidative stress status, inflammation, bone turnover, bone mineral density, and histological alterations in ovariectomized rats

Abstract

Osteoporosis (OP) is a serious health problem, and the most popular therapeutic strategy for OP is hormone replacement (estrogen); however, it increases the risk of reproductive cancers. Hydroxyapatite (HA) nanoparticles have a similar chemical structure to the bone mineral component and can be used as a new remedy for OP. This study was designed to investigate the osteoporosis-protective potential of nano zinc hydroxyapatite (ZnHA-NPs) and/or estradiol (E2) combined therapy. A total of 35 adult female rats were assigned into five groups (n = 7): 1) control group; 2) ovariectomized group (OVX); 3) OVX received oral estradiol replacement therapy (OVX/E2); 4) OVX received ZnHA replacement therapy (OVX/ZnHA); and 5) OVX received both estradiol and ZnHA-NPs combined therapy (OVX/E2+ZnHA). After 3 months of treatment, serum bone markers and estrogen level, oxidative/antioxidant, and inflammatory cytokines were determined. Additionally, femoral expression of estrogen receptors alpha and beta (ESR1; ESR2), receptor activator of nuclear factor-kappa B (RANKL) ligand, osteoprotegerin (OPG), bone mineral density (BMD), histological alterations, and immunohistochemical expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were assessed. ALP, PINP, Ca, and P concentrations improved significantly (p < 0.05) in all treatment groups, especially in the OVX/E + ZnHA group. MDA and NO were higher in OVX rats, while SOD activity and GSH were lower (p < 0.05). E2 alone or with ZnHA-NPs restored the estimated antioxidant molecules and cytokines toward normal levels in OVX rats (p < 0.05). On the other hand, E2 and ZnHA increased OPG and OC expression in femurs while decreasing ESR1, ESR2, and NF-kB expression (p < 0.05). The combination treatment was superior in the restoration of normal femoral histoarchitecture and both cortical and trabecular BMD (p < 0.05). Overall, the combined therapy of OVX/E2+ZnHA was more effective than the individual treatments in attenuating excessive bone turnover and preventing osteoporosis.

Keywords: Osteoporosis; PINP/VEGF/PCNA; estradiol; oxidative/inflammatory markers; zinc-doped nano hydroxyapatite.

FIGURE 1

Summary of the study.

FIGURE 2

Spectroscopic analyses of Zn-doped hydroxyapatite phase, (A) FT-IR spectrum, and (B) XRD diffraction pattern. TEM micrograph (C) and HR-TEM (D) of Zn-doped hydroxyapatite. The inset image on the right-hand side in (D) represents the Fast Fourier Transform (FFT) pattern of HR-TEM, showing three hkl-reflections (310, 211, and 202) of HA phase and three images of simulated lattice fringes calculated from the area inside the white-bordered square in HR-TEM image.

FIGURE 3

Effects of estrogen (E2) and/or zinc hydroxyapatite nanoparticles (ZnHA-NPs) on mRNA expression of femoral target genes in ovariectomized (OVX) rats. Data were expressed as mean ± SME (n = 5/group, triplicate). Bars carrying different superscripts are significantly different (p < 0.05). ER-α, Estrogen receptor alpha (A); ER-β, Estrogen receptor beta (B); OC, Osteocalcin (C); RANKL, receptor activator of nuclear factor-kappa B (NF-kB) ligand (D); OPG, osteoprtogrin (E).

FIGURE 4

Effects of estrogen (E2) and/or zinc hydroxyapatite nanoparticles (ZnHA-NPs) on serum concentrations of cytokines in ovariectomized (OVX) rats. Data were expressed as mean ± SME (n = 5/group, triplicate). Bars carrying different superscripts are significantly different (p < 0.05). TNF-α, tumor necrosis factor-alpha (A); IL1B, Interleukin-1β (B); IL-6, Interleukin-6 (C).

FIGURE 5

Photomicrograph of the bone tissue stained with H & E following estrogen (E2) and/or zinc hydroxyapatite nanoparticles (ZnHA-NPs) in ovariectomized (OVX) rats showing: articular cartilage (black arrow), anastomosing and thick bone trabeculae (TB), bone marrow cavity (BM), cortical bone (CB), lacunae containing osteocytes (black arrow head), distinct cement lines (green arrow head), smooth, continuous endosteal surface with osteogenic and osteoblast cells(yellow arrow head), Haversian canal (blue arrow head), blood vessel (tailed black arrow), overlying periosteum (blue arrow), thin bone trabeculae (tTB), distorted and cracked bone trabeculae (tailed green arrow), necrotic osteocytes (black tailed arrow), bone spicule island(s), distorted area of bone marrow (yellow tailed arrow), eroded endosteal surface with osteoblast absence (yellow arrow), cortical bone with disorganized bone matrix (discontinues green arrow) or with many resorption vacuoles (discontinuous black arrow), how ship’s (discontinues blue arrow) containing osteoclasts (green arrow). Note that some cracks (curved blue arrow) were detected in bones from OVX/E2 and OVX/ZnHA rats or a mosaic bony matrix appearance (curved green arrow) and discontinuous cement lines (red arrow head) were detected in bone from OVX/E2 rats. (a1,2) trabecular bone (a3-5) cortical bone of control group. (b1,2) trabecularbone (b3-5) cortical bone of OVX group. (c1,2) trabecular bone (c3-5) cortical bone of OVX/E group. (d1,2) trabecular bone (d3-5) cortical bone of OVX/ZnHA group. (e1,2) trabecular bone (e3-5) cortical bone of OVX/E+ZnHA group. Histomorphometric analysis (A-D) were expressed as mean ± SME (n = 5/group, triplicate). Bars carrying different superscripts are significantly different (p < 0.05).

FIGURE 6

Photomicrograph of bone tissue stained with Masson Trichrome stain following estrogen (E2) and/or zinc hydroxyapatite nanoparticles (ZnHA-NPs) in ovariectomized (OVX) rats showing: matrix of the trabecular bone (TB) or cortical bone (CB) with green colored collagen fibers (black arrowhead) or without green colored collagen fiber (green arrowhead). (a1,2) trabecular bone and (a3,4) cortical bone of control group. (b1,2) trabecular bone and (b3,4) cortical bone of OVX group. (c1,2) trabecular bone and (c3,4) cortical bone of OVX/E group. (d1,2) trabecular bone and (d3,4) cortical bone of OVX/ZnHA group. (e1,2) trabecular bone and (e3,4) cortical bone of OVX/E+ZnHA group. Note the green colored area was widely distributed in control group and OVX/E+ZnHA followed by OVX/ZnHA group then OVX/E group. Histomorphometric analysis (A,B) were expressed as mean ± SME (n = 5/group, triplicate). Bars carrying different superscripts are significantly different (p < 0.05).

FIGURE 7

Immunohistochemical analyses of bone tissues for VEGF protein (a1,2- b1,2- c1,2- d1,2 and e1,2- and PCNA (a3,4-b3,4-c3,4- d3,4 and e3,4-) following estrogen (E2) and/or zinc hydroxyapatite nanoparticles (ZnHA-NPs) in ovariectomized (OVX) rats. VEGF was detected in blood vessels of bone marrow (black arrow head) and some hematopoietic cells (green arrow head). PCNA expression was detected in osteoblasts or osteogenic cells (black arrow head) and some cells in bone marrow (blue arrow head). Note that there are some PCNA-negative bone marrow cells (green arrow head). (a1,3-b1,3-c1,3-d1,3- and e1,3) trabecular bone from control, OVX, OVX/E, OVX/ZnHA and OVX/E+ZnHA groups respectively. (a2,4-b2,4-c2,4-d2,4 and e2,4) cortical bone marrow from control, OVX, OVX/E, OVX/ZnHA and OVX/E+ZnHA groups, respectively. Histomorphometric analysis (A-D) were expressed as mean ± SME (n = 5/group, triplicate). Bars carrying different superscripts are significantly different (p < 0.05).

FIGURE 8

a1, b1, c1, d1 and e1 represent three-dimension image of femur within animal group. a2, b2, c2, d2 and e2 representing Average of bone mineral densities of both shafts and heads of femurs. (BMDs were measured in a region of interest [ROI) 5 x 5 voxels. Data of BMDs in head of femur (A) and shaft (B) expressed as Mean ± SEM. The different letters indicate significant differences (p < 0.05) among between experimental groups.