Osteoporosis is a disease that frequently presents asymptomatically, and is typically characterized by low bone mass, microarchitectural deterioration of bone tissue and decreased bone strength (1,2). Osteoporosis presents with symptoms such as low bone density and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture (3), and with a T-score of −2.5 standard deviations (SD) below the bone mineral density (BMD) of a healthy person of the same gender (4), which is most widely measured using dual energy X-ray absorptiometry (DXA) (5). In the United States, osteoporosis affects 2% of men and 10% of women aged ≥50 years old (6). Furthermore, 49% of older women and 30% of older men have osteopenia (7). Estimates indicate that 50% of women and 20% of men aged >50 years old will experience an osteoporosis-associated fracture. Among these osteoporosis-associated fractures, hip fracture is the most devastating of these, due to the consequent disability, mortality and costs (8). Owing to the aging of the global population, estimates suggest that the incidence of osteoporosis will double in the next 20 years (2). Consequently, an exponential increase in the numbers of fractures is anticipated, with an inevitable increased clinical and economic burden for healthcare systems.

Most strategies for treating bone loss have focused on pharmacological interventions (9); however, drug treatments may have adverse effects and poor long-term adherence, despite their effectiveness (10). Chinese herbal medicine has been used for thousands of years for the treatment of bone diseases (11). For postmenopausal women, Epimedium pubescens flavonoids are one of the most frequently used herbal compounds that is prescribed for the treatment of osteoporosis (12). Epimedium-derived phytoestrogenic flavonoids inhibit bone resorption, stimulate bone formation and prevent ovariectomy-induced osteoporosis, without resulting in uterine hyperplasia. It is suggested that these compounds have an anabolic effect on osteoporotic bone by concomitantly promoting the osteogenic differentiation of bone marrow stromal cells while suppressing adipogenic differentiation (13). Icariin (C₃₃H₄₀O₁₅; molecular weight: 676.65; Fig. 1), one of the primary active compounds within Epimedium, reportedly has an anabolic effect on the bone; it stimulates the proliferation of rat bone marrow stromal cells, increases the number that stain positive for osteocalcin (BGLAP) secretion, alkaline phosphatase (ALP) and enhances ALP activity, and calcium deposition levels in a dose-dependent manner (14,15). In previous work, we reported that icariin inhibits osteoporosis in vitro, potentially owing to its role in increasing bone morphogenetic protein-2 (BMP-2) protein expression (16), and that icariin promotes bone formation via the BMP-2/Smad4 signal transduction pathway in the hFOB 1.19 human osteoblastic cell line (17).

The present study investigated whether icariin promotes bone fracture healing in ovariectomized osteoporotic (OVX) rats in vivo, with the intention of determining a novel method to treat osteoporosis-associated fracture.

Osteoporosis, which results from a disturbance in normal bone remodeling by increasing bone resorption relative to bone formation, is identified by a low bone mass and leads to a high risk of fractures (18). Osteoporosis is often an undiagnosed disease prior to the occurrence of fracture. Bone fragility occurs due to an excess of resorption and reduced bone formation, resulting in an increased risk of hip and vertebral fractures (19). It is reported that osteoporosis affects about 25 million individuals in the United States alone, and it is estimated that women >50 years old possess an 11–18% risk of suffering a hip fracture (20). Therefore, to prevent and treat the osteoporosis, medication, exercise and additional treatments such as hormone replacement therapy are commonly used (21,22). Amongst these, hormone replacement therapy is the most widely used; however, previous evidence indicates that long-term treatments with these drugs may cause adverse reactions, such as an increased risk of ovarian and endometrial cancer (23,24). Thus, an alternative therapeutic strategy with a proven efficacy and safety is required to prevent and treat osteoporosis. Icariin, one of the primary active ingredients of Epimedium, reportedly has an anabolic effect on bones; this may contribute to its role in the induction of osteoblast proliferation and differentiation, which results in bone formation (25,26).

The ovariectomy model is a well-established animal model in osteoporosis studies (27). The OVX rat model was selected for the present study as it shares numerous similarities with postmenopausal bone loss and is recommended by the US Food and Drug Administration as a test species for evaluating the long-term skeletal safety and efficacy of osteoporosis therapies (20). Previous laboratory studies have reported that osteoporosis impairs fracture healing in early and late stages (28,29). In the present study, it was investigated whether icariin promotes bone fracture healing in OVX rats in vivo; this was determined 3 months after the ovariectomy, the unilateral cross-tibia fracture was made and the fracture had been fixed with an intramedullary nailing.

Decreased BMD is one of the major factors jeopardizing bone strength, resulting in increased susceptibility to fractures (30). The present study revealed that OVX reduced BMD in the distal femurs of female SD rats, which are rich in trabecular bone, whilst treatment with icariin prevented these decreases in BMD.

BGLAP is closely bonded with hydroxyapatite and calcium in the bone, and is an established bone formation marker; this was used to predict the bone loss rate as it is indirectly involved in the activation of osteoblasts during bone generation (31). ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids (32). ALP increases during active bone formation, as ALP is a byproduct of osteoblast activity (heightened levels of which appear in Paget's disease) (33). TRAP is a glycosylated monomeric metalloprotein enzyme expressed in mammals, which may be used as a bone resorption marker. Osteopontin and bone sialoprotein, which are bone matrix phosphoproteins, are highly efficient in vitro TRAP substrates that bind to osteoclasts when phosphorylated. Upon partial dephosphorylation, osteopontin and bone sialoprotein are incapable of binding to osteoclasts (34). From this effect, it has been hypothesized that TRAP is secreted from the ruffled membrane of osteoclasts, where it dephosphorylates osteopontin and allows osteoclast migration and additional resorption to occur (35). The OVX group significantly increased the blood BGLAP, ALP and TRAP levels, while the OVX + ICA group rescued these. These results may be due to decreased estrogen increasing the number of osteoblasts and osteoclasts, or altered activity of these cells.

The most common type of osteoporosis is the post-menopausal bone loss associated with ovarian hormone deficiency (36). Several previous studies report that estrogen is the most important hormone in maintaining bone mass and that a deficiency of this hormone is a major cause of bone loss associated with age in both genders (37–39). Notably, when the circulating estrogen level decreases, calcium in the bones rapidly decreases, and calcium loss occurs via an increase in its urinary excretion (40). In the current study, rat blood estradiol level was gauged using an ELISA kit. Those rats which experienced the menopause induced by ovariectomy demonstrated a significant decrease in blood estradiol compared with the SS group. Nian et al (41) suggested that icariin has an antiosteoporotic effect, similar to estrogen, and that it may be effective for prevention of bone fractures induced by estrogen deficiency. In the present study, the OVX + ICA group that was treated with icariin intragastrically for 5 weeks demonstrated a non-significant increase in blood estradiol compared with the OVX group. Ye et al (42) previously reported that nonconjugated forms of icaritin and desmethylicaritin, two derivatives of icariin, possess estrogen-like activity; however, icariin appeared to have no estrogenicity in the MCF-7 cell line model in vitro. Mok et al (43) revealed that icariin exerts anabolic effects in bone, possibly by activating the endoplasmic reticulum in a ligand-independent manner. In previous work, we reported that icariin inhibits osteoporosis in vitro, potentially owing to its role in increasing BMP-2 protein expression (16), and that icariin promotes bone formation via the BMP-2/Smad4 signal transduction pathway in the hFOB 1.19 human osteoblastic cell line (17).

The present study investigated the effects of icariin on callus formation, remodeling and bone union at 1, 3 and 5 weeks after treatment by examining X-rays. Callus formation and bone union increased every 2 weeks in the SS group and the fracture line was fuzzy at 5 weeks after sham surgery. In the OVX group, small calluses were formed and the fracture line was evident, revealing an absence of union. Callus formation and bone union increased temporally following icariin treatment, the fracture line was almost absent, and bone union and remodeling were observed 5 weeks after intragastric administration.

In conclusion, the present in vivo study reported that icariin attenuates the decrease in BMD in rats with osteopenia and that postfracture administration of icariin accelerates mineralization and osteogenesis and is associated with improved fracture healing. The current findings indicate that icariin has the potential to be developed as an alternative for fracture healing in postmenopausal osteoporosis. However, it should be noted that the mechanism by which icariin performs these roles remains to be examined.