Patients with osteoporosis are prone to developing OPF. There was 20 percent chance of death with complications, and 20 percent or so of patients with recurrent fractures at the proximal femur of OPF patients. PMOP results from a rapid decline in estrogen levels in women after menopause, in turn, resulting in a substantial increase in bone resorption that is mediated by osteoclasts, with a non-corresponding increase in osteoblasts. Therefore, osteoporosis develops when bone resorption is greater than bone formation. Osteoporosis and PMOPF have become critical public health problems globally. The present study investigated the relationships among variations in mRNA and protein expressions of LRP5, Runx2, Osterix, and RANKL in bone specimens collected from patients with PMOPF.
Osteogenesis and osteoclastogenesis are regulated by the Wnt/β-catenin, BMP-2/Runx2/Osterix, and RANKL/RANK signaling pathways.[8–9, 13–17] Based on histological and molecular analyses, the early stage of fracture healing can be divided into: early inflammatory response stage (1 day after fracture), non-specific bone tissue anabolic stage (3 days after fracture), nonspecific catabolic stage (3 days to 1 week after fracture), and specific bone anabolic stage (more than 1 week after fracture). The entire fracture healing phase can be divided into three stages: hematoma organization, original callus formation, and callus reconstruction molding stages. Hematoma organization stage is typically completed within 2–3 weeks after fracture.[18–19] Consequently, we stratified patients with PMOPF into several groups to reflect the healing stages: Group A (1–3 days after fracture), Group B (4–7 days after fracture), and Group C (8–14 days after fracture).
Subsequently, we assessed the correlations among the variations in the expression of several factors in bones of patients and PMOPF development. No statistically significant difference was observed between patients with PMOPF and the controls with regard to age, height, and weight.
In the Wnt/β-catenin signaling pathway, the Wnt, LRP5/6 and FZD complex recruits Dvl and degradative complexes, which inhibit the phosphorylation of β-catenin by GSK-3β. Accumulation of non-phosphorylated β-catenin in the nucleus activates downstream Runx2 and other factors, which result in osteogenesis.[8, 20] LRP5 exists on the surface membranes of numerous cells. Inhibition of LRP5 impairs proliferation and functioning of osteoblasts, in turn, affecting bone formation. Glinka et al. revealed that LGR5 regulated embryonic patterns and proliferation of stem cells through Wnt/β-catenin mediated agonist of R-cavernous signaling. In the present study, we observed substantial under-expression of LRP5 in patients with PMOPF, which impaired osteogenesis and could explain the observed osteoporosis in patients with PMOPF resulting from disrupted osteogenesis. Runx2 is a highly specific biomarker for osteogenesis. Expression of Runx2 gene is essential in bone formation and development. In particular, Runx2 up-regulates transcription of genes for several mineralization-related proteins in osteoblasts.[24, 25] The Wnt/β-catenin pathway directly regulates Runx2, strengthens osteogenic differentiation, and accelerates fracture healing. In the present study, we observed significant under-expression of Runx2 in patients with PMOPF.
LRP5 regulates osteoblastosis and bone formation by activating the expression of Runx2. LRP5 and Runx2 expression levels decreased considerably within 1–3 days after bone fracture but increased thereafter, which suggests that variations in the expressions of LRP5 and Runx2 were highly correlated with osteogenic stage.
BMP-2 modulates the transcription of BMP-2/Runx2/Osterix pathway-related osteogenic genes by activating the expression of Smads. Smads relays TGF-β signals to the nucleus, regulates transcription of target factors, and induces Runx2 expression. Osterix is a key osteogenic factor downstream of Runx2, and Runx2 up-regulates Osterix expression. Osterix was under-expressed in patients with PMOPF, suggesting that the protein is a critical downstream osteogenic factor that influences PMOPF development.
A study by Kaback et al., which used mice models revealed that cartilage and tissue formation commences 7 days after fracture, and is sustained until day 10. Osterix was generally expressed after 14 days of fracture in the osteoblasts adjacent to the injury site, which promoted hardening of cartilage at the injured site. Numerous studies have demonstrated that BMP exerts a unique osteogenic effect, which fixes the fiber junction within 2 weeks after fracture.[8, 9] In the present study, Osterix expression was lowest in Group C patients (8–14 days after fracture), which was consistent with the findings.
OPG/RANKL/RANK pathway is essential in regulating differentiation of osteoclasts, including the expression of RANKL and RANK on respective cell membranes and pseudoreceptors of OPG. OPG can competitively inhibit the interaction between RANKL and RANK due to the high affinity between OPG and RANKL, further disrupting the differentiation of osteoclasts, which, in turn, induces bone resorption and apoptosis. Differentiation and maturation of osteoclasts is exclusively stimulated by RANKL, which also inhibits apoptosis in PMOPF. We observed that RANKL was over-expressed in bone specimens obtained from patients with PMOPF, which was consistent with previous findings. In contrast, the highest increase in RANKL expression level was observed within 4–7 days after fracture, which decreased thereafter, reflecting the role of RANKL in osteoclasts and PMOPF healing process.
Although we did not investigate the mechanisms underlying abnormal variations in the expressions of LRP5, Runx2, Osterix, and RANKL in bone specimens associated with PMOPF, our findings provide strong evidence that the Wnt/β-catenin, BMP-2/Runx2/Osterix, and OPG/RANKL/RANK pathways regulate osteogenesis and osteoclastogenesis in patients with PMOPF. The expression of the four factors varied with time after fracture, which could be associated with the various stages of bone repair. The characteristic variations in the expression of the factors can inform on ideal interventions effective in the prevention of PMOP and management of PMOPF.