Transcription Factor Runx2 in Necrotic Femoral Head Tissue

Osteonecrosis of the femoral head (ONFH) is a destructive degenerative disease that can develop into subchondral and articular facet cartilage collapse. Among proposed pathological mechanisms underlying ONFH, much attention has been paid to the theory of increased intraosseous pressure. The core of this theory is an unbalance in osteoblast and adipocyte differentiation of bone marrow mesenchymal stem cells (BM-MSCs). The occurrence and development of osteonecrosis has been related to abnormal metabolism and differentiation of BM-MSCs. Normal adult BM-MSCs can differentiate into adipocytes and osteoblasts to maintain normal physical status. In response to exogenous stimuli (for example, use of exogenous hormones or alcohol), the bone marrow microenvironment changes to allow more BM-MSCs to differentiate into adipocytes, leading to increased bone marrow cavity pressure, thereby inducing ischemic ONFH.

Overexpression of Runx2, an osteoblast-specific transcription factor, can increase osteoblast differentiation of BM-MSCs, thereby strengthening the effects of BM-MSC transplantation to repair bone defects and necrosis. By controlling expression of osteocalcin, an osteoblast-specific gene, Runx2 also controls osteoblast differentiation and functioning. Osteocalcin, a common indicator used to evaluate bone formation and conversion rate, is a bone metabolism-regulating factor generated and secreted by osteoblasts; thus, it is considered to be a good marker for bone functioning. In a previous study by Chen et al., seven patients with glucocorticoid-induced ONFH at Association Research Circulation Osseous (ARCO) Stage IV were included as an experimental group and seven patients with femoral neck fracture served as a control group. Immunohistochemical staining and quantitative polymerase chain reaction (PCR) were used to detect osteocalcin immunoreactivity and Runx2 expression in femoral head and neck tissue. They found that glucocorticoid-induced ONFH is likely closely related to osteocalcin.

ONFH results from an interruption of the blood supply to the femoral head or injury-caused death of chondrocytes and bone marrow components. During subsequent repair processes, bone morphogenetic proteins (BMPs) not only stimulate BM-MSCs to differentiate into osteoblasts, but also promote osteoblast growth; BMP-2 is the key factor regulating bone tissue formation.

Sclerotin loss after ONFH is closely related to an unbalance in osteoclast activity and differentiation. Osteoprotegerin can inhibit bone absorption of mature osteoblasts and induce apoptosis of osteoblasts. Osteoprotegerin and its ligand system have been confirmed as key factors regulating osteoblast formation and differentiation, while bone absorption plays an important role in the pathogenesis and treatment of osteoporosis, osteoarthritis and bone tumors.

To the best of our knowledge, there have been no controlled clinical trials examining BMP, osteocalcin, osteoprotegerin or, in particular, Runx2 messenger RNA (mRNA) expression in femoral head tissue of patients with ONFH at Ficat Stage III-IV and osteoarthritis. In this non-randomized, parallel-controlled trial, we will use real-time PCR (RT-PCR) to detect Runx2, BMP-2, BMP-7 and osteoprotegerin mRNA expression in femoral head tissue from patients with ONFH at Ficat Stage III-IV. Simultaneously, we will detect osteocalcin immunoreactivity using an immunohistochemical staining method, and compare with osteoarthritis patients for the purpose of clarifying mechanisms of these factors in bone reconstruction post-ONFH.

Data collection, management, analysis and open access Data collection: According to the trial design, a table will be formulated for data collection. Collected data will be input into an electronic database by professional staff using a double-data entry strategy.

Data management: Information accuracy will be checked when all recruited patients are followed up. The database will be locked by the researcher in charge and will not be altered. All information relating to this trial will be preserved by Shengjing Hospital of China Medical University, China.

Data analysis: The electronic database will be fully disclosed to a professional statistician for statistical analysis.

Data open access: Published data will be available at www.figshare.com. Statistical analysis Statistical analysis will be performed by a professional statistician (blinded to grouping) using SPSS 19.0 software. If Runx2, BMP-2, BMP-7 and osteoprotegerin mRNA expression, as well as osteocalcin immunoreactivity in the femoral head and neck of patients with ONFH and osteoarthritis, are normally distributed, a two-sample t-test will be used to compare differences between groups. If, however, these data are not normally distributed, a Mann-Whitney U test will be used. A level of P < 0.05 will be considered statistically significant.