Steroid-associated osteonecrosis animal model in rats

Zheng, Li-Zhen; Wang, Jia-Li; Kong, Ling; Huang, Le; Tian, Li; Pang, Qian-Qian; Wang, Xin-Luan; Qin, Ling

The present study evaluated an SAON induction protocol in rats with combination injections of LPS and MPS with different evaluation approaches to examine the metabolic disorders on the bone and other affected cross-talking tissues. The endpoint evaluation is the ON incidence evaluated by histology, which is a gold standard to evaluate ON in an animal model [24] . We observed typical ON lesions in our histological slides for proximal and distal femur and proximal and distal tibia, with similar histopathological ON features seen in severe acute respiratory syndrome (SARS) patients who received large dose of corticosteroid with SAON developed at multiple skeletal sites, including hip, knee and ankle joint [26] . We found 100% ON incidence at week 2 attributed to combined effects of LPS and large dose of pulsed MPS treatment. Corticosteroid-induced ON was through its direct effects on bone cells to induce the apoptosis of osteoblasts and osteocytes so as to impair bone formation [27, 28] . LPS-induced inflammation could inhibit bone formation and increase bone resorption, which simulated the inflammation deceases [29, 30] . In this study, the Ob. S/BS was significantly lower at the time point of week 2, suggesting that the LPS and excess corticosteroid induced cell death with reduced osteoblast number. Correspondingly, the bone formation marker PINP was significantly lower, which suggested that the total bone formation function is inhibited and accompanied downregulation of another bone formation marker OC at week 2. OC is secreted by the osteoblast and is a marker of the activity of osteoblasts' activity of the entire skeleton within the body [31] ; it is also a bone-regulated endocrine hormone to regulate energy metabolism [31] . The significantly decreased OC could induce abnormal glucose and adipose metabolism [32, 33] . Our results showed that the bone marrow fat in the SAON group was significantly larger than that in the normal control group, which could decrease the blood supply in the bone marrow region. OC could also stimulate angiogenesis [34] . Corticosteroid has antiangiogenesis effect [35] , which consequently impairs blood supply. Our Microfil and angiography results showed that the volume of blood vessels in red bone marrow region of the proximal tibia of the SAON group was significantly lower than that in the normal control group, evidence of impaired blood supply in bone marrow, and consequently the induced hypoxia would eventually cause osteonecrosis. Oedema was appeared at bone marrow at week 2 after SAON induction. It was reported that LPS-induced inflammation could induce vascular dysfunction and blood vessel leakage [36] . Our Microfil and angiography results also showed more leakage particles in the distal tibia 2 weeks after SAON induction. More leakage particles found in the present study suggested higher vessel permeability that could induce oedema, and Figure 7 Serum markers. Bone turnover markers: serum PINP and OC in the SAON group were significantly lower at week 2 and week 6 than those in the normal group; serum CTX in the SAON group was significantly lower at week 6 than that in the normal group at the same time point and than that at week 2 in the SAON group. (*: p < 0.05, **: p < 0.01, n Z 6). CTX Z carboxy-terminal telopeptide; OC Z osteocalcin; PINP Z amino-terminal propeptide of type I collagen; SAON Z steroidassociated osteonecrosis. higher marrow pressure, in turn, reduced blood flow and induced hypoxi