AVN (also called osteonecrosis) of the femoral head is defined as a pathological state with death and/or necrosis of bone marrow cells and osteocytes due to interruption of blood supply to the bone Kerachian et al., 2010a. This leads to the collapse of the necrotic segment. Factors that may cause AVN include steroid administration, alcohol abuse, hemoglobinopathies, traumatic events, vascular injury and idiopathic origins J. Anthony Parker, 1988. In the early stage, patients do not show distinct symptoms but structural and cellular changes can be observed at the histological level. To date, treatment for this disease includes non-weight bearing therapy, bone grafting (for physical support) and tantalum implants Zalavras et al., 2003b.

Glucocorticoids are absorbed through the cell membrane by special receptors and affect lipid, protein and carbohydrate metabolism. The effect of glucocorticoids on bone can be summarized by three major mechanisms: (i) the imbalance between bone resorption and bone remodeling, (ii) vascular occlusion or vascular injury, and (iii) activation of apoptotic signaling pathway by some glucocorticoids Zalavras et al.,2003b. At the cellular level, glucocorticoids can decrease mitosis, increase apoptosis by Fas pathway and inhibit type II collagen synthetic ability in osteoblasts. For osteoclasts, glucocorticoids increase the expression of differentiation cytokines, e.g. G-CSF, RANK-L and IL-6. Similar to their effect on osteoblasts, glucocorticoids can stimulate apoptosis of osteocytes and change their elastic modulus. In addition, high levels of glucocorticoids can cause inhibition of lymphoid enhancer-binding factor 1/transcription factor 7 (LEF/TCF), associated with beta-catenin, in canonical Wnt signaling pathway MacDonald et al., 2009; therefore, the transition from G1 to S phase can be restricted (Ichiseki et al.). Moreover, glucocorticoids can cause reduced growth of cartilage and reduced synthesis of collagen and proteoglycans. Many animal models of AVN have been established using different glucocorticoids, such as prednisolone Qian et al.,2008, methylprednisolone (MPS) Ichiseki et al., 2004b, and dexamethasone Yin et al., 2006. Among them, MPS, a synthetic glucocorticoid, has been demonstrated to effectively increase osteonecrosis Miyanishi et al., 2005.

Lipopolysaccharides (LPS), molecules found in the outer membrane of gram-negative bacteria, have immunostimulatory properties; they can induce systemic inflammation and sepsis through Toll-like Receptor 4 Beutler and Rietschel, 2003. Therefore, application of LPS in our model should affect cartilage Bobacz et al., 2007Ichiseki et al., 2004a. Several studies have demonstrated that LPS activates many transcription factors and cytokines, including NF-κB, IL-33 Espinassous et al., 2009. In fact, the bone marrow and immune system are functionally interconnected Zupan et al., 2013. Thus, cellular immune and pro-inflammatory cytokines can impact bone cells. Recent studies have reported that LPS can enhance a glucocorticoidinduced disease progression model Qin et al., 2006Ryoo et al., 2014Wu et al., 2008.

In this study, AVN of the femoral head was induced by combining LPS and MPS in an effort to develop an experimental mouse model. Development of such a model will be useful for evaluation of effective therapeutic modalities for AVN.

A decrease in chondrocyte numbers may be the result of injury, induced by mechanical damage or steroid drug use Chen et al., 2001. Cartilage injury leads to apoptosis or necrosis and can contribute to changes in tissue structure. MPS and LPS have been demonstrated to induce change in cartilage structure. Many glycosaminoglycans attach to proteoglycans Esko JD et al., 2009, which are secreted into the cartilage matrix by chondrocytes. The interaction between chondrocytes and cartilage matrix play an important role in proliferation and cartilage remodeling.

MPS causes a reduction in size of aggrecans or aggregate proteoglycan in the cartilage matrix. This leads to the loss of glycosaminoglycans Todhunter et al., 1996. LPS activates TLR-4, a receptor for LPS which has been identified in chondrocytes. Upon LPS-induced TLR-4 activation, enhanced phosphorylation of p38 MAPK can occur; some studies have shown that activation of p38 MAPK leads to decrease of GAG synthesis in chondrocytes in vitro. In addition, enhanced regulation of IL-1β, controlled by p38 MAPK, inhibits the production of cartilage matrix Azuma et al., 2000and consequently to the loss of matrix structure.

Moreover, Nakazawa et al. showed that apoptosis increases in corticosteroid-induced chondrocytes Nakazawa et al., 2002. The membrane of chondrocytes partly expresses Fas antigen therefore stimulation of Fas receptor can induce apoptosis of chondrocytes Lotz et al., 1999.As a result, chondrocytes disappear and fibrochondrocytes develop in drug-treated cartilage.

Collagen is distributed in almost all connective tissue. Type I collagen is the major protein in bone and occupies approximately 80% of the total proteins present in bone Wu and Eyre, 1995. Collagen has mechanical functions and plays a substantial role in its toughness. Bone collagen abnormalities can lead to bone erosion Viguet-Carrin et al., 2006. LPS and MPS have been reported to inhibit collagen and non-collagen protein synthesis in bone matrix Larjava et al., 1987Millar et al., 1986. Moreover, researchers have shown that MPS inhibits synthesis of Wnt; since Wnt-mediated signals are involved in bone formation and resorption Kobayashi et al., 2008, the inhibition of Wnt can lead to decreased bone formation [24]. Bone erosion may be also caused by negative regulation of Fas expression, which extends the life span of osteoclasts Wu et al., 2003.

LPS and MPS impact the immune system as well as proliferation of bone cells. The bone marrow cell populations include many different types, such as stem cells, adipocytes, progenitor endothelial cells and immune cells. LPS and MPS can cause apoptosis in bone marrow-derived cells, particularly immune cells. LPS induces macrophage apoptosis through two mechanisms: secretion of TNF-α (early apoptosis) and production of nitric oxide (late apoptosis) Azuma et al., 2000. Through the Fas signaling pathway, MPS promotes apoptosis of bone cells Zalavras et al., 2003a. Glucocorticoids also rapidly induce apoptosis of T cells, B cells, neutrophils and eosinophils McManus, 2003Walsh, 2000. In this study, apoptosis was clearly induced in these cells after MPS and LPS administration.

Corticosteroids have been reported to stimulate osteoclast formation and lacunar resorption Hirayama et al.,2002. LPS and MPS stimulate immune cells to produce many different types of cytokines which affect osteoclasts Stamatos et al., 2010. There are three cytokine groups: (i) cytokines which activate osteoclasts Shaarawy et al., 2003, (ii) cytokines which inhibit osteoclast activity van't Hof and Ralston, 1997, and (iii) cytokines which have dual action on osteoclasts Zupan et al., 2013. Some cytokines, such as IL1, TNF-α, prostaglandin E2, G-CSF and RANK-L, induce the differentiation and activity of osteoclasts Azuma et al.,2000Sato and Takayanagi, 2006. In contrast, cytokines like interleukin-4, gamma-interferon and transforming factor-beta inhibit both osteoclast formation and osteoclast activity Roodman, 1993. LPS binds to osteoblasts in bone through CD14 receptor and stimulates them to produce cytokines and eicosanoids, thereby activating osteoclasts Itoh et al., 2003. Osteoclasts are formed by the fusion of many cells derived from blood, such as mature monocytes and macrophagesUdagawa et al., 1990.

In this study, LPS and MPS stimulated the division of bone marrow-derived mononuclear cells, beginning at week 3 after injection. This may supply the source for the formation of osteoclasts. In our study, expression of vascular-related factors demonstrated the importance of MPS and LPS on inducing necrosis of the femoral head. Cartilage is an important structural component of the body which does not have blood vessels and nerves Sophia Fox et al., 2009. VEGFR-2 is expressed during articular cartilage growth and becomes quiescent at maturity. During osteoarthritis, the VEGFR-2 expression is also induced Lingaraj et al., 2010. A study showed that VEGFR-2 was also expressed in osteoblasts, osteoclasts and chondrocytes during the endochondral bone formation Maes and Carmeliet, 2008. During the process, hypertrophic chondrocytes die through induction of apoptosis and are replaced by osteoblasts Shapiro et al., 2005. Another study has shown that VEGF and its receptor play an important role in the initial stages of femoral head necrosis. Moreover, expression of CD31 (PECAM), a marker on endothelial cells of the major organs and large vessels, were shown to be increased- a sign of invasion of vascular cells into cartilage Melinte et al., 2012Pusztaszeri et al., 2006. In our study, the increased expression of vascular-related factors in the drug-treated mice group is an indication of invasion of blood vessel structures into the cartilage matrix, resulting in a changed cartilage structure.

This study shows that MPS, combined with LPS, can be used in mice to induce a model of early AVN of femoral head. We are aware that the mouse model does not show standardized pathogenesis of AVN of the femoral head. Nevertheless, our findings contribute to a better understanding about the process of AVN. This basic model should be suitable for investigating effective therapeutic modalities for AVN.