Monotropein Protects Mesenchymal Stem Cells from Lipopolysaccharide-Induced Impairments and Promotes Fracture Healing in an Ovariectomized Mouse Model.

Monotropein is one of the active ingredients in Morinda Officinalis, which has been used for the treatment in multiple bone and joint diseases. This study aimed to observe the in vitro effects of Monotropein on osteogenic differentiation of lipopolysaccharide treated bone marrow mesenchymal stem cells (bMSCs), and the in vivo effects of local application of Monotropein on bone fracture healing in ovariectomized mice. Lipopolysaccharide was used to set up the inflammatory model in bMSCs, which were treated by Monotropein. Molecular docking analysis was performed to evaluate the potential interaction between Monotropein and p65. Transverse fractures of middle tibias were established in ovariectomized mice, and Monotropein was locally applied to the fracture site using injectable hydrogel. Monotropein enhanced the ability of primary bMSCs in chondro-osteogenic differentiation. Furthermore, Monotropein rescued lipopolysaccharide-induced osteogenic differentiation impairment and inhibited lipopolysaccharide-induced p65 phosphorylation in primary bMSCs. Docking analysis showed that the binding activity of Monotropein and p65/14-3-3 complex is stronger than the selective inhibitor of NF-κB (p65), DP-005. Local application of Monotropein partially rescued the decreased bone mass and biomechanical properties of callus or healed tibias in ovariectomized mice. The expressions of Runx2, Osterix and Collagen I in the 2-week callus were partially restored in Monotropein-treated ovariectomized mice. Taking together, local application of Monotropein promoted fracture healing in ovariectomized mice. Inhibition of p65 phosphorylation and enhancement in osteogenesis of mesenchymal stem cells could be partial of the effective mechanisms.

Changes in macrophage and inflammatory cytokine expressions during fracture healing in an ovariectomized mice model.

BACKGROUND: Macrophages and inflammatory cytokines play important roles in bone fracture healing. However, the expression patterns of macrophages and inflammatory cytokines during fracture healing under the condition of postmenopausal osteoporosis have not been fully revealed. METHODS: Tibia transverse fracture was established 12 weeks after ovariectomy or sham operation in 16-week old female mice. Tibias were harvested before fracture or 1, 3, 5, 7, 14, 21, 28 days after fracture for radiological and histological examinations. M1/M2 inflammatory macrophages, osteal macrophages and gene expressions of tumor necrosis factor-α, interleukin-6, interleukin-1ß and macrophage conversion related molecules in the fracture haematoma or callus were also detected. RESULTS: The processes of fracture healing, especially the phases of endochondral ossification and callus remodeling, were delayed in ovariectomized mice. The expressions of tumor necrosis factor-α and interleukin-6, but not interleukin-1ß, in the fracture haematoma or callus were disturbed. Expressions of tumor necrosis factor-α were decreased at 1, 14 and 21 days post-fracture (DPF), and were increased at 3, 5 and 7 DPF. Interleukin-6 expressions at 1, 3 and 21 DPF were significantly increased. We found the decreases in M1 and M2 macrophages at 1 DPF of the initial inflammatory stage. M2 macrophages at 14 DPF of the middle stage and osteal macrophages at 14, 21 and 28 DPF of the middle and late stages of fracture healing were also reduced in ovariectomized mice. CONCLUSIONS: The expressions of macrophages and inflammatory cytokines were impaired in ovariectomized mice, which might contribute partially to poor fracture healing.

Local Application of Tanshinone IIA protects mesenchymal stem cells from apoptosis and promotes fracture healing in ovariectomized mice.

BACKGROUND: Elderly patients suffering from osteoporotic fractures are more susceptible to delayed union or nonunion, and their bodies then are in a state of low-grade chronic inflammation with decreased antioxidant capacity. Tanshinone IIA is widely used in treating cardiovascular and cerebrovascular diseases in China and has anti-inflammatory and antioxidant effects. We aimed to observe the antioxidant effects of Tanshinone IIA on mesenchymal stem cells (MSCs), which play important roles in bone repair, and the effects of local application of Tanshinone IIA using an injectable biodegradable hydrogel on osteoporotic fracture healing. METHODS: MSCs were pretreated with or without different concentrations of Tanshinone IIA followed by H2O2 treatment. Ovariectomized (OVX) C57BL/6 mice received a mid-shaft transverse osteotomy fracture on the left tibia, and Tanshinone IIA was applied to the fracture site using an injectable hydrogel. RESULTS: Tanshinone IIA pretreatment promoted the expression of nuclear factor erythroid 2-related factor 2 and antioxidant enzymes, and inhibited H2O2-induced reactive oxygen species accumulation in MSCs. Furthermore, Tanshinone IIA reversed H2O2-induced apoptosis and decrease in osteogenic differentiation in MSCs. After 4 weeks of treatment with Tanshinone IIA in OVX mice, the bone mineral density of the callus was significantly increased and the biomechanical properties of the healed tibias were improved. Cell apoptosis was decreased and Nrf2 expression was increased in the early stage of callus formation. CONCLUSIONS: Taken together, these results indicate that Tanshinone IIA can activate antioxidant enzymes to protect MSCs from H2O2-induced cell apoptosis and osteogenic differentiation inhibition. Local application of Tanshinone IIA accelerates fracture healing in ovariectomized mice.