Curculigoside Protects against Titanium Particle-Induced Osteolysis through the Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Formation.

Wear particle-induced periprosthetic osteolysis is mainly responsible for joint replacement failure and revision surgery. Curculigoside is reported to have bone-protective potential, but whether curculigoside attenuates wear particle-induced osteolysis remains unclear. In this study, titanium particles (Ti) were used to stimulate osteoblastic MC3T3-E1 cells in the presence or absence of curculigoside, to determine their effect on osteoblast differentiation. Rat osteoclastic bone marrow stromal cells (BMSCs) were cocultured with Ti in the presence or absence of curculigoside, to evaluate its effect on osteoclast formation in vitro. Ti was also used to stimulate mouse calvaria to induce an osteolysis model, and curculigoside was administrated to evaluate its effect in the osteolysis model by micro-CT imaging and histopathological analyses. As the results indicated, in MC3T3-E1 cells, curculigoside treatment attenuated the Ti-induced inhibition on cell differentiation and apoptosis, increased alkaline phosphatase activity (ALP) and cell mineralization, and inhibited TNF-α, IL-1ß, and IL-6 production and ROS generation. In BMSCs, curculigoside treatment suppressed the Ti-induced cell formation and suppressed the TNF-α, IL-1ß, and IL-6 production and F-actin ring formation. In vivo, curculigoside attenuated Ti-induced bone loss and histological damage in murine calvaria. Curculigoside treatment also reversed the RANK/RANKL/OPG and NF-κB signaling pathways, by suppressing the RANKL and NF-κB expression, while activating the OPG expression. Our study demonstrated that curculigoside treatment was able to attenuate wear particle-induced periprosthetic osteolysis in in vivo and in vitro experiments, promoted osteoblastic MC3T3-E1 cell differentiation, and inhibited osteoclast BMSC formation. It suggests that curculigoside may be a potential pharmaceutical agent for wear particle-stimulated osteolysis therapy.

Culture and characterization of rat hair follicle stem cells.

The purpose of this study was to establish methods for isolation, culture, expansion, and characterization of rat hair follicle stem cells (rHFSCs). Hair follicles were harvested from 1-week-old Sprague-Dawley rats and digested with dispase and collagenase IV. The bulge of the hair follicle was dissected under a microscope and cultured in Dulbecco's modified Eagle's medium/F12 supplemented with KnockOut™ Serum Replacement serum substitute, penicillin-streptomycin, L-glutamine, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, polyhydric alcohol, and hydrocortisone. The rHFSCs were purified using adhesion to collagen IV. Cells were characterized by detecting marker genes with immunofluorescent staining and real-time polymerase chain reaction (PCR). The proliferation and vitality of rHFSCs at different passages were evaluated. The cultured rHFSCs showed typical cobblestone morphology with good adhesion and colony-forming ability. Expression of keratin 15, integrin α6, and integrin ß1 were shown by immunocytochemistry staining. On day 1-2, the cells were in the latent phase. On day 5-6, the cells were in the logarithmic phase. Cell vitality gradually decreased from the 7th passage. Real-time PCR showed that the purified rHFSCs had good vitality and proliferative capacity and contained no keratinocytes. Highly purified rHFSCs can be obtained using tissue culture and adhesion to collagen IV. The cultured cells had good proliferative capacity and could therefore be a useful cell source for tissue-engineered hair follicles, vessels, and skin.

[Effect of elongated-needle penetration intervention on spinal apoptosis and cell signal transduction in acute spinal cord injury rabbits].

OBJECTIVE: To observe the effect of elongated-needle penetration (ENP) stimulation of "Zhibian" (BL 54), "Shuidao" (ST 28), "Qihai" (CV 6) and "Zhongji" (CV 3) on spinal nerve cell apoptosis and cellular signal transduction in spinal cord injury rabbits, so as to reveal its mechanism underlying improvement of spinal injury. METHODS: A total of 80 adult Newzealand rabbits were randomized to control, model, ENP, ENP + LY 294002 (PI3K antagonist), ENP + PD 98059 (MEK antagonist) groups, with 16 rabbits in each group. The spinal cord injury model was established by using modified Allen's method (Gravity-drop device). Elongated-needle penetration was applied to bilateral BL 54, ST 28, CV 3 and CV 6, once daily for 3 times. For rabbits of the ENP+ LY 294002 and ENP+ PD 98059 groups, LY 294002 (10 microg, 20 microL), PD 98059 (3 microg, 20 microL) were separately given by intrathecal injection. Pathomorphological changes of the injured spinal cord (T13-L1) were observed after H.E. stain. Spinal cell apoptosis was detected by TUNEL,and phosphorylated (p)-Akt and p-ERK1/2 immunoactivity was detected by immunohistochemistry, and the expression levels of p-Akt, p-ERK1/2, cytochrome C (Cyt C) and Caspase-3 proteins were determined by Western blot (WB), and serum TNF-alpha content was assayed by ELISA. RESULTS: H. E. staining showed apparent structural changes as hemmorrhage, inflammatory cell infiltration, cellular edema and necrosis, and formation of vacuolation in the spinal cord in the model group, which was marked milder in the ENP group. TUNEL assay showed that the rate of apoptotic cells was notably increased in the model group than in the control group (P < 0.05), obviously decreased in the ENP group when compared with the model group (P < 0.05). Immunohistochemistry, WB and ELISA results showed that compared with the control group, spinal p-Akt and p-ERK1/2 protein expression levels in the model group were significantly decreased (P < 0.05), and Cyt C and Caspase-3 expression levels and serum TNF-a content were significantly increased in the model group (P < 0.05). Compared with the model group, the expression levels of p-Akt, p-ERK1/2 were significantly increased in the ENP group (P < 0.05), while Cyt C and Caspase-3 expression levels and TNF-alpha content were significantly down-regulated in the ENP group (P < 0.05). After intrathecal injection of PI3K and MEK antagonists, the effects of ENP were significantly weakened in reducing apoptosis rate, upregulating p-Akt and p-ERK1/2 expression and in down-regulating Cyt C and Caspase-3 expression and TNF-alpha content (P < 0.05), suggesting important roles of ERK1/2 mediated extracellular and PI3K/Akt mediated intracellular apoptotic signal transduction pathways in ENP induced repair of the traumatic tissues. CONCLUSION: ENP stimulation can decrease spinal injury and cell apoptosis in spinal injury rabbits, which may be closely related to its effects in up-regulating p-Akt and p-ERK1/2 and down-regulating Cyt C and Caspase-3 expression levels in the spinal cord and serum TNF-alpha content.

Short- and long-term effects of vertebroplastic bone cement on cancellous bone.

Vertebroplasty using poly(methyl methacrylate) (PMMA) bone cement is the most common method to treat osteoporotic vertebral fractures. However, several questions of interest remain to be clarified, including how does PMMA affect the cement-bone interface area and surrounding bone tissue, can damaged bone tissues be repaired, how will PMMA change the bone interface over the long-term, and what happens to PMMA itself? The purpose of this study is to investigate these concerns and provide a basis for clinical evaluation. We made bone defects in the lumbar vertebrae of New Zealand rabbits as a model of osteoporosis and injected them with bone cement. A mechanical testing machine was used to perform axial compression, three-point bending, and twisting resistance tests to observe and investigate the short- and long-term biomechanical properties of PMMA after implantation. Optical, fluorescence, scanning electron microscopy, and nanoindentation were used to observe the changes in the interface microstructure. PMMA can rapidly establish the strong support with stable function in the near future. Biomechanical experiments showed that biomechanical property of bone cement group was significantly higher than those in the other two groups (P<0.05) biomechanical property of bone cement group may decline with the time, but it's still better than that of OP in the control group (P<0.05). Histomorphological observation result shows that under osteoporosis state the bone grows slower, also bone's rebuilding time extended. And in the later period, main bone's continuous osteoporosis has some impact on the interface. Nano-indentation testing shows that the young modulus and stiffness of the interface among bone, material and interface were significantly differences (P<0.05). Bone cement had gave the best nano indentation hardness, then was interface and bone tissue. PMMA bone cement was able to quickly support and stabilize the defect in the short term, and bone growth restarted at the bone interface and was tightly integrated. However, over the long-term, fluorescent signal was weakened, osteoclasts appeared, the mechanical indicators for both the interface and the whole vertebra decreased, and bone resorption was eventually greater than bone formation, resulting in bone loss. Therefore, vertebroplasty is not the end of treatment, and we need to further study ways to improve the bone cement material, which is crucial for long-term vertebroplasty efficacy, to better treat osteoporosis.