Single-dose local simvastatin injection improves implant fixation via increased angiogenesis and bone formation in an ovariectomized rat model.

BACKGROUND: Statins have been reported to promote bone formation. However, taken orally, their bioavailability is low to the bones. Implant therapies require a local repair response, topical application of osteoinductive agents, or biomaterials that promote implant fixation. MATERIAL/METHODS: The present study evaluated the effect of a single local injection of simvastatin on screw fixation in an ovariectomized rat model of osteoporosis. RESULTS: Dual-energy X-ray absorptiometry, micro-computed tomography, histology, and biomechanical tests revealed that 5 and 10 mg simvastatin significantly improved bone mineral density by 18.2% and 22.4%, respectively (P<0.05); increased bone volume fraction by 51.0% and 57.9%, trabecular thickness by 16.4% and 18.9%, trabeculae number by 112.0% and 107.1%, and percentage of osseointegration by 115.7% and 126.3%; and decreased trabeculae separation by 34.1% and 36.6%, respectively (all P<0.01). Bone mineral apposition rate was significantly increased (P<0.01). Furthermore, implant fixation was significantly increased (P<0.05), and bone morphogenetic protein 2 (BMP2) expression was markedly increased. Local injection of a single dose of simvastatin also promoted angiogenesis. Vessel number, volume, thickness, surface area, and vascular volume per tissue volume were significantly increased (all P<0.01). Vascular endothelial growth factor (VEGF), VEGF receptor-2, von Willebrand factor, and platelet endothelial cell adhesion molecule-1 expression were enhanced. CONCLUSIONS: A single local injection of simvastatin significantly increased bone formation, promoted osseointegration, and enhanced implant fixation in ovariectomized rats. The underlying mechanism appears to involve enhanced BMP2 expression and angiogenesis in the target bone.

Femoral perfusion after pulsed electromagnetic field stimulation in a steroid-induced osteonecrosis model.

This study was designed to evaluate femoral perfusion after pulsed electromagnetic field (PEMF) stimulation in a steroid-induced osteonecrosis rabbit model by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Steroid-induced osteonecrosis was produced by single intramuscular injection of methylprednisolone in 15 rabbits. Eight rabbits underwent PEMF stimulation (PEMF group) and seven did not (control group). DCE-MRI was performed before PEMF stimulation, immediately before steroid administration, and 1, 5, 10, and 14 days after steroid administration. Regions of interest were set in the bilateral proximal femora. Enhancement ratio (ER), initial slope (IS), and area under the curve (AUC) were analyzed. ER, IS, and AUC in the control group significantly decreased after steroid administration compared with before administration (P<0.05). In PEMF group, IS significantly decreased; however, ER and AUC showed no significant differences after steroid administration compared with before. ER and IS in PEMF group were higher than in control group until 10th day, and AUC was higher until 5th day after steroid administration (P<0.05). PEMF stimulation restrains the decrease in blood flow after steroid administration.

Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model.

Plant-derived phytoestrogens have bone protective effects, but the molecular mechanism behind these effects remains unclear. This study is aimed at fully characterizing the fracture healing process of formononetin, and investigating the mechanism underlying angiogenesis in calluses of a rat fracture model. Femoral fractures were produced in 2-month-old Sprague-Dawley rats. A 20 microg/kg or 200 microg/kg dose of formononetin was orally administrated once a day during the healing period of 21 days. The results showed that in the early stage of chondrogenesis (days 3), formononetin significantly increased the number of vessels, and expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2/flk-1) compared with control. However, the larger dose of formononetin had no significant difference on expression of VEGF and VEGFR-2/Flk-1 compared with that of the smaller dose of formononetin. After 7 days of administration, formononetin markedly induced differentiation of mesenchymal stem cells in the fracture site. After 14 days, gene expression of mesenchymal progenitors such as alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), indicating osteogenic differentiation, was markedly stimulated by formononetin compared with control. These results suggest that formononetin promotes early fracture healing through angiogenesis activation in the early stage of fracture repair, and osteogenesis acceleration in the later stages, and thus may be beneficial for fracture healing.

Preventive effects of genistein on leukocyte adhesion in femur venules and on bone-loss induced in ovariectomized female rats.

This study was aimed to examine effects of genistein on leukocyte adhesion in femur microcirculation in relation to bone-loss induced in ovariectomized female rats. Sixty-four female Wistar rats were divided into 4 groups: sham (daily treated with vehicle; DMSO, sc; 100 microl/day), ovariectomized rat treated with vehicle (OVX(veh)), 17beta-estradiol treated-ovariectomized rat (OVX(E2), 5 microg/kg/day, s.c.) and genistein treated-ovariectomized rat (0.25 mg/kg/day, s.c.; OVX(gen)). One and three weeks after the ovariectomy, blood flow perfusion (BF) in femur tissue was measured using laser Doppler flowmetry. Leukocyte adhesion in femur venules (15-30 microm in diameter) of each group was evaluated by intravital fluorescence videomicroscopy. The bone mineral content (BMC) was measured and expressed in terms of ratio of ash-to-dry matter weight. Serum osteocalcin and alkaline phosphatase levels were determined using chemiluminescence immunoassay. In both one and three week-OVX(veh), leukocyte adhesion increased significantly, compared to their age-matched sham groups, but it decreased significantly in OVX(gen), compared to OVX(veh) (p<0.05). In three week-OVX(gen), both BF and BMC increased significantly, but osteocalcin and alkaline phosphatase levels decreased, compared to those of three week-OVX(veh). In conclusion, genistein supplementation could effectively prevent bone-loss and microvascular endothelial dysfunction induced by estrogen deficiency.

Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition.

OBJECTIVES: This study tested the effect of Epimedium-derived phytoestrogen (PE) on preventing steroid-associated osteonecrosis (ON) in rabbit model. METHODS: Thirty 28-week-old male New-Zealand white rabbits were divided into control group (CON; n=14) and PE group (PE; n=16; 5 mg/kg body weight/day) after receiving an established inductive protocol for inducing steroid-associated ON. Before and after inductive protocol, Dynamic-MRI was employed on bilateral femora for local intra-osseous perfusion, blood samples were examined for coagulation, fibrinolysis and lipid-transportation, and marrow samples were quantified for adipogenesis-gene mRNA expression. Six weeks later, bilateral femora were dissected for Micro-CT-based micro-angiography, and then ON lesion, intravascular thrombosis and extravascular fat-cell-size were examined histopathologically. RESULTS: The incidence of ON in the PE group (31%) was significantly lower than that in the CON group (93%). Compared to the CON group, local intra-osseous perfusion was maintained in the PE group. Blocked trunk vessels were seldom found in micro-angiography of the PE-treated rabbits. Thrombosis incidence and fat-cell-size were both significantly lower in the PE group than those in the CON group. During the early period after induction, indicator of coagulation, fibrinolysis, lipid-transportation and adipogenesis-gene expression were found with significantly changing pattern in the PE group compared to the CON group. CONCLUSION: PE was able to exert beneficial effect on preventing steroid-associated ON in rabbits with inhibition of both thrombosis and lipid deposition.

Effects of non-weight bearing and hyperbaric oxygen therapy in vascular deprivation-induced osteonecrosis of the rat femoral head.

We examined the role of hyperbaric oxygenation (HBO2) combined with non-weight bearing (NWB) in the treatment of vascular deprivation-induced osteonecrosis of the femoral head in the rat. Group 1 included 16 rats treated by a combination of NWB and HBO2. Twenty animals treated by NWB alone (group 2), and 18 rats which received no treatment (group 3), served as the control groups. Maximal benefit of HBO2 was observed on Day 30 of the study. The femoral heads were less deformed in group 1 animals (P = 0.07). Preservation of the femoral heads was observed in a larger proportion of the HBO2-treated animals (P = 0.06). A smaller proportion of high-grade new bone formation was observed, and more animals demonstrated well-regenerated hematopoietic tissue (P = 0.08). The tendency for less deformation of the femoral head in the HBO2-treated group might be a predictor of better function of the hipjoint.

Anti-thrombotic efficacies of enoxaparin, dalteparin, and unfractionated heparin in venous thrombo-embolism.

BACKGROUND: Few data exist by which the anti-thrombotic efficacy of different anticoagulants may be compared. We used a radiolabeled antibody specific for polymerizing fibrin to compare the in vivo anti-thrombotic potencies of different systemic anticoagulants (enoxaparin, dalteparin, and unfractionated heparin). METHODS AND RESULTS: Deep venous thrombi (DVTs) were induced in dogs' femoral veins. The dogs were then treated with one of the following subcutaneous regimens: enoxaparin 100 units/kg (1.0 mg/kg) every 12 hours (n=4), dalteparin 200 units/kg every 24 hours (n=4), or unfractionated heparin 240 units/kg every 8 hours with dose adjustment via aPTT (n=3). 111Indium-labeled anti-fibrin antibodies, specific for propagating thrombi, were given intravenously and nuclear scans of the legs were taken over the following 24 hours. Thrombus propagation was estimated by the ratio of gamma emissions from the legs containing DVTs divided by the emissions from the contralateral "control" legs. DVTs accumulated labeled anti-fibrin antibodies at the same rates in both the enoxaparin group and the dalteparin group (gamma emissions 171+/-6% and 168+/-36% of control by 24 hours, respectively). DVTs in the adjusted dose unfractionated heparin group tended to accumulate antibodies at a slower rate (129+/-19% of control by 24 hours). CONCLUSIONS: Enoxaparin and dalteparin inhibited propagation of pre-formed thrombi to the same degree. Subcutaneous unfractionated heparin, adjusted every 8 hours by aPTT, tended to suppress ongoing thrombosis more than either LMWH.

Hyperbaric oxygen: a means of decreasing ischemic epiphyseal damage in a pediatric rabbit model.

The effect of hyperbaric oxygen on epiphyseal ischemia was evaluated using a pediatric rabbit model. Forty-five animals were compared in this study: 23 from a control pilot study and 22 hyperbaric exposed animals. In each animal the right distal femoral and proximal tibial epiphyses were isolated on a popliteal vascular pedicle. The left leg acted as the control. The growth difference between the rabbit's hindlimbs was the means of comparison throughout the groups established. Warm ischemia was induced by applying a vascular clamp to the right popliteal artery for 12 hours (20 animals) and 7 hours (17 animals). The remaining 8 animals underwent a sham operation without interruption of epiphyseal perfusion. On completion of the ischemic period hyperbaric oxygen therapy (HBOT) was performed on 12 12-hour (12h-HBOT) and 10 7-hour (7h-HBOT) animals at 2 atmospheres for 90 minutes twice per day for 4 postoperative days. The animals were killed on either postoperative day 14 or 90. Measurement of longitudinal bone growth was performed on the 90-day animals from serial radiographs at the time of surgery and then at 1 month, 2 months, and 3 months after surgery. There was no significant difference in longitudinal bone growth between the sham-operated and the 7h-HBOT animals at 1, 2, and 3 months. There was a statistically significant difference, however, between the normal growth of the 7h-HBOT group compared with the abnormal growth of the 7-hour, 12-hour, and 12h-HBOT animals. Histology was consistent, with the bone growth data demonstrating relative normalcy of the 7h-HBOT group epiphyseal plates versus severe architectural aberrance and necrosis of the 12h-HBOT group epiphyses. Our experimental data indicate that a clinical trial should be instituted using HBO for pediatric replantation patients when warm ischemia exceeds 7 hours. (J Hand Surg 2000; 25A:159-165.

[Superficial blood flow (SBF) in 193 patients with traumatic paraplegia].

SBF in 15 regions (sacral spot and bilateral nail-fold, Zu-San-Li, San-Yin-Jiao, Tai-Xi, Xing-Jian, Guan-Yuan-Shu and femur) was measured by means of Laser Doppler Flowmeter in 193 patients with paraplegia resulted from trauma during the Tangshan earthquake, and the result was compared with that in 53 normal subjects. The results showed: (1) The SBF in region of the patients' limbs was more decreased than that in normal subjects (P less than 0.001). (2) There was also a declining of SBF in the right femur as compared with the normal (P less than 0.001). (3) The bilateral nail-fold blood flow in 55 patients with injury of vertebrae thoracales was significantly lower than that in 38 patients with lumbar vertebrae injury, indicating that having lived for 12 years since spinal cord injury, the patients still have microcirculatory disturbance over the whole body. Since SBF at the predilection site of decubital ulcer was decreased significantly more than that at non-ulcer site, it would be very important to improve local microcirculatory perfusion for prevention and treatment of decubital ulcer.

Functional changes in the vascularity of the irradiated rat femur. Implications for late effects.

Early and late changes in the extraction of 125I antipyrine was investigated in the rat femur after local irradiation with single doses of 5 to 25 Gy. The extraction of antipyrine by bone was generally reduced after irradiation, with the greatest effect being found 3 months after treatment. However, the effect was transient and by 7 months after less than or equal to 20 Gy, antipyrine extraction was similar to that in the normal femur. Extraction was still reduced after 25 Gy. The first significant reduction in dry bone weight occurred 7 months after 15, 20 and 25 Gy. The fall in the calcium and phosphorus content of bone was similar to that of the dry bone weight. Calcium/phosphorus ratio was not modified in irradiated bone. The likely role of the vascular changes in the subsequent development of bone atrophy is discussed.